Major Histocompatibility Complex-Dependent Cytotoxic T Lymphocyte Repertoire and Functional Avidity Contribute to Strain-Specific Disease Susceptibility after Murine Respiratory Syncytial Virus Infection ▿

PubMed Central

Jessen, Birthe; Faller, Simone; Krempl, Christine D.; Ehl, Stephan

2011-01-01

Susceptibility to respiratory syncytial virus (RSV) infection in mice is genetically determined. While RSV causes little pathology in C57BL/6 mice, pulmonary inflammation and weight loss occur in BALB/c mice. Using major histocompatibility complex (MHC)-congenic mice, we observed that the H-2d allele can partially transfer disease susceptibility to C57BL/6 mice. This was not explained by altered viral elimination or differences in the magnitude of the overall virus-specific cytotoxic T lymphocyte (CTL) response. However, H-2d mice showed a more focused response, with 70% of virus-specific CTL representing Vβ8.2+ CTL directed against the immunodominant epitope M2-1 82, while in H-2b mice only 20% of antiviral CTL were Vβ9+ CTL specific for the immunodominant epitope M187. The immunodominant H-2d-restricted CTL lysed target cells less efficiently than the immunodominant H-2b CTL, probably contributing to prolonged CTL stimulation and cytokine-mediated immunopathology. Accordingly, reduction of dominance of the M2-1 82-specific CTL population by introduction of an M187 response in the F1 generation of a C57BL/6N × C57BL/6-H-2d mating (C57BL/6-H-2dxb mice) attenuated disease. Moreover, disease in H-2d mice was less pronounced after infection with an RSV mutant failing to activate M2-1 82-specific CTL or after depletion of Vβ8.2+ cells. These data illustrate how the MHC-determined diversity and functional avidity of CTL responses contribute to disease susceptibility after viral infection. PMID:21795345

Characterization of choline transporters in the human placenta over gestation.

PubMed

Baumgartner, Heidi K; Trinder, Kinsey M; Galimanis, Carly E; Post, Annalisa; Phang, Tzu; Ross, Randal G; Winn, Virginia D

2015-12-01

The developing fetus relies on the maternal blood supply to provide the choline it requires for making membrane lipids, synthesizing acetylcholine, and performing important methylation reactions. It is vital, therefore, that the placenta is efficient at transporting choline from the maternal to the fetal circulation. Although choline transporters have been found in term placenta samples, little is known about what cell types express specific choline transporters and how expression of the transporters may change over gestation. The objective of this study was to characterize choline transporter expression levels and localization in the human placenta throughout placental development. We analyzed CTL1 and -2 expression over gestation in human placental biopsies from 6 to 40 weeks gestation (n = 6-10 per gestational window) by immunoblot analysis. To determine the cellular expression pattern of the choline transporters throughout gestation, immunofluorescence analysis was then performed. Both CTL1 and CTL2 were expressed in the chorionic villi from 6 weeks gestation to term. Labor did not alter expression levels of either transporter. CTL1 localized to the syncytial trophoblasts and the endothelium of the fetal vasculature within the chorionic villous structure. CTL2 localized mainly to the stroma early in gestation and by the second trimester co-localized with CTL1 at the fetal vasculature. The differential expression pattern of CTL1 and CTL2 suggests that CTL1 is the key transporter involved in choline transport from maternal circulation and both transporters are likely involved in stromal and endothelial cell choline transport. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of Choline Transporters in the Human Placenta over Gestation

PubMed Central

Baumgartner, Heidi K.; Trinder, Kinsey M.; Galimanis, Carly E.; Post, Annalisa; Phang, Tzu; Ross, Randal G.; Winn, Virginia D.

2015-01-01

INTRODUCTION The developing fetus relies on the maternal blood supply to provide the choline it requires for making membrane lipids, synthesizing acetylcholine, and performing important methylation reactions. It is vital, therefore, that the placenta is efficient at transporting choline from maternal to fetal circulation. Although choline transporters have been found in term placenta samples, little is known about what cell types express specific choline transporters and how expression of the transporters may change over gestation. The objective of this study was to characterize choline transporter expression levels and localization in the human placenta throughout placental development. METHODS We analyzed CTL1 and −2 expression over gestation in human placental biopsies from 6 to 40 weeks gestation (n=6–10 per gestational window) by immunoblot analysis. To determine the cellular expression pattern of the choline transporters throughout gestation, immunofluorescence analysis was then performed. RESULTS Both CTL1 and CTL2 were expressed in the chorionic villi from 6 weeks gestation to term. Labor did not alter expression levels of either transporter. CTL1 localized to the syncytial trophoblasts and the endothelium of the fetal vasculature within the chorionic villous structure. CTL2 localized mainly to the stroma early in gestation and by the second trimester co-localized with CTL1 at the fetal vasculature. DISCUSSION The differential expression pattern of CTL1 and CTL2 suggests that CTL1 is the key transporter involved in choline transport from maternal circulation and both transporters are likely involved in stromal and endothelial cell choline transport. PMID:26601765

Leukocyte function-associated antigen-1-dependent lysis of Fas+ (CD95+/Apo-1+) innocent bystanders by antigen-specific CD8+ CTL.

PubMed

Kojima, H; Eshima, K; Takayama, H; Sitkovsky, M V

1997-09-15

Exquisite specificity toward Ag-bearing cells (cognate targets) is one of the most important properties of CD8+ CTL-mediated cytotoxicity. Using highly Ag-specific CD8+ CTL lines and clones, which spare noncognate, Ag-free targets, we found that in the presence of Ag-bearing targets the CTL acquire the ability to lyse noncognate target cells (bystanders). It is shown that the unexpectedly rapid and efficient lysis of bystanders by Ag-activated CTL is mediated by a Fas ligand (FasL)/Fas-based mechanism and does not depend on perforin. The CTL lysed Fas-expressing bystanders, but spared the Fas-negative or anti-Fas mAb-resistant bystander cells. Accordingly, the FasL-deficient gld/gld CTL did not kill bystanders, while perforin-deficient CTL did. Unlike anti-Fas mAb-induced cell death, the lysis of bystanders was not only FasL/Fas dependent but also required adhesion molecule LFA-1 on the surface of the activated CTL. Lysis of bystanders is viewed as acceptable "collateral" damage, but the persistent presence of activated CTL could result in immunopathologies involving functional Fas-expressing tissues.

Detection of Wilms' tumor antigen--specific CTL in tumor-draining lymph nodes of patients with early breast cancer.

PubMed

Gillmore, Roopinder; Xue, Shao-An; Holler, Angelika; Kaeda, Jaspal; Hadjiminas, Dimitri; Healy, Vourneen; Dina, Roberto; Parry, Suzanne C; Bellantuono, Ilaria; Ghani, Yasmeen; Coombes, R Charles; Waxman, Jonathan; Stauss, Hans J

2006-01-01

The Wilms' tumor antigen (WT1) is overexpressed in approximately 90% of breast tumors and, thus, is a potential target antigen for the immunotherapy of breast cancer. We have tested the working hypotheses that WT1 can be immunogenic in patients with breast cancer and can stimulate CTL of sufficient avidity to kill tumor cells. Paired tumor-draining lymph node and peripheral blood samples were analyzed from five HLA-A2-positive patients with stage I/II breast cancer. Fluorescent HLA-A*0201/WT1 tetramers were used to quantify WT1-specific CTL and the functional capacity of the CTL was assessed using cytotoxicity assays and intracellular cytokine staining. WT1 tetramer-binding T cells expanded from all lymph node samples but none of the corresponding peripheral blood samples. Functional assays were carried out on T cells from the patient who had yielded the highest frequency of HLA-A*0201/WT1 tetramer-positive cells. The cytotoxicity assays showed WT1 peptide--specific killing activity of the CTL, whereas intracellular cytokine staining confirmed that the tetramer--positive T cells produced IFN-gamma after stimulation with WT1 peptide. These WT1-specific T cells killed HLA-A2-positive breast cancer cell lines treated with IFN-gamma but no killing was observed with untreated tumor cells. These results show that WT1-specific CTL can be expanded from the tumor-draining lymph nodes of breast cancer patients and that they can display peptide-specific effector function. However, the CTL only killed IFN-gamma-treated tumor targets expressing high levels of HLA-A2 and not tumor cells with low HLA expression. This suggests that induction of autologous WT1-specific CTL may offer only limited tumor protection and that strategies that allow a high level of peptide/MHC complex presentation and/or improve CTL avidity may be required.

A novel C-type lectin from the sea cucumber Apostichopus japonicus (AjCTL-2) with preferential binding of d-galactose.

PubMed

Wang, Hui; Xue, Zhuang; Liu, Zhaoqun; Wang, Weilin; Wang, Feifei; Wang, Ying; Wang, Lingling; Song, Linsheng

2018-05-15

C-type lectins (CTLs) are Ca 2+ dependent carbohydrate-binding proteins that share structural homology in their carbohydrate-recognition domains (CRDs). In the present study, a novel CTL was identified from sea cucumber Apostichopus japonicus (named as AjCTL-2). The deduced amino acid sequence of AjCTL-2 was homologous to CTLs from other animals with the identities ranging from 33% to 40%. It contained a canonical signal peptide at the N-terminus, a low density lipoprotein receptor class A (LDLa), a C1r/C1s/Uegf/bone morphogenetic protein 1 (CUB), and a CRD with two motifs Glu-Pro-Asn (EPN) and Trp-Asn-Asp (WND) in Ca 2+ binding site 2. The mRNA transcripts of AjCTL-2 were extensively expressed in all the tested tissues including respiratory tree, muscle, gut, coelomocyte, tube-foot, body wall and gonad, and the highest expression level of AjCTL-2 in coelomocyte was about 4.2-fold (p < 0.05) of that in body wall. The mRNA expression level of AjCTL-2 in coelomocyte increased significantly after Vibrio splendidus stimulation, and dramatically peaked at 12 h, which was 206.4-fold (p < 0.05) of that in control group. AjCTL-2 protein was mainly detected in cytoplasm of coelomocyte by immunofluorescence. The recombinant AjCTL-2 (rAjCTL-2) displayed binding activity to d-galactose independent of Ca 2+ , while the binding activity to other tested pathogen-associated molecular patterns (PAMPs) including lipopolysaccharide (LPS), peptidoglycan (PGN), and mannose (Man) could not be detected. Surface plasmon resonance (SPR) analysis further revealed the high binding specificity and moderate binding affinity of rAjCTL-2 to d-galactose (KD = 4.093 × 10 -6  M). After rAjCTL-2 was blocked by its polyclonal antibody, the binding activity to d-galactose could not be detected by using a blocking ELISA (B-ELISA). Moreover, rAjCTL-2 could bind various microorganisms including V. splendidus, V. anguillarum, Staphylococcus aureus, Bifidobacterium breve and Yarrowia lipolytica with the strongest binding activity to B. breve. These results collectively suggested that AjCTL-2 was a member of CTL superfamily (CTLs) with preferential binding of d-galactose and participated in the immune response of sea cucumber. Copyright © 2018. Published by Elsevier Ltd.

HIV-1 epitopes presented by MHC class I types associated with superior immune containment of viremia have highly constrained fitness landscapes.

PubMed

Gorin, Aleksandr M; Du, Yushen; Liu, Franklin Y; Zhang, Tian-Hao; Ng, Hwee L; Hofmann, Christian; Cumberland, William G; Sun, Ren; Yang, Otto O

2017-08-01

Certain Major Histocompatibility-I (MHC-I) types are associated with superior immune containment of HIV-1 infection by CD8+ cytotoxic T lymphocytes (CTLs), but the mechanisms mediating this containment are difficult to elucidate in vivo. Here we provide controlled assessments of fitness landscapes and CTL-imposed constraints for immunodominant epitopes presented by two protective (B*57 and B*27) and one non-protective (A*02) MHC-I types. Libraries of HIV-1 with saturation mutagenesis of CTL epitopes are propagated with and without CTL selective pressure to define the fitness landscapes for epitope mutation and escape from CTLs via deep sequencing. Immunodominant B*57- and B*27- present epitopes are highly limited in options for fit mutations, with most viable variants recognizable by CTLs, whereas an immunodominant A*02 epitope-presented is highly permissive for mutation, with many options for CTL evasion without loss of viability. Generally, options for evasion overlap considerably between CTL clones despite highly distinct T cell receptors. Finally, patterns of variant recognition suggest population-wide CTL selection for the A*02-presented epitope. Overall, these findings indicate that these protective MHC-I types yield CTL targeting of highly constrained epitopes, and underscore the importance of blocking public escape pathways for CTL-based interventions against HIV-1.

Fitness Costs and Diversity of the Cytotoxic T Lymphocyte (CTL) Response Determine the Rate of CTL Escape during Acute and Chronic Phases of HIV Infection▿†

PubMed Central

Ganusov, Vitaly V.; Goonetilleke, Nilu; Liu, Michael K. P.; Ferrari, Guido; Shaw, George M.; McMichael, Andrew J.; Borrow, Persephone; Korber, Bette T.; Perelson, Alan S.

2011-01-01

HIV-1 often evades cytotoxic T cell (CTL) responses by generating variants that are not recognized by CTLs. We used single-genome amplification and sequencing of complete HIV genomes to identify longitudinal changes in the transmitted/founder virus from the establishment of infection to the viral set point at 1 year after the infection. We found that the rate of viral escape from CTL responses in a given patient decreases dramatically from acute infection to the viral set point. Using a novel mathematical model that tracks the dynamics of viral escape at multiple epitopes, we show that a number of factors could potentially contribute to a slower escape in the chronic phase of infection, such as a decreased magnitude of epitope-specific CTL responses, an increased fitness cost of escape mutations, or an increased diversity of the CTL response. In the model, an increase in the number of epitope-specific CTL responses can reduce the rate of viral escape from a given epitope-specific CTL response, particularly if CD8+ T cells compete for killing of infected cells or control virus replication nonlytically. Our mathematical framework of viral escape from multiple CTL responses can be used to predict the breadth and magnitude of HIV-specific CTL responses that need to be induced by vaccination to reduce (or even prevent) viral escape following HIV infection. PMID:21835793

Fitness costs and diversity of the cytotoxic T lymphocyte (CTL) response determine the rate of CTL escape during acute and chronic phases of HIV infection.

PubMed

Ganusov, Vitaly V; Goonetilleke, Nilu; Liu, Michael K P; Ferrari, Guido; Shaw, George M; McMichael, Andrew J; Borrow, Persephone; Korber, Bette T; Perelson, Alan S

2011-10-01

HIV-1 often evades cytotoxic T cell (CTL) responses by generating variants that are not recognized by CTLs. We used single-genome amplification and sequencing of complete HIV genomes to identify longitudinal changes in the transmitted/founder virus from the establishment of infection to the viral set point at 1 year after the infection. We found that the rate of viral escape from CTL responses in a given patient decreases dramatically from acute infection to the viral set point. Using a novel mathematical model that tracks the dynamics of viral escape at multiple epitopes, we show that a number of factors could potentially contribute to a slower escape in the chronic phase of infection, such as a decreased magnitude of epitope-specific CTL responses, an increased fitness cost of escape mutations, or an increased diversity of the CTL response. In the model, an increase in the number of epitope-specific CTL responses can reduce the rate of viral escape from a given epitope-specific CTL response, particularly if CD8+ T cells compete for killing of infected cells or control virus replication nonlytically. Our mathematical framework of viral escape from multiple CTL responses can be used to predict the breadth and magnitude of HIV-specific CTL responses that need to be induced by vaccination to reduce (or even prevent) viral escape following HIV infection.

A novel immunization method to induce cytotoxic T-lymphocyte responses (CTL) against plasmid-encoded herpes simplex virus type-1 glycoprotein D.

PubMed

Cruz, P E; Khalil, P L; Dryden, T D; Chiou, H C; Fink, P S; Berberich, S J; Bigley, N J

1999-03-05

DNA molecules complexed with an asialoglycoprotein-polycation conjugate, consisting of asialoorosomucoid (ASOR) coupled to poly-L-lysine, can enter hepatocytes which bear receptors for ASOR. We used this receptor-mediated DNA delivery system to deliver plasmid DNA encoding glycoprotein D (gD) of herpes simplex virus type 1 to ASOR-positive cells. Maximum expression of gD protein was seen at 3 days after injection of this preparation in approximately 13% of cells from BALB/c mice [hepatocytes from mice injected intravenously (i.v.) or peritoneal exudate cells from mice injected intraperitoneally (i.p.)]. In comparison with mice injected with either the plasmid vector alone or the gD-containing plasmid uncomplexed to ASOR, mice immunized with gD-containing plasmid complexed with ASOR-poly-L-lysine induced marked antigen-specific CTL responses. BALB/c mice immunized with gD-DNA developed a T-cell-mediated CTL response against target cells expressing gD and MHC class II glycoproteins, but not against cells expressing only gD and MHC class I molecules. In C3H mice, gD-DNA induced a T-cell-mediated CTL response against target cells expressing gD and class I MHC molecules. Serum anti-gD antibody in low titers were produced in both strains of mice. DNA complexed with ASOR-poly-L-lysine induced CTL responses in mice.

Characterization of Gag and Nef-specific ELISpot-based CTL responses in HIV-1 infected Indian individuals.

PubMed

Mendiratta, Sanjay; Vajpayee, Madhu; Malhotra, Uma; Kaushik, Shweta; Dar, Lalit; Mojumdar, Kamalika; Chauhan, Neeraj Kumar; Sreenivas, Vishnubhatla

2009-02-01

Cytotoxic T lymphocyte (CTL) responses to Gag have been most frequently linked to control of viremia whereas CTL responses to Nef have direct relationship with viral load. IFN-gamma ELISpot assay was used to screen CTL responses at single peptide level directed at HIV-1 subtype C Gag and Nef proteins in 30 antiretroviral therapy naive HIV-1 infected Indian individuals. PBMCs from 73.3% and 90% of the study population showed response to Gag and Nef antigens, respectively. The magnitude of Gag-specific CTL responses was inversely correlated with plasma viral load (r = -0.45, P = 0.001), whereas magnitude of Nef-specific responses was directly correlated (r = 0.115). Thirteen immunodominant regions (6 in Gag, 7 in Nef) were identified in the current study. The identification of Gag and Nef-specific responses across HIV-1 infected Indian population and targeting epitopes from multiple immunodominant regions may provide useful insight into the designing of new immunotherapy and vaccines.

Cotransfection of DC with TLR4 and MART-1 RNA induces MART-1-specific responses.

PubMed

Abdel-Wahab, Zeinab; Cisco, Robin; Dannull, Jens; Ueno, Tomio; Abdel-Wahab, Omar; Kalady, Matthew F; Onaitis, Mark W; Tyler, Douglas S; Pruitt, Scott K

2005-04-01

Cotransfection of dendritic cells (DC) with MART-1 and constitutively active TLR4 (caTLR4) RNA enhances the maturation of DC. Immature DC were cotransfected with RNA constructs encoding MART-1 and caTLR4, and CTL responses were analyzed. Cotransfection of DC with MART-1 + caTLR4 enhanced the expression of CD80 and CD83 surface markers and increased the secretion of cytokines IL-6, IL-12, and TNFalpha. Neither the native nor the A27L-modified MART-1 RNA could induce significant DC maturation or cytokine secretion. More importantly, DC cotransfected with caTLR4 + MART-1 RNA induced MART-1-specific CTL responses of a higher magnitude than DC transfected with either the native or A27L MART-1 RNA. When the MART-1 RNA-transfected DC were treated with DC-maturing cytokines, the induced CTL were less frequent and less lytic than those induced with MART-1 + caTLR4. A 2- to 100-fold increase in MART-1 tetramer+ cells and 2- to 10-fold increases in IFNgamma secretion and cytotoxicity were seen in CTL induced with MART-1 + caTLR4 compared to CTL induced with either MART-1 or A27L RNA. CTL induced with the mixed RNA displayed high percentages of CD8+ cells coexpressing CD45RA, CD56, and 2B4 antigens. Transfection with caTLR4 alone induced DC maturation, but did not induce lytic CTL, suggesting that CTL responses were induced solely by MART-1 epitopes. caTLR4 increases the CTL-inducing capacity of DC generating a lytic response specific for the accompanying antigen. These results demonstrate the possibility of enhancing the immunogenicity of the native MART-1 and other RNA derived from weakly immunogenic tumors in DC-based immunotherapy.

Molecular cloning and characterization of a C-type lectin in roughskin sculpin (Trachidermus fasciatus).

PubMed

Yu, Shanshan; Yang, Hui; Chai, Yingmei; Liu, Yingying; Zhang, Qiuxia; Ding, Xinbiao; Zhu, Qian

2013-02-01

C-type lectins, as the members of pattern-recognition receptors (PRRs), play significant roles in innate immunity responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. In our study, a C-type lectin gene (TfCTL1) was cloned from the roughskin sculpin using expression sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length of TfCTL1 was 696 bp, consisting of a 95 bp 5' untranslated region (UTR), a 498 bp open reading frame (ORF) encoding a 165 amino acid protein, and a 103 bp 3' UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of TfCTL1 contained a signal peptide and a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys(61)-Cys(158), Cys(134)-Cys(150)) and a Ca(2+)/carbohydrate-binding site (QPD motif). Results from the qRT-PCR indicated that TfCTL1 mRNA was predominately expressed in the liver. The temporal expression of TfCTL1 was obviously up-regulated in the skin, blood, spleen and heart in time dependent manners by lipopolysaccharide (LPS) challenge, whereas in the liver, TfCTL1 was initially down-regulated from 2 h to 48 h followed by an abrupt up-regulation at 72 h. Recombinant TfCTL1 CRD purified from Escherichia coli BL21 was able to agglutinate some Gram-positive bacteria, Gram-negative bacteria and a yeast in a Ca(2+)-dependent manner. Further analysis showed that TfCTL1 can bind to several kinds of microorganisms selectively in a Ca(2+)-independent manner. These results suggested that TfCTL1 might be involved in the innate response as a PRR. Copyright © 2012 Elsevier Ltd. All rights reserved.

Autologous cellular vaccine overcomes cancer immunoediting in a mouse model of myeloma.

PubMed

Mazzocco, Marta; Martini, Matteo; Rosato, Antonio; Stefani, Elisabetta; Matucci, Andrea; Dalla Santa, Silvia; De Sanctis, Francesco; Ugel, Stefano; Sandri, Sara; Ferrarini, Giovanna; Cestari, Tiziana; Ferrari, Sergio; Zanovello, Paola; Bronte, Vincenzo; Sartoris, Silvia

2015-09-01

In the Sp6 mouse plasmacytoma model, a whole-cell vaccination with Sp6 cells expressing de novo B7-1 (Sp6/B7) induced anatomically localized and cytotoxic T cell (CTL)-mediated protection against wild-type (WT) Sp6. Both WT Sp6 and Sp6/B7 showed down-regulated expression of MHC H-2 L(d). Increase of H-2 L(d) expression by cDNA transfection (Sp6/B7/L(d)) raised tumour immune protection and shifted most CTL responses towards H-2 L(d)-restricted antigenic epitopes. The tumour-protective responses were not specific for the H-2 L(d)-restricted immunodominant AH1 epitope of the gp70 common mouse tumour antigen, although WT Sp6 and transfectants were able to present it to specific T cells in vitro. Gp70 transcripts, absent in secondary lymphoid organs of naive mice, were detected in immunized mice as well as in splenocytes from naive mice incubated in vitro with supernatants of CTL-lysed Sp6 cell cultures, containing damage-associated molecular patterns (DAMPs). It has been shown that Toll-like receptor triggering induces gp70 expression. Damage-associated molecular patterns are released by CTL-mediated killing of Sp6/B7-Sp6/B7/L(d) cells migrated to draining lymph nodes during immunization and may activate gp70 expression and presentation in most resident antigen-presenting cells. The same could also apply for Mus musculus endogenous ecotropic murine leukaemia virus 1 particles present in Sp6-cytosol, discharged by dying cells and superinfecting antigen-presenting cells. The outcome of such a massive gp70 cross-presentation would probably be tolerogenic for the high-affinity AH1-gp70-specific CTL clones. In this scenario, autologous whole-tumour-cell vaccines rescue tumour-specific immunoprotection by amplification of subdominant tumour antigen responses when those against the immune dominant antigens are lost. © 2015 The Authors. Immunology Published by John Wiley & Sons Ltd.

Autologous cellular vaccine overcomes cancer immunoediting in a mouse model of myeloma

PubMed Central

Mazzocco, Marta; Martini, Matteo; Rosato, Antonio; Stefani, Elisabetta; Matucci, Andrea; Dalla Santa, Silvia; De Sanctis, Francesco; Ugel, Stefano; Sandri, Sara; Ferrarini, Giovanna; Cestari, Tiziana; Ferrari, Sergio; Zanovello, Paola; Bronte, Vincenzo; Sartoris, Silvia

2015-01-01

In the Sp6 mouse plasmacytoma model, a whole-cell vaccination with Sp6 cells expressing de novo B7-1 (Sp6/B7) induced anatomically localized and cytotoxic T cell (CTL) -mediated protection against wild-type (WT) Sp6. Both WT Sp6 and Sp6/B7 showed down-regulated expression of MHC H-2 Ld. Increase of H-2 Ld expression by cDNA transfection (Sp6/B7/Ld) raised tumour immune protection and shifted most CTL responses towards H-2 Ld-restricted antigenic epitopes. The tumour-protective responses were not specific for the H-2 Ld-restricted immunodominant AH1 epitope of the gp70 common mouse tumour antigen, although WT Sp6 and transfectants were able to present it to specific T cells in vitro. Gp70 transcripts, absent in secondary lymphoid organs of naive mice, were detected in immunized mice as well as in splenocytes from naive mice incubated in vitro with supernatants of CTL-lysed Sp6 cell cultures, containing damage-associated molecular patterns (DAMPs). It has been shown that Toll-like receptor triggering induces gp70 expression. Damage-associated molecular patterns are released by CTL-mediated killing of Sp6/B7-Sp6/B7/Ld cells migrated to draining lymph nodes during immunization and may activate gp70 expression and presentation in most resident antigen-presenting cells. The same could also apply for Mus musculus endogenous ecotropic murine leukaemia virus 1 particles present in Sp6-cytosol, discharged by dying cells and superinfecting antigen-presenting cells. The outcome of such a massive gp70 cross-presentation would probably be tolerogenic for the high-affinity AH1-gp70-specific CTL clones. In this scenario, autologous whole-tumour-cell vaccines rescue tumour-specific immunoprotection by amplification of subdominant tumour antigen responses when those against the immune dominant antigens are lost. PMID:25959091

Casein expression in cytotoxic T lymphocytes.

PubMed Central

Grusby, M J; Mitchell, S C; Nabavi, N; Glimcher, L H

1990-01-01

A cDNA that expresses a mRNA restricted to cytotoxic T lymphocytes (CTL) and mammary tissue has been isolated and characterized. The deduced amino acid sequence from this cDNA shows extensive homology with the previously reported amino acid sequence for rat alpha-casein. Indeed, the presence of a six-residue-repeated motif that is specific for rodent alpha-caseins strongly supports the identification of this cDNA as mouse alpha-casein. Northern (RNA) blot analysis of many hematopoietic cell types revealed that this gene is restricted to CTL, being expressed in four of six CTL lines examined. Furthermore, CTL that express this gene were also found to express other members of the casein gene family, such as beta- and kappa-casein. These results suggest that caseins may be important in CTL function, and their potential role in CTL-mediated lysis is discussed. Images PMID:2395885

A Multiepitope of XBP1, CD138 and CS1 Peptides Induces Myeloma-Specific Cytotoxic T lymphocytes in T cells of Smoldering Myeloma Patients

PubMed Central

Bae, Jooeun; Prabhala, Rao; Voskertchian, Annie; Brown, Andrew; Maguire, Craig; Richardson, Paul; Dranoff, Glen; Anderson, Kenneth C.; Munshi, Nikhil C.

2014-01-01

We evaluated a cocktail of HLA-A2-specific peptides including heteroclitic XBP1 US184-192 (YISPWILAV), heteroclitic XBP1 SP367-375 (YLFPQLISV), native CD138260-268 (GLVGLIFAV) and native CS1239-247 (SLFVLGLFL), for their ability to elicit multipeptide specific cytotoxic T lymphocytes (MP-CTL) using T cells from smoldering multiple myeloma (SMM) patients. Our results demonstrate that MP-CTL generated from SMM patients’ T cells show effective anti-MM responses including CD137 (4-1BB) upregulation, CTL proliferation, IFN-γ production, and degranulation (CD107a) in an HLA-A2-restricted and peptide-specific manner. Phenotypically, we observed increased total CD3+CD8+ T cells (>80%) and cellular activation (CD69+) within the memory SMM MP-CTL (CD45RO+/CD3+CD8+) subset after repeated multipeptide stimulation. Importantly, SMM patients could be categorized into distinct groups by their level of MP-CTL expansion and anti-tumor activity. In high responders, the effector memory (CCR7-CD45RO+/CD3+CD8+) T cell subset was enriched, while the remaining responders’ CTL contained a higher frequency of the terminal effector (CCR7-CD45RO-/CD3+CD8+) subset. These results suggest that this multipeptide cocktail has the potential to induce effective and durable memory MP-CTL in SMM patients. Therefore, our findings provide the rationale for clinical evaluation of a therapeutic vaccine to prevent or delay progression of SMM to active disease. PMID:24935722

Increased Expression of Fatty-Acid and Calcium Metabolism Genes in Failing Human Heart

PubMed Central

Rodríguez-Penas, Diego; Feijóo-Bandín, Sandra; Noguera-Moreno, Teresa; Calaza, Manuel; Álvarez-Barredo, María; Mosquera-Leal, Ana; Parrington, John; Brugada, Josep; Portolés, Manuel; Rivera, Miguel; González-Juanatey, José Ramón; Lago, Francisca

2012-01-01

Background Heart failure (HF) involves alterations in metabolism, but little is known about cardiomyopathy-(CM)-specific or diabetes-independent alterations in gene expression of proteins involved in fatty-acid (FA) uptake and oxidation or in calcium-(Ca2+)-handling in the human heart. Methods RT-qPCR was used to quantify mRNA expression and immunoblotting to confirm protein expression in left-ventricular myocardium from patients with HF (n = 36) without diabetes mellitus of ischaemic (ICM, n = 16) or dilated (DCM, n = 20) cardiomyopathy aetiology, and non-diseased donors (CTL, n = 6). Results Significant increases in mRNA of genes regulating FA uptake (CD36) and intracellular transport (Heart-FA-Binding Protein (HFABP)) were observed in HF patients vs CTL. Significance was maintained in DCM and confirmed at protein level, but not in ICM. mRNA was higher in DCM than ICM for peroxisome-proliferator-activated-receptor-alpha (PPARA), PPAR-gamma coactivator-1-alpha (PGC1A) and CD36, and confirmed at the protein level for PPARA and CD36. Transcript and protein expression of Ca2+-handling genes (Two-Pore-Channel 1 (TPCN1), Two-Pore-Channel 2 (TPCN2), and Inositol 1,4,5-triphosphate Receptor type-1 (IP3R1)) increased in HF patients relative to CTL. Increases remained significant for TPCN2 in all groups but for TPCN1 only in DCM. There were correlations between FA metabolism and Ca2+-handling genes expression. In ICM there were six correlations, all distinct from those found in CTL. In DCM there were also six (all also different from those found in CTL): three were common to and three distinct from ICM. Conclusion DCM-specific increases were found in expression of several genes that regulate FA metabolism, which might help in the design of aetiology-specific metabolic therapies in HF. Ca2+-handling genes TPCN1 and TPCN2 also showed increased expression in HF, while HF- and CM-specific positive correlations were found among several FA and Ca2+-handling genes. PMID:22701570

Viral load and clinical disease enhancement associated with a lentivirus cytotoxic T lymphocyte vaccine regimen

PubMed Central

Mealey, Robert H.; Leib, Steven R.; Littke, Matt H.; Wagner, Bettina; Horohov, David W.; McGuire, Travis C.

2009-01-01

Effective DNA-based vaccines against lentiviruses will likely induce CTL against conserved viral proteins. Equine infectious anemia virus (EIAV) infects horses worldwide, and serves as a useful model for lentiviral immune control. Although attenuated live EIAV vaccines have induced protective immune responses, DNA-based vaccines have not. In particular, DNA-based vaccines have had limited success in inducing CTL responses against intracellular pathogens in the horse. We hypothesized that priming with a codon-optimized plasmid encoding EIAV Gag p15/p26 with co-administration of a plasmid encoding an equine IL-2/IgG fusion protein as a molecular adjuvant, followed by boosting with a vaccinia vector expressing Gag p15/p26, would induce protective Gag-specific CTL responses. Although the regimen induced Gag-specific CTL in four of seven vaccinated horses, CTL were not detected until after the vaccinia boost, and protective effects were not observed in EIAV challenged vaccinates. Unexpectedly, vaccinates had significantly higher viral loads and more severe clinical disease, associated with the presence of vaccine-induced CTL. It was concluded that 1.) further optimization of the timing and route of DNA immunization was needed for efficient CTL priming in vivo, 2.) co-administration of the IL-2/IgG plasmid did not enhance CTL priming by the Gag p15/p26 plasmid, 3.) vaccinia vectors are useful for lentivirus-specific CTL induction in the horse, 4.) Gag-specific CTL alone are either insufficient or a more robust Gag-specific CTL response is needed to limit EIAV viremia and clinical disease, and 5.) CTL-inducing vaccines lacking envelope immunogens can result in lentiviral disease enhancement. Although the mechanisms for enhancement associated with this vaccine regimen remain to be elucidated, these results have important implications for development of lentivirus T cell vaccines. PMID:19368787

Two distinct HLA-A0201-presented epitopes of the Wilms tumor antigen 1 can function as targets for leukemia-reactive CTL.

PubMed

Bellantuono, Ilaria; Gao, Liquan; Parry, Suzanne; Marley, Steve; Dazzi, Francesco; Apperley, Jane; Goldman, John M; Stauss, Hans J

2002-11-15

Using the allo-restricted T-cell approach to circumvent tolerance, we have previously identified a cytotoxic T-lymphocyte (CTL) epitope in the transcription factor Wilms tumor antigen 1 (WT1) presented by HLA-A0201 (A2) class I molecules. Here we describe an additional A2-presented epitope and show that CTLs against both epitopes kill WT1-expressing leukemia cell lines. Colony-forming assays demonstrated that both types of CTL killed CD34(+) progenitor cells from A2(+) leukemia patients, but not from A2(+) healthy individuals. The long-term culture-initiating cell (LTC-IC) assay was used to analyze the killing activity of WT1-specific CTLs against the more immature fraction of CD34(+) cells. The CTLs killed LTC-ICs of patients with chronic myelogenous leukemia (CML), whereas the function of normal CD34(+) progenitor/stem cells was not inhibited. Together, the data show that CTLs specific for 2 distinct peptide epitopes of WT1 can discriminate between normal and leukemia LTC-ICs, suggesting that such CTLs have the potential to selectively kill CML progenitor/stem cells.

Induction of anti-melanoma CTL response using DC transfected with mutated mRNA encoding full-length Melan-A/MART-1 antigen with an A27L amino acid substitution.

PubMed

Abdel-Wahab, Zeinab; Kalady, Matthew F; Emani, Sirisha; Onaitis, Mark W; Abdel-Wahab, Omar I; Cisco, Robin; Wheless, Lee; Cheng, Tsung-Yen; Tyler, Douglas S; Pruitt, Scott K

2003-08-01

Modification of the parental immunodominant Melan-A/MART-1 peptide (MART-1(26-35)) by replacing the alanine with leucine (A27L) enhances its immunogenicity. Because of the reported advantages of RNA over peptides in DC vaccines, we sought to mutate the MART-1 gene to encode a full-length MART-1 antigen with an A27L amino acid substitution. Human DC were transfected with A27L-mutated MART-1 RNA (A27L RNA) or native MART-1 RNA, and then used to stimulate autologous T cells from a series of 8 HLA-A2+ volunteers. After three stimulations, all CTL induced with DC/A27L RNA exhibited more tetramer+ cells, and demonstrated stronger antigen-specific IFNgamma-secreting activity compared to CTL induced with DC/native RNA. A potent MART-1-specific, and predominantly class-I-restricted lysis was detected in most CTL induced with DC/A27L RNA, while native RNA-induced CTL showed minimal and non-specific lysis. HLA-A2+ DC and MART-1 negative/A2+ melanoma cells transfected with the A27L RNA were recognized and killed by MART-1-specific CTL, suggesting that these APC efficiently processed the A27L RNA and presented correct MART-1-specific epitope(s). In summary, introducing an A27L mutation into the MART-1 full-length mRNA sequence enhanced the immunogenicity of the encoded MART-1 Ag. The ease with which such a mutation can be made in RNA presents another potential advantage of using RNA for immunotherapy. Our results support considering this strategy for enhancing the immunogenicity of DC-based RNA vaccines.

Cytomegalovirus-Specific CD8+ T-Cells With Different T-Cell Receptor Affinities Segregate T-Cell Phenotypes and Correlate With Chronic Graft-Versus-Host Disease in Patients Post-Hematopoietic Stem Cell Transplantation

PubMed Central

Poiret, Thomas; Axelsson-Robertson, Rebecca; Remberger, Mats; Luo, Xiao-Hua; Rao, Martin; Nagchowdhury, Anurupa; Von Landenberg, Anna; Ernberg, Ingemar; Ringden, Olle; Maeurer, Markus

2018-01-01

Virus-specific T-cell responses are crucial to control cytomegalovirus (CMV) infections/reactivation in immunocompromised individuals. Adoptive cellular therapy with CMV-specific T-cells has become a viable treatment option. High-affinity anti-viral cellular immune responses are associated with improved long-term immune protection against CMV infection. To date, the characterization of high-affinity T-cell responses against CMV has not been achieved in blood from patients after allogeneic hematopoietic stem cell transplantation (HSCT). Therefore, the purpose of this study was to describe and analyze the phenotype and clinical impact of different CMV-specific CD8+ cytotoxic T-lymphocytes (CMV-CTL) classes based on their T-cell receptor (TCR) affinity. T-cells isolated from 23 patients during the first year following HSCT were tested for the expression of memory markers, programmed cell death 1 (PD-1), as well as TCR affinity, using three different HLA-A*02:01 CMVNLVPMVATV-Pp65 tetramers (wild-type, a245v and q226a mutants). High-affinity CMV-CTL defined by q226a tetramer binding, exhibited a higher frequency in CD8+ T-cells in the first month post-HSCT and exhibited an effector memory phenotype associated with strong PD-1 expression as compared to the medium- and low-affinity CMV-CTLs. High-affinity CMV-CTL was found at higher proportion in patients with chronic graft-versus-host disease (p < 0.001). This study provides a first insight into the detailed TCR affinities of CMV-CTL. This may be useful in order to improve current immunotherapy protocols using isolation of viral-specific T-cell populations based on their TCR affinity. PMID:29692783

The Memory Cytotoxic T-Lymphocyte (CTL) Response to Human Cytomegalovirus Infection Contains Individual Peptide-Specific CTL Clones That Have Undergone Extensive Expansion In Vivo

PubMed Central

Weekes, Michael P.; Wills, Mark R.; Mynard, Kim; Carmichael, Andrew J.; Sissons, J. G. Patrick

1999-01-01

Human cytomegalovirus (HCMV)-specific CD8+ cytotoxic T lymphocytes (CTL) appear to play an important role in the control of virus replication and in protection against HCMV-related disease. We have previously reported high frequencies of memory CTL precursors (CTLp) specific to the HCMV tegument protein pp65 in the peripheral blood of healthy virus carriers. In some individuals, the CTL response to this protein is focused on only a single epitope, whereas in other virus carriers CTL recognized multiple epitopes which we identified by using synthetic peptides. We have analyzed the clonal composition of the memory CTL response to four of these pp65 epitopes by sequencing the T-cell receptors (TCR) of multiple independently derived epitope-specific CTL clones, which were derived by formal single-cell cloning or from clonal CTL microcultures. In all cases, we have observed a high degree of clonal focusing: the majority of CTL clones specific to a defined pp65 peptide from any one virus carrier use only one or two different TCRs at the level of the nucleotide sequence. Among virus carriers who have the same major histocompatibility complex (MHC) class I allele, we observed that CTL from different donors that recognize the same peptide-MHC complex often used the same Vβ segment, although other TCR gene segments and CDR3 length were not in general conserved. We have also examined the clonal composition of CTL specific to pp65 peptides in asymptomatic human immunodeficiency virus-infected individuals. We have observed a similarly focused peptide-specific CTL response. Thus, the large population of circulating HCMV peptide-specific memory CTLp in virus carriers in fact contains individual CTL clones that have undergone extensive clonal expansion in vivo. PMID:9971792

Two roles for CD4 cells in the control of the generation of cytotoxic T lymphocytes.

PubMed

Cassell, D; Forman, J

1991-01-01

The generation of CTL against Qa-1 Ag in C57BL/6 (B6) (Qa-1b) and B6.Tlaa (Qa-1a) congenic strains requires in vivo priming with the Qa-1 alloantigen together with a helper Ag, such as H-Y. The primed precursors obtained from these female mice generate Qa-1-specific CTL activity upon culture in vitro. Although the presence of the H-Y helper Ag is not required for the in vitro sensitization, no response occurs in the absence of CD4 cells. Addition of unprimed B6.Tlaa CD4 cells from Qa-1 incompatible radiation bone marrow chimeras (B6.Tlaa----B6), that are presumably tolerant to Qa-1b, provide helper activity for Qa-1b-specific CTL. This indicates that although CD4 cells are obligatory for the Qa-1 response, they need not be specific for alloantigens on the APC to generate helper activity in in vitro cultures. Addition of unirradiated B6 CD8-depleted spleen cells to CD4-depleted B6.Tlaa anti-B6 cultures in the presence of either B6.Tlaa CD4 cells or rIL-2 prevents the generation of Qa-1 specific CTL. This inhibition is not due to an anti-idiotypic Ts cell since B6.Tlaa----B6 chimeric cells do not suppress an anti-Qa-1b response. Rather, this finding is consistent with that of a veto cell mechanism. To determine whether CD4 cells themselves exhibit veto activity, highly purified CD4 populations were tested for their ability to inhibit the generation of Qa-1-specific CTL. CD4 cells precultured for 2 to 3 days with Con A and rIL-2 specifically inhibit CTL activity whereas resting cells do not, similar to that noted for CD8 veto cells. The relative efficiency of activated CD4 cells is greater than that of resting NK cells but is less than that of activated CD8 or NK cells. Thus, CD4 cells not only provide helper activity for CTL precursors, but also act as veto cells to prevent the generation of CTL activity.

Astrocyte-specific expression of human T-cell lymphotropic virus type 1 (HTLV-1) Tax: induction of tumor necrosis factor alpha and susceptibility to lysis by CD8+ HTLV-1-specific cytotoxic T cells.

PubMed

Méndez, E; Kawanishi, T; Clemens, K; Siomi, H; Soldan, S S; Calabresi, P; Brady, J; Jacobson, S

1997-12-01

Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with a chronic neurological disease termed HTLV-1-associated myelopathy/tropical spastic paraperesis (HAM/TSP). Although the pathogenesis of this disease remains to be elucidated, the evidence suggests that immunopathological mechanisms are involved. Since HTLV-1 tax mRNA was colocalized with glial acidic fibrillary protein, a marker for astrocytes, we developed an in vitro model to assess whether HTLV-1 infection activates astrocytes to secrete cytokines or present viral immunodominant epitopes to virus-specific T cells. Two human astrocytic glioma cell lines, U251 and U373, were transfected with the 3' portion of the HTLV-1 genome and with the HTLV-1 tax gene under astrocyte-specific promoter control. In this study, we report that Tax-expressing astrocytic glioma transfectants activate the expression of tumor necrosis factor alpha mRNA in vitro. Furthermore, these Tax-expressing glioma transfectants can serve as immunological targets for HTLV-1-specific cytotoxic T lymphocytes (CTL). We propose that these events could contribute to the neuropathology of HAM/TSP, since infected astrocytes can become a source for inflammatory cytokines upon HTLV-1 infection and serve as targets for HTLV-1-specific CTL, resulting in parenchymal damage by direct lysis and/or cytokine release.

Identification and transcriptional analysis of two types of lectins (SgCTL-1 and SgGal-1) from mollusk Solen grandis.

PubMed

Wei, Xiumei; Yang, Jianmin; Liu, Xiangquan; Yang, Dinglong; Xu, Jie; Fang, Jinghui; Wang, Weijun; Yang, Jialong

2012-08-01

C-type lectin and galectin are two types of animal carbohydrate-binding proteins which serve as pathogen recognition molecules and play crucial roles in the innate immunity of invertebrates. In the present study, a C-type lectin (designated as SgCTL-1) and galectin (designated as SgGal-1) were identified from mollusk Solen grandis, and their expression patterns, both in tissues and toward three pathogen-associated molecular patterns (PAMPs) stimulation were characterized. The full-length cDNA of SgCTL-1 and SgGal-1 was 1280 and 1466 bp, containing an open reading frame (ORF) of 519 and 1218 bp, respectively. Their deduced amino acid sequences showed high similarity to other members of C-type lectin and galectin superfamily, respectively. SgCTL-1 encoded a single carbohydrate-recognition domain (CRD), and the motif of Ca(2+)-binding site 2 was EPN (Glu(135)-Pro(136)-Asn(137)). While SgGal-1 encoded two CRDs, and the amino acid residues constituted the carbohydrate-binding motifs were well conserved in CRD1 but partially conserved in CRD2. Although SgCTL-1 and SgGal-1 exhibited different tissue expression pattern, they were both constitutively expressed in all tested tissues, including hemocytes, gonad, mantle, muscle, gill and hepatopancreas, and they were both highly expressed in hepatopancreas and gill. Furthermore, the mRNA expression of two lectins in hemocytes was significantly (P < 0.01) up-regulated with different levels after S. grandis were stimulated by lipopolysaccharide (LPS), peptidoglycan (PGN) or β-1,3-glucan. Our results suggested that SgCTL-1 and SgGal-1 from razor clam were two novel members of animal lectins, and they might function as pattern recognition receptors (PRRs) taking part in the process of pathogen recognition. Copyright © 2012 Elsevier Ltd. All rights reserved.

Diverse Heterologous Primary Infections Radically Alter Immunodominance Hierarchies and Clinical Outcomes Following H7N9 Influenza Challenge in Mice

PubMed Central

Duan, Susu; Meliopoulos, Victoria A.; McClaren, Jennifer L.; Guo, Xi-Zhi J.; Sanders, Catherine J.; Smallwood, Heather S.; Webby, Richard J.; Schultz-Cherry, Stacey L.; Doherty, Peter C.; Thomas, Paul G.

2015-01-01

The recent emergence of a novel H7N9 influenza A virus (IAV) causing severe human infections in China raises concerns about a possible pandemic. The lack of pre-existing neutralizing antibodies in the broader population highlights the potential protective role of IAV-specific CD8+ cytotoxic T lymphocyte (CTL) memory specific for epitopes conserved between H7N9 and previously encountered IAVs. In the present study, the heterosubtypic immunity generated by prior H9N2 or H1N1 infections significantly, but variably, reduced morbidity and mortality, pulmonary virus load and time to clearance in mice challenged with the H7N9 virus. In all cases, the recall of established CTL memory was characterized by earlier, greater airway infiltration of effectors targeting the conserved or cross-reactive H7N9 IAV peptides; though, depending on the priming IAV, each case was accompanied by distinct CTL epitope immunodominance hierarchies for the prominent KbPB1703, DbPA224, and DbNP366 epitopes. While the presence of conserved, variable, or cross-reactive epitopes between the priming H9N2 and H1N1 and the challenge H7N9 IAVs clearly influenced any change in the immunodominance hierarchy, the changing patterns were not tied solely to epitope conservation. Furthermore, the total size of the IAV-specific memory CTL pool after priming was a better predictor of favorable outcomes than the extent of epitope conservation or secondary CTL expansion. Modifying the size of the memory CTL pool significantly altered its subsequent protective efficacy on disease severity or virus clearance, confirming the important role of heterologous priming. These findings establish that both the protective efficacy of heterosubtypic immunity and CTL immunodominance hierarchies are reflective of the immunological history of the host, a finding that has implications for understanding human CTL responses and the rational design of CTL-mediated vaccines. PMID:25668410

Ex vivo PD-L1/PD-1 pathway blockade reverses dysfunction of circulating CEA specific T cells in pancreatic cancer patients

PubMed Central

Chen, Y; Xue, SA; Behboudi, S; Mohammad, GH; Pereira, SP; Morris, EC

2017-01-01

Carcinoembryonic antigen (CEA) is a candidate target for cellular immunotherapy of pancreatic cancer (PC). In this study, we have characterised the antigen-specific function of autologous cytotoxic T lymphocytes (CTL) specific for the HLA-A2 restricted peptide, pCEA691–699, isolated from the peripheral T cell repertoire of PC patients and sought to determine if ex vivo PD-L1 & TIM3 blockade could enhance CTL function. CD8+ T cell lines were generated from peripheral blood mononuclear cells (PBMCs) of 18 HLA-A2+ patients with PC and from 15 healthy controls. In vitro peptide specific responses were evaluated by flow cytometry after staining for intracellular cytokine production and CSFE cytotoxicity assays using pancreatic cancer cell lines as targets. Cytokine secreting functional CEA691-specific CTL lines were successfully generated from 10 of 18PC patients, with two CTL lines able to recognise and kill both CEA691 peptide-loaded T2 cells and CEA+ HLA-A2+ pancreatic cancer cell lines. In the presence of ex vivo PD-L1 blockade, functional CEA691-specific CD8+ T cell responses, including IFN-γ secretion and proliferation, were enhanced and this effect was more pronounced on Ag-specific T cells isolated from tumor draining lymph nodes. These data demonstrate that CEA691-specific CTL can be readily expanded from the self-restricted T cell repertoire of PC patients and that their function can be enhanced by PD-L1 blockade. PMID:28710313

Adaptation to Human Populations Is Revealed by Within-Host Polymorphisms in HIV-1 and Hepatitis C Virus

PubMed Central

Poon, Art F. Y; Kosakovsky Pond, Sergei L.; Bennett, Phil; Richman, Douglas D; Leigh Brown, Andrew J.; Frost, Simon D. W

2007-01-01

CD8+ cytotoxic T-lymphocytes (CTLs) perform a critical role in the immune control of viral infections, including those caused by human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). As a result, genetic variation at CTL epitopes is strongly influenced by host-specific selection for either escape from the immune response, or reversion due to the replicative costs of escape mutations in the absence of CTL recognition. Under strong CTL-mediated selection, codon positions within epitopes may immediately “toggle” in response to each host, such that genetic variation in the circulating virus population is shaped by rapid adaptation to immune variation in the host population. However, this hypothesis neglects the substantial genetic variation that accumulates in virus populations within hosts. Here, we evaluate this quantity for a large number of HIV-1– (n ≥ 3,000) and HCV-infected patients (n ≥ 2,600) by screening bulk RT-PCR sequences for sequencing “mixtures” (i.e., ambiguous nucleotides), which act as site-specific markers of genetic variation within each host. We find that nonsynonymous mixtures are abundant and significantly associated with codon positions under host-specific CTL selection, which should deplete within-host variation by driving the fixation of the favored variant. Using a simple model, we demonstrate that this apparently contradictory outcome can be explained by the transmission of unfavorable variants to new hosts before they are removed by selection, which occurs more frequently when selection and transmission occur on similar time scales. Consequently, the circulating virus population is shaped by the transmission rate and the disparity in selection intensities for escape or reversion as much as it is shaped by the immune diversity of the host population, with potentially serious implications for vaccine design. PMID:17397261

Elicitation of anti-vesicular stomatitis virus cytotoxic T lymphocytes by using purified viral and cellular antigens incorporated into phospholipid vesicles.

PubMed

Ruebush, M J; Hale, A H; Harris, D T

1981-05-01

We evaluated the minimal molecular and cellular requirements for elicitation of anti-vesicular stomatitis virus (VSV) cytotoxic T lymphocytes (CTL). The results indicated that lipid vesicles containing the purified major surface glyco-protein of VSV (G protein) and purified H-2K(k) glycoproteins elicited specific H-2K(k)-restricted anti-VSV CTL. These antiviral CTL were shown to be Ly 1(-),2(+). However, both Ly 1(+),2(-) and Ly1(-),2(+) T-cell subpopulations were shown to be required for elicitation of these CTL.

Cancer regression induced by modified CTL therapy is regulated by HLA class II and class I antigens in Japanese patients with advanced cancer.

PubMed

Araki, K; Noguchi, Y; Hirouchi, T; Yoshikawa, E; Kataoka, S; Silverni, L; Miyazawa, H; Kuzuhara, H; Suzuki, C; Shimada, Y; Hamasato, S; Maeda, N; Shimamura, Y; Ogawa, Y; Ohtsuki, Y; Fujimoto, S

2000-12-01

Autologous cancer-specific bulk CTLs are unlikely to be induced by in vitro CTL generation (ivtCTLG) using peripheral blood mononuclear cells (PBMCs) of cancer patients when autologous cancer cells are used as in vitro stimulators. However, autologous cancer-specific bulk CTLs are frequently activated when allogeneic cancer cells are used as in vitro stimulators, regardless of the type of cancer cell. We have developed a cancer-specific immunotherapy called modified CTL therapy, which involves adoptive immunotherapy of autologous cancer-specific bulk CTLs after active immunization of autologous or allogeneic cancer cells screened as in vitro stimulators according to their ability to induce autologous cancer-specific CTLs (ACS. CTLs). Cancer did not regress in patients in whom ACS.CTLs were not induced by ivtCTLG using the patients' PBMCs in therapy. Cancer regression, albeit temporary, occurred solely in patients under the immunological condition that ACS.CTLs were induced by ivtCTLG using PBMCs through the therapy. The induction of ACS.CTLs by ivtCTLG using patient PBMCs in therapy was related to patients' HLA class II antigens. HLA DR8 was seen more frequently in ACS.CTL-inducible patients than in ACS.CTL-uninducible patients (P=0.051). On the contrary, HLA DQ3 was seen more frequently in ACS.CTL-uninducible patients (P=0.055). On the other hand, the success in therapy, albeit temporary, was related mainly to patients' HLA class I antigens. HLA B61 was seen more frequently in patients whose therapy proved effective than in patients whose therapy proved ineffective (P=0.018). HLA Cw7 was seen more frequently in therapy-ineffective patients (P=0.040).

Potential contribution of a novel Tax epitope-specific CD4+ T cells to graft-versus-Tax effect in adult T cell leukemia patients after allogeneic hematopoietic stem cell transplantation.

PubMed

Tamai, Yotaro; Hasegawa, Atsuhiko; Takamori, Ayako; Sasada, Amane; Tanosaki, Ryuji; Choi, Ilseung; Utsunomiya, Atae; Maeda, Yasuhiro; Yamano, Yoshihisa; Eto, Tetsuya; Koh, Ki-Ryang; Nakamae, Hirohisa; Suehiro, Youko; Kato, Koji; Takemoto, Shigeki; Okamura, Jun; Uike, Naokuni; Kannagi, Mari

2013-04-15

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for adult T cell leukemia/lymphoma (ATL) caused by human T cell leukemia virus type 1 (HTLV-1). We previously reported that Tax-specific CD8(+) cytotoxic T lymphocyte (CTL) contributed to graft-versus-ATL effects in ATL patients after allo-HSCT. However, the role of HTLV-1-specific CD4(+) T cells in the effects remains unclear. In this study, we showed that Tax-specific CD4(+) as well as CD8(+) T cell responses were induced in some ATL patients following allo-HSCT. To further analyze HTLV-1-specific CD4(+) T cell responses, we identified a novel HLA-DRB1*0101-restricted epitope, Tax155-167, recognized by HTLV-1-specific CD4(+) Th1-like cells, a major population of HTLV-1-specific CD4(+) T cell line, which was established from an ATL patient at 180 d after allo-HSCT from an unrelated seronegative donor by in vitro stimulation with HTLV-1-infected cells from the same patient. Costimulation of PBMCs with both the identified epitope (Tax155-167) and known CTL epitope peptides markedly enhanced the expansion of Tax-specific CD8(+) T cells in PBMCs compared with stimulation with CTL epitope peptide alone in all three HLA-DRB1*0101(+) patients post-allo-HSCT tested. In addition, direct detection using newly generated HLA-DRB1*0101/Tax155-167 tetramers revealed that Tax155-167-specific CD4(+) T cells were present in all HTLV-1-infected individuals tested, regardless of HSCT. These results suggest that Tax155-167 may be the dominant epitope recognized by HTLV-1-specific CD4(+) T cells in HLA-DRB1*0101(+)-infected individuals and that Tax-specific CD4(+) T cells may augment the graft-versus-Tax effects via efficient induction of Tax-specific CD8(+) T cell responses.

Mathematical modeling of escape of HIV from cytotoxic T lymphocyte responses

NASA Astrophysics Data System (ADS)

Ganusov, Vitaly V.; Neher, Richard A.; Perelson, Alan S.

2013-01-01

Human immunodeficiency virus (HIV-1 or simply HIV) induces a persistent infection, which in the absence of treatment leads to AIDS and death in almost all infected individuals. HIV infection elicits a vigorous immune response starting about 2-3 weeks postinfection that can lower the amount of virus in the body, but which cannot eradicate the virus. How HIV establishes a chronic infection in the face of a strong immune response remains poorly understood. It has been shown that HIV is able to rapidly change its proteins via mutation to evade recognition by virus-specific cytotoxic T lymphocytes (CTLs). Typically, an HIV-infected patient will generate 4-12 CTL responses specific for parts of viral proteins called epitopes. Such CTL responses lead to strong selective pressure to change the viral sequences encoding these epitopes so as to avoid CTL recognition. Indeed, the viral population ‘escapes’ from about half of the CTL responses by mutation in the first year. Here we review experimental data on HIV evolution in response to CTL pressure, mathematical models developed to explain this evolution, and highlight problems associated with the data and previous modeling efforts. We show that estimates of the strength of the epitope-specific CTL response depend on the method used to fit models to experimental data and on the assumptions made regarding how mutants are generated during infection. We illustrate that allowing CTL responses to decay over time may improve the model fit to experimental data and provides higher estimates of the killing efficacy of HIV-specific CTLs. We also propose a novel method for simultaneously estimating the killing efficacy of multiple CTL populations specific for different epitopes of HIV using stochastic simulations. Lastly, we show that current estimates of the efficacy at which HIV-specific CTLs clear virus-infected cells can be improved by more frequent sampling of viral sequences and by combining data on sequence evolution with experimentally measured CTL dynamics.

Quantitative T-cell repertoire analysis of peripheral blood mononuclear cells from lung cancer patients following long-term cancer peptide vaccination.

PubMed

Takeda, Kazuyoshi; Kitaura, Kazutaka; Suzuki, Ryuji; Owada, Yuki; Muto, Satoshi; Okabe, Naoyuki; Hasegawa, Takeo; Osugi, Jun; Hoshino, Mika; Tsunoda, Takuya; Okumura, Ko; Suzuki, Hiroyuki

2018-06-01

Therapeutic cancer peptide vaccination is an immunotherapy designed to elicit cytotoxic T-lymphocyte (CTL) responses in patients. A number of therapeutic vaccination trials have been performed, nevertheless there are only a few reports that have analyzed the T-cell receptors (TCRs) expressed on tumor antigen-specific CTLs. Here, we use next-generation sequencing (NGS) to analyze TCRs of vaccine-induced CTL clones and the TCR repertoire of bulk T cells in peripheral blood mononuclear cells (PBMCs) from two lung cancer patients over the course of long-term vaccine therapy. In both patients, vaccination with two epitope peptides derived from cancer/testis antigens (upregulated lung cancer 10 (URLC10) and cell division associated 1 (CDCA1)) induced specific CTLs expressing various TCRs. All URLC10-specific CTL clones tested showed Ca 2+ influx, IFN-γ production, and cytotoxicity when co-cultured with URLC10-pulsed tumor cells. Moreover, in CTL clones that were not stained with the URLC10/MHC-multimer, the CD3 ζ chain was not phosphorylated. NGS of the TCR repertoire of bulk PBMCs demonstrated that the frequency of vaccine peptide-specific CTL clones was near the minimum detectable threshold level. These results demonstrate that vaccination induces antigen-specific CTLs expressing various TCRs at different time points in cancer patients, and that some CTL clones are maintained in PBMCs during long-term treatment, including some with TCRs that do not bind peptide/MHC-multimer.

Encapsulating Immunostimulatory CpG Oligonucleotides in Listeriolysin O-Liposomes Promotes a Th1-Type Response and CTL Activity

PubMed Central

Andrews, Chasity D.; Huh, Myung-Sook; Patton, Kathryn; Higgins, Debbie; Van Nest, Gary; Ott, Gary; Lee, Kyung-Dall

2013-01-01

Immunostimulatory sequences (ISS) are short DNA sequences containing unmethylated CpG dimers that have multiple effects on the host immune system, including the ability to stimulate antigen-specific cytotoxic T lymphocytes (CTLs) and drive Th1-type immune responses. Listeriolysin O (LLO)-containing pH-sensitive liposomes have been shown to efficiently deliver macromolecules to the cytosol of APCs and efficiently stimulate CTLs. We hypothesized that encapsulating ISS-oligodeoxyribonucleotides (ODNs) in this delivery system would enhance the cell-mediated immune response and skew Th1-type responses in protein antigen-based vaccination utilizing LLO-liposomes. In vitro studies indicated that co-encapsulation of ISS in LLO-liposomes engendered activation of the NF-κB pathway while maintaining the efficient cytosolic delivery of antigen mediated by the co-encapsulated LLO. Antigen-specific CTL responses monitored by using the model antigen ovalbumin (OVA) in mice were enhanced when mice were immunized with OVA and ISS-ODN-containing LLO-liposomes compared with those immunized with either OVA-containing LLO-liposomes or OVA-ISS conjugates. The enhanced immune responses were of the Th1-type as monitored by the robust OVA-specific IgG2a induction and the OVA CD8 peptide-stimulated IFN-γ secretion. Our study suggests that including ISS-ODN in LLO-containing pH-sensitive liposomes yields a vaccine delivery system that enhances the cell-mediated immune response and skews this response toward the Th1-type. PMID:22376145

Short communication an interferon-γ ELISPOT assay with two cytotoxic T cell epitopes derived from HTLV-1 tax region 161-233 discriminates HTLV-1-associated myelopathy/tropical spastic paraparesis patients from asymptomatic HTLV-1 carriers in a Peruvian population.

PubMed

Best, Ivan; López, Giovanni; Talledo, Michael; MacNamara, Aidan; Verdonck, Kristien; González, Elsa; Tipismana, Martín; Asquith, Becca; Gotuzzo, Eduardo; Vanham, Guido; Clark, Daniel

2011-11-01

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic and progressive disorder caused by the human T-lymphotropic virus type 1 (HTLV-1). In HTLV-1 infection, a strong cytotoxic T cell (CTL) response is mounted against the immunodominant protein Tax. Previous studies carried out by our group reported that increased IFN-γ enzyme-linked immunospot (ELISPOT) responses against the region spanning amino acids 161 to 233 of the Tax protein were associated with HAM/TSP and increased HTLV-1 proviral load (PVL). An exploratory study was conducted on 16 subjects with HAM/TSP, 13 asymptomatic carriers (AC), and 10 HTLV-1-seronegative controls (SC) to map the HAM/TSP-associated CTL epitopes within Tax region 161-233. The PVL of the infected subjects was determined and the specific CTL response was evaluated with a 6-h incubation IFN-γ ELISPOT assay using peripheral blood mononuclear cells (PBMCs) stimulated with 16 individual overlapping peptides covering the Tax region 161-233. Other proinflammatory and Th1/Th2 cytokines were also quantified in the supernatants by a flow cytometry multiplex assay. In addition, a set of human leukocyte antigen (HLA) class I alleles that bind with high affinity to the CTL epitopes of interest was determined using computational tools. Univariate analyses identified an association between ELISPOT responses to two new CTL epitopes, Tax 173-185 and Tax 181-193, and the presence of HAM/TSP as well as an increased PVL. The HLA-A*6801 allele, which is predicted to bind to the Tax 181-193 peptide, was overpresented in the HAM/TSP patients tested.

Quantifying factors determining the rate of CTL escape and reversion during acute and chronic phases of HIV infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganusov, Vitaly V; Korber, Bette M; Perelson, Alan S

Human immunodeficiency virus (HIV) often evades cytotoxic T cell (CTL) responses by generating variants that are not recognized by CTLs. However, the importance and quantitative details of CTL escape in humans are poorly understood. In part, this is because most studies looking at escape of HIV from CTL responses are cross-sectional and are limited to early or chronic phases of the infection. We use a novel technique of single genome amplification (SGA) to identify longitudinal changes in the transmitted/founder virus from the establishment of infection to the viral set point at 1 year after the infection. We find that HIVmore » escapes from virus-specific CTL responses as early as 30-50 days since the infection, and the rates of viral escapes during acute phase of the infection are much higher than was estimated in previous studies. However, even though with time virus acquires additional escape mutations, these late mutations accumulate at a slower rate. A poor correlation between the rate of CTL escape in a particular epitope and the magnitude of the epitope-specific CTL response suggests that the lower rate of late escapes is unlikely due to a low efficacy of the HIV-specific CTL responses in the chronic phase of the infection. Instead, our results suggest that late and slow escapes are likely to arise because of high fitness cost to the viral replication associated with such CTL escapes. Targeting epitopes in which virus escapes slowly or does not escape at all by CTL responses may, therefore, be a promising direction for the development of T cell based HIV vaccines.« less

Primary Human Immunodeficiency Virus Type 1 (HIV-1) Infection during HIV-1 Gag Vaccination▿

PubMed Central

Balamurugan, Arumugam; Lewis, Martha J.; Kitchen, Christina M. R.; Robertson, Michael N.; Shiver, John W.; Daar, Eric S.; Pitt, Jacqueline; Ali, Ayub; Ng, Hwee L.; Currier, Judith S.; Yang, Otto O.

2008-01-01

Vaccination for human immunodeficiency virus type 1 (HIV-1) remains an elusive goal. Whether an unsuccessful vaccine might not only fail to provoke detectable immune responses but also could actually interfere with subsequent natural immunity upon HIV-1 infection is unknown. We performed detailed assessment of an HIV-1 gag DNA vaccine recipient (subject 00015) who was previously uninfected but sustained HIV-1 infection before completing a vaccination trial and another contemporaneously acutely infected individual (subject 00016) with the same strain of HIV-1. Subject 00015 received the vaccine at weeks 0, 4, and 8 and was found to have been acutely HIV-1 infected around the time of the third vaccination. Subject 00016 was a previously HIV-1-seronegative sexual contact who had symptoms of acute HIV-1 infection approximately 2 weeks earlier than subject 00015 and demonstrated subsequent seroconversion. Both individuals reached an unusually low level of chronic viremia (<1,000 copies/ml) without treatment. Subject 00015 had no detectable HIV-1-specific cytotoxic T-lymphocyte (CTL) responses until a borderline response was noted at the time of the third vaccination. The magnitude and breadth of Gag-specific CTL responses in subject 00015 were similar to those of subject 00016 during early chronic infection. Viral sequences from gag, pol, and nef confirmed the common source of HIV-1 between these individuals. The diversity and divergence of sequences in subjects 00015 and 00016 were similar, indicating similar immune pressure on these proteins (including Gag). As a whole, the data suggested that while the gag DNA vaccine did not prime detectable early CTL responses in subject 00015, vaccination did not appreciably impair his ability to contain viremia at levels similar to those in subject 00016. PMID:18199650

CD8 down-regulation and functional impairment of SIV-specific cytotoxic T lymphocytes in lymphoid and mucosal tissues during SIV infection.

PubMed

Xu, Huanbin; Wang, Xiaolei; Lackner, Andrew A; Veazey, Ronald S

2013-06-01

Functional impairment of virus-specific T cells is a hallmark of HIV/SIV infection, but the underlying mechanisms of this dysfunction are not well understood. To address this, we simultaneously analyzed the expression and intensity of CD8 and inhibitory PD-1 on CTL in blood and lymphoid tissues in SIV-infected rhesus macaques. The intensity (mean channel fluorescence) of CD8 expression was transiently down-regulated in early SIV infection (10-14 dpi), despite an increase in CD8(+) T cell proliferation. In chronic infection, CD8 expression was maintained at low levels on CD8(+) T cells in all tissues. Interestingly, Gag-specific CTLs were clearly divided into CD8high- and CD8low-expressing populations in SIV-infected macaques, and CD8low Gag-specific cells increased with disease progression, especially in lymphoid tissues when compared with peripheral blood or in Gag-vaccinated controls. Moreover, the CD8low CTL population secreted lower levels of cytokines upon SIV antigen stimulation and exhibited lower proliferative capacity during infection compared with the CD8high CTL population. Meanwhile, intensity of PD-1 expression on Gag-specific CTL in chronic infection was significantly higher than in acute SIV infection, although the frequencies of PD-1+ Gag-specific cells were similar in acute and chronic stages. In summary, down-regulation of CD8 expression and higher expression of PD-1 on SIV-specific CTLs could coordinately attenuate SIV-specific CTL responses and their ability to recognize virus-infected target cells, especially in lymphoid tissues, resulting in failure to contain viremia, and continued persistence and replication of HIV in lymphoid tissue reservoirs.

Suppression of antitumour protective cytotoxic T lymphocyte responses to a human papillomavirus 16 E7 DNA vaccine by coinjection of interleukin-12 complementary DNA: involvement of nitric oxide in immune suppression

PubMed Central

Sin, Jeong-Im

2009-01-01

Interleukin-12 (IL-12) has been shown to enhance cellular immunity in vitro and in vivo. The beneficial roles of IL-12 as a DNA vaccine adjuvant have been commonly observed. Here the impact of IL-12 complementary DNA (cDNA) as an adjuvant for a human papillomavirus (HPV) type 16 E7 DNA vaccine is investigated in a mouse tumour model. Coinjection of E7 DNA vaccine with IL-12 cDNA completely suppressed antigen-specific cytotoxic T-lymphocyte (CTL) responses, leading to a complete loss of antitumour protection from a tumour cell challenge. In addition, antigen-specific antibody and T helper cell proliferative responses were also suppressed by IL-12 cDNA coinjection. This inhibition was observed over different IL-12 cDNA doses. Furthermore, separate leg injections of IL-12 and E7 cDNAs suppressed antigen-specific CTL and tumour protective responses, but not antibody and T helper cell proliferative responses, suggesting different pathways for suppression of these two separate responses. Further knockout animal studies demonstrated that interferon-γ and nitric oxide are not directly associated with suppression of antigen-specific antibody responses by IL-12 cDNA coinjection. However, nitric oxide was found to be involved in suppression of antigen-specific CTL and tumour protective responses by IL-12 cDNA coinjection. These data suggest that coinjection of IL-12 cDNA results in suppression of E7-specific CTL responses through nitric oxide, leading to a loss of antitumour resistance in this DNA vaccine model. This study further shows that the adjuvant effect of IL-12 is dependent on the antigen types tested. PMID:19740332

Suppression of antitumour protective cytotoxic T lymphocyte responses to a human papillomavirus 16 E7 DNA vaccine by coinjection of interleukin-12 complementary DNA: involvement of nitric oxide in immune suppression.

PubMed

Sin, Jeong-Im

2009-09-01

Interleukin-12 (IL-12) has been shown to enhance cellular immunity in vitro and in vivo. The beneficial roles of IL-12 as a DNA vaccine adjuvant have been commonly observed. Here the impact of IL-12 complementary DNA (cDNA) as an adjuvant for a human papillomavirus (HPV) type 16 E7 DNA vaccine is investigated in a mouse tumour model. Coinjection of E7 DNA vaccine with IL-12 cDNA completely suppressed antigen-specific cytotoxic T-lymphocyte (CTL) responses, leading to a complete loss of antitumour protection from a tumour cell challenge. In addition, antigen-specific antibody and T helper cell proliferative responses were also suppressed by IL-12 cDNA coinjection. This inhibition was observed over different IL-12 cDNA doses. Furthermore, separate leg injections of IL-12 and E7 cDNAs suppressed antigen-specific CTL and tumour protective responses, but not antibody and T helper cell proliferative responses, suggesting different pathways for suppression of these two separate responses. Further knockout animal studies demonstrated that interferon-gamma and nitric oxide are not directly associated with suppression of antigen-specific antibody responses by IL-12 cDNA coinjection. However, nitric oxide was found to be involved in suppression of antigen-specific CTL and tumour protective responses by IL-12 cDNA coinjection. These data suggest that coinjection of IL-12 cDNA results in suppression of E7-specific CTL responses through nitric oxide, leading to a loss of antitumour resistance in this DNA vaccine model. This study further shows that the adjuvant effect of IL-12 is dependent on the antigen types tested.

A scallop C-type lectin from Argopecten irradians (AiCTL5) with activities of lipopolysaccharide binding and Gram-negative bacteria agglutination.

PubMed

Mu, Changkao; Song, Xiaoyan; Zhao, Jianmin; Wang, Lingling; Qiu, Limei; Zhang, Huan; Zhou, Zhi; Wang, Mengqiang; Song, Linsheng; Wang, Chunlin

2012-05-01

C-type lectins are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a C-type lectin (designated as AiCTL5) was identified and characterized from Argopecten irradians. The full-length cDNA of AiCTL5 was of 673 bp, containing a 5' untranslated region (UTR) of 24 bp, a 3' UTR of 130 bp with a poly (A) tail, and an open reading frame (ORF) of 519 bp encoding a polypeptide of 172 amino acids with a putative signal peptide of 17 amino acids. A C-type lectin-like domain (CRD) containing 6 conserved cysteines and a putative glycosylation sites were identified in the deduced amino acid sequence of AiCTL5. AiCTL5 shared 11%-27.5% identity with the previous reported C-type lectin from A. irradians. The cDNA fragment encoding the mature peptide of AiCTL5 was recombined into pET-21a (+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli Origami (DE3). The recombinant AiCTL5 (rAiCTL5) agglutinated Gram-negative E. coli TOP10F' and Listonella anguillarum, but did not agglutinate Gram-positive bacteria Bacillus thuringiensis and Micrococcus luteus, and the agglutination could be inhibited by EDTA, indicating that AiCTL5 was a Ca(2+)-dependent lectin. rAiCTL5 exhibited a significantly strong activity to bind LPS from E. coli, which conformed to the agglutinating activity toward Gram-negative bacteria. Moreover, rAiCTL5 also agglutinated rabbit erythrocytes. These results indicated that AiCTL5 could function as a pattern recognition receptor to protect bay scallop from Gram-negative bacterial infection, and also provide evidence to understand the structural and functional diverse of lectin. Copyright © 2012 Elsevier Ltd. All rights reserved.

Diverse cross-reactive potential and Vbeta gene usage of an epitope-specific cytotoxic T-lymphocyte population in monkeys immunized with diverse human immunodeficiency virus type 1 Env immunogens.

PubMed

Hulot, Sandrine L; Seaman, Michael S; Sen, Pritha; Autissier, Patrick A; Manuel, Edwin R; Letvin, Norman L

2009-10-01

An ideal human immunodeficiency virus type 1 (HIV-1) vaccine would elicit potent cellular and humoral immune responses that recognize diverse strains of the virus. In the present study, combined methodologies (flow cytometry, Vbeta repertoire analysis, and complementarity-determining region 3 sequencing) were used to determine the clonality of CD8(+) T lymphocytes taking part in the recognition of variant epitope peptides elicited in Mamu-A*01-positive rhesus monkeys immunized with vaccines encoding diverse HIV-1 envelopes (Envs). Monkeys immunized with clade B Envs generated CD8(+) T lymphocytes that cross-recognized both clade B- and clade C-p41A epitope peptides using a large degree of diversity in Vbeta gene usage. However, with two monkeys immunized with clade C Env, one monkey exhibited p41A-specific cytotoxic T-lymphocytes (CTL) with the capacity for cross-recognition of variant epitopes, while the other monkey did not. These studies demonstrate that the cross-reactive potential of variant p41A epitope peptide-specific CTL populations can differ between monkeys that share the same restricting major histocompatibility complex class I molecule and receive the same vaccine immunogens.

Loss of the ex vivo but not the reinducible CD8+ T-cell response to Tax in human T-cell leukemia virus type 1-infected patients with adult T-cell leukemia/lymphoma.

PubMed

Arnulf, B; Thorel, M; Poirot, Y; Tamouza, R; Boulanger, E; Jaccard, A; Oksenhendler, E; Hermine, O; Pique, C

2004-01-01

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy (HAM). In asymptomatic carriers and HAM patients, HTLV-1 infection leads to a vigorous cytotoxic T-cell (CTL) response mainly directed to the regulatory Tax protein. In contrast, initial studies showed that anti-HTLV-1 CTL activities were not reproductively detected in ATLL patients, neither ex vivo, nor after in vitro restimulation. To better understand this discrepancy, we explored the anti-HTLV-1 CD8+ T-cell response of eight ATLL patients by using in vitro restimulated or freshly isolated CD8+ T cells. In all the ATLL patients, we found that mitogenic activation allowed the induction of CD8+ T cells able to lyse autologous HTLV-1-infected cells and/or to produce IFNgamma in response to Tax peptides. In contrast, only a minority of the patients possessed CD8+ cells able to respond ex vivo to the same epitopes. These findings indicate that although a restimulatable anti-HTLV-1 CTL activity persists during ATLL, the specific ex vivo response is not constantly maintained. This provides definitive evidence that the CD8+ T-cell response to HTLV-1 is affected by ATLL development and reveals that a major defect concerns the generation and/or the functionality of CD8+ effectors.

A monoclonal cytolytic T-lymphocyte response observed in a melanoma patient vaccinated with a tumor-specific antigenic peptide encoded by gene MAGE-3

PubMed Central

Coulie, Pierre G.; Karanikas, Vaios; Colau, Didier; Lurquin, Christophe; Landry, Claire; Marchand, Marie; Dorval, Thierry; Brichard, Vincent; Boon, Thierry

2001-01-01

Vaccination of melanoma patients with tumor-specific antigens recognized by cytolytic T lymphocytes (CTL) produces significant tumor regressions in a minority of patients. These regressions appear to occur in the absence of massive CTL responses. To detect low-level responses, we resorted to antigenic stimulation of blood lymphocyte cultures in limiting dilution conditions, followed by tetramer analysis, cloning of the tetramer-positive cells, and T-cell receptor (TCR) sequence analysis of the CTL clones that showed strict specificity for the tumor antigen. A monoclonal CTL response against a MAGE-3 antigen was observed in a melanoma patient, who showed partial rejection of a large metastasis after treatment with a vaccine containing only the tumor-specific antigenic peptide. Tetramer analysis after in vitro restimulation indicated that about 1/40,000 postimmunization CD8+ blood lymphocytes were directed against the antigen. The same TCR was present in all of the positive microcultures. TCR evaluation carried out directly on blood lymphocytes by PCR amplification led to a similar frequency estimate after immunization, whereas the TCR was not found among 2.5 × 106 CD8+ lymphocytes collected before immunization. Our results prove unambiguously that vaccines containing only a tumor-specific antigenic peptide can elicit a CTL response. Even though they provide no information about the effector mechanisms responsible for the observed reduction in tumor mass in this patient, they would suggest that low-level CTL responses can initiate tumor rejection. PMID:11517302

Detection of Tax-specific CTLs in lymph nodes of adult T-cell leukemia/lymphoma patients and its association with Foxp3 positivity of regulatory T-cell function.

PubMed

Ichikawa, Ayako; Miyoshi, Hiroaki; Arakawa, Fumiko; Kiyasu, Junichi; Sato, Kensaku; Niino, Daisuke; Kimura, Yoshizo; Yoshida, Maki; Kawano, Riko; Muta, Hiroko; Sugita, Yasuo; Ohshima, Koichi

2017-06-01

Human T-cell lymphotropic virus type (HTLV)-1 Tax is a viral protein that has been reported to be important in the proliferation of adult T-cell leukemia/lymphoma (ATLL) cells and to be a target of HTLV-1-specific cytotoxic T lymphocytes (CTLs). However, it is not clear how Tax-specific CTLs behave in lymph nodes of ATLL patients. The present study analyzed the immunostaining of Tax-specific CTLs. Furthermore, ATLL tumor cells are known to be positive for forkhead box P3 (Foxp3)and to have a regulatory T (Treg)-cell-like function. The association between T-reg function and number and activity of Tax-specific CTLs was also investigated. A total of 15 ATLL lymphoma cases with human leukocyte antigen (HLA)-A24, for which Tax has a high affinity, were selected from the files of the Department of Pathology, School of Medicine, Kurume University (Kurume, Japan) using a polymerase chain reaction (PCR) method. Immunostaining was performed for cluster of differentiation (CD) 20, CD3, CD4, CD8, T-cell intracellular antigen-1 and Foxp3 in paraffin sections, and for Tax, interferon γ and HLA-A24 in frozen sections. In addition, the staining of Tax-specific CTLs (HLA-A24-restricted) was analyzed by MHC Dextramer ® assay in frozen sections. In addition, the messenger RNA expression of Tax and HTLV-1 basic leucine zipper factor were also evaluated by reverse transcription-PCR. Immunohistochemical staining of Tax protein in lymphoma tissue revealed the presence of positive lymphoma cells ranging from 5 to 80%, and immunohistochemical staining of HLA-A24 revealed the presence of positive lymphoma cells ranging from 1 to 95%. The expression of Tax and HLA-A24 was downregulated by viral function. Foxp3, a marker for Treg cells, was expressed in 0-90% of cells. Several cases exhibited Tax-specific CTL (HLA-A24-restricted)-positive cells, and there was an inverse correlation between Tax-specific CTLs and Foxp3. However, neither Tax nor HLA-A24 expression was associated with CTL or Foxp3. Our study indicated the possibility that ATLL cells, which expressed Tax, target of CTL, evade the CTL-mediated immune control by expression of Foxp3 as a Treg function.

Detection of Tax-specific CTLs in lymph nodes of adult T-cell leukemia/lymphoma patients and its association with Foxp3 positivity of regulatory T-cell function

PubMed Central

Ichikawa, Ayako; Miyoshi, Hiroaki; Arakawa, Fumiko; Kiyasu, Junichi; Sato, Kensaku; Niino, Daisuke; Kimura, Yoshizo; Yoshida, Maki; Kawano, Riko; Muta, Hiroko; Sugita, Yasuo; Ohshima, Koichi

2017-01-01

Human T-cell lymphotropic virus type (HTLV)-1 Tax is a viral protein that has been reported to be important in the proliferation of adult T-cell leukemia/lymphoma (ATLL) cells and to be a target of HTLV-1-specific cytotoxic T lymphocytes (CTLs). However, it is not clear how Tax-specific CTLs behave in lymph nodes of ATLL patients. The present study analyzed the immunostaining of Tax-specific CTLs. Furthermore, ATLL tumor cells are known to be positive for forkhead box P3 (Foxp3)and to have a regulatory T (Treg)-cell-like function. The association between T-reg function and number and activity of Tax-specific CTLs was also investigated. A total of 15 ATLL lymphoma cases with human leukocyte antigen (HLA)-A24, for which Tax has a high affinity, were selected from the files of the Department of Pathology, School of Medicine, Kurume University (Kurume, Japan) using a polymerase chain reaction (PCR) method. Immunostaining was performed for cluster of differentiation (CD) 20, CD3, CD4, CD8, T-cell intracellular antigen-1 and Foxp3 in paraffin sections, and for Tax, interferon γ and HLA-A24 in frozen sections. In addition, the staining of Tax-specific CTLs (HLA-A24-restricted) was analyzed by MHC Dextramer® assay in frozen sections. In addition, the messenger RNA expression of Tax and HTLV-1 basic leucine zipper factor were also evaluated by reverse transcription-PCR. Immunohistochemical staining of Tax protein in lymphoma tissue revealed the presence of positive lymphoma cells ranging from 5 to 80%, and immunohistochemical staining of HLA-A24 revealed the presence of positive lymphoma cells ranging from 1 to 95%. The expression of Tax and HLA-A24 was downregulated by viral function. Foxp3, a marker for Treg cells, was expressed in 0–90% of cells. Several cases exhibited Tax-specific CTL (HLA-A24-restricted)-positive cells, and there was an inverse correlation between Tax-specific CTLs and Foxp3. However, neither Tax nor HLA-A24 expression was associated with CTL or Foxp3. Our study indicated the possibility that ATLL cells, which expressed Tax, target of CTL, evade the CTL-mediated immune control by expression of Foxp3 as a Treg function. PMID:28599462

The role and mechanics of dendritic cells in tumor antigen acquisition and presentation following laser immunotherapy

NASA Astrophysics Data System (ADS)

Laverty, Sean M.; Dawkins, Bryan A.; Chen, Wei R.

2018-02-01

We extend our model of the antitumor immune response initiated by laser-immunotherapy treatment to more closely examine key steps in the immune response 1) tumor antigen acquisition by antigen-presenting dendritic cells (DCs) and 2) cytotoxic T cell (CTL) priming by lymphatic DCs. Specifically we explore the formation of DC-CTL complexes that lead to CTL priming. We find that the bias in the dissociation rate of the complex influences the outcome of treatment. In particular, a bias towards priming favors a rapid activated CTL response and the clearance of tumors.

Acute Morphine Administration Reduces Cell-Mediated Immunity and Induces Reactivation of Latent Herpes Simplex Virus Type 1 in BALB/c Mice

PubMed Central

Mojadadi, Shafi; Jamali, Abbas; Khansarinejad, Behzad; Soleimanjahi, Hoorieh; Bamdad, Taravat

2009-01-01

Acute morphine administration is known to alter the course of herpes simplex virus infection. In this study, the effect of acute morphine administration on the reactivation of latent herpes was investigated in a mouse model. Because of the important role of cytolytic T lymphocyte (CTL) activity in the inhibition of herpes simplex virus type 1 (HSV-1) reactivation, the effect of acute morphine administration on CTL responses was also evaluated. Furthermore, lymphocyte proliferation and IFN-γ production were evaluated for their roles in the induction of the CTL response. The findings showed that acute morphine administration significantly reduced CTL responses, lymphocyte proliferation, and IFN-γ production. Furthermore, acute morphine administration has been shown to reactivate latent HSV-1. Previous studies have shown that cellular immune responses have important roles in the inhibition of HSV reactivation. These findings suggest that suppression of a portion of the cellular immune response after acute morphine administration may constitute one part of the mechanism that induces HSV reactivation. PMID:19403060

Low doses of allergen and probiotic supplementation separately or in combination alleviate allergic reactions to cow β-lactoglobulin in mice.

PubMed

Thang, Cin L; Boye, Joyce I; Zhao, Xin

2013-02-01

Probiotic supplementation and oral tolerance induction can reduce certain types of food allergy. The objectives of this study were to investigate the allergy-reducing effects of probiotics (VSL#3) and/or oral tolerance induction via low doses of an allergen supplementation in β-lactoglobulin (BLG)-sensitized mice. Three-week-old, male BALB/c mice were divided into 6 groups (n = 8/group): sham-sensitized negative control (CTL-), BLG-sensitized positive control (CTL+), oral tolerance-induced and BLG-sensitized group (OT), probiotic-supplemented OT group (OTP), probiotic-supplemented CTL- (PRO), and probiotic-supplemented and BLG-sensitized (PROC) groups. Mice were i.p. sensitized with BLG and alum and then orally challenged with BLG. Immunological responses were assessed by monitoring hypersensitivity scores and measuring levels of BLG-specific serum Igs, total serum IgE and fecal IgA, and cytokines from serum and spleen lysates. Hypersensitivity scores were significantly lower in the PROC (2.00 ± 0.53), OT (0.75 ± 0.46), and OTP mice (1.00 ± 0.53) than in the CTL+ mice (2.63 ± 0.52) as were BLG-specific serum IgE concentrations (34.3 ± 10, 0.442 ± 0.36, 3.54 ± 3.5, and 78.5 ± 8.7 μg/L for PROC, OT, OTP, and CTL+, respectively). Our results suggest that supplementation of VSL#3 suppressed the allergic reaction mainly through increased intestinal secretary IgA (sIgA) in PROC mice, and oral tolerance offered allergen-specific protective effects to BLG-induced allergy, probably through CD4+CD25+ regulatory T cell-mediated active suppression. In OTP mice, probiotics did not induce a further reduction of hypersensitivity score compared with OT mice but may provide additional protection to unforeseen nonspecific challenges through increased intestinal sIgA.

Sanger and Next-Generation Sequencing data for characterization of CTL epitopes in archived HIV-1 proviral DNA.

PubMed

Tumiotto, Camille; Riviere, Lionel; Bellecave, Pantxika; Recordon-Pinson, Patricia; Vilain-Parce, Alice; Guidicelli, Gwenda-Line; Fleury, Hervé

2017-01-01

One of the strategies for curing viral HIV-1 is a therapeutic vaccine involving the stimulation of cytotoxic CD8-positive T cells (CTL) that are Human Leucocyte Antigen (HLA)-restricted. The lack of efficiency of previous vaccination strategies may have been due to the immunogenic peptides used, which could be different from a patient's virus epitopes and lead to a poor CTL response. To counteract this lack of specificity, conserved epitopes must be targeted. One alternative is to gather as many data as possible from a large number of patients on their HIV-1 proviral archived epitope variants, taking into account their genetic background to select the best presented CTL epitopes. In order to process big data generated by Next-Generation Sequencing (NGS) of the DNA of HIV-infected patients, we have developed a software package called TutuGenetics. This tool combines an alignment derived either from Sanger or NGS files, HLA typing, target gene and a CTL epitope list as input files. It allows automatic translation after correction of the alignment obtained between the HxB2 reference and the reads, followed by automatic calculation of the MHC IC50 value for each epitope variant and the HLA allele of the patient by using NetMHCpan 3.0, resulting in a csv file as output result. We validated this new tool by comparing Sanger and NGS (454, Roche) sequences obtained from the proviral DNA of patients at success of ART included in the Provir Latitude 45 study and showed a 90% correlation between the quantitative results of NGS and Sanger. This automated analysis combined with complementary samples should yield more data regarding the archived CTL epitopes according to the patients' HLA alleles and will be useful for screening epitopes that in theory are presented efficiently to the HLA groove, thus constituting promising immunogenic peptides for a therapeutic vaccine.

Identification of a C-type lectin with antiviral and antibacterial activity from pacific white shrimp Litopenaeus vannamei.

PubMed

Li, Ming; Li, Chaozheng; Ma, Chunxia; Li, Haoyang; Zuo, Hongliang; Weng, Shaoping; Chen, Xiaohan; Zeng, Digang; He, Jianguo; Xu, Xiaopeng

2014-10-01

C-type lectins (CTLs) play crucial roles in innate immune responses in invertebrates by recognizing and eliminating microinvaders. In this study, a CTL from pacific white shrimp Litopenaeus vannamei (LvCTL3) was identified. LvCTL3 contains a single C-type lectin-like domain (CTLD), which shows similarities to those of other shrimp CTLs and has a mutated 'EPD' motif in Ca(2+)-binding site 2. LvCTL3 mRNA can be detected in all tested tissues and expression of LvCTL3 in gills was up-regulated after Lipopolysaccharides, poly (I:C), Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenges, suggesting activation responses of LvCTL3 to bacterial, virus and immune stimulant challenges. The 5'flanking regulatory region of LvCTL3 was cloned and we identified a NF-κB binding motif in the LvCTL3 promoter region. Dual-luciferase reporter assays indicated that over-expression of L. vannamei dorsal can dramatically up regulate the promoter activity of LvCTL3, suggesting that LvCTL3 expression could be regulated through NF-κB signaling pathway. As far as we know, this is the first report on signaling pathway involve in shrimp CTLs expression. The recombinant LvCTL3 protein was expressed in Escherichia coli and purified by Ni-affinity chromatography. The purified LvCTL3 can agglutinate Gram-negative microbe Vibrio alginolyticus and V. parahaemolyticus and Gram-positive bacteria Bacillus subtilis in the presence of calcium ions, but cannot agglutinate Gram-positive bacteria Streptococcus agalactiae. The agglutination activity of LvCTL3 was abolished when Ca(2+) was chelated with EDTA, suggesting the function of LvCTL3 is Ca(2+)-dependent. In vivo challenge experiments showed that the recombinant LvCTL3 protein can significantly reduce the mortalities of V. parahemolyticus and WSSV infection, indicating LvCTL3 might play significant roles in shrimp innate immunity defense against bacterial and viral infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Antigen-specific CTLs: to produce autologous cells product for adoptive cellular therapy.

PubMed

Liu, Sai; Shao, Yi; Xu, Jie; Jiang, Na; Dai, Yanchao; Wang, Yu; Sun, Huanqing; Sun, Jianping; Zhang, Yonghong

2017-06-01

As antiretroviral therapy provides long term viral suppression but no cure, alternative therapies such as adoptive cellular therapy have thus been investigated in the anti-AIDS field. This study sought to establish a HLA-A02 specific CTL cell culture method with comparison of the effects of different cytokines used in CTL cultivation to decide the best cultivation environment. In order to produce CTLs with targeted HLA-A02 restricted antigen specificity for adoptive cellular therapy, we evaluated autologous PBMC cultivation in different cytokine environment to select a better expansion condition to produce qualified CTL production. We co-cultivated PBMC and peptides of these patients with HLA-A02 allele with different cytokines. After cultivation, multiple parameters were tested. 1) Cytokines IL-2 alone can effectively amplify HLA-A02 specific CTL cells, and the count of CTLs was >85% all through the process. 2) The HLA-A02 specific cells at the end of the cultivation were mainly CD3+CD8+ cells. 3) The interferon stimulation test had shown that the expanded CTLs secreted more IFN-γ than before cultivation (0.9% -11.70%). This model of CTL cultivation is successful in redirecting the specificity of antigen recognition and safely for HLA-A02+ patients cell adoptive therapy. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Identification and characterization of two novel C-type lectins from the larvae of housefly, Musca domestica L.

PubMed

Zhou, Jing; Fang, Nai-Nai; Zheng, Ya; Liu, Kai-Yu; Mao, Bin; Kong, Li-Na; Chen, Ya; Ai, Hui

2018-04-20

Lectins and antimicrobial peptides (AMPs) are widely distributed in various insects and play crucial roles in primary host defense against pathogenic microorganisms. Two AMPs (cecropin and attacin) have been identified and characterized in the larvae of housefly. In this study, two novel C-type lectins (CTLs) were obtained from Musca domestica, while their agglutinating and antiviral properties were evaluated. Real-time PCR analysis showed that the mRNA levels of four immune genes (MdCTL1, MdCTL2, Cecropin, and Attacin) from M. domestica were significantly upregulated after injection with killed Gram-negative Escherichia coli. Moreover, purified MdCTL1-2 proteins can agglutinate E. coli and Staphylococcus aureus in the presence of calcium ions, suggesting their immune function is Ca 2+ dependent. Sequence analysis indicated that typical WND and QPD motifs were found in the Ca 2+ -binding site 2 of carbohydrate recognition domain from MdCTL1-2, which was consistent with their agglutinating activities. Subsequently, antiviral experiments indicated that MdCTL1-2 proteins could significantly reduce the infection rate of Spodoptera frugiperda 9 cells by the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, indicating they might play important roles in insect innate immunity against microbial pathogens. In addition, MdCTL1-2 proteins could effectively inhibit the replication of influenza H 1 N 1 virus, which was similar to the effect of ribavirin. These results suggested that two novel CTLs could be considered a promising drug candidate for the treatment of influenza. Moreover, it is believed that the discovery of the CTLs with antiviral effects in M. domestica will improve our understanding of the molecular mechanism of insect immune response against viruses. © 2018 Wiley Periodicals, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsao, Y.-P.; Lin, J.-Y.; Jan, J.-T.

The immunogenicity of HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) peptide in severe acute respiratory syndrome coronavirus (SARS-CoV) nuclear capsid (N) and spike (S) proteins was determined by testing the proteins' ability to elicit a specific cellular immune response after immunization of HLA-A2.1 transgenic mice and in vitro vaccination of HLA-A2.1 positive human peripheral blood mononuclearcytes (PBMCs). First, we screened SARS N and S amino acid sequences for allele-specific motif matching those in human HLA-A2.1 MHC-I molecules. From HLA peptide binding predictions (http://thr.cit.nih.gov/molbio/hla{sub b}ind/), ten each potential N- and S-specific HLA-A2.1-binding peptides were synthesized. The high affinity HLA-A2.1 peptides were validated bymore » T2-cell stabilization assays, with immunogenicity assays revealing peptides N223-231, N227-235, and N317-325 to be First identified HLA-A*0201-restricted CTL epitopes of SARS-CoV N protein. In addition, previous reports identified three HLA-A*0201-restricted CTL epitopes of S protein (S978-986, S1203-1211, and S1167-1175), here we found two novel peptides S787-795 and S1042-1050 as S-specific CTL epitopes. Moreover, our identified N317-325 and S1042-1050 CTL epitopes could induce recall responses when IFN-{gamma} stimulation of blood CD8{sup +} T-cells revealed significant difference between normal healthy donors and SARS-recovered patients after those PBMCs were in vitro vaccinated with their cognate antigen. Our results would provide a new insight into the development of therapeutic vaccine in SARS.« less

Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors.

PubMed Central

Chen, W; Qin, H; Chesebro, B; Cheever, M A

1996-01-01

FBL-3 is a highly immunogenic murine leukemia of C57BL/6 origin induced by Friend murine leukemia virus (MuLV). Immunization of C57BL/6 mice with FBL-3 readily elicits CD8+ cytotoxic T lymphocytes (CTL) capable of lysing FBL-3 as well as syngeneic leukemias induced by Moloney and Rauscher MuLV. The aim of this current study was to identify the immunogenic epitope(s) recognized by the FBL-3-specific CD8+ CTL. A series of FBL-3-specific CD8+ CTL clones were generated from C57BL/6 mice immunized to FBL-3. The majority of CTL clones (32 of 38) were specific for F-MuLV gag-encoded antigen. By using a series of recombinant vaccinia viruses expressing full-length and truncated F-MuLV gag genes, the antigenic epitope recognized by the FBL-3 gag-specific CTL clones, as well as by bulk-cultured CTL from spleens of mice immune to FBL-3, was localized to the leader sequence of gPr80gag protein. The precise amino acid sequence of the CTL epitope in the leader sequence was identified as CCLCLTVFL (positions 85-93) by examining lysis of targets incubated with a series of synthetic leader sequence peptides. No evidence of other CTL epitopes in the gPr80gag or Pr65gag core virion structural polyproteins was found. The identity of CCLCLTVFL as the target peptide was validated by showing that immunization with the peptide elicited CTL that lysed FBL-3. The CTL elicited by the Gag peptide also specifically lysed syngeneic leukemia cells induced by Moloney and Rauscher MuLV (MBL-2 and RBL-5). The transmembrane peptide was shown to be the major gag-encoded antigenic epitope recognized by bulk-cultured CTL derived from C57BL/6 mice immunized to MBL-2 or RBL-5. Thus, the CTL epitope of FBL-3 is localized to the transmembrane anchor domain of the nonstructural Gag polyprotein and is shared by leukemia/lymphoma cell lines induced by Friend, Moloney, and Rauscher MuLV. PMID:8892898

HIV-specific CD8+ T cells: serial killers condemned to die?

PubMed

Petrovas, Constantinos; Mueller, Yvonne M; Katsikis, Peter D

2004-04-01

An increasing body of evidence supports a key role for cytotoxic CD8+ T cells (CTL) in controlling HIV infection. Although a vigorous HIV-specific CD8+ T cell response is raised during the primary infection, these cells ultimately fail to control virus and prevent disease progression. The failure of CTL to control HIV infection has been attributed to a number of strategies HIV employs to evade the immune system. Recently, intrinsic defects in the CTL themselves have been proposed to contribute to the failure of CTL to control HIV. HIV-specific CD8+ T cells differ in their effector/memory phenotype from other virus-specific CD8+ T cells indicating that their differentiation status differs. This altered differentiation may affect effector functions as well as homing properties of these cells. Other studies have indicated that activation of HIV-specific CTL may be impaired and this contributes to their dysfunction. The effector function of these CTL may also be affected. There are conflicting reports about their ability to kill, whereas IFNgamma production does not appear to be impaired in these cells. In this review we focus on recent work indicating that apoptosis may be an important mechanism through which HIV evades the CTL response. In particular, HIV-specific CD8+ T cells are highly susceptible to CD95/Fas-induced apoptosis. This leads to the hypothesis that virus-specific cytotoxic T cells can be eliminated upon binding CD95L/FasL on HIV-infected cells. Understanding the intrinsic defects of CTL in HIV infection could lead to new therapeutic strategies and optimized vaccination protocols that enhance the HIV-specific cytotoxic response.

High frequencies of circulating IFN-gamma-secreting CD8 cytotoxic T cells specific for a novel MHC class I-restricted Mycobacterium tuberculosis epitope in M. tuberculosis-infected subjects without disease.

PubMed

Pathan, A A; Wilkinson, K A; Wilkinson, R J; Latif, M; McShane, H; Pasvol, G; Hill, A V; Lalvani, A

2000-09-01

MHC class I-restricted CD8 cytotoxic T lymphocytes (CTL) are essential for protective immunity to Mycobacterium tuberculosis in animal models but their role in humans remains unclear. We therefore studied subjects who had successfully contained M. tuberculosis infection in vivo, i.e. exposed healthy household contacts and individuals with inactive self-healed pulmonary tuberculosis. Using the ELISPOT assay for IFN-gamma, we screened peptides from ESAT-6, a secreted antigen that is highly specific for M. tuberculosis. We identified a novel nonamer epitope: unstimulated peripheral blood-derived CD8 T cells displayed peptide-specific IFN-gamma release ex vivo while CD8 T cell lines and clones exhibited HLA-A68.02-restricted cytolytic activity and recognized endogenously processed antigen. The frequency of CD8 CTL specific for this single M. tuberculosis epitope, 1/2500 peripheral blood lymphocytes, was equivalent to the combined frequency of all IFN-gamma-secreting purified protein derivative-reactive T cells ex vivo. This highly focused CTL response was maintained in an asymptomatic contact over 2 years and is the most potent antigen-specific antimycobacterial CD8 CTL response hitherto described. Thus, human M. tuberculosis-specific CD8 CTL are not necessarily associated with active disease per se. Rather, our results are consistent with a protective role for these ESAT-6-specific CD8 T cells in the long-term control of M. tuberculosis in vivo in humans.

Role of HIV-specific CD8+ T cells in pediatric HIV cure strategies after widespread early viral escape.

PubMed

Leitman, Ellen M; Thobakgale, Christina F; Adland, Emily; Ansari, M Azim; Raghwani, Jayna; Prendergast, Andrew J; Tudor-Williams, Gareth; Kiepiela, Photini; Hemelaar, Joris; Brener, Jacqui; Tsai, Ming-Han; Mori, Masahiko; Riddell, Lynn; Luzzi, Graz; Jooste, Pieter; Ndung'u, Thumbi; Walker, Bruce D; Pybus, Oliver G; Kellam, Paul; Naranbhai, Vivek; Matthews, Philippa C; Gall, Astrid; Goulder, Philip J R

2017-11-06

Recent studies have suggested greater HIV cure potential among infected children than adults. A major obstacle to HIV eradication in adults is that the viral reservoir is largely comprised of HIV-specific cytotoxic T lymphocyte (CTL) escape variants. We here evaluate the potential for CTL in HIV-infected slow-progressor children to play an effective role in "shock-and-kill" cure strategies. Two distinct subgroups of children were identified on the basis of viral load. Unexpectedly, in both groups, as in adults, HIV-specific CTL drove the selection of escape variants across a range of epitopes within the first weeks of infection. However, in HIV-infected children, but not adults, de novo autologous variant-specific CTL responses were generated, enabling the pediatric immune system to "corner" the virus. Thus, even when escape variants are selected in early infection, the capacity in children to generate variant-specific anti-HIV CTL responses maintains the potential for CTL to contribute to effective shock-and-kill cure strategies in pediatric HIV infection. © 2017 Leitman et al.

Multiple mechanisms underlie defective recognition of melanoma cells cultured in three-dimensional architectures by antigen-specific cytotoxic T lymphocytes.

PubMed

Feder-Mengus, C; Ghosh, S; Weber, W P; Wyler, S; Zajac, P; Terracciano, L; Oertli, D; Heberer, M; Martin, I; Spagnoli, G C; Reschner, A

2007-04-10

Cancer cells' growth in three-dimensional (3D) architectures promotes resistance to drugs, cytokines, or irradiation. We investigated effects of 3D culture as compared to monolayers (2D) on melanoma cells' recognition by tumour-associated antigen (TAA)-specific HLA-A(*)0201-restricted cytotoxic T-lymphocytes (CTL). Culture of HBL, D10 (both HLA-A(*)0201+, TAA+) and NA8 (HLA-A(*)0201+, TAA-) melanoma cells on polyHEMA-coated plates, resulted in generation of 3D multicellular tumour spheroids (MCTS). Interferon-gamma (IFN-gamma) production by HLA-A(*)0201-restricted Melan-A/MART-1(27-35) or gp 100(280-288)-specific CTL clones served as immunorecognition marker. Co-culture with melanoma MCTS, resulted in defective TAA recognition by CTL as compared to 2D as witnessed by decreased IFN-gamma production and decreased Fas Ligand, perforin and granzyme B gene expression. A multiplicity of mechanisms were potentially involved. First, MCTS per se limit CTL capacity of recognising HLA class I restricted antigens by reducing exposed cell surfaces. Second, expression of melanoma differentiation antigens is downregulated in MCTS. Third, expression of HLA class I molecules can be downregulated in melanoma MCTS, possibly due to decreased interferon-regulating factor-1 gene expression. Fourth, lactic acid production is increased in MCTS, as compared to 2D. These data suggest that melanoma cells growing in 3D, even in the absence of immune selection, feature characteristics capable of dramatically inhibiting TAA recognition by specific CTL.

Development of oral CTL vaccine using a CTP-integrated Sabin 1 poliovirus-based vector system.

PubMed

Han, Seung-Soo; Lee, Jinjoo; Jung, Yideul; Kang, Myeong-Ho; Hong, Jung-Hyub; Cha, Min-Suk; Park, Yu-Jin; Lee, Ezra; Yoon, Cheol-Hee; Bae, Yong-Soo

2015-09-11

We developed a CTL vaccine vector by modification of the RPS-Vax system, a mucosal vaccine vector derived from a poliovirus Sabin 1 strain, and generated an oral CTL vaccine against HIV-1. A DNA fragment encoding a cytoplasmic transduction peptide (CTP) was integrated into the RPS-Vax system to generate RPS-CTP, a CTL vaccine vector. An HIV-1 p24 cDNA fragment was introduced into the RPS-CTP vector system and a recombinant poliovirus (rec-PV) named vRPS-CTP/p24 was produced. vRPS-CTP/p24 was genetically stable and efficiently induced Th1 immunity and p24-specific CTLs in immunized poliovirus receptor-transgenic (PVR-Tg) mice. In challenge experiments, PVR-Tg mice that were pre-immunized orally with vRPS-CTP/p24 were resistant to challenge with a lethal dose of p24-expressing recombinant vaccinia virus (rMVA-p24). These results suggested that the RPS-CTP vector system had potential for developing oral CTL vaccines against infectious diseases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Immunostimulatory activities of dendritic cells loaded with adenovirus vector carrying HBcAg/HBsAg

PubMed Central

Jia, Hongyu; Li, Chunling; Zhang, Yimin; Yu, Liang; Xiang, Dairong; Liu, Jun; Chen, Fengzhe; Han, Xiaochun

2015-01-01

Objective: This study is to investigate the immunostimulatory activities of dendritic cells (DCs) transfected with HBcAg and/or HBsAg recombinant adenovirus (rAd). Methods: DCs were transfected with rAd (DC/Ad-C+Ad-S, DC/Ad-C, and DC/Ad-S), or pulsed with HBcAg antigen (DC/HBcAg). Flow cytometry was used to detect the phenotype of DCs and the cytokine production of T lymphocytes. Mice were vaccinated with DCs transfected with rAd or pulsed with antigen, and DNA vaccine. Mixed lymphocyte reaction (MLR) was used to evaluate the T-cell stimulatory capacity, and HBcAg-specific cytotoxic T lymphocyte (CTL) activity was assessed. Results: Phenotypic analysis showed that DCs transfected with rAd or pulsed with HBcAg antigen exhibited mature phenotypes. MLR indicated no significant differences in stimulating T-cell proliferation between the DC/rAd and DC/HBcAg groups. When mixed with DCs, Th and Tc cells mainly secreted IFN-γ, indicating type I immune responses. In vaccinated mice, DCs transduced with rAd and pulsed with HBcAg induced significantly more IFN-γ secretion from Th cells, compared with DNA vaccine, indicating stronger Th1 response. Moreover, DCs transduced with rAd stimulated Tc cells to produce more IFN-γ, indicating stronger Tc1 response. In vaccinated mice, HBcAg-specific CTL activities were decreased in the following order: the DC/Ad-C+Ad-S, DC/Ad-C, DC/Ad-S, DC/HBcAg, and DNA vaccine groups. Conclusion: DCs transfected with rAd induce stronger Th1/Tc1 (type I) cell immune responses and specific CTL response than HBcAg-pulsed DCs or DNA vaccine. Our findings suggest that DCs transfected with rAd-C/rAd-S might provide an effective approach in the treatment of persistent hepatitis B virus infection. PMID:26064236

Susceptibility to cytotoxic T cell lysis of cancer stem cells derived from cervical and head and neck tumor cell lines.

PubMed

Liao, Tian; Kaufmann, Andreas M; Qian, Xu; Sangvatanakul, Voramon; Chen, Chao; Kube, Tina; Zhang, Guoyou; Albers, Andreas E

2013-01-01

To explore cancer stem cell susceptibility to a host's cytotoxic T lymphocyte (CTL)-mediated immune response. We compared the susceptibility of putative CSC generated from cancer cell lines to immunologic recognition and killing by alloantigen-specific CD8(+) CTL. CSC-enriched spheroid culture-derived cells (SDC) exhibited higher expression of ALDH, ICAM1 and of stem/progenitor cell markers on all 3 tumor cell lines investigated and lower MHC class I on the cervical cancer cell line as compared to their monolayer-derived cells (MDC). The expression of ICAM1 and MHCI was upregulated by IFN-γ treatment. CSC populations were less sensitive to MHC class I-restricted alloantigen-specific CD8(+) CTL lysis as compared to matched MDC. IFN-γ pretreatment resulted in over-proportionally enhanced lysis of SDC. Finally, the subset of ALDH(high) expressing SDC presented more sensitivity toward CD8(+) CTL killing than the ALDH(low) SDC. Tumor therapy resistance has been attributed to cancer stem cells (CSC). We show in vitro susceptibility of CSC to CTL-mediated lysis. Immunotherapy targeting of ALDH(+) CSC may therefore be a promising approach. Our results and method may be helpful for the development and optimization of adjuvants, as here exemplified for INF-γ, for CSC-targeted vaccines, independent of the availability of CSC-specific antigens.

Differences in antigen presentation to MHC class I-and class II- restricted influenza virus-specific cytolytic T lymphocyte clones

PubMed Central

1986-01-01

We have examined requirements for antigen presentation to a panel of MHC class I-and class II-restricted, influenza virus-specific CTL clones by controlling the form of virus presented on the target cell surface. Both H-2K/D- and I region-restricted CTL recognize target cells exposed to infectious virus, but only the I region-restricted clones efficiently lysed histocompatible target cells pulsed with inactivated virus preparations. The isolated influenza hemagglutinin (HA) polypeptide also could sensitize target cells for recognition by class II-restricted, HA-specific CTL, but not by class I-restricted, HA- specific CTL. Inhibition of nascent viral protein synthesis abrogated the ability of target cells to present viral antigen relevant for class I-restricted CTL recognition. Significantly, presentation for class II- restricted recognition was unaffected in target cells exposed to preparations of either inactivated or infectious virus. This differential sensitivity suggested that these H-2I region-restricted CTL recognized viral polypeptides derived from the exogenously introduced virions, rather than viral polypeptides newly synthesized in the infected cell. In support of this contention, treatment of the target cells with the lysosomotropic agent chloroquine abolished recognition of infected target cells by class II-restricted CTL without diminishing class I-restricted recognition of infected target cells. Furthermore, when the influenza HA gene was introduced into target cells without exogenous HA polypeptide, the target cells that expressed the newly synthesized protein product of the HA gene were recognized only by H-2K/D-restricted CTL. These observations suggest that important differences may exist in requirements for antigen presentation between H-2K/D and H-2I region-restricted CTL. These differences may reflect the nature of the antigenic epitopes recognized by these two CTL subsets. PMID:3485173

Antigen-Specific CD8 Lytic Phenotype Induced by Sipuleucel-T in Hormone-Sensitive or Castration-Resistant Prostate Cancer and Association With Overall Survival.

PubMed

Antonarakis, Emmanuel S; Small, Eric J; Petrylak, Daniel; Quinn, David I; Kibel, Adam S; Chang, Nancy; Dearstyne, Erica; Harmon, Matthew; Campogan, Dwayne; Haynes, Heather; Vu, Tuyen; Sheikh, Nadeem A; Drake, Charles G

2018-06-01

Sipuleucel-T is FDA-approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC) based on the IMPACT trial showing a 4.1-month benefit in median overall survival (OS) for patients receiving sipuleucel-T vs control. Although efficacy of sipuleucel-T is well-established, its mechanism remains incompletely understood. Patient samples from three sipuleucel-T trials were assessed for peripheral cellular immune responses to the immunogen PA2024 and the target antigen prostatic acid phosphatase (PAP). PAP- and PA2024-specific proliferative and cytolytic responses were characterized to delineate sipuleucel-T-induced immune responses. To quantify potential cytotoxic T lymphocyte (CTL) activity, cell-surface CD107a expression on PAP- or PA2024-specific CD8+ T cells was measured in sipuleucel-T-treated patient and healthy volunteer samples. Increased PA2024-specific CD4+ (p=0.030) and CD8+ (p=0.052) T-cell proliferation from baseline to week 6 was observed (N=14) post-sipuleucel-T, with greater magnitude of PA2024-specific responses compared to PAP. PAP- and PA2024-CTL activity (CD107a positivity) significantly increased at weeks 6 and 26 after sipuleucel-T treatment (p<0.0001; N=22). At 26 weeks post-sipuleucel-T, OS correlated with the magnitude of PAP (Pearson's R, 0.52; p=0.013) or PA2024 (Pearson's R, 0.67; p=0.0006) CTL activity. Higher PA2024-CTL activity at week 26 was significantly associated with longer OS using tertile analysis (p=0.0005; N=22), with PA2024 responses correlating with PAP responses at week 26 (R=0.90; p=1.53E -08 ). This study is the first to report PAP-specific CD8+ T-cell responses elicited by sipuleucel-T treatment. Increased and persistent potential PA2024-specific CTL activity correlated with PAP-specific CTL activity and associated with improved OS following sipuleucel-T treatment. Copyright ©2018, American Association for Cancer Research.

Viral evolution in HLA-B27-restricted CTL epitopes in human immunodeficiency virus type 1-infected individuals.

PubMed

Setiawan, Laurentia C; Gijsbers, Esther F; van Nuenen, Adrianus C; Kootstra, Neeltje A

2015-08-01

The HLA-B27 allele is over-represented among human immunodeficiency virus type 1-infected long-term non-progressors. In these patients, strong CTL responses targeting HLA-B27-restricted viral epitopes have been associated with long-term asymptomatic survival. Indeed, loss of control of viraemia in HLA-B27 patients has been associated with CTL escape at position 264 in the immunodominant KK10 epitope. This CTL escape mutation in the viral Gag protein has been associated with severe viral attenuation and may require the presence of compensatory mutations before emerging. Here, we studied sequence evolution within HLA-B27-restricted CTL epitopes in the viral Gag protein during the course of infection of seven HLA-B27-positive patients. Longitudinal gag sequences obtained at different time points around the time of AIDS diagnosis were obtained and analysed for the presence of mutations in epitopes restricted by HLA-B27, and for potential compensatory mutations. Sequence variations were observed in the HLA-B27-restricted CTL epitopes IK9 and DR11, and the immunodominant KK10 epitope. However, the presence of sequence variations in the HLA-B27-restricted CTL epitopes could not be associated with an increase in viraemia in the majority of the patients studied. Furthermore, we observed low genetic diversity in the gag region of the viral variants throughout the course of infection, which is indicative of low viral replication and corresponds to the low viral load observed in the HLA-B27-positive patients. These data indicated that control of viral replication can be maintained in HLA-B27-positive patients despite the emergence of viral mutations in HLA-B27-restricted epitopes.

Proteasome inhibition blocks NF-κB and ERK1/2 pathways, restores antigen expression and sensitizes resistant human melanoma to TCR-engineered CTLs

PubMed Central

Jazirehi, Ali R.; Economou, James S.

2012-01-01

Adoptive cell transfer (ACT) of ex vivo engineered autologous lymphocytes encoding high-affinity MART-1/HLA-A*0201-specific T-cell receptor (TCR) α/β chains (F5 CTL), densely infiltrate into sites of metastatic disease, mediating dramatic but partial clinical responses in melanoma patients. We hypothesized that MART-1 down-modulation in addition to aberrant apoptotic/survival signaling could confer resistance to death signals delivered by transgenic CTLs. To explore this hypothesis, we established an in vitro model of resistant (R) lines from MART-1+/HLA-A*0201+ F5 CTL-sensitive parental (P) lines under serial F5 CTL-selective pressure. We have recently reported that several melanoma R lines, while retaining MART-1 expression, exhibited constitutive NF-κB activation and over-expression of NF-κB-dependent resistance factors. Another established melanoma cell line M244, otherwise sensitive to F5 CTL, yielded R lines after serial F5 CTL selective pressure which had both reduced MART-1 expression levels, thus, could not be recognized, and were resistant to CTL-delivered apoptotic death signals. The proteasome inhibitor bortezomib blocked NF-κB activity, decreased phopspho-ERK1/2, increased phospho-JNK levels, reduced expression of resistance-factors, restored MART-1 expression to sufficient levels, which in combination allowed M244R lines be sensitized to F5 CTL-killing. These findings suggest that proteasome inhibition in immune resistant tumors can restore proapoptotic signaling and improve tumor antigen expression. PMID:22532603

A T-cell response to a liver-stage Plasmodium antigen is not boosted by repeated sporozoite immunizations

PubMed Central

Murphy, Sean C.; Kas, Arnold; Stone, Brad C.; Bevan, Michael J.

2013-01-01

Development of an antimalarial subunit vaccine inducing protective cytotoxic T lymphocyte (CTL)-mediated immunity could pave the way for malaria eradication. Experimental immunization with sporozoites induces this type of protective response, but the extremely large number of proteins expressed by Plasmodium parasites has so far prohibited the identification of sufficient discrete T-cell antigens to develop subunit vaccines that produce sterile immunity. Here, using mice singly immunized with Plasmodium yoelii sporozoites and high-throughput screening, we identified a unique CTL response against the parasite ribosomal L3 protein. Unlike CTL responses to the circumsporozoite protein (CSP), the population of L3-specific CTLs was not expanded by multiple sporozoite immunizations. CSP is abundant in the sporozoite itself, whereas L3 expression does not increase until the liver stage. The response induced by a single immunization with sporozoites reduces the parasite load in the liver so greatly during subsequent immunizations that L3-specific responses are only generated during the primary exposure. Functional L3-specific CTLs can, however, be expanded by heterologous prime-boost regimens. Thus, although repeat sporozoite immunization expands responses to preformed antigens like CSP that are present in the sporozoite itself, this immunization strategy may not expand CTLs targeting parasite proteins that are synthesized later. Heterologous strategies may be needed to increase CTL responses across the entire spectrum of Plasmodium liver-stage proteins. PMID:23530242

Immune Selection In Vitro Reveals Human Immunodeficiency Virus Type 1 Nef Sequence Motifs Important for Its Immune Evasion Function In Vivo

PubMed Central

Lee, Patricia; Ng, Hwee L.; Yang, Otto O.

2012-01-01

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates major histocompatibility complex class I (MHC-I), impairing the clearance of infected cells by CD8+ cytotoxic T lymphocytes (CTLs). While sequence motifs mediating this function have been determined by in vitro mutagenesis studies of laboratory-adapted HIV-1 molecular clones, it is unclear whether the highly variable Nef sequences of primary isolates in vivo rely on the same sequence motifs. To address this issue, nef quasispecies from nine chronically HIV-1-infected persons were examined for sequence evolution and altered MHC-I downregulatory function under Gag-specific CTL immune pressure in vitro. This selection resulted in decreased nef diversity and strong purifying selection. Site-by-site analysis identified 13 codons undergoing purifying selection and 1 undergoing positive selection. Of the former, only 6 have been reported to have roles in Nef function, including 4 associated with MHC-I downregulation. Functional testing of naturally occurring in vivo polymorphisms at the 7 sites with no previously known functional role revealed 3 mutations (A84D, Y135F, and G140R) that ablated MHC-I downregulation and 3 (N52A, S169I, and V180E) that partially impaired MHC-I downregulation. Globally, the CTL pressure in vitro selected functional Nef from the in vivo quasispecies mixtures that predominately lacked MHC-I downregulatory function at the baseline. Overall, these data demonstrate that CTL pressure exerts a strong purifying selective pressure for MHC-I downregulation and identifies novel functional motifs present in Nef sequences in vivo. PMID:22553319

A novel strategy for the identification of antigens that are recognised by bovine MHC class I restricted cytotoxic T cells in a protozoan infection using reverse vaccinology.

PubMed

Graham, Simon P; Honda, Yoshikazu; Pellé, Roger; Mwangi, Duncan M; Glew, E Jane; de Villiers, Etienne P; Shah, Trushar; Bishop, Richard; van der Bruggen, Pierre; Nene, Vishvanath; Taracha, Evans L N

2007-02-09

Immunity against the bovine protozoan parasite Theileria parva has previously been shown to be mediated through lysis of parasite-infected cells by MHC class I restricted CD8+ cytotoxic T lymphocytes. It is hypothesized that identification of CTL target schizont antigens will aid the development of a sub-unit vaccine. We exploited the availability of the complete genome sequence data and bioinformatics tools to identify genes encoding secreted or membrane anchored proteins that may be processed and presented by the MHC class I molecules of infected cells to CTL. Of the 986 predicted open reading frames (ORFs) encoded by chromosome 1 of the T. parva genome, 55 were selected based on the presence of a signal peptide and/or a transmembrane helix domain. Thirty six selected ORFs were successfully cloned into a eukaryotic expression vector, transiently transfected into immortalized bovine skin fibroblasts and screened in vitro using T. parva-specific CTL. Recognition of gene products by CTL was assessed using an IFN-gamma ELISpot assay. A 525 base pair ORF encoding a 174 amino acid protein, designated Tp2, was identified by T. parva-specific CTL from 4 animals. These CTL recognized and lysed Tp2 transfected skin fibroblasts and recognized 4 distinct epitopes. Significantly, Tp2 specific CD8+ T cell responses were observed during the protective immune response against sporozoite challenge. The identification of an antigen containing multiple CTL epitopes and its apparent immunodominance during a protective anti-parasite response makes Tp2 an attractive candidate for evaluation of its vaccine potential.

CD40 agonist converting CTL exhaustion via the activation of the mTORC1 pathway enhances PD-1 antagonist action in rescuing exhausted CTLs in chronic infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Aizhang; Wang, Rong; Department of Oncology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan

Expansion of PD-1-expressing CD8{sup +} cytotoxic T lymphocytes (CTLs) and associated CTL exhaustion are chief issues for ineffective virus-elimination in chronic infectious diseases. PD-1 blockade using antagonistic anti-PD-L1 antibodies results in a moderate conversion of CTL exhaustion. We previously demonstrated that CD40L signaling of ovalbumin (OVA)-specific vaccine, OVA-Texo, converts CTL exhaustion via the activation of the mTORC1 pathway in OVA-expressing adenovirus (AdVova)-infected B6 mice showing CTL inflation and exhaustion. Here, we developed AdVova-infected B6 and transgenic CD11c-DTR (termed AdVova-B6 and AdVova-CD11c-DTR) mice with chronic infection, and assessed a potential effect of CD40 agonist on the conversion of CTL exhaustion andmore » on a potential enhancement of PD-1 antagonist action in rescuing exhausted CTLs in our chronic infection models. We demonstrate that a single dose of anti-CD40 alone can effectively convert CTL exhaustion by activating the mTORC1 pathway, leading to CTL proliferation, up-regulation of an effector-cytokine IFN-γ and the cytolytic effect in AdVova-B6 mice. Using anti-CD4 antibody and diphtheria toxin (DT) to deplete CD4{sup +} T-cells and dendritic cells (DCs), we discovered that the CD40 agonist-induced conversion in AdVova-B6 and AdVova-CD11c-DTR mice is dependent upon host CD4{sup +} T-cell and DC involvements. Moreover, CD40 agonist significantly enhances PD-1 antagonist effectiveness in rescuing exhausted CTLs in chronic infection. Taken together, our data demonstrate the importance of CD40 signaling in the conversion of CTL exhaustion and its ability to enhance PD-1 antagonist action in rescuing exhausted CTLs in chronic infection. Therefore, our findings may positively impact the design of new therapeutic strategies for chronic infectious diseases. - Highlights: • Anti-CD40 agonistic Ab can convert CTL exhaustion in chronically infected mice. • The conversion relies on the activation of the mTORC1 pathway in exhausted CTLs. • The conversion depends on the involvement of host DCs and CD4{sup +} T cells. • Anti-CD40 Ab enhances the effect of PD-1 blockade in rescuing CTL exhaustion.« less

Molecular cloning and characterization of a C-type lectin in yellow catfish Tachysurus fulvidraco.

PubMed

Ke, F; Zhang, H B; Wang, Y; Hou, L F; Dong, H J; Wang, Z F; Pan, G W; Cao, X Y

2016-09-01

This study represents the first report of a C-type lectin (ctl) in yellow catfish Tachysurus fulvidraco. The complete sequence of ctl complementary (c)DNA consisted of 685 nucleotides. The open reading frame potentially encoded a protein of 177 amino acids with a calculated molecular mass of c.y 20.204 kDa. The deduced amino-acid sequence contained a signal peptide and a single carbohydrate recognition domain with four cysteine residues and GlnProAsp (QPD) and TrpAsnAsp (WND) motifs. Ctl showed the highest identity (56.0%) to the predicted lactose binding lectin from channel catfish Ictalurus punctatus. Quantitative real-time (qrt)-PCR analysis showed that ctl messenger (m)RNA was constitutively expressed in all examined tissues in normal fish, with high expression in trunk kidney and head kidney, which was increased following Aeromonas hydrophila challenge in a duration-dependent manner. Purified recombinant Ctl (rCtl) from Escherichia coli BL21 was able to bind and agglutinate Gram-positive and Gram-negative bacteria in a calcium-dependent manner. These results suggested that Ctl might be a C-type lectin of T. fulvidraco involved in innate immune responses as receptors (PRR). © 2016 The Fisheries Society of the British Isles.

Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity.

PubMed

Davenport, A J; Cross, R S; Watson, K A; Liao, Y; Shi, W; Prince, H M; Beavis, P A; Trapani, J A; Kershaw, M H; Ritchie, D S; Darcy, P K; Neeson, P J; Jenkins, M R

2018-02-27

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell. Copyright © 2018 the Author(s). Published by PNAS.

Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity

PubMed Central

Davenport, A. J.; Cross, R. S.; Watson, K. A.; Liao, Y.; Shi, W.; Prince, H. M.; Beavis, P. A.; Trapani, J. A.; Kershaw, M. H.; Ritchie, D. S.; Darcy, P. K.; Jenkins, M. R.

2018-01-01

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell. PMID:29440406

Response of ELA-A1 horses immunized with lipopeptide containing an equine infectious anemia virus ELA-A1-restricted CTL epitope to virus challenge.

PubMed

Ridgely, Sherritta L; Zhang, Baoshan; McGuire, Travis C

2003-01-17

Lipopeptide containing an ELA-A1-restricted cytotoxic T lymphocyte (CTL) epitope from the envelope surface unit (SU) protein of the EIAV(WSU5) strain was used to immunize three horses having the ELA-A1 haplotype. Peptide-specific ELA-A1-restricted CTL were induced in all three horses, although these were present transiently in PBMC. These horses were further immunized with lipopeptide containing the corresponding CTL epitope from the EIAV(PV) strain. Then, the three immunized horses and three non-immunized horses were challenged by intravenous inoculation with 300 TCID(50) EIAV(PV). All horses developed cell free viremia, fever and thrombocytopenia. However, there was a statistically lower fever and thrombocytopenia severity score in the immunized group. Shorter duration of plasma viral load in two of the three immunized horses likely explains the less severe clinical disease in this group. Results indicate that lipopeptide immunization had a protective effect against development of clinical disease following virus challenge.

Antibody-peptide-MHC fusion conjugates target non-cognate T cells to kill tumour cells.

PubMed

King, Ben C; Hamblin, Angela D; Savage, Philip M; Douglas, Leon R; Hansen, Ted H; French, Ruth R; Johnson, Peter W M; Glennie, Martin J

2013-06-01

Attempts to generate robust anti-tumour cytotoxic T lymphocyte (CTL) responses using immunotherapy are frequently thwarted by exhaustion and anergy of CTL recruited to tumour. One strategy to overcome this is to retarget a population of virus-specific CTL to kill tumour cells. Here, we describe a proof-of-principle study using a bispecific conjugate designed to retarget ovalbumin (OVA)-specific CTL to kill tumour cells via CD20. A single-chain trimer (SCT) consisting of MHCI H-2K(b)/SIINFEKL peptide/beta 2 microglobulin/BirA was expressed in bacteria, refolded and chemically conjugated to one (1:1; F2) or two (2:1; F3) anti-hCD20 Fab' fragments. In vitro, the [SCT × Fab'] (F2 and F3) redirected SIINFEKL-specific OT-I CTL to kill CD20(+) target cells, and in the presence of CD20(+) target cells to provide crosslinking, they were also able to induce proliferation of OT-I cells. In vivo, activated OT-I CTL could be retargeted to kill [SCT × Fab']-coated B cells from hCD20 transgenic (hCD20 Tg) mice and also EL4 and B16 mouse tumour cells expressing human CD20 (hCD20). Importantly, in a hCD20 Tg mouse model, [SCT × Fab'] administered systemically were able to retarget activated OT-I cells to deplete normal B cells, and their performance matched that of a bispecific antibody (BsAb) comprising anti-CD3 and anti-CD20. [SCT × Fab'] were also active therapeutically in an EL4 tumour model. Furthermore, measurement of serum cytokine levels suggests that [SCT × Fab'] are associated with a lower level of inflammatory cytokine release than the BsAb and so may be advantageous clinically in terms of reduced toxicity.

Generation of TCR-Expressing Innate Lymphoid-like Helper Cells that Induce Cytotoxic T Cell-Mediated Anti-leukemic Cell Response.

PubMed

Ueda, Norihiro; Uemura, Yasushi; Zhang, Rong; Kitayama, Shuichi; Iriguchi, Shoichi; Kawai, Yohei; Yasui, Yutaka; Tatsumi, Minako; Ueda, Tatsuki; Liu, Tian-Yi; Mizoro, Yasutaka; Okada, Chihiro; Watanabe, Akira; Nakanishi, Mahito; Senju, Satoru; Nishimura, Yasuharu; Kuzushima, Kiyotaka; Kiyoi, Hitoshi; Naoe, Tomoki; Kaneko, Shin

2018-06-05

CD4 + T helper (Th) cell activation is essential for inducing cytotoxic T lymphocyte (CTL) responses against malignancy. We reprogrammed a Th clone specific for chronic myelogenous leukemia (CML)-derived b3a2 peptide to pluripotency and re-differentiated the cells into original TCR-expressing T-lineage cells (iPS-T cells) with gene expression patterns resembling those of group 1 innate lymphoid cells. CD4 gene transduction into iPS-T cells enhanced b3a2 peptide-specific responses via b3a2 peptide-specific TCR. iPS-T cells upregulated CD40 ligand (CD40L) expression in response to interleukin-2 and interleukin-15. In the presence of Wilms tumor 1 (WT1) peptide, antigen-specific dendritic cells (DCs) conditioned by CD4-modified CD40L high iPS-T cells stimulated WT1-specific CTL priming, which eliminated WT1 peptide-expressing CML cells in vitro and in vivo. Thus, CD4 modification of CD40L high iPS-T cells generates innate lymphoid helper-like cells inducing bcr-abl-specific TCR signaling that mediates effectiveanti-leukemic CTL responses via DC maturation, showing potential for adjuvant immunotherapy against leukemia. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Wnt signaling inhibits CTL memory programming

PubMed Central

Xiao, Zhengguo; Sun, Zhifeng; Smyth, Kendra; Li, Lei

2013-01-01

Induction of functional CTLs is one of the major goals for vaccine development and cancer therapy. Inflammatory cytokines are critical for memory CTL generation. Wnt signaling is important for CTL priming and memory formation, but its role in cytokine-driven memory CTL programming is unclear. We found that wnt signaling inhibited IL-12-driven CTL activation and memory programming. This impaired memory CTL programming was attributed to up-regulation of eomes and down-regulation of T-bet. Wnt signaling suppressed the mTOR pathway during CTL activation, which was different to its effects on other cell types. Interestingly, the impaired memory CTL programming by wnt was partially rescued by mTOR inhibitor rapamycin. In conclusion, we found that crosstalk between wnt and the IL-12 signaling inhibits T-bet and mTOR pathways and impairs memory programming which can be recovered in part by rapamycin. In addition, direct inhibition of wnt signaling during CTL activation does not affect CTL memory programming. Therefore, wnt signaling may serve as a new tool for CTL manipulation in autoimmune diseases and immune therapy for certain cancers. PMID:23911398

Establishment of anti-tumor memory in humans using in vitro-educated CD8+ T cells

PubMed Central

Butler, Marcus O.; Friedlander, Philip; Milstein, Matthew I.; Mooney, Mary M.; Metzler, Genita; Murray, Andrew P.; Tanaka, Makito; Berezovskaya, Alla; Imataki, Osamu; Drury, Linda; Brennan, Lisa; Flavin, Marisa; Neuberg, Donna; Stevenson, Kristen; Lawrence, Donald; Hodi, F. Stephen; Velazquez, Elsa F.; Jaklitsch, Michael T.; Russell, Sara E.; Mihm, Martin; Nadler, Lee M.; Hirano, Naoto

2013-01-01

While advanced stage melanoma patients have a median survival of less than a year, adoptive T cell therapy can induce durable clinical responses in some patients. Successful adoptive T cell therapy to treat cancer requires engraftment of anti-tumor T lymphocytes that not only retain specificity and function in vivo but also display an intrinsic capacity to survive. To date, adoptively transferred anti-tumor CD8+ T lymphocytes (CTL) have had limited life spans unless the host has been manipulated. To generate CTL that possess an intrinsic capacity to persist in vivo, we developed a human artificial antigen presenting cell system that can educate anti-tumor CTL to acquire both a central memory and effector memory phenotype as well as the capacity to survive in culture for prolonged periods of time. In the present report, we examined whether anti-tumor CTL generated using this system could function and persist in patients. Here, we showed that MART1-specific CTL, educated and expanded using our artificial antigen presenting cell system, could survive for prolonged periods in advanced stage melanoma patients without previous conditioning or cytokine treatment. Moreover, these CTL trafficked to the tumor, mediated biological and clinical responses, and established anti-tumor immunologic memory. Therefore, this approach may broaden the availability of adoptive cell therapy to patients both alone and in combination with other therapeutic modalities. PMID:21525398

Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion.

PubMed

Engelhard, V H; Powers, G A; Moore, L C; Holterman, M J; Correa-Freire, M C

1984-01-01

HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.

CADM1/TSLC1 Identifies HTLV-1-Infected Cells and Determines Their Susceptibility to CTL-Mediated Lysis

PubMed Central

Tanaka, Yuetsu; Taylor, Graham P.; Bangham, Charles R. M.

2016-01-01

Human T cell lymphotropic virus-1 (HTLV-1) primarily infects CD4+ T cells, causing inflammatory disorders or a T cell malignancy in 5% to 10% of carriers. The cytotoxic T lymphocyte (CTL) response is a key factor that controls the viral load and thus the risk of disease. The ability to detect the viral protein Tax in primary cells has made it possible to estimate the rate at which Tax-expressing infected cells are eliminated by CTLs in persistently infected people. However, most HTLV-1-infected cells are Tax–at a given time, and their immunophenotype is poorly defined. Here, we aimed to identify a cell-surface molecule expressed by both Tax+ and Tax–HTLV-1-infected cells and use it to analyse the CTL response in fresh peripheral blood mononuclear cells. Cell adhesion molecule 1 (CADM1/TSLC1) was the best single marker of HTLV-1 infection, identifying HTLV-1-infected cells with greater sensitivity and specificity than CD25, CCR4 or ICAM-1. CADM1+CD4+ T cells carried a median of 65% of proviral copies in peripheral blood. In a cohort of 23 individuals, we quantified the rate of CTL-mediated killing of Tax+ and Tax−CADM1+ cells. We show that CADM1 expression is associated with enhanced susceptibility of infected cells to CTL lysis: despite the immunodominance of Tax in the CTL response, Tax+CADM1– cells were inefficiently lysed by CTLs. Upregulation of the CADM1 ligand CRTAM on CD8+ T cells correlated with efficient lysis of infected cells. Tax–CADM1+ cells were lysed at a very low rate by autologous CTLs, however, were efficiently killed when loaded with exogenous peptide antigen. High expression of CADM1 on most HTLV-1-infected cells in the face of enhanced CTL counterselection implies that CADM1 confers a strong benefit on the virus. PMID:27105228

Use of human antigen presenting cell gene array profiling to examine the effect of human T-cell leukemia virus type 1 Tax on primary human dendritic cells.

PubMed

Ahuja, Jaya; Kampani, Karan; Datta, Suman; Wigdahl, Brian; Flaig, Katherine E; Jain, Pooja

2006-02-01

Human T-cell leukemia virus type 1 (HTLV-1) is etiologically linked to adult T-cell leukemia and a progressive demyelinating disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the most striking features of the immune response in HAM/TSP centers on the expansion of HTLV-1-specific CD8(+) cytotoxic T lymphocyte (CTL) compartment in the peripheral blood and cerebrospinal fluid. More than 90% of the HTLV-1-specific CTLs are directed against the viral Tax (11-19) peptide implying that Tax is available for immune recognition by antigen presenting cells, such as dendritic cells (DCs). DCs obtained from HAM/TSP patients have been shown to be infected with HTLV-1 and exhibit rapid maturation. Therefore, we hypothesized that presentation of Tax peptides by activated DCs to naIve CD8(+) T cells may play an important role in the induction of a Tax-specific CTL response and neurologic dysfunction. In this study, a pathway-specific antigen presenting cell gene array was used to study transcriptional changes induced by exposure of monocyte-derived DCs to extracellular HTLV-1 Tax protein. Approximately 100 genes were differentially expressed including genes encoding toll-like receptors, cell surface receptors, proteins involved in antigen uptake and presentation and adhesion molecules. The differential regulation of chemokines and cytokines characteristic of functional DC activation was also observed by the gene array analyses. Furthermore, the expression pattern of signal transduction genes was also significantly altered. These results have suggested that Tax-mediated DC gene regulation might play a critical role in cellular activation and the mechanisms resulting in HTLV-1-induced disease.

The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints

PubMed Central

Llosa, Nicolas J.; Cruise, Michael; Tam, Ada; Wick, Elizabeth C.; Hechenbleikner, Elizabeth M.; Taube, Janis M.; Blosser, Lee; Fan, Hongni; Wang, Hao; Luber, Brandon; Zhang, Ming; Papadopoulos, Nickolas; Kinzler, Kenneth W.; Vogelstein, Bert; Sears, Cynthia L.; Anders, Robert A.; Pardoll, Drew M.; Housseau, Franck

2014-01-01

We examined the immune microenvironment of primary colorectal cancer (CRC) using immunohistochemistry, laser capture microdissection/qRT-PCR, flow cytometry and functional analysis of tumor infiltrating lymphocytes. A subset of CRC displayed high infiltration with activated CD8+ CTL as well as activated Th1 cells characterized by IFN-γ production and the Th1 transcription factor Tbet. Parallel analysis of tumor genotypes revealed that virtually all of the tumors with this active Th1/CTL microenvironment had defects in mismatch repair, as evidenced by microsatellite instability (MSI). Counterbalancing this active Th1/CTL microenvironment, MSI tumors selectively demonstrated highly up-regulated expression of multiple immune checkpoints, including five – PD-1, PD-L1, CTLA-4, LAG-3 and IDO – currently being targeted clinically with inhibitors. These findings link tumor genotype with the immune microenvironment, and explain why MSI tumors are not naturally eliminated despite a hostile Th1/CTL microenvironment. They further suggest that blockade of specific checkpoints may be selectively efficacious in the MSI subset of CRC. PMID:25358689

Molecular Mechanism of MART-1+/A*0201+ Human Melanoma Resistance to Specific CTL-Killing Despite Functional Tumor-CTL Interaction

PubMed Central

Jazirehi, Ali R.; Baritaki, Stavroula; Koya, Richard C.; Bonavida, Benjamin; Economou, James S.

2014-01-01

Durable responses in metastatic melanoma patients remain generally difficult to achieve. Adoptive cell therapy with ex vivo engineered lymphocytes expressing high affinity T cell receptors TCRα/β for the melanoma antigen MART-127-35/HLA A*0201 (recognized by F5 cytotoxic T lymphocytes [F5 CTLs]) has been found to benefit certain patients. However, many other patients are inherently unresponsive and/or relapse for unknown reasons. To analyze the basis for the acquired-resistance and strategies to reverse it, we established F5 CTLresistant (R) human melanoma clones from relatively sensitive parental lines under selective F5 CTL pressure. Surface MART-127-35/HLA-A*0201 in these clones was unaltered and F5 CTLs recognized and interacted with them similarly to the parental lines. Nevertheless, the R clones were resistant to F5 CTL killing, exhibited hyperactivation of the NF-κB survival pathway, and overexpression of the anti-apoptotic genes Bcl-2, Bcl-xL and Mcl-1. Sensitivity to F5 CTL-killing could be increased by pharmacological inhibition of the NF-κB pathway, Bcl-2 family members, or the proteasome, the latter of which reduced NF-κB activity and diminished anti-apoptotic gene expression. Specific gene-silencing (by siRNA) confirmed the protective role of anti-apoptotic factors by reversing R clone resistance. Together, our findings suggest that long-term immunotherapy may impose a selection for the development of resistant cells that are unresponsive to highly avid and specific melanoma-reactive CTLs, despite maintaining expression of functional peptide:MHC complexes, due to activation of anti-apoptotic signaling pathways. Though unresponsive to CTL, our results argue that resistant cells can be re-sensitized to immunotherapy with co-administration of targeted inhibitors to anti-apoptotic survival pathways. PMID:21159666

Balancing Immune Protection and Immune Pathology by CD8+ T-Cell Responses to Influenza Infection

PubMed Central

Duan, Susu; Thomas, Paul G.

2016-01-01

Influenza A virus (IAV) is a significant human pathogen causing annual epidemics and periodic pandemics. CD8+ cytotoxic T lymphocyte (CTL)-mediated immunity contributes to the clearance of virus-infected cells, and CTL immunity targeting the conserved internal proteins of IAVs is a key protection mechanism when neutralizing antibodies are absent during heterosubtypic IAV infection. However, CTL infiltration into the airways, its cytotoxicity, and the effects of produced proinflammatory cytokines can cause severe lung tissue injury, thereby contributing to immunopathology. Studies have discovered complicated and exquisite stimulatory and inhibitory mechanisms that regulate CTL magnitude and effector activities during IAV infection. Here, we review the state of knowledge on the roles of IAV-specific CTLs in immune protection and immunopathology during IAV infection in animal models, highlighting the key findings of various requirements and constraints regulating the balance of immune protection and pathology involved in CTL immunity. We also discuss the evidence of cross-reactive CTL immunity as a positive correlate of cross-subtype protection during secondary IAV infection in both animal and human studies. We argue that the effects of CTL immunity on protection and immunopathology depend on multiple layers of host and viral factors, including complex host mechanisms to regulate CTL magnitude and effector activity, the pathogenic nature of the IAV, the innate response milieu, and the host historical immune context of influenza infection. Future efforts are needed to further understand these key host and viral factors, especially to differentiate those that constrain optimally effective CTL antiviral immunity from those necessary to restrain CTL-mediated non-specific immunopathology in the various contexts of IAV infection, in order to develop better vaccination and therapeutic strategies for modifying protective CTL immunity. PMID:26904022

Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines

PubMed Central

Gasper, David J.; Neldner, Brandon; Plisch, Erin H.; Rustom, Hani; Imai, Hirotaka; Kawaoka, Yoshihiro; Suresh, M.

2016-01-01

CD8+ cytotoxic T lymphocytes (CTLs) are critical for clearing many viral infections, and protective CTL memory can be induced by vaccination with attenuated viruses and vectors. Non-replicating vaccines are typically potentiated by the addition of adjuvants that enhance humoral responses, however few are capable of generating CTL responses. Adjuplex is a carbomer-lecithin-based adjuvant demonstrated to elicit robust humoral immunity to non-replicating antigens. We report that mice immunized with non-replicating Adjuplex-adjuvanted vaccines generated robust antigen-specific CTL responses. Vaccination by the subcutaneous or the intranasal route stimulated systemic and mucosal CTL memory respectively. However, only CTL memory induced by intranasal vaccination was protective against influenza viral challenge, and correlated with an enhancement of memory CTLs in the airways and CD103+ CD69+ CXCR3+ resident memory-like CTLs in the lungs. Mechanistically, Myd88-deficient mice mounted primary CTL responses to Adjuplex vaccines that were similar in magnitude to wild-type mice, but exhibited altered differentiation of effector cell subsets. Immune potentiating effects of Adjuplex entailed alterations in the frequency of antigen-presenting-cell subsets in vaccine draining lymph nodes, and in the lungs and airways following intranasal vaccination. Further, Adjuplex enhanced the ability of dendritic cells to promote antigen-induced proliferation of naïve CD8 T cells by modulating antigen uptake, its intracellular localization, and rate of processing. Taken together, we have identified an adjuvant that elicits both systemic and mucosal CTL memory to non-replicating antigens, and engenders protective CTL-based heterosubtypic immunity to influenza A virus in the respiratory tract. Further, findings presented in this manuscript have provided key insights into the mechanisms and factors that govern the induction and programming of systemic and protective memory CTLs in the respiratory tract. PMID:27997610

Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines.

PubMed

Gasper, David J; Neldner, Brandon; Plisch, Erin H; Rustom, Hani; Carrow, Emily; Imai, Hirotaka; Kawaoka, Yoshihiro; Suresh, M

2016-12-01

CD8+ cytotoxic T lymphocytes (CTLs) are critical for clearing many viral infections, and protective CTL memory can be induced by vaccination with attenuated viruses and vectors. Non-replicating vaccines are typically potentiated by the addition of adjuvants that enhance humoral responses, however few are capable of generating CTL responses. Adjuplex is a carbomer-lecithin-based adjuvant demonstrated to elicit robust humoral immunity to non-replicating antigens. We report that mice immunized with non-replicating Adjuplex-adjuvanted vaccines generated robust antigen-specific CTL responses. Vaccination by the subcutaneous or the intranasal route stimulated systemic and mucosal CTL memory respectively. However, only CTL memory induced by intranasal vaccination was protective against influenza viral challenge, and correlated with an enhancement of memory CTLs in the airways and CD103+ CD69+ CXCR3+ resident memory-like CTLs in the lungs. Mechanistically, Myd88-deficient mice mounted primary CTL responses to Adjuplex vaccines that were similar in magnitude to wild-type mice, but exhibited altered differentiation of effector cell subsets. Immune potentiating effects of Adjuplex entailed alterations in the frequency of antigen-presenting-cell subsets in vaccine draining lymph nodes, and in the lungs and airways following intranasal vaccination. Further, Adjuplex enhanced the ability of dendritic cells to promote antigen-induced proliferation of naïve CD8 T cells by modulating antigen uptake, its intracellular localization, and rate of processing. Taken together, we have identified an adjuvant that elicits both systemic and mucosal CTL memory to non-replicating antigens, and engenders protective CTL-based heterosubtypic immunity to influenza A virus in the respiratory tract. Further, findings presented in this manuscript have provided key insights into the mechanisms and factors that govern the induction and programming of systemic and protective memory CTLs in the respiratory tract.

A Unique T-Cell Receptor Amino Acid Sequence Selected by Human T-Cell Lymphotropic Virus Type 1 Tax301-309-Specific Cytotoxic T Cells in HLA-A24:02-Positive Asymptomatic Carriers and Adult T-Cell Leukemia/Lymphoma Patients.

PubMed

Ishihara, Yuko; Tanaka, Yukie; Kobayashi, Seiichiro; Kawamura, Koji; Nakasone, Hideki; Gomyo, Ayumi; Hayakawa, Jin; Tamaki, Masaharu; Akahoshi, Yu; Harada, Naonori; Kusuda, Machiko; Kameda, Kazuaki; Ugai, Tomotaka; Wada, Hidenori; Sakamoto, Kana; Sato, Miki; Terasako-Saito, Kiriko; Kikuchi, Misato; Kimura, Shun-Ichi; Tanihara, Aki; Kako, Shinichi; Uchimaru, Kaoru; Kanda, Yoshinobu

2017-10-01

We previously reported that the T-cell receptor (TCR) repertoire of human T-cell lymphotropic virus type 1 (HTLV-1) Tax 301-309 -specific CD8 + cytotoxic T cells (Tax 301-309 -CTLs) was highly restricted and a particular amino acid sequence motif, the PDR motif, was conserved among HLA-A*24:02-positive (HLA-A*24:02 + ) adult T-cell leukemia/lymphoma (ATL) patients who had undergone allogeneic hematopoietic cell transplantation (allo-HSCT). Furthermore, we found that donor-derived PDR + CTLs selectively expanded in ATL long-term HSCT survivors with strong CTL activity against HTLV-1. On the other hand, the TCR repertoires in Tax 301-309 -CTLs of asymptomatic HTLV-1 carriers (ACs) remain unclear. In this study, we directly identified the DNA sequence of complementarity-determining region 3 (CDR3) of the TCR-β chain of Tax 301-309 -CTLs at the single-cell level and compared not only the TCR repertoires but also the frequencies and phenotypes of Tax 301-309 -CTLs between ACs and ATL patients. We did not observe any essential difference in the frequencies of Tax 301-309 -CTLs between ACs and ATL patients. In the single-cell TCR repertoire analysis of Tax 301-309 -CTLs, 1,458 Tax 301-309 -CTLs and 140 clones were identified in this cohort. Tax 301-309 -CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was exclusively observed in all ACs and ATL patients. However, there was no correlation between PDR + CTL frequencies and HTLV-1 proviral load (PVL). In conclusion, we have identified, for the first time, a unique amino acid sequence, PDR, as a public TCR-CDR3 motif against Tax in HLA-A*24:02 + HTLV-1-infected individuals. Further investigations are warranted to elucidate the role of the PDR + CTL response in the progression from carrier state to ATL. IMPORTANCE ATL is an aggressive T-cell malignancy caused by HTLV-1 infection. The HTLV-1 regulatory protein Tax aggressively promotes the proliferation of HTLV-1-infected lymphocytes and is also a major target antigen for CD8 + CTLs. In our previous evaluation of Tax 301-309 -CTLs, we found that a unique amino acid sequence motif, PDR, in CDR3 of the TCR-β chain of Tax 301-309 -CTLs was conserved among ATL patients after allo-HSCT. Furthermore, the PDR + Tax 301-309 -CTL clones selectively expanded and showed strong cytotoxic activities against HTLV-1. On the other hand, it remains unclear how Tax 301-309 -CTL repertoire exists in ACs. In this study, we comprehensively compared Tax-specific TCR repertoires at the single-cell level between ACs and ATL patients. Tax 301-309 -CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was conserved in all ACs and ATL patients, regardless of clinical subtype in HTLV-1 infection. Copyright © 2017 American Society for Microbiology.

A Unique T-Cell Receptor Amino Acid Sequence Selected by Human T-Cell Lymphotropic Virus Type 1 Tax301-309-Specific Cytotoxic T Cells in HLA-A24:02-Positive Asymptomatic Carriers and Adult T-Cell Leukemia/Lymphoma Patients

PubMed Central

Ishihara, Yuko; Tanaka, Yukie; Kobayashi, Seiichiro; Kawamura, Koji; Nakasone, Hideki; Gomyo, Ayumi; Hayakawa, Jin; Tamaki, Masaharu; Akahoshi, Yu; Harada, Naonori; Kusuda, Machiko; Kameda, Kazuaki; Ugai, Tomotaka; Wada, Hidenori; Sakamoto, Kana; Sato, Miki; Terasako-Saito, Kiriko; Kikuchi, Misato; Kimura, Shun-ichi; Tanihara, Aki; Kako, Shinichi; Uchimaru, Kaoru

2017-01-01

ABSTRACT We previously reported that the T-cell receptor (TCR) repertoire of human T-cell lymphotropic virus type 1 (HTLV-1) Tax301-309-specific CD8+ cytotoxic T cells (Tax301-309-CTLs) was highly restricted and a particular amino acid sequence motif, the PDR motif, was conserved among HLA-A*24:02-positive (HLA-A*24:02+) adult T-cell leukemia/lymphoma (ATL) patients who had undergone allogeneic hematopoietic cell transplantation (allo-HSCT). Furthermore, we found that donor-derived PDR+ CTLs selectively expanded in ATL long-term HSCT survivors with strong CTL activity against HTLV-1. On the other hand, the TCR repertoires in Tax301-309-CTLs of asymptomatic HTLV-1 carriers (ACs) remain unclear. In this study, we directly identified the DNA sequence of complementarity-determining region 3 (CDR3) of the TCR-β chain of Tax301-309-CTLs at the single-cell level and compared not only the TCR repertoires but also the frequencies and phenotypes of Tax301-309-CTLs between ACs and ATL patients. We did not observe any essential difference in the frequencies of Tax301-309-CTLs between ACs and ATL patients. In the single-cell TCR repertoire analysis of Tax301-309-CTLs, 1,458 Tax301-309-CTLs and 140 clones were identified in this cohort. Tax301-309-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was exclusively observed in all ACs and ATL patients. However, there was no correlation between PDR+ CTL frequencies and HTLV-1 proviral load (PVL). In conclusion, we have identified, for the first time, a unique amino acid sequence, PDR, as a public TCR-CDR3 motif against Tax in HLA-A*24:02+ HTLV-1-infected individuals. Further investigations are warranted to elucidate the role of the PDR+ CTL response in the progression from carrier state to ATL. IMPORTANCE ATL is an aggressive T-cell malignancy caused by HTLV-1 infection. The HTLV-1 regulatory protein Tax aggressively promotes the proliferation of HTLV-1-infected lymphocytes and is also a major target antigen for CD8+ CTLs. In our previous evaluation of Tax301-309-CTLs, we found that a unique amino acid sequence motif, PDR, in CDR3 of the TCR-β chain of Tax301-309-CTLs was conserved among ATL patients after allo-HSCT. Furthermore, the PDR+ Tax301-309-CTL clones selectively expanded and showed strong cytotoxic activities against HTLV-1. On the other hand, it remains unclear how Tax301-309-CTL repertoire exists in ACs. In this study, we comprehensively compared Tax-specific TCR repertoires at the single-cell level between ACs and ATL patients. Tax301-309-CTLs showed highly restricted TCR repertoires with a strongly biased usage of BV7, and PDR, the unique motif in TCR-β CDR3, was conserved in all ACs and ATL patients, regardless of clinical subtype in HTLV-1 infection. PMID:28724766

Mannosylated Mucin-Type Immunoglobulin Fusion Proteins Enhance Antigen-Specific Antibody and T Lymphocyte Responses

PubMed Central

Johansson, Tomas; Nilsson, Anki; Chatzissavidou, Nathalie; Sjöblom, Magnus; Rova, Ulrika; Holgersson, Jan

2012-01-01

Targeting antigens to antigen-presenting cells (APC) improve their immunogenicity and capacity to induce Th1 responses and cytotoxic T lymphocytes (CTL). We have generated a mucin-type immunoglobulin fusion protein (PSGL-1/mIgG2b), which upon expression in the yeast Pichia pastoris became multivalently substituted with O-linked oligomannose structures and bound the macrophage mannose receptor (MMR) and dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) with high affinity in vitro. Here, its effects on the humoral and cellular anti-ovalbumin (OVA) responses in C57BL/6 mice are presented. OVA antibody class and subclass responses were determined by ELISA, the generation of anti-OVA CTLs was assessed in 51Cr release assays using in vitro-stimulated immune spleen cells from the different groups of mice as effector cells and OVA peptide-fed RMA-S cells as targets, and evaluation of the type of Th cell response was done by IFN-γ, IL-2, IL-4 and IL-5 ELISpot assays. Immunizations with the OVA − mannosylated PSGL-1/mIgG2b conjugate, especially when combined with the AbISCO®-100 adjuvant, lead to faster, stronger and broader (with regard to IgG subclass) OVA IgG responses, a stronger OVA-specific CTL response and stronger Th1 and Th2 responses than if OVA was used alone or together with AbISCO®-100. Also non-covalent mixing of mannosylated PSGL-1/mIgG2b, OVA and AbISCO®-100 lead to relatively stronger humoral and cellular responses. The O-glycan oligomannoses were necessary because PSGL-1/mIgG2b with mono- and disialyl core 1 structures did not have this effect. Mannosylated mucin-type fusion proteins can be used as versatile APC-targeting molecules for vaccines and as such enhance both humoral and cellular immune responses. PMID:23071675

Multiple mechanisms underlie defective recognition of melanoma cells cultured in three-dimensional architectures by antigen-specific cytotoxic T lymphocytes

PubMed Central

Feder-Mengus, C; Ghosh, S; Weber, W P; Wyler, S; Zajac, P; Terracciano, L; Oertli, D; Heberer, M; Martin, I; Spagnoli, G C; Reschner, A

2007-01-01

Cancer cells' growth in three-dimensional (3D) architectures promotes resistance to drugs, cytokines, or irradiation. We investigated effects of 3D culture as compared to monolayers (2D) on melanoma cells' recognition by tumour-associated antigen (TAA)-specific HLA-A*0201-restricted cytotoxic T-lymphocytes (CTL). Culture of HBL, D10 (both HLA-A*0201+, TAA+) and NA8 (HLA-A*0201+, TAA−) melanoma cells on polyHEMA-coated plates, resulted in generation of 3D multicellular tumour spheroids (MCTS). Interferon-gamma (IFN-γ) production by HLA-A*0201-restricted Melan-A/MART-127–35 or gp100280–288-specific CTL clones served as immunorecognition marker. Co-culture with melanoma MCTS, resulted in defective TAA recognition by CTL as compared to 2D as witnessed by decreased IFN-γ production and decreased Fas Ligand, perforin and granzyme B gene expression. A multiplicity of mechanisms were potentially involved. First, MCTS per se limit CTL capacity of recognising HLA class I restricted antigens by reducing exposed cell surfaces. Second, expression of melanoma differentiation antigens is downregulated in MCTS. Third, expression of HLA class I molecules can be downregulated in melanoma MCTS, possibly due to decreased interferon-regulating factor-1 gene expression. Fourth, lactic acid production is increased in MCTS, as compared to 2D. These data suggest that melanoma cells growing in 3D, even in the absence of immune selection, feature characteristics capable of dramatically inhibiting TAA recognition by specific CTL. PMID:17342088

Patterns of Immunodominance in HIV-1–specific Cytotoxic T Lymphocyte Responses in Two Human Histocompatibility Leukocyte Antigens (HLA)-identical Siblings with HLA-A*0201 Are Influenced by Epitope Mutation

PubMed Central

Goulder, P.J.R.; Sewell, A.K.; Lalloo, D.G.; Price, D.A.; Whelan, J.A.; Evans, J.; Taylor, G.P.; Luzzi, G.; Giangrande, P.; Phillips, R.E.; McMichael, A.J.

1997-01-01

Primary human immunodeficiency virus (HIV) infection is controlled principally by HIV-specific cytotoxic T lymphocytes (CTL) to a steady-state level of virus load, which strongly influences the ultimate rate of progression to disease. Epitope selection by CTL may be an important determinant of the degree of immune control over the virus. This report describes the CTL responses of two HLA-identical hemophiliac brothers who were exposed to identical batches of Factor VIII and became seropositive within 10 wk of one another. Both have HLA-A*0201. The CTL responses of the two siblings were very dissimilar, one donor making strong responses to two epitopes within p17 Gag (HLA-A*0201–restricted SLYNTVATL and HLA-A3–restricted RLRPGGKKK). The sibling responded to neither epitope, but made strong responses to two epitopes presented by HLA-B7. This was not the result of differences in presentation of the epitopes. However, mutations in both immunodominant epitopes of the p17 Gag responder were seen in proviral sequences of the nonresponder. We then documented the CTL responses to two HLA-A*0201–restricted epitopes, in Gag (SLYNTVATL) and Pol (ILKEPVHGV) in 22 other HIV-infected donors with HLA-A*0201. The majority (71%) generated responses to the Gag epitope. In the 29% of donors failing to respond to the Gag epitope in standard assays, there was evidence of low frequency memory CTL responses using peptide stimulation of PBMC, and most of these donors also showed mutations in or around the Gag epitope. We concluded that HLA class I genotype determines epitope selection initially but that mutation in immunodominant epitopes can profoundly alter the pattern of CTL response. PMID:9126923

Anti-idiotype antibody induced cellular immunity in mice transgenic for human carcinoembryonic antigen.

PubMed

Saha, Asim; Chatterjee, Sunil K; Foon, Kenneth A; Bhattacharya-Chatterjee, Malaya

2006-08-01

In the present study, we have analysed the detailed cellular immune mechanisms involved in tumour rejection in carcinoembryonic antigen (CEA) transgenic mice after immunization with dendritic cells (DC) pulsed with an anti-idiotype (Id) antibody, 3H1, which mimics CEA. 3H1-pulsed DC vaccinations resulted in induction of CEA specific cytotoxic T lymphocyte (CTL) responses in vitro and the rejection of CEA-transfected MC-38 murine colon carcinoma cells, C15, in vivo (Saha et al.,Cancer Res 2004; 64: 4995-5003). These CTL mediated major histocompatibility complex (MHC) class I-restricted tumour cell lysis, production of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), and expression of Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) in response to C15 cells. CTL used perforin-, FasL-, and TRAIL-mediated death pathways to lyse C15 cells, although perforin-mediated killing was the predominant lytic mechanism in vitro. The cytokines IFN-gamma and TNF-alpha synergistically enhanced surface expression of Fas, TRAIL receptor, MHC class I and class II on C15 cells that increased the sensitivity of tumour cells to CTL lysis. CTL activity generated in 3H1-pulsed DC immunized mice was directed against an epitope defined by the idio-peptide LCD-2, derived from 3H1. In vivo lymphocyte depletion experiments demonstrated that induction of CTL response and antitumour immunity was dependent on both CD4+ and CD8+ T cells. The analysis of splenocytes of immunized mice that had rejected C15 tumour growth revealed up-regulated surface expression of memory phenotype Ly-6C and CD44 on both CD4+ and CD8+ T cells. The adoptive transfer experiments also suggested the role of both CD4+ and CD8+ T cells in this model system. Furthermore, mice that had rejected C15 tumour growth, developed tumour-specific immunological memory.

Recombinant modified vaccinia virus Ankara–simian immunodeficiency virus gag pol elicits cytotoxic T lymphocytes in rhesus monkeys detected by a major histocompatibility complex class I/peptide tetramer

PubMed Central

Seth, Aruna; Ourmanov, Ilnour; Kuroda, Marcelo J.; Schmitz, Jörn E.; Carroll, Miles W.; Wyatt, Linda S.; Moss, Bernard; Forman, Meryl A.; Hirsch, Vanessa M.; Letvin, Norman L.

1998-01-01

The utility of modified vaccinia virus Ankara (MVA) as a vector for eliciting AIDS virus-specific cytotoxic T lymphocytes (CTL) was explored in the simian immunodeficiency virus (SIV)/rhesus monkey model. After two intramuscular immunizations with recombinant MVA-SIVSM gag pol, the monkeys developed a Gag epitope-specific CTL response readily detected in peripheral blood lymphocytes by using a functional killing assay. Moreover, those immunizations also elicited a population of CD8+ T lymphocytes in the peripheral blood that bound a specific major histocompatibility complex class I/peptide tetramer. These Gag epitope-specific CD8+ T lymphocytes also were demonstrated by using both functional and tetramer-binding assays in lymph nodes of the immunized monkeys. These observations suggest that MVA may prove a useful vector for an HIV-1 vaccine. They also suggest that tetramer staining may be a useful technology for monitoring CTL generation in vaccine trials in nonhuman primates and in humans. PMID:9707609

Accelerated production of antigen-specific T-cells for pre-clinical and clinical applications using Gas-permeable Rapid Expansion cultureware (G-Rex)

PubMed Central

Vera, Juan F.; Brenner, Lara J.; Gerdemann, Ulrike; Ngo, Minhtran C.; Sili, Uluhan; Liu, Hao; Wilson, John; Dotti, Gianpietro; Heslop, Helen E.; Leen, Ann M.; Rooney, Cliona M.

2009-01-01

The clinical manufacture of antigen-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy is limited by the complexity and time required to produce large numbers with the desired function and specificity. The culture conditions required are rigorous, and in some cases only achieved in 2cm2 wells in which cell growth is limited by gas exchange, nutrients and waste accumulation. Bioreactors developed to overcome these issues tend to be complex, expensive and not always conducive to CTL growth. We observed that antigen-specific CTL undergo seven to ten divisions post-stimulation. However the expected CTL numbers were achieved only in the first week of culture. By recreating the culture conditions present during this first week - low frequency of antigen-specific T-cells and high frequency of feeder cells - we were able to increase CTL expansion to expected levels which could be sustained for several weeks without affecting phenotype or function. However, the number of 24-well plates needed was excessive and cultures required frequent media changes, increasing complexity and manufacturing costs. Therefore, we evaluated novel gas-permeable culture devices (G-Rex) with a silicone membrane at the base allowing gas exchange to occur uninhibited by depth of medium above. This system effectively supports the expansion of CTL and actually increases output by up to 20-fold while decreasing required technician time. Importantly, this amplified cell expansion is not due to more cell divisions but to reduced cell death. This bioprocess optimization increased T-cell output while decreasing the complexity and cost of CTL manufacture, making cell therapy more accessible. PMID:20445351

Perceived patient control over personal health information in the presence of context-specific concerns

NASA Astrophysics Data System (ADS)

Nanayakkara, Prabhashi A.

Information privacy issues have plagued the world of electronic media since its inception. This research focused mainly on factors that increase or decrease perceived patient control over personal health information (CTL) in the presence of context-specific concerns. Control agency theory was used for the paper's theoretical contributions. Personal and proxy control agencies acted as the independent variables, and context-specific concerns for information privacy (CFIP) were used as the moderator between proxy control agency, healthcare provider, and CTL. Demographic data and three control variables-- the desire for information control, privacy experience, and trust propensity--were also included in the model to gauge the contribution to CTL from external factors. Only personal control agency and desire for information control were found to impact CTL.

Tumor vessel-injuring ability improves antitumor effect of cytotoxic T lymphocytes in adoptive immunotherapy.

PubMed

Kanagawa, N; Yanagawa, T; Nakagawa, T; Okada, N; Nakagawa, S

2013-01-01

Angiogenesis is required for normal physiologic processes, but it is also involved in tumor growth, progression and metastasis. Here, we report the development of an immune-based antiangiogenic strategy based on the generation of T lymphocytes that possess killing specificity for cells expressing vascular endothelial growth factor receptor 2 (VEGFR2). To target VEGFR2-expressing cells, we engineered cytotoxic T lymphocyte (CTL) expressing chimeric T-cell receptors (cTCR-CTL) comprised of a single-chain variable fragment (scFv) against VEGFR2 linked to an intracellular signaling sequence derived from the CD3ζ chain of the TCR and CD28 by retroviral gene transduction methods. The cTCR-CTL exhibited efficient killing specificity against VEGFR2 and a tumor-targeting function in vitro and in vivo. Reflecting such abilities, we confirmed that the cTCR-CTL strongly inhibited the growth of a variety of syngeneic tumors after adoptive transfer into tumor-bearing mice without consequent damage to normal tissue. In addition, CTL expressing both cTCR and tumor-specific TCR induced complete tumor regression due to enhanced tumor infiltration by the CTL and long-term antigen-specific function. These findings provide evidence that the tumor vessel-injuring ability improved the antitumor effect of CTLs in adoptive immunotherapy for a broad range of cancers by inducing immune-mediated destruction of the tumor neovasculature.

Exosomal pMHC-I complex targets T cell-based vaccine to directly stimulate CTL responses leading to antitumor immunity in transgenic FVBneuN and HLA-A2/HER2 mice and eradicating trastuzumab-resistant tumor in athymic nude mice.

PubMed

Wang, Lu; Xie, Yufeng; Ahmed, Khawaja Ashfaque; Ahmed, Shahid; Sami, Amer; Chibbar, Rajni; Xu, Qingyong; Kane, Susan E; Hao, Siguo; Mulligan, Sean J; Xiang, Jim

2013-07-01

One of the major obstacles in human epidermal growth factor receptor 2 (HER2)-specific trastuzumab antibody immunotherapy of HER2-positive breast cancer is the development of trastuzumab resistance, warranting the search for other therapeutic strategies. Using mouse models, we previously demonstrated that ovalbumin (OVA)-specific dendritic cell (DC)-released exosome (EXOOVA)-targeted CD4(+) T cell-based (OVA-TEXO) vaccine stimulates efficient cytotoxic T lymphocyte (CTL) responses via exosomal peptide/major histocompatibility complex (pMHC)-I, exosomal CD80 and endogenous IL-2 signaling; and long-term CTL memory by means of via endogenous CD40L signaling. In this study, using two-photon microscopy, we provide the first visual evidence on targeting OVA-TEXO to cognate CD8(+) T cells in vivo via exosomal pMHC-I complex. We prepared HER2/neu-specific Neu-TEXO and HER2-TEXO vaccines using adenoviral vector (AdVneu and AdVHER2)-transfected DC (DCneu and DCHER2)-released EXOs (EXOneu and EXOHER2), and assessed their stimulatory effects on HER2/neu-specific CTL responses and antitumor immunity. We demonstrate that Neu-TEXO vaccine is capable of stimulating efficient neu-specific CTL responses, leading to protective immunity against neu-expressing Tg1-1 breast cancer in all 6/6 transgenic (Tg) FVBneuN mice with neu-specific self-immune tolerance. We also demonstrate that HER2-TEXO vaccine is capable of inducing HER2-specific CTL responses and protective immunity against transgene HLA-A2(+)HER2(+) BL6-10A2/HER2 B16 melanoma in 2/8 double Tg HLA-A2/HER2 mice with HER2-specific self-immune tolerance. The remaining 6/8 mice had significantly prolonged survival. Furthermore, we demonstrate that HER2-TEXO vaccine stimulates responses of CD8(+) T cells capable of not only inducing killing activity to HLA-A2(+)HER2(+) BL6-10A2/HER2 melanoma and trastuzumab-resistant BT474A2 breast cancer cells in vitro but also eradicating 6-day palpable HER2(+) BT474A2 breast cancer (3-4 mm in diameter) in athymic nude mice. Therefore, the novel T cell-based HER2-TEXO vaccine may provide a new therapeutic alternative for women with HER2(+) breast cancer, especially for trastuzumab-resistant HER2(+) breast cancer patients.

Expression of Interleukin-4 by Recombinant Respiratory Syncytial Virus Is Associated with Accelerated Inflammation and a Nonfunctional Cytotoxic T-Lymphocyte Response following Primary Infection but Not following Challenge with Wild-Type Virus

PubMed Central

Bukreyev, Alexander; Belyakov, Igor M.; Prince, Gregory A.; Yim, Kevin C.; Harris, Katie K.; Berzofsky, Jay A.; Collins, Peter L.

2005-01-01

The outcome of a viral infection or of immunization with a vaccine can be influenced by the local cytokine environment. In studies of experimental vaccines against respiratory syncytial virus (RSV), an increased stimulation of Th2 (T helper 2) lymphocytes was associated with increased immunopathology upon subsequent RSV infection. For this study, we investigated the effect of increased local expression of the Th2 cytokine interleukin-4 (IL-4) from the genome of a recombinant RSV following primary infection and after a challenge with wild-type (wt) RSV. Mice infected with RSV/IL-4 exhibited an accelerated pulmonary inflammatory response compared to those infected with wt RSV, although the wt RSV group caught up by day 8. In the first few days postinfection, RSV/IL-4 was associated with a small but significant acceleration in the expansion of pulmonary T lymphocytes specific for an RSV CD8+ cytotoxic T-lymphocyte (CTL) epitope presented as a major histocompatibility complex class I tetramer. However, by day 7 the response of tetramer-positive T lymphocytes in the wt RSV group caught up and exceeded that of the RSV/IL-4 group. At all times, the CTL response of the RSV/IL-4 group was deficient in the production of gamma interferon and was nonfunctional for in vitro cell killing. The accelerated inflammatory response coincided with an accelerated accumulation and activation of pulmonary dendritic cells early in infection, but thereafter the dendritic cells were deficient in the expression of B7-1, which governs the acquisition of cytolytic activity by CTL. Following a challenge with wt RSV, there was an increase in Th2 cytokines in the animals that had previously been infected with RSV/IL-4 compared to those previously infected with wt RSV, but the CD8+ CTL response and the amount of pulmonary inflammation were not significantly different. Thus, a strong Th2 environment during primary pulmonary immunization with live RSV resulted in early inflammation and a largely nonfunctional primary CTL response but had a minimal effect on the secondary response. PMID:16014914

Pseudorabies virus glycoprotein gIII is a major target antigen for murine and swine virus-specific cytotoxic T lymphocytes.

PubMed Central

Zuckermann, F A; Zsak, L; Mettenleiter, T C; Ben-Porat, T

1990-01-01

Pseudorabies virus (PrV) is the etiological agent of Aujeszky's disease, a disease that causes heavy economic losses in the swine industry. A rational approach to the generation of an effective vaccine against this virus requires an understanding of the immune response induced by it and of the role of the various viral antigens in inducing such a response. We have constructed mutants of PrV [strain PrV (Ka)] that differ from each other only in expression of the viral nonessential glycoproteins gI, gp63, gX, and gIII (i.e., are otherwise isogenic). These mutants were used to ascertain the importance of each of the nonessential glycoproteins in eliciting a PrV-specific cytotoxic T-lymphocyte (CTL) response in mice and pigs. Immunization of DBA/2 mice and pigs with a thymidine kinase-deficient (TK-) mutant of PrV elicits the formation of cytotoxic cells that specifically lyse syngeneic infected target cells. These PrV-specific cytolytic cells have the phenotype of major histocompatibility complex class I antigen-restricted CTLs. The relative number of CTLs specific for glycoproteins gI, gp63, gX, and gIII induced in mice vaccinated with a TK- mutant of PrV was ascertained by comparing their levels of cytotoxicity against syngeneic cells infected with either wild-type virus or gI-/gp63-, gX-, or gIII- virus deletion mutants. The PrV-specific CLTs were significantly less effective in lysing gIII(-)-infected targets than in lysing gI-/gp63-, gX-, or wild-type-infected targets. The in vitro secondary CTL response of lymphocytes obtained from either mice or pigs 6 or more weeks after immunization with a TK- mutant of PrV was also tested. Lymphocytes obtained from these animals were cultured with different glycoprotein-deficient mutants of PrV, and their cytolytic activities against wild-type-infected targets were ascertained. The importance of each of the nonessential viral glycoproteins in eliciting CTLs was assessed from the effectiveness of each of the virus mutants to stimulate the secondary anti-PrV CTL response. Cultures of both murine or swine lymphocytes that had been stimulated with gIII- virus contained only approximately half as many lytic units as did those stimulated with either wild-type virus, a gX- virus mutant, or a gI-/gp63- virus mutant. Thus, a large proportion of the PrV-specific CTLs that are induced by immunization with PrV of both mice and pigs are directed against gIII. Furthermore, glycoproteins gI, gp63, and gX play at most a minor role in the CTL response of these animals to PrV. PMID:2153244

C-type lectin B (SpCTL-B) regulates the expression of antimicrobial peptides and promotes phagocytosis in mud crab Scylla paramamosain.

PubMed

Wei, Xiaoyuan; Wang, Limin; Sun, Wanwei; Zhang, Ming; Ma, Hongyu; Zhang, Yueling; Zhang, Xinxu; Li, Shengkang

2018-07-01

As pattern recognition receptors, C-type lectins (CTLs) play important roles in immune system of crustaceans through identifying and binding to the conservative pathogen-associated molecular patterns (PAMPs) on pathogen surfaces. In this study, a new CTL, SpCTL-B, was identified from the hemocytes of mud crab Scylla paramamosain. The full-length of SpCTL-B cDNA was 1278 bp with an open reading frame (ORF) of 348 bp. The predicted SpCTL-B protein contains a single carbohydrate-recognition domain (CRD). SpCTL-B transcripts were distributed in all examined tissues with the highest levels in hepatopancreas. After challenged with Vibrio parahaemolyticus, LPS, polyI:C and white spot syndrome virus (WSSV), the mRNA levels of SpCTL-B in hemocytes and hepatopancreas were up-regulated. The recombinant SpCTL-B (rSpCTL-B) purified by Ni-affinity chromatography showed stronger binding activities with Staphylococcus aureus, β-hemolytic Streptococcus, Escherichia coli, Aeromonas hydrophila, Vibrio alginolyticus than those with V. parahaemolyticus and Saccharomyces cerevisiae. rSpCTL-B exhibited a broad spectrum of microorganism-agglutination activities against Gram-positive bacteria (S. aureus, β-hemolytic Streptococcus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, A. hydrophila, V. alginolyticus) in a Ca 2+ -dependent manner. The agglutination activities of rSpCTL-B could be inhibited by D-mannose and LPS, but not by d-fructose and galactose. The antimicrobial assay showed that rSpCTL-B exhibited the growth inhibition against all examined gram-positive bacteria and gram-negative bacteria. When SpCTL-B was silenced by RNAi, the bacterial clearance ability in mud crab was decreased and the transcript levels of five antimicrobial peptides (AMPs) (SpCrustin, SpHistin, SpALF4 (anti-lipopolysaccharide factor), SpALF5 and SpALF6) were significantly decreased in hemocytes. In our study, knockdown of SpCTL-B could down-regulate the expression of SpSTAT at mRNA transcriptional level and protein translational level in mud crab. Meantime, the phagocytosis rate and the expression of three phagocytosis related genes were declined after RNAi of SpCTL-B in hemocytes in mud crab. Collectively, our results suggest that SpCTL-B might play its roles as a pattern recognition receptor (PRR) in immune response towards pathogens infection through influencing the expression of AMPs and the phagocytosis of hemocytes in mud crab S. paramamosain. Copyright © 2018 Elsevier Ltd. All rights reserved.

Natural variants of cytotoxic epitopes are T-cell receptor antagonists for antiviral cytotoxic T cells

NASA Astrophysics Data System (ADS)

Bertoletti, Antonio; Sette, Alessandro; Chisari, Francis V.; Penna, Amalia; Levrero, Massimo; Carli, Marco De; Fiaccadori, Franco; Ferrari, Carlo

1994-06-01

IT has been suggested that mutations within immunodominant cytotoxic T-lymphocyte (CTL) epitopes may be exploited by viruses to evade protective immune responses critical for clearance1-4. Viral escape could originate from passive mechanisms, such as mutations within crucial CTL epitopes, either affecting major histocompatibility complex binding or T-cell antigen receptor (TCR) recognition. Additionally, it has recently been shown that substitutions of TCR contact sites can yield analogue peptides that can still interact with the T-cell receptor but be unable to deliver a full stimulatory signal, thus inducing anergy5 or acting as an antagonist for the TCR6-8. We report here that hepatitis B virus isolates derived from two chronically infected patients display variant epitopes that act as natural TCR antagonists with the capacity to inhibit the CTL response to the wild-type epitope. During natural infection, TCR antagonist mutations of CTL epitopes could contribute to the development of viral persistence, especially if the antiviral CTL response is monospecific or the epitope is strongly immunodominant.

The complex and specific pMHC interactions with diverse HIV-1 TCR clonotypes reveal a structural basis for alterations in CTL function

NASA Astrophysics Data System (ADS)

Xia, Zhen; Chen, Huabiao; Kang, Seung-Gu; Huynh, Tien; Fang, Justin W.; Lamothe, Pedro A.; Walker, Bruce D.; Zhou, Ruhong

2014-02-01

Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function.

Pancreatic cancer counterattack: MUC4 mediates Fas-independent apoptosis of antigen-specific cytotoxic T lymphocyte.

PubMed

Zhu, Yi; Zhang, Jing-Jing; Liang, Wen-Biao; Zhu, Rong; Wang, Bin; Miao, Yi; Xu, Ze-Kuan

2014-04-01

Tumor-associated MUC4 mucin has considerable potential as an immunotherapy target for pancreatic cancer. In previous studies, we developed dendritic cell (DC) vaccines which elicited MUC4 antigen-specific cytotoxic T lymphocyte (MS-CTL) response against tumor cells in vitro. Due to the observation that MS-CTL apoptotic rate increased significantly when co-cultured with MUC4+ tumor cells compared with T2 cells, we investigated whether high expression levels of MUC4 in pancreatic cancer cells would have an effect on the significant increase of apoptosis rate of MS-CTLs. First, the adverse influence of regulatory T cells (Tregs) was eliminated by CD8+ T lymphocyte sorting before the induction of MS-CTLs. Then, we constructed clonal MUC4-knockdown HPAC pancreatic cancer sublines with different MUC4 expression for co-incubation system. By utilizing appropriate control to rule out the possible apoptosis-induced pathway of intrinsic activated cell-autonomous death (ACAD) and analogous antigen-dependent apoptosis of CTL (ADAC) in our study system, further analysis of the effect of MUC4 membrane-expression, supernatants and blockade of CTL surface Fas receptor on MS-CTL apoptosis was carried out. The results demonstrated that the level of MUC4 membrane expression strongly positively correlated with MS-CTL apoptosis and the influence of supernatants and Fas-blockade did not significantly correlate with MS-CTL apoptosis. This evidence suggested that there may be a novel counterattack pathway of pancreatic cancer cells, which is a MUC4-mediated, cell contact-dependent and Fas-independent process, to induce CTL apoptosis. Therefore, further exploration and understanding of the potential counterattack mechanisms is beneficial to enhance the efficacy of MUC4 specific tumor vaccines.

Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26.

PubMed

Anderson, John P; Rascoe, Lisa N; Levert, Keith; Chastain, Holly M; Reed, Matthew S; Rivera, Hilda N; McAuliffe, Isabel; Zhan, Bin; Wiegand, Ryan E; Hotez, Peter J; Wilkins, Patricia P; Pohl, Jan; Handali, Sukwan

2015-01-01

The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag.

Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26

PubMed Central

Anderson, John P.; Rascoe, Lisa N.; Levert, Keith; Chastain, Holly M.; Reed, Matthew S.; Rivera, Hilda N.; McAuliffe, Isabel; Zhan, Bin; Wiegand, Ryan E.; Hotez, Peter J.; Wilkins, Patricia P.; Pohl, Jan; Handali, Sukwan

2015-01-01

The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag. PMID:26485145

Therapeutic limitations in tumor-specific CD8+ memory T cell engraftment

PubMed Central

Bathe, Oliver F; Dalyot-Herman, Nava; Malek, Thomas R

2003-01-01

Background Adoptive immunotherapy with cytotoxic T lymphocytes (CTL) represents an alternative approach to treating solid tumors. Ideally, this would confer long-term protection against tumor. We previously demonstrated that in vitro-generated tumor-specific CTL from the ovalbumin (OVA)-specific OT-I T cell receptor transgenic mouse persisted long after adoptive transfer as memory T cells. When recipient mice were challenged with the OVA-expressing E.G7 thymoma, tumor growth was delayed and sometimes prevented. The reasons for therapeutic failures were not clear. Methods OT-I CTL were adoptively transferred to C57BL/6 mice 21 – 28 days prior to tumor challenge. At this time, the donor cells had the phenotypical and functional characteristics of memory CD8+ T cells. Recipients which developed tumor despite adoptive immunotherapy were analyzed to evaluate the reason(s) for therapeutic failure. Results Dose-response studies demonstrated that the degree of tumor protection was directly proportional to the number of OT-I CTL adoptively transferred. At a low dose of OT-I CTL, therapeutic failure was attributed to insufficient numbers of OT-I T cells that persisted in vivo, rather than mechanisms that actively suppressed or anergized the OT-I T cells. In recipients of high numbers of OT-I CTL, the E.G7 tumor that developed was shown to be resistant to fresh OT-I CTL when examined ex vivo. Furthermore, these same tumor cells no longer secreted a detectable level of OVA. In this case, resistance to immunotherapy was secondary to selection of clones of E.G7 that expressed a lower level of tumor antigen. Conclusions Memory engraftment with tumor-specific CTL provides long-term protection against tumor. However, there are several limitations to this immunotherapeutic strategy, especially when targeting a single antigen. This study illustrates the importance of administering large numbers of effectors to engraft sufficiently efficacious immunologic memory. It also demonstrates the importance of targeting several antigens when developing vaccine strategies for cancer. PMID:12882650

MUC1-specific CTLs are non-functional within a pancreatic tumor microenvironment.

PubMed

Mukherjee, P; Ginardi, A R; Madsen, C S; Tinder, T L; Jacobs, F; Parker, J; Agrawal, B; Longenecker, B M; Gendler, S J

2001-01-01

Pancreatic cancer is a highly aggressive, treatment refractory disease and is the fourth leading cause of death in the United States. In humans, 90% of pancreatic adenocarcinomas over-express altered forms of a tumor-associated antigen, MUC1 (an epithelial mucin glycoprotein), which is a target for immunotherapy. Using a clinically relevant mouse model of pancreas cancer that demonstrates peripheral and central tolerance to human MUC1 and develops spontaneous tumors of the pancreas, we have previously reported the presence of functionally active, low affinity, MUC1-specific precursor cytotoxic T cells (pCTLs). Hypothesis for this study is that MUC1-based immunization may enhance the low level MUC1-specific immunity that may lead to an effective anti-tumor response. Data demonstrate that MUC1 peptide-based immunization elicits mature MUC1-specific CTLs in the peripheral lymphoid organs. The mature CTLs secrete IFN-gamma and are cytolytic against MUC1-expressing tumor cells in vitro. However, active CTLs that infiltrate the pancreas tumor microenvironment become cytolytically anergic and are tolerized to MUC1 antigen, allowing the tumor to grow. We demonstrate that the CTL tolerance could be reversed at least in vitro with the use of anti-CD40 co-stimulation. The pancreas tumor cells secrete immunosuppressive cytokines, including IL-10 and TGF-beta that are partly responsible for the down-regulation of CTL activity. In addition, they down-regulate their MHC class I molecules to avoid immune recognition. CD4+ CD25+ T regulatory cells, which secrete IL-10, were also found in the tumor environment. Together these data indicate the use of several immune evasion mechanisms by tumor cells to evade CTL killing. Thus altering the tumor microenvironment to make it more conducive to CTL killing may be key in developing a successful anti-cancer immunotherapy.

Experimental Rhodococcus equi and equine infectious anemia virus DNA vaccination in adult and neonatal horses: Effect of IL-12, dose, and route

PubMed Central

Mealey, R.H.; Stone, D.M.; Hines, M.T.; Alperin, D.C.; Littke, M.H.; Leib, S.R.; Leach, S.E.; Hines, S.A.

2012-01-01

Improving the ability of DNA-based vaccines to induce potent Type1/Th1 responses against intracellular pathogens in large outbred species is essential. Rhodoccocus equi and equine infectious anemia virus (EIAV) are two naturally occurring equine pathogens that also serve as important large animal models of neonatal immunity and lentiviral immune control. Neonates present a unique challenge for immunization due to their diminished immunologic capabilities and apparent Th2 bias. In an effort to augment R. equi- and EIAV-specific Th1 responses induced by DNA vaccination, we hypothesized that a dual promoter plasmid encoding recombinant equine IL-12 (rEqIL-12) would function as a molecular adjuvant. In adult horses, DNA vaccines induced R. equi- and EIAV-specific antibody and lymphoproliferative responses, and EIAV-specific CTL and tetramer-positive CD8+ T lymphocytes. These responses were not enhanced by the rEqIL-12 plasmid. In neonatal foals, DNA immunization induced EIAV-specific antibody and lymphoproliferative responses, but not CTL. The R. equi vapA vaccine was poorly immunogenic in foals even when co-administered with the IL-12 plasmid. It was concluded that DNA immunization was capable of inducing Th1 responses in horses; dose and route were significant variables, but rEqIL-12 was not an effective molecular adjuvant. Additional work is needed to optimize DNA vaccine-induced Th1 responses in horses, especially in neonates. PMID:17889970

High Rate of Induction of Human Autologous Cytotoxic T Lymphocytes against Renal Carcinoma Cells Cultured with an Interleukin Cocktail

PubMed Central

Liu, Shu Qin; Kawai, Koji; Shiraiwa, Hiroshi; Hayashi, Hitoshi; Akaza, Hideyuki; Hashizaki, Kazuko; Shiba, Reiko; Saijo, Kaoru

1998-01-01

A high rate of induction (9 of 10 cases) of human autologous cytotoxic T lymphocytes (CTL) was achieved in vitro from peripheral blood mononuclear cells of renal carcinoma patients by applying an interleukin (IL)‐cocktail consisting of IL‐1, ‐2, ‐4, and ‐6. The CTL specifically lysed their own target carcinoma cells within 24 h but did not kill neighboring autologous normal kidney cells or allogeneic renal cancer cell lines. In the case of TUHR4TKB, for which autologous CTL were not induced, no expression of MHC class‐I molecules was observed on the surface of these carcinoma cells, although they were sensitive to autologous natural killer cells. The results imply that adoptive immunotherapy for metastasized renal carcinoma will be feasible with autologous CTL in combination with natural killer cells. PMID:9914789

Frequent associations between CTL and T-Helper epitopes in HIV-1 genomes and implications for multi-epitope vaccine designs

PubMed Central

2010-01-01

Background Epitope vaccines have been suggested as a strategy to counteract viral escape and development of drug resistance. Multiple studies have shown that Cytotoxic T-Lymphocyte (CTL) and T-Helper (Th) epitopes can generate strong immune responses in Human Immunodeficiency Virus (HIV-1). However, not much is known about the relationship among different types of HIV epitopes, particularly those epitopes that can be considered potential candidates for inclusion in the multi-epitope vaccines. Results In this study we used association rule mining to examine relationship between different types of epitopes (CTL, Th and antibody epitopes) from nine protein-coding HIV-1 genes to identify strong associations as potent multi-epitope vaccine candidates. Our results revealed 137 association rules that were consistently present in the majority of reference and non-reference HIV-1 genomes and included epitopes of two different types (CTL and Th) from three different genes (Gag, Pol and Nef). These rules involved 14 non-overlapping epitope regions that frequently co-occurred despite high mutation and recombination rates, including in genomes of circulating recombinant forms. These epitope regions were also highly conserved at both the amino acid and nucleotide levels indicating strong purifying selection driven by functional and/or structural constraints and hence, the diminished likelihood of successful escape mutations. Conclusions Our results provide a comprehensive systematic survey of CTL, Th and Ab epitopes that are both highly conserved and co-occur together among all subtypes of HIV-1, including circulating recombinant forms. Several co-occurring epitope combinations were identified as potent candidates for inclusion in multi-epitope vaccines, including epitopes that are immuno-responsive to different arms of the host immune machinery and can enable stronger and more efficient immune responses, similar to responses achieved with adjuvant therapies. Signature of strong purifying selection acting at the nucleotide level of the associated epitopes indicates that these regions are functionally critical, although the exact reasons behind such sequence conservation remain to be elucidated. PMID:20696039

Antigen delivery by α2-macroglobulin enhances the cytotoxic T lymphocyte response

PubMed Central

Bowers, Edith V.; Horvath, Jeffrey J.; Bond, Jennifer E.; Cianciolo, George J.; Pizzo, Salvatore V.

2009-01-01

α2M* targets antigens to APCs for rapid internalization, processing, and presentation. When used as an antigen-delivery vehicle, α2M* amplifies MHC class II presentation, as demonstrated by increased antibody titers. Recent evidence, however, suggests that α2M* encapsulation may also enhance antigen-specific CTL immunity. In this study, we demonstrate that α2M*-delivered antigen (OVA) enhances the production of specific in vitro and in vivo CTL responses. Murine splenocytes expressing a transgenic TCR specific for CTL peptide OVA257–264 (SIINFEKL) demonstrated up to 25-fold greater IFN-γ and IL-2 secretion when treated in vitro with α2M*-OVA compared with soluble OVA. The frequency of IFN-γ-producing cells was increased ∼15-fold, as measured by ELISPOT. Expansion of the OVA-specific CD8+ T cell population, as assayed by tetramer binding and [3H]thymidine incorporation, and OVA-specific cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also enhanced significantly by α2M*-OVA. Furthermore, significant CTL responses were observed at antigen doses tenfold lower than those required with OVA alone. Finally, we also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with α2M*-OVA. These α2M*-OVA-immunized mice demonstrated increased protection against a s.c.-implanted, OVA-expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The observation that α2M*-mediated antigen delivery elicits specific CTL responses suggests the cross-presentation of antigen onto MHC class I. These results support α2M* as an effective antigen-delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing. PMID:19652028

A phase I-II trial to examine the toxicity of CMV- and EBV-specific cytotoxic T lymphocytes when used for prophylaxis against EBV and CMV disease in recipients of CD34-selected/T cell-depleted stem cell transplants.

PubMed

Lucas, K G; Sun, Q; Burton, R L; Tilden, A; Vaughan, W P; Carabasi, M; Salzman, D; Ship, A

2000-07-01

Epstein-Barr virus (EBV)-induced lymphoproliferative disease and cytomegalovirus (CMV) infection are major causes of morbidity and mortality in individuals with compromised cellular immunity. Although anti-viral pharmacological agents exist, severe side effects such as myelosuppression often limit the application of these medications. Infusion of ex vivo-expanded, virus-specific cytotoxic T-lymphocytes (CTL) has been proven to be safe and efficacious for the prophylaxis and treatment of EBV and CMV complications. While EBV-specific CTL can be readily and reliably produced with EBV-immortalized B-lymphoblastoid cell lines (BLCL) as stimulators, current protocols for CMV-specific CTL, which use CMV-infected fibroblasts as stimulators, may be associated with alloreactivity and the need for cloning, as well as the potential for exposure to human blood-born infectious agents. Our laboratory has developed a novel system to generate EBV/CMV-bi-specific CTL by co-culturing PBMC with autologous BLCL expressing a CMV protein pp65 (BLCLpp65) (Sun et al., 1999). pp65, an immunodominant CMV antigen, is transduced into BLCL by a recombinant retrovirus MSCVpp65. While low in alloreactivity, BLCLpp65-stimulated CTL are cytolytic to autologous cells infected with EBV or CMV, and this cytotoxicity is mediated by polyclonal, CD8+, MHC Class I-restricted T-cells. Further experiments revealed that retroviral transduction and expression of pp65 do not compromise the capacity of presenting EBV antigens, and T cells stimulated by BLCLpp65 recognize clinical strains of CMV (Sun et al., 2000). These data indicated that BLCLpp65 could substitute for BLCL as antigen presenting cells in adoptive immunotherapy against EBV-LPD, with the benefit of providing protection against CMV reactivation. This protocol is a Phase I/II study to examine the toxicity associated with and the immunologic effects of ex vivo simultaneously expanded EBV- and CMV-specific CTL for prophylaxis against EBV and CMV complications in recipients of CD34 selected/T-cell depleted stem cell transplants (SCT). EBV/CMV-specific CTL will be generated from peripheral blood mononuclear cells (PBMC) of EBV/CMV-seropositive donors in a course of from 21-28 days by weekly stimulation with autologous BLCLpp65. Qualified CTL will be administered to consenting patients at 40, 60, and 80 days post-transpOFF criteria of molecular virology and immunological reconstitution, which include blood levels of pp65 antigen and EBV viral DNA, and virus-specific CTL precursor frequency. Patients will also be tested for replication-competent retrovirus at 3, 6, and 12 month intervals post-transplant to ensure bio-safety.

Positive Selection of γδ CTL by TL Antigen Expressed in the Thymus

PubMed Central

Tsujimura, Kunio; Takahashi, Toshitada; Morita, Akimichi; Hasegawa-Nishiwaki, Hitomi; Iwase, Shigeru; Obata, Yuichi

1996-01-01

To elucidate the function of the mouse TL antigen in the thymus, we have derived two TL transgenic mouse strains by introducing Tla a -3 of A strain origin with its own promoter onto a C3H background with no expression of TL in the thymus. These transgenic mouse strains, both of which express high levels of Tlaa-3-TL antigen in their thymus, were analyzed for their T cell function with emphasis on cytotoxic T lymphocyte (CTL) generation. A T cell response against TL was induced in Tg.Tlaa-3-1, Tg.Tlaa-3-2, and control C3H mice by skin grafts from H-2K b/T3 b transgenic mice, Tg.Con.3-1, expressing T3b-TL ubiquitously. Spleen cells from mice that had rejected the T3b-TL positive skin grafts were restimulated in vitro with Tg.Con.3-1 irradiated spleen cells. In mixed lymphocyte cultures (MLC), approximately 20% and 15% of Thy-1+ T cells derived from Tg.Tlaa-3-1 and Tg.Tlaa-3-2, respectively, expressed TCRγδ, whereas almost all those from C3H expressed TCRαβ. The MLC from Tg.Tlaa-3-2 and C3H demonstrated high CTL activity against TL, while those from Tg.Tlaa-3-1 had little or none. The generation of γδ CTL recognizing TL in Tg.Tlaa-3-2, but not C3H mice, was confirmed by the establishment of CTL clones. A total of 14 γδ CTL clones were established from Tg.Tlaa-3-2, whereas none were obtained from C3H. Of the 14 γδ CTL clones, 8 were CD8+ and 6 were CD4−CD8− double negative. The CTL activity of all these clones was TL specific and inhibited by anti-TL, but not by anti-H-2 antibodies, demonstrating that they recognize TL directly without antigen presentation by H-2. The CTL activity was blocked by antibodies to TCRγδ and CD3, and also by antibodies to CD8α and CD8β in CD8+ clones, showing that the activity was mediated by TCRγδ and coreceptors. The thymic origin of these γδ CTL clones was indicated by the expression of Thy-1 and Ly-1 (CD5), and also CD8αβ heterodimers in CD8+ clones on their surfaces and by the usage of TCR Vγ4 chains in 12 of the 14 clones. Taken together, these results suggest that Tlaa-3-TL antigen expressed in the thymus engages in positive selection of a sizable population of γδ T cells. PMID:8976173

DOE studies on coal-to-liquids

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

2007-07-01

The US DOE National Energy Technology Laboratory has issued reports that examine the feasibility of coal-to-liquids (CTL) facilities, both general and site specific, which are available at www.netl.gov/energy-analyses/ref-shelf.html. The US Department of Defence has been investigating use of Fischer-Tropsch fuels. Congress is considering various CTL proposals while the private sector is building pilot plants and performing feasibility studies for proposed plants. The article includes a table listing 14 coal-to-liquids plants under consideration. The private sector has formed the coal-to-liquids coalition (www.futurecoalfuels.org). The article mentions other CTL projects in South Africa, China, Indonesia, the Philippines and New Zealand. 1 tab.

FLT3-regulated antigens as targets for leukemia-reactive cytotoxic T lymphocytes

PubMed Central

Brackertz, B; Conrad, H; Daniel, J; Kast, B; Krönig, H; Busch, D H; Adamski, J; Peschel, C; Bernhard, H

2011-01-01

The FMS-like tyrosine kinase 3 (FLT3) is highly expressed in acute myeloid leukemia (AML). Internal tandem duplications (ITD) of the juxtamembrane domain lead to the constitutive activation of the FLT3 kinase inducing the activation of multiple genes, which may result in the expression of leukemia-associated antigens (LAAs). We analyzed the regulation of LAA in FLT3-wild-type (WT)- and FLT3-ITD+ myeloid cells to identify potential targets for antigen-specific immunotherapy for AML patients. Antigens, such as PR-3, RHAMM, Survivin, WT-1 and PRAME, were upregulated by constitutively active FLT3-ITD as well as FLT3-WT activated by FLT3 ligand (FL). Cytotoxic T-cell (CTL) clones against PR-3, RHAMM, Survivin and an AML-directed CTL clone recognized AML cell lines and primary AML blasts expressing FLT3-ITD, as well as FLT3-WT+ myeloid dendritic cells in the presence of FL. Downregulation of FLT3 led to the abolishment of CTL recognition. Comparing our findings concerning LAA upregulation by the FLT3 kinase with those already made for the Bcr-Abl kinase, we found analogies in the LAA expression pattern. Antigens upregulated by both FLT3 and Bcr-Abl may be promising targets for the development of immunotherapeutical approaches against myeloid leukemia of different origin. PMID:22829124

Deficiency of activated STAT1 in head and neck cancer cells mediates TAP1-dependent escape from cytotoxic T lymphocytes

PubMed Central

Leibowitz, Michael S.; Filho, Pedro A. Andrade; Ferrone, Soldano; Ferris, Robert L.

2012-01-01

Squamous cell carcinoma of the head and neck (SCCHN) cells can escape recognition by tumor antigen (TA)-specific cytotoxic T lymphocytes (CTL) by downregulation of antigen processing machinery (APM) components, such as the transporter associated with antigen processing (TAP)-1/2 heterodimer. APM component upregulation by interferon gamma (IFN-γ) restores SCCHN cell recognition and susceptibility to lysis by CTL, but the mechanism underlying TAP1/2 downregulation in SCCHN cells is not known. Because IFN-γ activates signal transducer and activator of transcription (STAT)-1, we investigated phosphorylated (p)-STAT1 as a mediator of low basal TAP1/2 expression in SCCHN cells. SCCHN cells were found to express basal total STAT1 but low to undetectable levels of activated STAT1. The association of increased pSTAT1 levels and APM components likely reflects a cause–effect relationship, since STAT1 knockdown significantly reduced both IFN-γ-mediated APM component expression and TA-specific CTL recognition of IFN-γ-treated SCCHN cells. On the other hand, since oncogenic pSTAT3 is overexpressed in SCCHN cells and was found to heterodimerize with pSTAT1, we also tested whether pSTAT3 and pSTAT1:pSTAT3 heterodimers inhibited IFN-γ-induced STAT1 activation and APM component expression. First, STAT3 activation or depletion did not affect basal or IFN-γ-induced expression of pSTAT1 and APM components or recognition of SCCHN cells by TA-specific CTL. Second, pSTAT1:pSTAT3 heterodimers did not interfere with IFN-γ-induced STAT1 binding to the TAP1 promoter or APM protein expression. These findings demonstrate that APM component downregulation is regulated primarily by an IFN-γ-pSTAT1-mediated signaling pathway, independent of oncogenic STAT3 overexpression in SCCHN cells. PMID:21207025

Understanding MHC Class I Presentation of Viral Antigens by Human Dendritic Cells as a Basis for Rational Design of Therapeutic Vaccines

PubMed Central

van Montfoort, Nadine; van der Aa, Evelyn; Woltman, Andrea M.

2014-01-01

Effective viral clearance requires the induction of virus-specific CD8+ cytotoxic T lymphocytes (CTL). Since dendritic cells (DC) have a central role in initiating and shaping virus-specific CTL responses, it is important to understand how DC initiate virus-specific CTL responses. Some viruses can directly infect DC, which theoretically allow direct presentation of viral antigens to CTL, but many viruses target other cells than DC and thus the host depends on the cross-presentation of viral antigens by DC to activate virus-specific CTL. Research in mouse models has highly enhanced our understanding of the mechanisms underlying cross-presentation and the dendritic cells (DC) subsets involved, however, these results cannot be readily translated toward the role of human DC in MHC class I-antigen presentation of human viruses. Here, we summarize the insights gained in the past 20 years on MHC class I presentation of viral antigen by human DC and add to the current debate on the capacities of different human DC subsets herein. Furthermore, possible sources of viral antigens and essential DC characteristics for effective induction of virus-specific CTL are evaluated. We conclude that cross-presentation is not only an efficient mechanism exploited by DC to initiate immunity to viruses that do not infect DC but also to viruses that do infect DC, because cross-presentation has many conceptual advantages and bypasses direct immune modulatory effects of the virus on its infected target cells. Since knowledge on the mechanism of viral antigen presentation and the preferred DC subsets is crucial for rational vaccine design, the obtained insights are very instrumental for the development of effective anti-viral immunotherapy. PMID:24795724

Mutation of a chitinase-like gene causes ectopic deposition of lignin, aberrant cell shapes, and overproduction of ethylene.

PubMed

Zhong, Ruiqin; Kays, Stanley J; Schroeder, Betty P; Ye, Zheng-Hua

2002-01-01

Chitinase-like proteins have long been proposed to play roles in normal plant growth and development, but no mutations in chitinase-like genes have been obtained previously to support this hypothesis. In this study, we have shown that the gene responsible for the elp1 mutation in Arabidopsis encodes a chitinase-like protein (AtCTL1). Mutation of this chitinase-like gene caused ectopic deposition of lignin and aberrant shapes of cells with incomplete cell walls in the pith of inflorescence stems. The AtCTL1 gene was expressed in all organs during normal plant growth and development, but it was not induced by wounding, salicylic acid, pectin fragments, or ethylene. Consistent with its ubiquitous expression pattern, mutation of the AtCTL1 gene affected many aspects of plant growth and development, including exaggerated hook curvature, reduced length and increased diameter of hypocotyls in dark-grown seedlings, and reduced root length and increased number of root hairs in light-grown seedlings. The mutant phenotypes could be rescued partially by ethylene inhibitors, and ethylene production in the mutant was significantly greater than in the wild type. Together, these results suggest that AtCTL1, a chitinase-like gene, is essential for normal plant growth and development in Arabidopsis.

The complex and specific pMHC interactions with diverse HIV-1 TCR clonotypes reveal a structural basis for alterations in CTL function

PubMed Central

Xia, Zhen; Chen, Huabiao; Kang, Seung-gu; Huynh, Tien; Fang, Justin W.; Lamothe, Pedro A.; Walker, Bruce D.; Zhou, Ruhong

2014-01-01

Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function. PMID:24522437

Choline transporter-like protein 4 (CTL4) links to non-neuronal acetylcholine synthesis

PubMed Central

Song, Pingfang; Rekow, Stephen S.; Singleton, Corey-Ayne; Sekhon, Harmanjatinder S.; Dissen, Gregory A.; Zhou, Minerva; Campling, Barbara; Lindstrom, Jon; Spindel, Eliot R.

2013-01-01

Synthesis of acetylcholine (ACh) by non-neuronal cells is now well established and plays diverse physiologic roles. In neurons, the Na+-dependent, high affinity choline transporter (CHT1) is absolutely required for ACh synthesis. By contrast, some non-neuronal cells synthesize ACh in the absence of CHT1 indicating a fundamental difference in ACh synthesis compared to neurons. The aim of this study was to identify choline transporters, other than CHT1, that play a role in non-neuronal ACh synthesis. ACh synthesis was studied in lung and colon cancer cell lines focusing on the choline transporter-like proteins, a five gene family (CTL1-5). Supporting a role for CTLs in choline transport in lung cancer cells, choline transport was Na+-independent and CTL1-5 were expressed in all cells examined. CTL1,2,&5 were expressed at highest levels and knockdown of CTL1,2&5 decreased choline transport in H82 lung cancer cells. Knockdowns of CTL1,2,3&5 had no effect on ACh synthesis in H82 cells. By contrast, knockdown of CTL4 significantly decreased ACh secretion by both lung and colon cancer cells. Conversely, increasing expression of CTL4 increased ACh secretion. These results indicate that CTL4 mediates ACh synthesis in non-neuronal cell lines and presents a mechanism to target non-neuronal ACh synthesis without affecting neuronal ACh synthesis. PMID:23651124

Quantitative Expression and Immunogenicity of MAGE-3 and -6 in Upper Aerodigestive Tract Cancer

PubMed Central

Andrade Filho, Pedro A.; López-Albaitero, Andrés; Xi, Liqiang; Gooding, William; Godfrey, Tony; Ferris, Robert L.

2009-01-01

The MAGE antigens are frequently expressed cancer vaccine targets. However, quantitative analysis of MAGE expression in upper aero-digestive tract (UADT) tumor cells and its association with T cell recognition has not been performed, hindering the selection of appropriate candidates for MAGE specific immunotherapy. Using quantitative RT-PCR (QRT-PCR), we evaluated the expression of MAGE-3/6 in 65 UADT cancers, 48 normal samples from tumor matched sites and 7 HLA-A*0201+squamous cell carcinoma of the head and neck (SCCHN) cell lines. Expression results were confirmed using western blot. HLA-A*0201:MAGE-3(271–279) specific cytotoxic T lymphocytes (MAGE-CTL) from SCCHN patients and healthy donors showed that MAGE-3/6 expression was highly associated with CTL recognition in vitro. Based on MAGE-3/6 expression we could identify 31 (47%) of the 65 UADT tumors which appeared to express MAGE-3/6 at levels that correlated with efficient CTL recognition. To confirm that the level of MAGE-3 expression was responsible for CTL recognition, two MAGE-3/6 mRNAhigh SCCHN cell lines, PCI-13 and PCI-30, were subjected to MAGE-3/6 specific knockdown. RNAi–transfected cells showed that MAGE expression, and MAGE-CTL recognition, were significantly reduced. Furthermore, treatment of cells expressing low MAGE-3/6 mRNA with a demethylating agent, 5-aza-2'-deoxycytidine (DAC), increased the expression of MAGE-3/6 and CTL recognition. Thus, using QRT-PCR UADT cancers frequently express MAGE-3/6 at levels sufficient for CTL recognition, supporting the use of a QRT-PCR based assay for the selection of candidates likely to respond to MAGE-3/6 immunotherapy. Demethylating agents could increase the number of patients amenable for targeting epigenetically modified tumor antigens in vaccine trials. PMID:19610063

Quantitative expression and immunogenicity of MAGE-3 and -6 in upper aerodigestive tract cancer.

PubMed

Filho, Pedro A Andrade; López-Albaitero, Andrés; Xi, Liqiang; Gooding, William; Godfrey, Tony; Ferris, Robert L

2009-10-15

The MAGE antigens are frequently expressed cancer vaccine targets. However, quantitative analysis of MAGE expression in upper aerodigestive tract (UADT) tumor cells and its association with T-cell recognition has not been performed, hindering the selection of appropriate candidates for MAGE-specific immunotherapy. Using quantitative RT-PCR (QRT-PCR), we evaluated the expression of MAGE-3/6 in 65 UADT cancers, 48 normal samples from tumor matched sites and 7 HLA-A*0201+ squamous cell carcinoma of the head and neck (SCCHN) cell lines. Expression results were confirmed using Western blot. HLA-A*0201:MAGE-3- (271-279) specific cytotoxic T lymphocytes (MAGE-CTL) from SCCHN patients and healthy donors showed that MAGE-3/6 expression was highly associated with CTL recognition in vitro. On the basis of the MAGE-3/6 expression, we could identify 31 (47%) of the 65 UADT tumors, which appeared to express MAGE-3/6 at levels that correlated with efficient CTL recognition. To confirm that the level of MAGE-3 expression was responsible for CTL recognition, 2 MAGE-3/6 mRNA(high) SCCHN cell lines, PCI-13 and PCI-30, were subjected to MAGE-3/6-specific knockdown. RNAi-transfected cells showed that MAGE expression and MAGE-CTL recognition were significantly reduced. Furthermore, treatment of cells expressing low MAGE-3/6 mRNA with a demethylating agent, 5-aza-2'-deoxycytidine (DAC), increased the expression of MAGE-3/6 and CTL recognition. Thus, using QRT-PCR UADT cancers frequently express MAGE-3/6 at levels sufficient for CTL recognition, supporting the use of a QRT-PCR-based assay for the selection of candidates likely to respond to MAGE-3/6 immunotherapy. Demethylating agents could increase the number of patients amenable for targeting epigenetically modified tumor antigens in vaccine trials.

A parathyroid-hormone-related-protein (PTH-rP)-specific cytotoxic T cell response induced by in vitro stimulation of tumour-infiltrating lymphocytes derived from prostate cancer metastases, with epitope peptide-loaded autologous dendritic cells and low-dose IL-2

PubMed Central

Correale, P; Micheli, L; Vecchio, M T Del; Sabatino, M; Petrioli, R; Pozzessere, D; Marsili, S; Giorgi, G; Lozzi, L; Neri, P; Francini, G

2001-01-01

Bone metastases are one of the most common events in patients with prostate carcinoma. PTH-rP, a protein produced by prostate carcinoma and other epithelial cancers, is a key agent for the development of bone metastases. A PTH-rP-derived peptide, designated PTR-4 was identified, which is capable to bind HLA-A2.1 molecules and to generate PTH-rP-specific cytotoxic T cell (CTL) lines from healthy HLA-A2.1+ individual peripheral-blood-mononuclear-cells (PBMC). In this model, we investigated the in vitro possibility of generating an efficient PTH-rP specific CTL response by cyclical stimulations with IL-2 and PTR-4 peptide-pulsed autologous dendritic cells (DC), of HLA-A2.1+ tumour infiltrating lymphocytes (TIL) derived from a patient with metastatic prostate carcinoma. A T cell line generated in this way (called TM-PTR-4) had a CD3+, CD5+, CD4−, CD8+, CD45Ro+, CD56− immunophenotype and a HLA-A2.1 restricted cytotoxic activity to PTR-4-peptide pulsed CIR-A2 (HLA-A2.1+) target cells, PTH-rP+/HLA-A2.1+ CIR-A2 transfected with PTH-rP gene, prostate carcinoma LNCaP cells, and autologous metastatic prostate cancer cells (M-CaP). These lymphocytes were not cytotoxic to HLA-A2.1+ targets not producing PTH-rP, such as peptide-unpulsed CIR-A2 and colon carcinoma SW-1463, cell lines. Our results provide evidence that PTR-4 peptide-pulsed autologous DC may break the tolerance of human TIL against the autologous tumour by inducing a PTH-rP-specific CTL immune reaction. In conclusion PTR-4 peptide-pulsed autologous DC may be a promising approach for vaccine-therapy and antigen-specific CTL adoptive immunotherapy of hormone-resistant prostrate cancer. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11742494

Generals die in friendly fire, or modeling immune response to HIV

NASA Astrophysics Data System (ADS)

Rouzine, Igor M.; Murali-Krishna, Kaja; Ahmed, Rafi

2005-12-01

We develop a kinetic model for CD8 T lymphocytes (CTL) whose purpose is to kill cells infected with viruses and intracellular parasites. Using a set of first-order nonlinear differential equations, the model predicts how numbers of different cell types involved in CTL response depend on time. The model postulates that CTL response requires continuous presence of professional antigen-presenting cells (APC) comprised of macrophages and dendritic cells. It assumes that any virus present in excess of a threshold level activates APC that, in turn, activate CTL that expand in number and become armed "effector" cells. In the end, APC are deactivated after virus is cleared. The lack of signal from APC causes effector cells to differentiate, by default, into "transitory cells" that either die, or, in a small part, become long-lived memory cells. Viruses capable of infecting APC will cause premature retirement of effector CTL. If transitory cells encounter virus, which takes place after the premature depletion, CTL become anergic (unresponsive to external stimuli). The model is designed to fit recent experiments on primary CTL response to simian immunodeficiency virus closely related to HIV and lymphocytic choriomeningitis virus. The two viruses are known to infect APC and make them targets for CTL they are supposed to control. Both viruses cause premature depletion and anergy of CTL and persist in the host for life.

Potent CD4+ T-cell epitope P30 enhances HER2/neu-engineered dendritic cell-induced immunity against Tg1-1 breast cancer in transgenic FVBneuN mice by enhanced CD4+ T-cell-stimulated CTL responses.

PubMed

Xie, Y; Chen, Y; Ahmed, K A; Li, W; Ahmed, S; Sami, A; Chibbar, R; Tang, X; Tao, M; Xu, J; Xiang, J

2013-10-01

One of the major obstacles in human epidermal growth factor receptor (HER)-2/neu-specific trastuzumab immunotherapy of HER2/neu-positive breast cancer is the development of trastuzumab resistance, warranting the search for other therapeutic strategies. Although dendritic cell (DC) vaccines have been extensively applied in clinical trials for cancer treatment, the vaccination efficacy is still limited, mostly because DC vaccines are not sufficient to break tumor-associated antigen-specific self-immune tolerance in cancer patients. P30 (FNNFTVSFWLRVPKVSASHLE) derived from tetanus toxin is a universally potent CD4(+) T helper epitope capable of enhancing CD8(+) cytotoxic T-lymphocyte (CTL) responses. In this study, we constructed two recombinant adenoviral vectors (AdVs), AdVOVA-P30 and AdVHER2/neu-P30, expressing ovalbumin (OVA)-P30 and HER2/neu-P30. In order to enhance DC vaccine efficacy, we transfected mouse bone marrow (BM)-derived DCs with AdVOVA-P30 and AdVHER2/neu-P30 to generate engineered DCOVA-P30 and DCHER2/neu-P30 vaccines, respectively. We, then, compared CD4(+) and CD8(+) T-cell responses and antitumor immunity derived from DCOVA-P30 and DCHER2/neu-P30 vaccination in wild-type C57BL/6 and transgenic FVBneuN mice, respectively. We demonstrate that engineered DCOVA-P30 vaccine stimulates more efficient CD4(+) and CD8(+) T-cell responses than DCOVA in C57BL/6 mice. Interestingly, the increased DCOVA-P30-induced CTL responses are mainly contributed by enhanced CD4(+) T-cell-stimulated CTL proliferation. We show that DCOVA-P30 vaccine also stimulates more efficient therapeutic immunity against OVA-expressing BL6-10OVA melanoma than DCOVA in C57BL/6 mice. In addition, we demonstrate that DCHER2/neu-P30 vaccine stimulates more efficient CD4(+) and CD8(+) T-cell responses and protective immunity against HER2/neu-expressing Tg1-1 breast cancer than DCHER2/neu in transgenic FVBneuN mice with HER2/neu-specific self-immune tolerance. Therefore, the engineered DCHER2/neu-P30 vaccine may provide a new immunotherapy alternative for women with HER2/neu(+) breast cancer, especially for trastuzumab-resistant HER2/neu(+) breast cancer patients.

Identification of a new C-type lectin, TES-70, secreted by infective larvae of Toxocara canis, which binds to host ligands.

PubMed

Loukas, A; Doedens, A; Hintz, M; Maizels, R M

2000-11-01

Infective larvae of the dog roundworm Toxocara canis survive in the tissues of their hosts for extended periods in a state of developmental arrest, successfully evading immune destruction. This survival strategy is thought to be mediated by T. canis excretory/secretory (TES) products which downregulate or divert the immune response. We purified one of the major TES products, TES-70 and gained amino acid sequence from 4 tryptic peptides. These peptides were matched to a predicted protein from a cDNA that was isolated by expression screening a T. canis cDNA library with mouse anti-TES serum. The predicted protein (Tc-CTL-4) is similar to, but larger than, Tc-CTL-1, a 32-kDa C-type lectin secreted by T. canis larvae. Tc-CTL-4 has a signal peptide, 2 Cys-rich domains and a C-terminal calcium-dependent C-type lectin domain that shares sequence similarity with host immune cell receptors such as macrophage mannose receptor and CD23. The lectin domain was expressed in bacteria and antiserum to the purified recombinant protein was used to confirm that Tc-ctl-4 did encode the native TES-70 glycoprotein. TES-70 selectively bound to ligands on the surface of Madin-Darby Canine Kidney cells in vitro in a calcium-dependent manner, inhibitable by mammalian serum, indicating that a host glycan is the native ligand for this new parasite lectin.

Novel Adjuvant Based on the Pore-Forming Protein Sticholysin II Encapsulated into Liposomes Effectively Enhances the Antigen-Specific CTL-Mediated Immune Response

PubMed Central

Laborde, Rady J.; Sanchez-Ferras, Oraly; Luzardo, María C.; Cruz-Leal, Yoelys; Fernández, Audry; Mesa, Circe; Oliver, Liliana; Canet, Liem; Abreu-Butin, Liane; Nogueira, Catarina V.; Tejuca, Mayra; Pazos, Fabiola; Álvarez, Carlos; Alonso, María E.; Longo-Maugéri, Ieda M.; Starnbach, Michael N.; Higgins, Darren E.; Fernández, Luis E.; Lanio, María E.

2017-01-01

Vaccine strategies to enhance CD8+ CTL responses remain a current challenge because they should overcome the plasmatic and endosomal membranes for favoring exogenous Ag access to the cytosol of APCs. As a way to avoid this hurdle, sticholysin (St) II, a pore-forming protein from the Caribbean Sea anemone Stichodactyla helianthus, was encapsulated with OVA into liposomes (Lp/OVA/StII) to assess their efficacy to induce a CTL response. OVA-specific CD8+ T cells transferred to mice immunized with Lp/OVA/StII experienced a greater expansion than when the recipients were injected with the vesicles without St, mostly exhibiting a memory phenotype. Consequently, Lp/OVA/StII induced a more potent effector function, as shown by CTLs, in vivo assays. Furthermore, treatment of E.G7-OVA tumor-bearing mice with Lp/OVA/StII significantly reduced tumor growth being more noticeable in the preventive assay. The contribution of CD4+ and CD8+ T cells to CTL and antitumor activity, respectively, was elucidated. Interestingly, the irreversibly inactive variant of the StI mutant StI W111C, encapsulated with OVA into Lp, elicited a similar OVA-specific CTL response to that observed with Lp/OVA/StII or vesicles encapsulating recombinant StI or the reversibly inactive StI W111C dimer. These findings suggest the relative independence between StII pore-forming activity and its immuno-modulatory properties. In addition, StII-induced in vitro maturation of dendritic cells might be supporting these properties. These results are the first evidence, to our knowledge, that StII, a pore-forming protein from a marine eukaryotic organism, encapsulated into Lp functions as an adjuvant to induce a robust specific CTL response. PMID:28258198

Immune-tolerant elastin-like polypeptides (iTEPs) and their application as CTL vaccine carriers.

PubMed

Cho, S; Dong, S; Parent, K N; Chen, M

2016-01-01

Cytotoxic T lymphocyte (CTL) vaccine carriers are known to enhance the efficacy of vaccines, but a search for more effective carriers is warranted. Elastin-like polypeptides (ELPs) have been examined for many medical applications but not as CTL vaccine carriers. We aimed to create immune tolerant ELPs using a new polypeptide engineering practice and create CTL vaccine carriers using the ELPs. Four sets of novel ELPs, termed immune-tolerant elastin-like polypeptide (iTEP) were generated according to the principles dictating humoral immunogenicity of polypeptides and phase transition property of ELPs. The iTEPs were non-immunogenic in mice. Their phase transition feature was confirmed through a turbidity assay. An iTEP nanoparticle (NP) was assembled from an amphiphilic iTEP copolymer plus a CTL peptide vaccine, SIINFEKL. The NP facilitated the presentation of the vaccine by dendritic cells (DCs) and enhanced vaccine-induced CTL responses. A new ELP design and development practice was established. The non-canonical motif and the immune tolerant nature of the iTEPs broaden our insights about ELPs. ELPs, for the first time, were successfully used as carriers for CTL vaccines. It is feasible to concurrently engineer both immune-tolerant and functional peptide materials. ELPs are a promising type of CTL vaccine carriers.

Prior Population Immunity Reduces the Expected Impact of CTL-Inducing Vaccines for Pandemic Influenza Control

PubMed Central

Bolton, Kirsty J.; McCaw, James M.; Brown, Lorena; Jackson, David; Kedzierska, Katherine; McVernon, Jodie

2015-01-01

Vaccines that trigger an influenza-specific cytotoxic T cell (CTL) response may aid pandemic control by limiting the transmission of novel influenza A viruses (IAV). We consider interventions with hypothetical CTL-inducing vaccines in a range of epidemiologically plausible pandemic scenarios. We estimate the achievable reduction in the attack rate, and, by adopting a model linking epidemic progression to the emergence of IAV variants, the opportunity for antigenic drift. We demonstrate that CTL-inducing vaccines have limited utility for modifying population-level outcomes if influenza-specific T cells found widely in adults already suppress transmission and prove difficult to enhance. Administration of CTL-inducing vaccines that are efficacious in "influenza-experienced" and "influenza-naive" hosts can likely slow transmission sufficiently to mitigate a moderate IAV pandemic. However if neutralising cross-reactive antibody to an emerging IAV are common in influenza-experienced hosts, as for the swine-variant H3N2v, boosting CTL immunity may be ineffective at reducing population spread, indicating that CTL-inducing vaccines are best used against novel subtypes such as H7N9. Unless vaccines cannot readily suppress transmission from infected hosts with naive T cell pools, targeting influenza-naive hosts is preferable. Such strategies are of enhanced benefit if naive hosts are typically intensively mixing children and when a subset of experienced hosts have pre-existing neutralising cross-reactive antibody. We show that CTL-inducing vaccination campaigns may have greater power to suppress antigenic drift than previously suggested, and targeting adults may be the optimal strategy to achieve this when the vaccination campaign does not have the power to curtail the attack rate. Our results highlight the need to design interventions based on pre-existing cellular immunity and knowledge of the host determinants of vaccine efficacy, and provide a framework for assessing the performance requirements of high-impact CTL-inducing vaccines. PMID:25811654

Prior population immunity reduces the expected impact of CTL-inducing vaccines for pandemic influenza control.

PubMed

Bolton, Kirsty J; McCaw, James M; Brown, Lorena; Jackson, David; Kedzierska, Katherine; McVernon, Jodie

2015-01-01

Vaccines that trigger an influenza-specific cytotoxic T cell (CTL) response may aid pandemic control by limiting the transmission of novel influenza A viruses (IAV). We consider interventions with hypothetical CTL-inducing vaccines in a range of epidemiologically plausible pandemic scenarios. We estimate the achievable reduction in the attack rate, and, by adopting a model linking epidemic progression to the emergence of IAV variants, the opportunity for antigenic drift. We demonstrate that CTL-inducing vaccines have limited utility for modifying population-level outcomes if influenza-specific T cells found widely in adults already suppress transmission and prove difficult to enhance. Administration of CTL-inducing vaccines that are efficacious in "influenza-experienced" and "influenza-naive" hosts can likely slow transmission sufficiently to mitigate a moderate IAV pandemic. However if neutralising cross-reactive antibody to an emerging IAV are common in influenza-experienced hosts, as for the swine-variant H3N2v, boosting CTL immunity may be ineffective at reducing population spread, indicating that CTL-inducing vaccines are best used against novel subtypes such as H7N9. Unless vaccines cannot readily suppress transmission from infected hosts with naive T cell pools, targeting influenza-naive hosts is preferable. Such strategies are of enhanced benefit if naive hosts are typically intensively mixing children and when a subset of experienced hosts have pre-existing neutralising cross-reactive antibody. We show that CTL-inducing vaccination campaigns may have greater power to suppress antigenic drift than previously suggested, and targeting adults may be the optimal strategy to achieve this when the vaccination campaign does not have the power to curtail the attack rate. Our results highlight the need to design interventions based on pre-existing cellular immunity and knowledge of the host determinants of vaccine efficacy, and provide a framework for assessing the performance requirements of high-impact CTL-inducing vaccines.

Preexisting CD8+ T-cell immunity to the H7N9 influenza A virus varies across ethnicities

PubMed Central

Quiñones-Parra, Sergio; Grant, Emma; Loh, Liyen; Nguyen, Thi H. O.; Campbell, Kristy-Anne; Tong, Steven Y. C.; Miller, Adrian; Doherty, Peter C.; Vijaykrishna, Dhanasekaran; Rossjohn, Jamie; Gras, Stephanie; Kedzierska, Katherine

2014-01-01

The absence of preexisting neutralizing antibodies specific for the novel A (H7N9) influenza virus indicates a lack of prior human exposure. As influenza A virus–specific CD8+ T lymphocytes (CTLs) can be broadly cross-reactive, we tested whether immunogenic peptides derived from H7N9 might be recognized by memory CTLs established following infection with other influenza strains. Probing across multiple ethnicities, we identified 32 conserved epitopes derived from the nucleoprotein (NP) and matrix-1 (M1) proteins. These NP and M1 peptides are presented by HLAs prevalent in 16–57% of individuals. Remarkably, some HLA alleles (A*0201, A*0301, B*5701, B*1801, and B*0801) elicit robust CTL responses against any human influenza A virus, including H7N9, whereas ethnicities where HLA-A*0101, A*6801, B*1501, and A*2402 are prominent, show limited CTL response profiles. By this criterion, some groups, especially the Alaskan and Australian Indigenous peoples, would be particularly vulnerable to H7N9 infection. This dissection of CTL-mediated immunity to H7N9 thus suggests strategies for both vaccine delivery and development. PMID:24395804

The Addition of the BTK Inhibitor Ibrutinib to Anti-CD19 Chimeric Antigen Receptor T Cells (CART19) Improves Responses against Mantle Cell Lymphoma.

PubMed

Ruella, Marco; Kenderian, Saad S; Shestova, Olga; Fraietta, Joseph A; Qayyum, Sohail; Zhang, Qian; Maus, Marcela V; Liu, Xiaobin; Nunez-Cruz, Selene; Klichinsky, Michael; Kawalekar, Omkar U; Milone, Michael; Lacey, Simon F; Mato, Anthony; Schuster, Stephen J; Kalos, Michael; June, Carl H; Gill, Saar; Wasik, Mariusz A

2016-06-01

Responses to therapy with chimeric antigen receptor T cells recognizing CD19 (CART19, CTL019) may vary by histology. Mantle cell lymphoma (MCL) represents a B-cell malignancy that remains incurable despite novel therapies such as the BTK inhibitor ibrutinib, and where data from CTL019 therapy are scant. Using MCL as a model, we sought to build upon the outcomes from CTL019 and from ibrutinib therapy by combining these in a rational manner. MCL cell lines and primary MCL samples were combined with autologous or normal donor-derived anti-CD19 CAR T cells along with ibrutinib. The effect of the combination was studied in vitro and in mouse xenograft models. MCL cells strongly activated multiple CTL019 effector functions, and MCL killing by CTL019 was further enhanced in the presence of ibrutinib. In a xenograft MCL model, we showed superior disease control in the CTL019- as compared with ibrutinib-treated mice (median survival not reached vs. 95 days, P < 0.005) but most mice receiving CTL019 monotherapy eventually relapsed. Therefore, we added ibrutinib to CTL019 and showed that 80% to 100% of mice in the CTL019 + ibrutinib arm and 0% to 20% of mice in the CTL019 arm, respectively, remained in long-term remission (P < 0.05). Combining CTL019 with ibrutinib represents a rational way to incorporate two of the most recent therapies in MCL. Our findings pave the way to a two-pronged therapeutic strategy in patients with MCL and other types of B-cell lymphoma. Clin Cancer Res; 22(11); 2684-96. ©2016 AACR. ©2016 American Association for Cancer Research.

Chitinase-like1/pom-pom1 and its homolog CTL2 are glucan-interacting proteins important for cellulose biosynthesis in Arabidopsis.

PubMed

Sánchez-Rodríguez, Clara; Bauer, Stefan; Hématy, Kian; Saxe, Friederike; Ibáñez, Ana Belén; Vodermaier, Vera; Konlechner, Cornelia; Sampathkumar, Arun; Rüggeberg, Markus; Aichinger, Ernst; Neumetzler, Lutz; Burgert, Ingo; Somerville, Chris; Hauser, Marie-Theres; Persson, Staffan

2012-02-01

Plant cells are encased by a cellulose-containing wall that is essential for plant morphogenesis. Cellulose consists of β-1,4-linked glucan chains assembled into paracrystalline microfibrils that are synthesized by plasma membrane-located cellulose synthase (CESA) complexes. Associations with hemicelluloses are important for microfibril spacing and for maintaining cell wall tensile strength. Several components associated with cellulose synthesis have been identified; however, the biological functions for many of them remain elusive. We show that the chitinase-like (CTL) proteins, CTL1/POM1 and CTL2, are functionally equivalent, affect cellulose biosynthesis, and are likely to play a key role in establishing interactions between cellulose microfibrils and hemicelluloses. CTL1/POM1 coincided with CESAs in the endomembrane system and was secreted to the apoplast. The movement of CESAs was compromised in ctl1/pom1 mutant seedlings, and the cellulose content and xyloglucan structures were altered. X-ray analysis revealed reduced crystalline cellulose content in ctl1 ctl2 double mutants, suggesting that the CTLs cooperatively affect assembly of the glucan chains, which may affect interactions between hemicelluloses and cellulose. Consistent with this hypothesis, both CTLs bound glucan-based polymers in vitro. We propose that the apoplastic CTLs regulate cellulose assembly and interaction with hemicelluloses via binding to emerging cellulose microfibrils.

CHITINASE-LIKE1/POM-POM1 and Its Homolog CTL2 Are Glucan-Interacting Proteins Important for Cellulose Biosynthesis in Arabidopsis[W][OA

PubMed Central

Sánchez-Rodríguez, Clara; Bauer, Stefan; Hématy, Kian; Saxe, Friederike; Ibáñez, Ana Belén; Vodermaier, Vera; Konlechner, Cornelia; Sampathkumar, Arun; Rüggeberg, Markus; Aichinger, Ernst; Neumetzler, Lutz; Burgert, Ingo; Somerville, Chris; Hauser, Marie-Theres; Persson, Staffan

2012-01-01

Plant cells are encased by a cellulose-containing wall that is essential for plant morphogenesis. Cellulose consists of β-1,4-linked glucan chains assembled into paracrystalline microfibrils that are synthesized by plasma membrane–located cellulose synthase (CESA) complexes. Associations with hemicelluloses are important for microfibril spacing and for maintaining cell wall tensile strength. Several components associated with cellulose synthesis have been identified; however, the biological functions for many of them remain elusive. We show that the chitinase-like (CTL) proteins, CTL1/POM1 and CTL2, are functionally equivalent, affect cellulose biosynthesis, and are likely to play a key role in establishing interactions between cellulose microfibrils and hemicelluloses. CTL1/POM1 coincided with CESAs in the endomembrane system and was secreted to the apoplast. The movement of CESAs was compromised in ctl1/pom1 mutant seedlings, and the cellulose content and xyloglucan structures were altered. X-ray analysis revealed reduced crystalline cellulose content in ctl1 ctl2 double mutants, suggesting that the CTLs cooperatively affect assembly of the glucan chains, which may affect interactions between hemicelluloses and cellulose. Consistent with this hypothesis, both CTLs bound glucan-based polymers in vitro. We propose that the apoplastic CTLs regulate cellulose assembly and interaction with hemicelluloses via binding to emerging cellulose microfibrils. PMID:22327741

Doxycycline induces bone repair and changes in Wnt signalling

PubMed Central

Gomes, Kátia do Nascimento; Alves, Ana Paula Negreiros Nunes; Dutra, Paula Góes Pinheiro; Viana, Glauce Socorro de Barros

2017-01-01

Doxycycline (DOX) exhibits anti-inflammatory and MMP inhibitory properties. The objectives of this study were to evaluate the effects of DOX on alveolar bone repair. Controls (CTL) and DOX-treated (10 and 25 mg·kg−1) molars were extracted, and rats were killed 7 or 14 days later. The maxillae were processed and subjected to histological and immunohistochemical assays. Hematoxylin-eosin staining (7th day) revealed inflammation in the CTL group that was partly reversed after DOX treatment. On the 14th day, the CTL group exhibited bone neoformation, conjunctive tissue, re-epithelization and the absence of inflammatory infiltrate. DOX-treated groups exhibited complete re-epithelization, tissue remodelling and almost no inflammation. Picrosirius red staining in the DOX10 group (7th and 14th days) revealed an increased percentage of type I and III collagen fibres compared with the CTL and DOX25 groups. The DOX10 and DOX25 groups exhibited increases in osteoblasts on the 7th and 14th days. However, there were fewer osteoclasts in the DOX10 and DOX25 groups on the 7th and 14th days. Wnt-10b-immunopositive cells increased by 130% and 150% on the 7th and 14th days, respectively, in DOX-treated groups compared with the CTL group. On the 7th day, Dickkopf (Dkk)-1 immunostaining was decreased by 63% and 46% in the DOX10 and DOX25 groups, respectively. On the 14th day, 69% and 42% decreases in immunopositive cells were observed in the DOX10 and DOX25 groups, respectively, compared with the CTL group. By increasing osteoblasts, decreasing osteoclasts, activating Wnt 10b and neutralising Dkk, DOX is a potential candidate for bone repair in periodontal diseases. PMID:28960195

Correlates of Protective Cellular Immunity Revealed by Analysis of Population-Level Immune Escape Pathways in HIV-1

PubMed Central

Brumme, Chanson J.; Martin, Eric; Listgarten, Jennifer; Brockman, Mark A.; Le, Anh Q.; Chui, Celia K. S.; Cotton, Laura A.; Knapp, David J. H. F.; Riddler, Sharon A.; Haubrich, Richard; Nelson, George; Pfeifer, Nico; DeZiel, Charles E.; Heckerman, David; Apps, Richard; Carrington, Mary; Mallal, Simon; Harrigan, P. Richard; John, Mina

2012-01-01

HLA class I-associated polymorphisms identified at the population level mark viral sites under immune pressure by individual HLA alleles. As such, analysis of their distribution, frequency, location, statistical strength, sequence conservation, and other properties offers a unique perspective from which to identify correlates of protective cellular immunity. We analyzed HLA-associated HIV-1 subtype B polymorphisms in 1,888 treatment-naïve, chronically infected individuals using phylogenetically informed methods and identified characteristics of HLA-associated immune pressures that differentiate protective and nonprotective alleles. Over 2,100 HLA-associated HIV-1 polymorphisms were identified, approximately one-third of which occurred inside or within 3 residues of an optimally defined cytotoxic T-lymphocyte (CTL) epitope. Differential CTL escape patterns between closely related HLA alleles were common and increased with greater evolutionary distance between allele group members. Among 9-mer epitopes, mutations at HLA-specific anchor residues represented the most frequently detected escape type: these occurred nearly 2-fold more frequently than expected by chance and were computationally predicted to reduce peptide-HLA binding nearly 10-fold on average. Characteristics associated with protective HLA alleles (defined using hazard ratios for progression to AIDS from natural history cohorts) included the potential to mount broad immune selection pressures across all HIV-1 proteins except Nef, the tendency to drive multisite and/or anchor residue escape mutations within known CTL epitopes, and the ability to strongly select mutations in conserved regions within HIV's structural and functional proteins. Thus, the factors defining protective cellular immune responses may be more complex than simply targeting conserved viral regions. The results provide new information to guide vaccine design and immunogenicity studies. PMID:23055555

Dendritic cell-tumor coculturing vaccine can induce antitumor immunity through both NK and CTL interaction.

PubMed

Kim, K D; Choi, S C; Kim, A; Choe, Y K; Choe, I S; Lim, J S

2001-11-01

Immunization of dendritic cells (DC) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL) that are responsible for protection and regression. We show here that immunization with bone marrow-derived DC cocultured with tumor cells can induce a protective immunity against challenges to viable tumor cells. In this study, we further investigated the mechanism by which the antitumor activity was induced. Immunization of mice with DC cocultured with murine colon carcinoma. CT-26 cells, augmented CTL activity against the tumor cells. Concomitantly, an increase in natural killer (NK) cell activity was also detected in the same mice. When DC were fixed with paraformaldehyde prior to coculturing with tumor cells, most of the CTL and NK cell activity diminished, indicating that DC are involved in the process of presenting the tumor antigen(s) to CTL. NK cell depletion in vivo produced markedly low tumor-specific CTL activity responsible for tumor prevention. In addition, RT-PCR analysis confirmed the high expression of INF-gamma mRNA in splenocytes after vaccination with DC cocultured with tumors, but low expression in splenocytes from NK-depleted mice. Most importantly, the tumor protective effect rendered to DC by the coculturing with CT-26 cells was not observed in NK-depleted mice, which suggests that DC can induce an antitumor immune response by enhancing NK cell-dependent CTL activation. Collectively, our results indicate that NK cells are required during the priming of cytotoxic T-cell response by DC-based tumor vaccine and seem to delineate a mechanism by which DC vaccine can provide the desired immunity.

In vitro generation of helper T cells and suppressor T cells that regulate the cytolytic T lymphocyte response to trinitrophenyl-modified syngeneic cells.

PubMed

Gualde, N; Weinberger, O; Ratnofsky, S; Benacerraf, B; Burakoff, S J

1982-04-01

Helper T cells and suppressor T cells have been generated in vitro that regulate the cytolytic T lymphocyte (CTL) response to trinitrophenyl (TNP)-modified syngeneic cells. B6D2F1 helper cells generated to TNP-modified parental (P1) cells augment the CTL response to those P1-TNP-modified antigens but not to P2-TNP-modified antigens. The generation of these helper T cells requires the presence of splenic adherent cells and these helper T cells are radioresistant. A soluble factor can be obtained from the helper T cell cultures that can also augment the CTL response. The suppressor T cells generated in culture do not demonstrate the specificity observed with the helper T cells; however, they are antigen-dependent in their induction. Whether helper or suppressor activity is obtained depends upon the length of time cells are cultured in vitro.

In vitro generation of helper T cells and suppressor T cells that regulate the cytolytic T lymphocyte response to trinitrophenyl-modified syngeneic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gualde, N.; Weinberger, O.; Ratnofsky, S.

1982-04-01

Helper T cells and suppressor T cells have been generated in vitro that regulate the cytolytic T lymphocyte (CTL) response to trinitrophenyl (TNP)-modified syngeneic cells. B6D2F1 helper cells generated to TNP-modified parental (P1) cells augment the CTL response to those P1-TNP-modified antigens but not to P2-TNP-modified antigens. The generation of these helper T cells requires the presence of splenic adherent cells and these helper T cells are radioresistant. A soluble factor can be obtained from the helper T cell cultures that can also augment the CTL response. The suppressor T cells generated in culture do not demonstrate the specificity observedmore » with the helper T cells; however, they are antigen-dependent in their induction. Whether helper or suppressor activity is obtained depends upon the length of time cells are cultured in vitro.« less

Identification of a novel HLA-A*02:01-restricted cytotoxic T lymphocyte epitope derived from the EML4-ALK fusion gene

PubMed Central

YOSHIMURA, MAYUKO; TADA, YOSHITAKA; OFUZI, KAZUYA; YAMAMOTO, MASAKAZU; NAKATSURA, TETSUYA

2014-01-01

Cancer immunotherapy is a promising new approach to cancer treatment. It has been demonstrated that a high number of tumor-specific cytotoxic T cells (CTLs) is associated with increased disease-specific survival in lung cancer patients. Identification of superior CTL epitopes from tumor antigens is essential for the development of immunotherapy for malignant tumors. The EML4-ALK fusion gene was recently identified in a subset of non-small cell lung cancers (NSCLCs). In this study we searched for HLA-A*02:01- and HLA-A*24:02-restricted epitopes derived from EML4-ALK by screening predicted EML4-ALK-derived candidate peptides for the induction of tumor-reactive CTLs. Nine EML4-ALK-derived peptides were selected by a computer algorithm based on a permissive HLA-A*02:01 or HLA-A*24:02 binding motif. One of the nine peptides induced peptide-specific CTLs from human peripheral blood mononuclear cells. We were able to generate a peptide-specific CTL clone. This CTL clone specifically recognized peptide-pulsed T2 cells and H2228 cells expressing HLA-A*02:01 and EML4-ALK that had been treated with IFN-γ 48 h prior to examination. CTL activity was inhibited by an anti-HLA-class I monoclonal antibody (W6/32), consistent with a class I-restricted mechanism of cytotoxicity. These results suggest that this peptide (RLSALESRV) is a novel HLA-A*02:01-restricted CTL epitope and that it may be a new target for antigen-specific immunotherapy against EML4-ALK-positive cancers. PMID:24842630

Identification of a novel HLA-A 02:01-restricted cytotoxic T lymphocyte epitope derived from the EML4-ALK fusion gene.

PubMed

Yoshimura, Mayuko; Tada, Yoshitaka; Ofuzi, Kazuya; Yamamoto, Masakazu; Nakatsura, Tetsuya

2014-07-01

Cancer immunotherapy is a promising new approach to cancer treatment. It has been demonstrated that a high number of tumor-specific cytotoxic T cells (CTLs) is associated with increased disease-specific survival in lung cancer patients. Identification of superior CTL epitopes from tumor antigens is essential for the development of immunotherapy for malignant tumors. The EML4-ALK fusion gene was recently identified in a subset of non-small cell lung cancers (NSCLCs). In this study we searched for HLA-A 02:01- and HLA-A 24:02‑restricted epitopes derived from EML4-ALK by screening predicted EML4-ALK‑derived candidate peptides for the induction of tumor‑reactive CTLs. Nine EML4-ALK‑derived peptides were selected by a computer algorithm based on a permissive HLA-A 02:01 or HLA-A 24:02 binding motif. One of the nine peptides induced peptide-specific CTLs from human peripheral blood mononuclear cells. We were able to generate a peptide‑specific CTL clone. This CTL clone specifically recognized peptide‑pulsed T2 cells and H2228 cells expressing HLA-A 02:01 and EML4-ALK that had been treated with IFN-γ 48 h prior to examination. CTL activity was inhibited by an anti-HLA‑class I monoclonal antibody (W6/32), consistent with a class I-restricted mechanism of cytotoxicity. These results suggest that this peptide (RLSALESRV) is a novel HLA-A 02:01-restricted CTL epitope and that it may be a new target for antigen-specific immunotherapy against EML4‑ALK-positive cancers.

Presentation of human T cell leukemia virus type 1 (HTLV-1) Tax protein by dendritic cells: the underlying mechanism of HTLV-1-associated neuroinflammatory disease.

PubMed

Manuel, Sharrón L; Schell, Todd D; Acheampong, Edward; Rahman, Saifur; Khan, Zafar K; Jain, Pooja

2009-11-01

HTLV-1 is the etiologic agent of a debilitating neurologic disorder, HAM/TSP. This disease features a robust immune response including the oligoclonal expansion of CD8+ CTLs specific for the viral oncoprotein Tax. The key pathogenic process resulting in the proliferation of CTLs and the presentation of Tax peptide remains uncharacterized. We have investigated the role of APCs, particularly DCs, in priming of the anti-Tax CTL response under in vitro and in vivo conditions. We investigated two routes (direct vs. indirect) of Tax presentation using live virus, infected primary CD4+/CD25+ T cells, and the CD4+ T cell line (C8166, a HTLV-1-mutated line that only expresses Tax). Our results indicated that DCs are capable of priming a pronounced Tax-specific CTL response in cell cultures consisting of naïve PBLs as well as in HLA-A*0201 transgenic mice (line HHD II). DCs were able to direct the presentation of Tax successfully through infected T cells, live virus, and cell-free Tax. These observations were comparable with those made with a known stimulant of DC maturation, a combination of CD40L and IFN-gamma. Our studies clearly establish a role for this important immune cell component in HTLV-1 immuno/neuropathogenesis and suggest that modulation of DC functions could be an important tool for therapeutic interventions.

Presentation of human T cell leukemia virus type 1 (HTLV-1) Tax protein by dendritic cells: the underlying mechanism of HTLV-1-associated neuroinflammatory disease

PubMed Central

Manuel, Sharrón L.; Schell, Todd D.; Acheampong, Edward; Rahman, Saifur; Khan, Zafar K.; Jain, Pooja

2009-01-01

HTLV-1 is the etiologic agent of a debilitating neurologic disorder, HAM/TSP. This disease features a robust immune response including the oligoclonal expansion of CD8+ CTLs specific for the viral oncoprotein Tax. The key pathogenic process resulting in the proliferation of CTLs and the presentation of Tax peptide remains uncharacterized. We have investigated the role of APCs, particularly DCs, in priming of the anti-Tax CTL response under in vitro and in vivo conditions. We investigated two routes (direct vs. indirect) of Tax presentation using live virus, infected primary CD4+/CD25+ T cells, and the CD4+ T cell line (C8166, a HTLV-1-mutated line that only expresses Tax). Our results indicated that DCs are capable of priming a pronounced Tax-specific CTL response in cell cultures consisting of naïve PBLs as well as in HLA-A*0201 transgenic mice (line HHD II). DCs were able to direct the presentation of Tax successfully through infected T cells, live virus, and cell-free Tax. These observations were comparable with those made with a known stimulant of DC maturation, a combination of CD40L and IFN-γ. Our studies clearly establish a role for this important immune cell component in HTLV-1 immuno/neuropathogenesis and suggest that modulation of DC functions could be an important tool for therapeutic interventions. PMID:19656902

NetCTLpan: pan-specific MHC class I pathway epitope predictions

PubMed Central

Larsen, Mette Voldby; Lundegaard, Claus; Nielsen, Morten

2010-01-01

Reliable predictions of immunogenic peptides are essential in rational vaccine design and can minimize the experimental effort needed to identify epitopes. In this work, we describe a pan-specific major histocompatibility complex (MHC) class I epitope predictor, NetCTLpan. The method integrates predictions of proteasomal cleavage, transporter associated with antigen processing (TAP) transport efficiency, and MHC class I binding affinity into a MHC class I pathway likelihood score and is an improved and extended version of NetCTL. The NetCTLpan method performs predictions for all MHC class I molecules with known protein sequence and allows predictions for 8-, 9-, 10-, and 11-mer peptides. In order to meet the need for a low false positive rate, the method is optimized to achieve high specificity. The method was trained and validated on large datasets of experimentally identified MHC class I ligands and cytotoxic T lymphocyte (CTL) epitopes. It has been reported that MHC molecules are differentially dependent on TAP transport and proteasomal cleavage. Here, we did not find any consistent signs of such MHC dependencies, and the NetCTLpan method is implemented with fixed weights for proteasomal cleavage and TAP transport for all MHC molecules. The predictive performance of the NetCTLpan method was shown to outperform other state-of-the-art CTL epitope prediction methods. Our results further confirm the importance of using full-type human leukocyte antigen restriction information when identifying MHC class I epitopes. Using the NetCTLpan method, the experimental effort to identify 90% of new epitopes can be reduced by 15% and 40%, respectively, when compared to the NetMHCpan and NetCTL methods. The method and benchmark datasets are available at http://www.cbs.dtu.dk/services/NetCTLpan/. Electronic supplementary material The online version of this article (doi:10.1007/s00251-010-0441-4) contains supplementary material, which is available to authorized users. PMID:20379710

P53 Immune Responses in Breast Cancer Patients: Assessment of CTL Recognizing the HLA-A2.1 Restricted, Wild-type Sequence p53 (264-272) Epitope; Frequencies of Tetramer+ T Cells Specific for the Wild-Type Sequence P53 (264-272) Peptide in the Circulation of Patients with Head and Neck Cancer; The Ability of Variant Peptides to Reverse the Nonresponsiveness of T Lymphocytes to the Wild-Type Sequence P53 (264-272) Epitope

DTIC Science & Technology

2002-10-01

This document contains three papers focusing on the analysis of anti-p53 cellular immune responses of breast, head, neck, and oral cancer patients...variants were generated by amino acid exchanges at positions 6 (6T) and 7 (7W) of the peptide. The 7W variant peptide has potential for immunotherapy of nonresponsive oral cancer patients.

Infection of mice with a human influenza A/H3N2 virus induces protective immunity against lethal infection with influenza A/H5N1 virus.

PubMed

Kreijtz, J H C M; Bodewes, R; van den Brand, J M A; de Mutsert, G; Baas, C; van Amerongen, G; Fouchier, R A M; Osterhaus, A D M E; Rimmelzwaan, G F

2009-08-06

The transmission of highly pathogenic avian influenza (HPAI) A viruses of the H5N1 subtype from poultry to man and the high case fatality rate fuels the fear for a pandemic outbreak caused by these viruses. However, prior infections with seasonal influenza A/H1N1 and A/H3N2 viruses induce heterosubtypic immunity that could afford a certain degree of protection against infection with the HPAI A/H5N1 viruses, which are distantly related to the human influenza A viruses. To assess the protective efficacy of such heterosubtypic immunity mice were infected with human influenza virus A/Hong Kong/2/68 (H3N2) 4 weeks prior to a lethal infection with HPAI virus A/Indonesia/5/05 (H5N1). Prior infection with influenza virus A/Hong Kong/2/68 reduced clinical signs, body weight loss, mortality and virus replication in the lungs as compared to naive mice infected with HPAI virus A/Indonesia/5/05. Priming by infection with respiratory syncytial virus, a non-related virus did not have a beneficial effect on the outcome of A/H5N1 infections, indicating that adaptive immune responses were responsible for the protective effect. In mice primed by infection with influenza A/H3N2 virus cytotoxic T lymphocytes (CTL) specific for NP(366-374) epitope ASNENMDAM and PA(224-232) SCLENFRAYV were observed. A small proportion of these CTL was cross-reactive with the peptide variant derived from the influenza A/H5N1 virus (ASNENMEVM and SSLENFRAYV respectively) and upon challenge infection with the influenza A/H5N1 virus cross-reactive CTL were selectively expanded. These CTL, in addition to those directed to conserved epitopes, shared by the influenza A/H3N2 and A/H5N1 viruses, most likely contributed to accelerated clearance of the influenza A/H5N1 virus infection. Although also other arms of the adaptive immune response may contribute to heterosubtypic immunity, the induction of virus-specific CTL may be an attractive target for development of broad protective vaccines. Furthermore the existence of pre-existing heterosubtypic immunity may dampen the impact a future influenza pandemic may have.

Clinical outcomes of a novel therapeutic vaccine with Tax peptide-pulsed dendritic cells for adult T cell leukaemia/lymphoma in a pilot study.

PubMed

Suehiro, Youko; Hasegawa, Atsuhiko; Iino, Tadafumi; Sasada, Amane; Watanabe, Nobukazu; Matsuoka, Masao; Takamori, Ayako; Tanosaki, Ryuji; Utsunomiya, Atae; Choi, Ilseung; Fukuda, Tetsuya; Miura, Osamu; Takaishi, Shigeo; Teshima, Takanori; Akashi, Koichi; Kannagi, Mari; Uike, Naokuni; Okamura, Jun

2015-05-01

Adult T cell leukaemia/lymphoma (ATL) is a human T cell leukaemia virus type-I (HTLV-I)-infected T cell malignancy with poor prognosis. We herein developed a novel therapeutic vaccine designed to augment an HTLV-I Tax-specific cytotoxic T lymphocyte (CTL) response that has been implicated in anti-ATL effects, and conducted a pilot study to investigate its safety and efficacy. Three previously treated ATL patients, classified as intermediate- to high-risk, were subcutaneously administered with the vaccine, consisting of autologous dendritic cells (DCs) pulsed with Tax peptides corresponding to the CTL epitopes. In all patients, the performance status improved after vaccination without severe adverse events, and Tax-specific CTL responses were observed with peaks at 16-20 weeks. Two patients achieved partial remission in the first 8 weeks, one of whom later achieved complete remission, maintaining their remission status without any additional chemotherapy 24 and 19 months after vaccination, respectively. The third patient, whose tumour cells lacked the ability to express Tax at biopsy, obtained stable disease in the first 8 weeks and later developed slowly progressive disease although additional therapy was not required for 14 months. The clinical outcomes of this pilot study indicate that the Tax peptide-pulsed DC vaccine is a safe and promising immunotherapy for ATL. © 2015 John Wiley & Sons Ltd.

Zinc deficiency reduces bone mineral density in the spine of young adult rats: a pilot study.

PubMed

Ryz, Natasha R; Weiler, Hope A; Taylor, Carla G

2009-01-01

The objective of this study was to investigate the effects of zinc deficiency initiated during adolescence on skeletal densitometry, serum markers of bone metabolism, femur minerals and morphometry in young adult rats. Ten-week-old male rats were fed a <1-mg Zn/kg diet (9ZD), a 5-mg Zn/kg diet (9MZD) or a 30-mg Zn/kg diet (9CTL) for up to 9 weeks. Analyses included bone mineral density, serum osteocalcin and C-terminal peptides of type I collagen, serum zinc, femur zinc, calcium and phosphorus, and femur morphometry. Bone mineral density was 14% lower in the spine of 9ZD, but was not altered in the whole body, tibia or femur, or in any of the aforementioned sites in 9MZD, compared to 9CTL. When adjusted for size, spine bone mineral apparent density was still 8% lower in 9ZD than 9CTL. Serum osteocalcin, a marker for bone formation, was approximately 33% lower in 9ZD compared to both 9MZD and 9CTL. The 9ZD and 9MZD had 57% lower femur zinc and 56-88% lower serum zinc concentrations compared to 9CTL. These findings indicate that severe zinc deficiency initiated during adolescence may have important implications for future bone health, especially with regards to bone consolidation in the spine. 2009 S. Karger AG, Basel.

TNF-induced target cell killing by CTL activated through cross-presentation.

PubMed

Wohlleber, Dirk; Kashkar, Hamid; Gärtner, Katja; Frings, Marianne K; Odenthal, Margarete; Hegenbarth, Silke; Börner, Carolin; Arnold, Bernd; Hämmerling, Günter; Nieswandt, Bernd; van Rooijen, Nico; Limmer, Andreas; Cederbrant, Karin; Heikenwalder, Mathias; Pasparakis, Manolis; Protzer, Ulrike; Dienes, Hans-Peter; Kurts, Christian; Krönke, Martin; Knolle, Percy A

2012-09-27

Viruses can escape cytotoxic T cell (CTL) immunity by avoiding presentation of viral components via endogenous MHC class I antigen presentation in infected cells. Cross-priming of viral antigens circumvents such immune escape by allowing noninfected dendritic cells to activate virus-specific CTLs, but they remain ineffective against infected cells in which immune escape is functional. Here, we show that cross-presentation of antigen released from adenovirus-infected hepatocytes by liver sinusoidal endothelial cells stimulated cross-primed effector CTLs to release tumor necrosis factor (TNF), which killed virus-infected hepatocytes through caspase activation. TNF receptor signaling specifically eliminated infected hepatocytes that showed impaired anti-apoptotic defense. Thus, CTL immune surveillance against infection relies on two similarly important but distinct effector functions that are both MHC restricted, requiring either direct antigen recognition on target cells and canonical CTL effector function or cross-presentation and a noncanonical effector function mediated by TNF. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

The impact of acute high-intensity interval exercise on biomarkers of cardiovascular health in type 2 diabetes.

PubMed

Francois, Monique E; Little, Jonathan P

2017-08-01

High-intensity interval training (HIIT) interventions improve cardiovascular health, yet the acute effects on circulating and functional biomarkers of cardiovascular function are unclear in individuals with type 2 diabetes (T2D). To explore this, we conducted two investigations to examine the acute response to HIIT in individuals with T2D. Study 1 measured blood pressure, endothelial-dependent dilation, circulating measures of endothelial activation, and troponin T, 30 min and 2 h after HIIT (7 × 1-min intervals) in T2D (n = 8) and age-matched normoglycemic controls (CTL; n = 8). Study 2 assessed circulating measures of endothelial activation and troponin T, 30 min, and 24 h after HIIT (10 × 1-min intervals) in ten previously trained T2D men. In study 1, markers of endothelial function and activation within the first 2 h after HIIT did not differ from baseline between T2D and CTL participants, except at 30 min after HIIT for glucose, which was reduced more in T2D than CTL (by -0.8 ± 1.2 mmol/L, p = 0.04), and VCAM-1, which was reduced more 30 min after HIIT in CTL compared to T2D (by -187 ± 221 ng/mL, p = 0.05). Study 2 saw no significant difference in any circulating markers of endothelial activation and troponin T, 30 min, and 24 h after HIIT in trained T2D males. Exploratory findings from these two studies suggest that acute HIIT does not substantially alter circulating and functional markers of cardio(vascular) health in individuals with T2D who are unaccustomed (study 1) and accustomed to HIIT (study 2).

Induction of Protective CTL Responses in Newborn Mice by a Murine Retrovirus

NASA Astrophysics Data System (ADS)

Sarzotti, Marcella; Robbins, Deanna S.; Hoffman, Paul M.

1996-03-01

The susceptibility of neonates to virus-induced disease is thought to reflect, in part, the immaturity of their immune systems. However, inoculation of newborn mice with low doses of Cas-Br-M murine leukemia virus induced a protective cytotoxic T lymphocyte (CTL) response. The inability of neonates to develop a CTL response to high doses of virus was not the result of immunological immaturity but correlated with the induction of a nonprotective type 2 cytokine response. Thus, the initial viral dose is critical in the development of protective immunity in newborns.

Identification of a Polymorphic Gene, BCL2A1, Encoding Two Novel Hematopoietic Lineage-specific Minor Histocompatibility Antigens

PubMed Central

Akatsuka, Yoshiki; Nishida, Tetsuya; Kondo, Eisei; Miyazaki, Mikinori; Taji, Hirohumi; Iida, Hiroatsu; Tsujimura, Kunio; Yazaki, Makoto; Naoe, Tomoki; Morishima, Yasuo; Kodera, Yoshihisa; Kuzushima, Kiyotaka; Takahashi, Toshitada

2003-01-01

We report the identification of two novel minor histocompatibility antigens (mHAgs), encoded by two separate single nucleotide polymorphisms on a single gene, BCL2A1, and restricted by human histocompatibility leukocyte antigen (HLA)-A*2402 (the most common HLA-A allele in Japanese) and B*4403, respectively. Two cytotoxic T lymphocyte (CTL) clones specific for these mHAgs were first isolated from two distinct recipients after hematopoietic cell transplantation. Both clones lyse only normal and malignant cells within the hematopoietic lineage. To localize the gene encoding the mHAgs, two-point linkage analysis was performed on the CTL lytic patterns of restricting HLA-transfected B lymphoblastoid cell lines obtained from Centre d'Etude du Polymorphisme Humain. Both CTL clones showed a completely identical lytic pattern for 4 pedigrees and the gene was localized within a 3.6-cM interval of 15q24.3–25.1 region that encodes at least 46 genes. Of those, only BCL2A1 has been reported to be expressed in hematopoietic cells and possess three nonsynonymous nucleotide changes. Minigene transfection and epitope reconstitution assays with synthetic peptides identified both HLA-A*2402– and B*4403-restricted mHAg epitopes to be encoded by distinct polymorphisms within BCL2A1. PMID:12771180

Design of an Adaptive Human-Machine System Based on Dynamical Pattern Recognition of Cognitive Task-Load.

PubMed

Zhang, Jianhua; Yin, Zhong; Wang, Rubin

2017-01-01

This paper developed a cognitive task-load (CTL) classification algorithm and allocation strategy to sustain the optimal operator CTL levels over time in safety-critical human-machine integrated systems. An adaptive human-machine system is designed based on a non-linear dynamic CTL classifier, which maps a set of electroencephalogram (EEG) and electrocardiogram (ECG) related features to a few CTL classes. The least-squares support vector machine (LSSVM) is used as dynamic pattern classifier. A series of electrophysiological and performance data acquisition experiments were performed on seven volunteer participants under a simulated process control task environment. The participant-specific dynamic LSSVM model is constructed to classify the instantaneous CTL into five classes at each time instant. The initial feature set, comprising 56 EEG and ECG related features, is reduced to a set of 12 salient features (including 11 EEG-related features) by using the locality preserving projection (LPP) technique. An overall correct classification rate of about 80% is achieved for the 5-class CTL classification problem. Then the predicted CTL is used to adaptively allocate the number of process control tasks between operator and computer-based controller. Simulation results showed that the overall performance of the human-machine system can be improved by using the adaptive automation strategy proposed.

Identification and functional analysis of choline transporter in tongue cancer: A novel molecular target for tongue cancer therapy.

PubMed

Nishiyama, Ryohta; Nagashima, Fumiaki; Iwao, Beniko; Kawai, Yuiko; Inoue, Kana; Midori, Arisa; Yamanaka, Tsuyoshi; Uchino, Hiroyuki; Inazu, Masato

2016-06-01

We examined the functional characteristics of choline uptake in human tongue carcinoma using the cell line HSC-3. Furthermore, we explored the possible correlation between the inhibition of choline uptake and apoptotic cell death. Both choline transporter-like protein 1 (CTL1) and CTL2 mRNAs and proteins were expressed, and were located in plasma membrane and mitochondria, respectively. Choline uptake was saturable and mediated by a single transport system, which is pH-dependent. Several cationic drugs inhibited cell viability and [(3)H]choline uptake. Choline uptake inhibitors and choline deficiency inhibited cell viability and increased caspase-3/7 activity. We conclude that extracellular choline is mainly transported via a CTL1 that relies on a directed H(+) gradient as a driving force. The functional inhibition of CTL1 by cationic drugs could promote apoptotic cell death. Furthermore, CTL2 may be the major site for the control of choline oxidation in mitochondria and hence for the supply of endogenous betaine and S-adenosyl methionine, which serves as a major methyl donor. Identification of this CTL1- and CTL2-mediated choline transport system provides a potential new target for tongue cancer therapy. Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Harmful Gas Recognition Exploiting a CTL Sensor Array

PubMed Central

Wang, Qihui; Xie, Lijun; Zhu, Bo; Zheng, Yao; Cao, Shihua

2013-01-01

In this paper, a novel cataluminescence (CTL)-based sensor array consisting of nine types of catalytic materials is developed for the recognition of several harmful gases, namely carbon monoxide, acetone, chloroform and toluene. First, the experimental setup is constructed by using sensing nanomaterials, a heating plate, a pneumatic pump, a gas flow meter, a digital temperature device, a camera and a BPCL Ultra Weak Chemiluminescence Analyzer. Then, unique CTL patterns for the four types of harmful gas are obtained from the sensor array. The harmful gases are successful recognized by the PCA method. The optimal conditions are also investigated. Finally, experimental results show high sensitivity, long-term stability and good linearity of the sensor array, which combined with simplicity, make our system a promising application in this field. PMID:24113681

Regulation of CTL responses to MHC-restricted class I peptide of the gp70 tumour antigen by splenic parenchymal CD4+ T cells in mice failing immunotherapy with DISC-mGM-CSF.

PubMed

Ahmad, Murrium; Rees, Robert C; McArdle, Stephanie E; Li, Geng; Mian, Shahid; Entwisle, Claire; Loudon, Peter; Ali, Selman A

2005-07-20

Direct intratumour injection of the disabled infectious single-cycle-herpes simplex virus-encoding murine granulocyte/macrophage colony-stimulating factor (DISC-HSV-mGM-CSF) into established colon carcinoma CT26 tumours induced complete tumour rejection in up to 70% of treated animals (regressors), while the remaining mice developed progressive tumours (progressors). This murine Balb/c model was used to dissect the cellular mechanisms involved in tumour regression or progression following immunotherapy. CTLs were generated by coculturing lymphocytes and parenchymal cells from the same spleens of individual regressor or progressor animals in the presence of the relevant AH-1 peptide derived from the gp70 tumour-associated antigens expressed by CT26 tumours. Tumour regression was correlated with potent CTL responses, spleen weight and cytokine (IFN-gamma) production. Conversely, progressor splenocytes exhibited weak to no CTL activity and poor IFN-gamma production, concomitant with the presence of a suppressor cell population in the progressor splenic parenchymal cell fraction. Further fractionation of this parenchymal subpopulation demonstrated that cells inhibitory to the activation of AH-1-specific CTLs, restimulated in vitro with peptide, were present in the nonadherent parenchymal fraction. In vitro depletion of progressor parenchymal CD3+/CD4+ T cells restored the CTL response of the cocultured splenocytes (regressor lymphocytes and progressor parenchymal cells) and decreased the production of IL-10, suggesting that CD3+CD4+ T lymphocytes present in the parenchymal fraction regulated the CTL response to AH-1. We examined the cellular responses associated with tumour rejection and progression, identifying regulatory pathways associated with failure to respond to immunotherapy. Copyright 2005 Wiley-Liss, Inc.

Candidate mosaic proteins for a pan-filoviral cytotoxic T-Cell lymphocyte vaccine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fenimore, Paul W; Fischer, William M; Kuiken, Carla

The extremely high fatality rates of many filovirus (FILV) strains the recurrent but rarely identified origin of human epidemics, the only partly identified viral reservoirs and the continuing non-human primate epizootics in Africa make a broadly-protective filovirus vaccine highly desirable. Cytotoxic T-cells (CTL) have been shown to be protective in mice, guinea pigs and non-human primates. In murine models the cytotoxic T-cell epitopes that are protective against Ebola virus have been mapped and in non-human primates CTL-mediated protection between viral strains (John Dye: specify) has been demonstrated using two filoviral proteins, nucleoprotein (NP) and glycoprotein (GP). These immunological results suggestmore » that the CTL avenue of immunity deserves consideration for a vaccine. The poorly-understood viral reservoirs means that it is difficult to predict what strains are likely to cause epidemics. Thus, there is a premium on developing a pan-filoviral vaccine. The genetic diversity of FILV is large, roughly the same scale as human immunodeficiency virus (HIV). This presents a serious challenge for the vaccine designer because a traditional vaccine aspiring to pan-filoviral coverage is likely to require the inclusion of many antigenic reagents. A recent method for optimizing cytotoxic T-cell lymphocyte epitope coverage with mosaic antigens was successful in improving potential CTL epitope coverage against HIV and may be useful in the context of very different viruses, such as the filoviruses discussed here. Mosaic proteins are recombinants composed of fragments of wild-type proteins joined at locations resulting in exclusively natural k-mers, 9 {le} k {le} 15, and having approximately the same length as the wild-type proteins. The use of mosaic antigens is motivated by three conjectures: (1) optimizing a mosaic protein to maximize coverage of k-mers found in a set of reference proteins will give better odds of including broadly-protective CTL epitopes in a vaccine than is possible with a wild-type protein, (2) reducing the number of low-prevalence k-mers minimizes the likelihood of undesirable immunodominance, and (3) excluding exogenous k-mers will result in mosaic proteins whose processing for presentation is close to what occurs with wild-type proteins. The first and second applications of the mosaic method were to HIV and Hepatitis C Virus (HCV). HIV is the virus with the largest number of known sequences, and consequently a plethora of information for the CTL vaccine designer to incorporate into their mosaics. Experience with HIV and HCV mosaics supports the validity of the three conjectures above. The available FILV sequences are probably closer to the minimum amount of information needed to make a meaningful mosaic vaccine candidate. There were 532 protein sequences in the National Institutes of Health GenPept database in November 2007 when our reference set was downloaded. These sequences come from both Ebola and Marburg viruses (EBOV and MARV), representing transcripts of all 7 genes. The coverage of viral diversity by the 7 genes is variable, with genes 1 (nucleoprotein, NP), 4 (glycoprotein, GP; soluble glycoprotein, sGP) and 7 (polymerase, L) giving the best coverage. Broadly-protective vaccine candidates for diverse viruses, such as HIV or Hepatitis C virus (HCV) have required pools of antigens. FILV is similar in this regard. While we have designed CTL mosaic proteins using all 7 types of filoviral proteins, only NP, GP and L proteins are reported here. If it were important to include other proteins in a mosaic CTL vaccine, additional sequences would be required to cover the space of known viral diversity.« less

The Transcription Factor T-Bet Is Regulated by MicroRNA-155 in Murine Anti-Viral CD8+ T Cells via SHIP-1.

PubMed

Hope, Jennifer L; Stairiker, Christopher J; Spantidea, Panagiota I; Gracias, Donald T; Carey, Alison J; Fike, Adam J; van Meurs, Marjan; Brouwers-Haspels, Inge; Rijsbergen, Laurine C; Fraietta, Joseph A; Mueller, Yvonne M; Klop, Rosemarieke C; Stelekati, Erietta; Wherry, E John; Erkeland, Stefan J; Katsikis, Peter D

2017-01-01

We report here that the expression of the transcription factor T-bet, which is known to be required for effector cytotoxic CD8 + T lymphocytes (CTL) generation and effector memory cell formation, is regulated in CTL by microRNA-155 (miR-155). Importantly, we show that the proliferative effect of miR-155 on CD8 + T cells is mediated by T-bet. T-bet levels in CTL were controlled in vivo by miR-155 via SH2 (Src homology 2)-containing inositol phosphatase-1 (SHIP-1), a known direct target of miR-155, and SHIP-1 directly downregulated T-bet. Our studies reveal an important and unexpected signaling axis between miR-155, T-bet, and SHIP-1 in in vivo CTL responses and suggest an important signaling module that regulates effector CTL immunity.

Structuration and acquisition of medical knowledge. Using UMLS in the conceptual graph formalism.

PubMed Central

Volot, F.; Zweigenbaum, P.; Bachimont, B.; Ben Said, M.; Bouaud, J.; Fieschi, M.; Boisvieux, J. F.

1993-01-01

The use of a taxonomy, such as the concept type lattice (CTL) of Conceptual Graphs, is a central structuring piece in a knowledge-based system. The knowledge it contains is constantly used by the system, and its structure provides a guide for the acquisition of other pieces of knowledge. We show how UMLS can be used as a knowledge resource to build a CTL and how the CTL can help the process of acquisition for other kinds of knowledge. We illustrate this method in the context of the MENELAS natural language understanding project. PMID:8130568

Antigen sensitivity of CD22-specific chimeric TCR is modulated by target epitope distance from the cell membrane.

PubMed

James, Scott E; Greenberg, Philip D; Jensen, Michael C; Lin, Yukang; Wang, Jinjuan; Till, Brian G; Raubitschek, Andrew A; Forman, Stephen J; Press, Oliver W

2008-05-15

We have targeted CD22 as a novel tumor-associated Ag for recognition by human CTL genetically modified to express chimeric TCR (cTCR) recognizing this surface molecule. CD22-specific cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR(+) CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR(+) CTL exhibited lower levels of maximum lysis and lower Ag sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of Ag engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope, but constructed as a truncated CD22 molecule to approximate the length of a TCR:peptide-MHC complex. The reduced sensitivity of CD22-specific cTCR(+) CTL for Ag-induced triggering of effector functions has potential therapeutic applications, because such cells selectively lysed B cell lymphoma lines expressing high levels of CD22, but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength, and consequently Ag sensitivity, can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate Ag density.

Differential requirements of CD4(+) T-cell signals for effector cytotoxic T-lymphocyte (CTL) priming and functional memory CTL development at higher CD8(+) T-cell precursor frequency.

PubMed

Umeshappa, Channakeshava S; Nanjundappa, Roopa H; Xie, Yufeng; Freywald, Andrew; Xu, Qingyong; Xiang, Jim

2013-04-01

Increased CD8(+) T-cell precursor frequency (PF) precludes the requirement of CD4(+) helper T (Th) cells for primary CD8(+) cytotoxic T-lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) -pulsed dendritic cells (DC(OVA)) derived from C57BL/6, CD40 knockout (CD40(-/-)) or CD40 ligand knockout (CD40L(-/-)) mice were used to immunize C57BL/6, Ia(b-/-), CD40(-/-) or CD40L(-/-) mice, whose PF was previously increased with transfer of 1 × 10(6) CD8(+) T cells derived from OVA-specific T-cell receptor (TCR) transgenic OTI, OTI(CD40(-/-)) or OTI(CD40L(-/-)) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4(+) T-cell help and Th-provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA-expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4(+) T-cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4(+) T-cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4(+) T-cell functions. © 2012 Blackwell Publishing Ltd.

Cytotoxic T cell recognition of an endogenous class I HLA peptide presented by a class II HLA molecule.

PubMed

Chen, B P; Madrigal, A; Parham, P

1990-09-01

Human leukocytes were stimulated in vitro with peptides corresponding in sequence to the highly variable helix of the alpha 1 domain of various HLA-B and -C molecules. A CD4+ CD8- cytotoxic T cell line, CTL-AV, that is specific for the HLA-B7 peptide presented by HLA-DR11.1 was obtained. The HLA-DR11.2 molecule, which only differs at three residues from HLA-DR11.1, did not present the HLA-B7 peptide to CTL-AV. Peptides from the alpha 1 domain helix of other HLA-A and HLA-B molecules, but not HLA-C molecules, competed with the HLA-B7 peptide for binding to HLA-DR11.1. A cell line (WT50) that coexpresses HLA-B7 and HLA-DR11.1 was killed by CTL-AV in the absence of any added HLA-B7 peptide. The processing and presentation of HLA-B7 in these cells appears to be through the endogenous, and not the exogenous, pathway of antigen presentation. Thus, Brefeldin A inhibits presentation and chloroquine does not. Furthermore, introduction of purified HLA-B7 molecules into HLA-DR11.1+, HLA-B7- cells by cytoplasmic loading via osmotic lysis of pinosomes, but not by simple incubation, rendered them susceptible to CTL-AV killing. These results provide an example of class II major histocompatibility complex (MHC) presentation of a constitutively synthesized self protein that uses the endogenous pathway of antigen presentation. They also emphasize the capacity for presentation of MHC peptides by MHC molecules.

Exposure to Melan-A/MART-126-35 tumor epitope specific CD8+T cells reveals immune escape by affecting the ubiquitin-proteasome system (UPS)

PubMed Central

Ebstein, Frédéric; Keller, Martin; Paschen, Annette; Walden, Peter; Seeger, Michael; Bürger, Elke; Krüger, Elke; Schadendorf, Dirk; Kloetzel, Peter-M.; Seifert, Ulrike

2016-01-01

Efficient processing of target antigens by the ubiquitin-proteasome-system (UPS) is essential for treatment of cancers by T cell therapies. However, immune escape due to altered expression of IFN-γ-inducible components of the antigen presentation machinery and consequent inefficient processing of HLA-dependent tumor epitopes can be one important reason for failure of such therapies. Here, we show that short-term co-culture of Melan-A/MART-1 tumor antigen-expressing melanoma cells with Melan-A/MART-126-35-specific cytotoxic T lymphocytes (CTL) led to resistance against CTL-induced lysis because of impaired Melan-A/MART-126-35 epitope processing. Interestingly, deregulation of p97/VCP expression, which is an IFN-γ-independent component of the UPS and part of the ER-dependent protein degradation pathway (ERAD), was found to be essentially involved in the observed immune escape. In support, our data demonstrate that re-expression of p97/VCP in Melan-A/MART-126-35 CTL-resistant melanoma cells completely restored immune recognition by Melan-A/MART-126-35 CTL. In conclusion, our experiments show that impaired expression of IFN-γ-independent components of the UPS can exert rapid immune evasion of tumor cells and suggest that tumor antigens processed by distinct UPS degradation pathways should be simultaneously targeted in T cell therapies to restrict the likelihood of immune evasion due to impaired antigen processing. PMID:27143649

Immune Response to Recombinant Adenovirus in Humans: Capsid Components from Viral Input Are Targets for Vector-Specific Cytotoxic T Lymphocytes

PubMed Central

Molinier-Frenkel, Valérie; Gahery-Segard, Hanne; Mehtali, Majid; Le Boulaire, Christophe; Ribault, Sébastien; Boulanger, Pierre; Tursz, Thomas; Guillet, Jean-Gérard; Farace, Françoise

2000-01-01

We previously demonstrated that a single injection of 109 PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218–2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8+ CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses. PMID:10906225

Changes in Cardiac Variability after REM Sleep Deprivation in Recurrent Nightmares

PubMed Central

Nielsen, Tore; Paquette, Tyna; Solomonova, Elizaveta; Lara-Carrasco, Jessica; Colombo, Roberto; Lanfranchi, Paola

2010-01-01

Study Objectives: To assess whether dysfunctional autonomic regulation during REM sleep as indexed by heart rate variability (HRV) is a pathophysiological factor in frequent nightmares (NMs). Design: Monitoring with polysomnography (PSG) and electrocardiography (ECG) for 3 consecutive nights: Night 1 (N1), adaptation night; N2, administration of partial REM sleep deprivation; N3, recovery night. Differences between NM and control (CTL) groups assessed for ECG measures drawn from wakefulness, REM sleep, and Stage 2 sleep on both N1 and N3. Setting: Hospital-based sleep laboratory Participants: Sixteen subjects with frequent NMs ( ≥ 1 NM/week; mean age = 26.1 ± 8.7 years) but no other medical or psychiatric disorders and 11 healthy comparison subjects ( < 1 NM/month; mean age = 27.1±5.6 years). Results: NM and CTL groups differed on 2 REM sleep measures only on N1; the NM group had longer REM latencies and REM/NREM cycle durations than did the CTL group. No differences were found on time domain and absolute frequency domain ECG measures for either N1 or N3. However, altered HRV for the NM group was suggested by significantly higher LFnu, lower HFnu, and higher LF/HF ratio than for the CTL group. Conclusions: Results are consistent with a higher than normal sympathetic drive among NM subjects which is unmasked by high REM sleep propensity. Results also support a growing literature linking anxiety disorders of several types (panic disorder, posttraumatic stress disorder (PTSD), generalized anxiety disorder) to altered HR variability. Citation: Nielsen T; Paquette T; Solomonova E; Lara-Carrasco J; Colombo R; Lanfranchi P. Changes in cardiac variability after rem sleep deprivation in recurrent nightmares. SLEEP 2010;33(1):113-122. PMID:20120628

Synthetic peptides coupled to the surface of liposomes effectively induce SARS coronavirus-specific cytotoxic T lymphocytes and viral clearance in HLA-A*0201 transgenic mice.

PubMed

Ohno, Satoshi; Kohyama, Shunsuke; Taneichi, Maiko; Moriya, Osamu; Hayashi, Hidenori; Oda, Hiroshi; Mori, Masahito; Kobayashi, Akiharu; Akatsuka, Toshitaka; Uchida, Tetsuya; Matsui, Masanori

2009-06-12

We investigated whether the surface-linked liposomal peptide was applicable to a vaccine based on cytotoxic T lymphocytes (CTLs) against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV). We first identified four HLA-A*0201-restricted CTL epitopes derived from SARS-CoV using HLA-A*0201 transgenic mice and recombinant adenovirus expressing predicted epitopes. These peptides were coupled to the surface of liposomes, and inoculated into mice. Two of the liposomal peptides were effective for peptide-specific CTL induction, and one of them was efficient for the clearance of vaccinia virus expressing epitopes of SARS-CoV, suggesting that the surface-linked liposomal peptide might offer an effective CTL-based vaccine against SARS.

Predicting the impact of blocking human immunodeficiency virus type 1 Nef in vivo.

PubMed

Wick, W David; Gilbert, Peter B; Yang, Otto O

2009-03-01

Human immunodeficiency virus type 1 (HIV-1) Nef is a multifunctional protein that confers an ability to evade killing by cytotoxic T lymphocytes (CTLs) as well as other advantages to the virus in vivo. Here we exploited mathematical modeling and related statistical methods to estimate the impact of Nef activity on viral replication in vivo in relation to CTLs. Our results indicate that downregulation of major histocompatibility complex class I (MHC-I) A and B by wild-type Nef confers an advantage to the virus of about 82% in decreased CTL killing efficiency on average, meaning that abolishing the MHC-I downregulation function of Nef would increase killing by more than fivefold. We incorporated this estimate, as well as prior estimates of replicative enhancement by Nef, into a previously published model of HIV-1 and CTLs in vivo (W. D. Wick, O. O. Yang, L. Corey, and S. G. Self, J. Virol. 79:13579-13586, 2005), generalized to permit CTL recognition of multiple epitopes. A sequence database analysis revealed that 92.9% of HIV-1 epitopes are A or B restricted, and a previous study found an average of about 19 epitopes recognized (M. M. Addo et al., J. Virol. 77:2081-2092, 2003). We combined these estimates in the model in order to predict the impact of inhibiting Nef function in the general (chronically infected) population by a drug. The predicted impact on viral load ranged from negligible to 2.4 orders of magnitude, depending on the effects of the drug and the CTL dynamical scenario assumed. We conclude that inhibiting Nef could make a substantial reduction in disease burden, lengthening the time before the necessity of undertaking combination therapy with other antiretroviral drugs.

Use of an In Vivo FTA Assay to Assess the Magnitude, Functional Avidity and Epitope Variant Cross-Reactivity of T Cell Responses Following HIV-1 Recombinant Poxvirus Vaccination

PubMed Central

Wijesundara, Danushka K.; Ranasinghe, Charani; Jackson, Ronald J.; Lidbury, Brett A.; Parish, Christopher R.; Quah, Benjamin J. C.

2014-01-01

Qualitative characteristics of cytotoxic CD8+ T cells (CTLs) are important in measuring the effectiveness of CTLs in controlling HIV-1 infections. Indeed, in recent studies patients who are naturally resistant to HIV-1 infections have been shown to possess CTLs that are of high functional avidity and have a high capacity to recognize HIV epitope variants, when compared to HIV-1 infection progressors. When developing efficacious vaccines, assays that can effectively measure CTL quality specifically in vivo are becoming increasingly important. Here we report the use of a recently developed high-throughput multi-parameter technique, known as the fluorescent target array (FTA) assay, to simultaneously measure CTL killing magnitude, functional avidity and epitope variant cross-reactivity in real time in vivo. In the current study we have applied the FTA assay as a screening tool to assess a large cohort of over 20 different HIV-1 poxvirus vaccination strategies in mice. This screen revealed that heterologous poxvirus prime-boost vaccination regimes (i.e., recombinant fowlpox (FPV)-HIV prime followed by a recombinant vaccinia virus (VV)-HIV booster) were the most effective in generating high quality CTL responses in vivo. In conclusion, we have demonstrated how the FTA assay can be utilized as a cost effective screening tool (by reducing the required number of animals by >100 fold), to evaluate a large range of HIV-1 vaccination strategies in terms of CTL avidity and variant cross-reactivity in an in vivo setting. PMID:25170620

Use of an in vivo FTA assay to assess the magnitude, functional avidity and epitope variant cross-reactivity of T cell responses following HIV-1 recombinant poxvirus vaccination.

PubMed

Wijesundara, Danushka K; Ranasinghe, Charani; Jackson, Ronald J; Lidbury, Brett A; Parish, Christopher R; Quah, Benjamin J C

2014-01-01

Qualitative characteristics of cytotoxic CD8+ T cells (CTLs) are important in measuring the effectiveness of CTLs in controlling HIV-1 infections. Indeed, in recent studies patients who are naturally resistant to HIV-1 infections have been shown to possess CTLs that are of high functional avidity and have a high capacity to recognize HIV epitope variants, when compared to HIV-1 infection progressors. When developing efficacious vaccines, assays that can effectively measure CTL quality specifically in vivo are becoming increasingly important. Here we report the use of a recently developed high-throughput multi-parameter technique, known as the fluorescent target array (FTA) assay, to simultaneously measure CTL killing magnitude, functional avidity and epitope variant cross-reactivity in real time in vivo. In the current study we have applied the FTA assay as a screening tool to assess a large cohort of over 20 different HIV-1 poxvirus vaccination strategies in mice. This screen revealed that heterologous poxvirus prime-boost vaccination regimes (i.e., recombinant fowlpox (FPV)-HIV prime followed by a recombinant vaccinia virus (VV)-HIV booster) were the most effective in generating high quality CTL responses in vivo. In conclusion, we have demonstrated how the FTA assay can be utilized as a cost effective screening tool (by reducing the required number of animals by >100 fold), to evaluate a large range of HIV-1 vaccination strategies in terms of CTL avidity and variant cross-reactivity in an in vivo setting.

Slp1 and Slp2-a Localize to the Plasma Membrane of CTL and Contribute to Secretion from the Immunological Synapse

PubMed Central

Holt, Oliver; Kanno, Eiko; Bossi, Giovanna; Booth, Sarah; Daniele, Tiziana; Santoro, Alessandra; Arico, Maurizio; Saegusa, Chika; Fukuda, Mitsunori; Griffiths, Gillian M

2008-01-01

Rab27a is required for polarized secretion of lysosomes from cytotoxic T lymphocytes (CTLs) at the immunological synapse. A series of Rab27a-interacting proteins have been identified; however, only Munc13-4 has been found to be expressed in CTL. In this study, we screened for expression of the synaptotagmin-like proteins (Slps): Slp1/JFC1, Slp2-a/exophilin4, Slp3-a, Slp4/granuphilin, Slp5 and rabphilin in CTL. We found that both Slp1 and Slp2-a are expressed in CTL. Isoforms of Slp2-a in CTL showed variation of the linker region but conserved the C2A and C2B and Slp homology (SHD) domains. Both Slp1 and Slp2-a interact with Rab27a in CTL, and Slp2-a, but not Slp1, is rapidly degraded when Rab27a is absent. Slp2-a contains PEST-like sequences within its linker region, which render it susceptible to degradation. Both Slp1 and Slp2-a localize predominantly to the plasma membrane of both human and mouse CTLs, and we show that Slp2-a can focus tightly at the immunological synapse formed with a target cell. Individual knockouts of either Slp2-a or Slp1 fail to impair CTL-mediated killing of targets; however, overexpression of a dominant-negative construct consisting of the SHD of Slp2-a, which is 56% identical to that of Slp1, reduces target cell death, suggesting that both Slp1 and Slp2-a contribute to secretory lysosome exocytosis from CTL. These results suggest that both Slp1 and Slp2-a may form part of a docking complex, capturing secretory lysosomes at the immunological synapse. PMID:18266782

A new vesicle trafficking regulator CTL1 plays a crucial role in ion homeostasis.

PubMed

Gao, Yi-Qun; Chen, Jiu-Geng; Chen, Zi-Ru; An, Dong; Lv, Qiao-Yan; Han, Mei-Ling; Wang, Ya-Ling; Salt, David E; Chao, Dai-Yin

2017-12-01

Ion homeostasis is essential for plant growth and environmental adaptation, and maintaining ion homeostasis requires the precise regulation of various ion transporters, as well as correct root patterning. However, the mechanisms underlying these processes remain largely elusive. Here, we reported that a choline transporter gene, CTL1, controls ionome homeostasis by regulating the secretory trafficking of proteins required for plasmodesmata (PD) development, as well as the transport of some ion transporters. Map-based cloning studies revealed that CTL1 mutations alter the ion profile of Arabidopsis thaliana. We found that the phenotypes associated with these mutations are caused by a combination of PD defects and ion transporter misregulation. We also established that CTL1 is involved in regulating vesicle trafficking and is thus required for the trafficking of proteins essential for ion transport and PD development. Characterizing choline transporter-like 1 (CTL1) as a new regulator of protein sorting may enable researchers to understand not only ion homeostasis in plants but also vesicle trafficking in general.

A new vesicle trafficking regulator CTL1 plays a crucial role in ion homeostasis

PubMed Central

Gao, Yi-Qun; Chen, Jiu-Geng; Chen, Zi-Ru; An, Dong; Lv, Qiao-Yan; Han, Mei-Ling; Wang, Ya-Ling; Salt, David E.; Chao, Dai-Yin

2017-01-01

Ion homeostasis is essential for plant growth and environmental adaptation, and maintaining ion homeostasis requires the precise regulation of various ion transporters, as well as correct root patterning. However, the mechanisms underlying these processes remain largely elusive. Here, we reported that a choline transporter gene, CTL1, controls ionome homeostasis by regulating the secretory trafficking of proteins required for plasmodesmata (PD) development, as well as the transport of some ion transporters. Map-based cloning studies revealed that CTL1 mutations alter the ion profile of Arabidopsis thaliana. We found that the phenotypes associated with these mutations are caused by a combination of PD defects and ion transporter misregulation. We also established that CTL1 is involved in regulating vesicle trafficking and is thus required for the trafficking of proteins essential for ion transport and PD development. Characterizing choline transporter-like 1 (CTL1) as a new regulator of protein sorting may enable researchers to understand not only ion homeostasis in plants but also vesicle trafficking in general. PMID:29284002

Induction of tolerance towards TNP entails down-regulation of an autoimmune attack.

PubMed Central

Zöller, M; Andrighetto, G

1988-01-01

In order to follow the process of induction and maintainance of tolerance, BALB/c mice were tolerized by free hapten, and effector and regulatory cell interactions were analysed by limiting-dilution (LD) cultures. Injection of trinitrobenzenesulphonic acid (TNBS) resulted, predominantly, in the activation and expansion of self-reactive cytotoxic T cells (CTL), which were observed transiently at frequencies comparable to allo-specific CTL. In addition, self-reactive helper T cells (Th) were activated and expanded in tolerized mice. TNP-specific reactivity was difficult to evaluate, since cytotoxic activity against haptenized self followed the pattern of self-reactivity throughout the test period. But in LD cultures determining proliferation, two populations of Th responding to TNP-self were observed, while only one Th population could be detected in response to self. Expansion/activation of Th and CTL precursors (CTLp) was followed by activation of suppressor T cells (Ts). The suppressor population could be divided into two subpopulations, one interfering with Th, the second interacting directly with CTL (veto cells). The results indicate that during the induction of tolerance, animals pass through an autoimmune attack, with expansion and activation of self-reactive clones (CTL, Th). The final status of non-responsiveness towards TNP is not due to the deletion of effector or regulatory cells, but results from the establishment of a steady state of dominance of self-reactive and TNP-self-reactive suppression. PMID:2965095

Identification of HLA-A2–restricted CD8+ Cytotoxic T Cell Responses in Primary Biliary Cirrhosis

PubMed Central

Kita, Hiroto; Lian, Zhe-Xiong; Van de Water, Judy; He, Xiao-Song; Matsumura, Shuji; Kaplan, Marshall; Luketic, Velimir; Coppel, Ross L.; Ansari, Aftab A.; Gershwin, M. Eric

2002-01-01

Primary biliary cirrhosis (PBC) is characterized by an intense biliary inflammatory CD4+ and CD8+ T cell response. Very limited information on autoantigen-specific cytotoxic T lymphocyte (CTL) responses is available compared with autoreactive CD4+ T cell responses. Using peripheral blood mononuclear cells (PBMCs) from PBC, we identified an HLA-A2–restricted CTL epitope of the E2 component of pyruvate dehydrogenase (PDC-E2), the immunodominant mitochondrial autoantigen. This peptide, amino acids 159–167 of PDC-E2, induces specific MHC class I–restricted CD8+ CTL lines from 10/12 HLA-A2+ PBC patients, but not controls, after in vitro stimulation with antigen-pulsed dendritic cells (DCs). PDC-E2–specific CTLs could also be generated by pulsing DCs with full-length recombinant PDC-E2 protein. Furthermore, using soluble PDC-E2 complexed with either PDC-E2–specific human monoclonal antibody or affinity-purified autoantibodies against PDC-E2, the generation of PDC-E2–specific CTLs, occurred at 100-fold and 10-fold less concentration, respectively, compared with soluble antigen alone. Collectively, these data demonstrate that autoantibody, helper, and CTL epitopes all contain a shared peptide sequence. The finding that autoantigen–immune complexes can not only cross-present but also that presentation of the autoantigen is of a higher relative efficiency, for the first time defines a unique role for autoantibodies in the pathogenesis of an autoimmune disease. PMID:11781370

Microcinematographic and electron microscopic analysis of target cell lysis induced by cytotoxic T lymphocytes.

PubMed Central

Matter, A

1979-01-01

A study was carried out to determine the sequence of events of T-cell mediated target cell lysis in microcinematography and electron microscopy. Highly efficient cytotoxic T lymphocytes (CTL) were generated in vivo and in vitro using preimmunized spleen cells and purification procedures. Such CTL were highly specific. This specificity correlated well with the number of adhesions formed between CTL and targets and this criterion was used to study killer-target cell interaction. Microcinematography showed that target cell lysis at the single cell level, despite time variations, could be clearly separated into three phases: (a) a recognition phase, visible by random crawling of CTL over the target cell surface until firm contact was established; (b) a post-recognition phase, during which firm contact between CTL and target was maintained without gross modification of either cell; (c) a phase of target cell disintegration, mainly characterized by vigorous blebbing of the cell membrane resulting in a motionless carcass of the target cell but not in its total dissolution. Only later this carcass decayed and formed a necrotic ghost. Electron microscopic observations were put into sequence according to microcinematography. Post-recognition phase was characterized by a tight apposition of the membranes of CTL and target cell. No gap junctions could be observed. During target cell disintegration, profound cytoplasmic and nuclear changes occurred simultaneous with surface blebbing. Most noticeable were extensive internal vacuolization, mitochondrial swelling, nuclear pycnosis and dissolution of the nucleolus. These observations suggested that target cell lysis does not start with a surface phenomenon similar to complement lysis, but a process involving practically the whole cell simultaneously. It is conceivable, therefore, that the signal from the CTL is transmitted across the target cell, and that the switch to sudden cell death is manipulated deep inside the cell. Images Figure 3 Figures 4-7 Figures 8-11 Figure 12 Figures 13-14 Figure 15 PMID:312256

Abundant tax protein expression in CD4+ T cells infected with human T-cell lymphotropic virus type I (HTLV-I) is prevented by cytotoxic T lymphocytes.

PubMed

Hanon, E; Hall, S; Taylor, G P; Saito, M; Davis, R; Tanaka, Y; Usuku, K; Osame, M; Weber, J N; Bangham, C R

2000-02-15

The role of the cellular immune response in human T-cell leukemia virus type I (HTLV-I) infection is not fully understood. A persistently activated cytotoxic T lymphocyte (CTL) response to HTLV-I is found in the majority of infected individuals. However, it remains unclear whether this CTL response is protective or causes tissue damage. In addition, several observations paradoxically suggest that HTLV-I is transcriptionally silent in most infected cells and, therefore, not detectable by virus-specific CTLs. With the use of a new flow cytometric procedure, we show here that a high proportion of naturally infected CD4+ peripheral blood mononuclear cells (PBMC) (between 10% and 80%) are capable of expressing Tax, the immunodominant target antigen recognized by virus-specific CTLs. Furthermore, we provide direct evidence that autologous CD8+ T cells rapidly kill CD4+ cells naturally infected with HTLV-I and expressing Tax in vitro by a perforin-dependent mechanism. Consistent with these observations, we observed a significant negative correlation between the frequency of Tax(11-19)-specific CD8+ T cells and the percentage of CD4+ T cells in peripheral blood of patients infected with HTLV-I. Those results are in accordance with the view that virus-specific CTLs participate in a highly efficient immune surveillance mechanism that persistently destroys Tax-expressing HTLV-I-infected CD4+ T cells in vivo. (Blood. 2000;95:1386-1392)

Deficiency in Th2 cytokine responses exacerbate orthopoxvirus infection.

PubMed

Sakala, Isaac G; Chaudhri, Geeta; Eldi, Preethi; Buller, R Mark; Karupiah, Gunasegaran

2015-01-01

Ectromelia virus (ECTV) causes mousepox in mice, a disease very similar to smallpox in humans. ECTV and variola virus (VARV), the agent of smallpox, are closely related orthopoxviruses. Mousepox is an excellent small animal model to study the genetic and immunologic basis for resistance and susceptibility of humans to smallpox. Resistance to mousepox is dependent on a strong polarized type 1 immune response, associated with robust natural killer (NK) cell, cytotoxic T lymphocyte (CTL) and gamma interferon (IFN-γ) responses. In contrast, ECTV-susceptible mice generate a type 2 response, associated with weak NK cell, CTL and IFN-γ responses but robust IL-4 responses. Nonetheless, susceptible strains infected with mutant ECTV lacking virus-encoded IFN-γ binding protein (vIFN-γbp) (ECTV-IFN-γbpΔ) control virus replication through generation of type 1 response. Since the IL-4/IL-13/STAT-6 signaling pathways polarize type 2/T helper 2 (Th2) responses with a corresponding suppression of IFN-γ production, we investigated whether the combined absence of vIFN-γbp, and one or more host genes involved in Th2 response development, influence generation of protective immunity. Most mutant mouse strains infected with wild-type (WT) virus succumbed to disease more rapidly than WT animals. Conversely, the disease outcome was significantly improved in WT mice infected with ECTV-IFN-γbpΔ but absence of IL-4/IL-13/STAT-6 signaling pathways did not provide any added advantage. Deficiency in IL-13 or STAT-6 resulted in defective CTL responses, higher mortality rates and accelerated deaths. Deficiencies in IL-4/IL-13/STAT-6 signaling pathways significantly reduced the numbers of IFN-γ producing CD4 and CD8 T cells, indicating an absence of a switch to a Th1-like response. Factors contributing to susceptibility or resistance to mousepox are far more complex than a balance between Th1 and Th2 responses.

Epstein-Barr virus-positive cytotoxic T-cell lymphoma followed by chronic active Epstein-Barr virus infection-associated T/NK-cell lymphoproliferative disorder: a case report.

PubMed

Kato, Seiichi; Miyata, Tomoko; Takata, Katsuyoshi; Shimada, Satoko; Ito, Yoshinori; Tomita, Akihiro; Elsayed, Ahmed Ali; Takahashi, Emiko; Asano, Naoko; Kinoshita, Tomohiro; Kimura, Hiroshi; Nakamura, Shigeo

2013-12-01

A 30-year-old female patient presented with intestinal Epstein-Barr virus (EBV)-positive cytotoxic T-cell lymphoma (EBV+ CTL), which was surgically resected. Fourteen years later, she returned to our hospital with hypersensitivity to mosquito bites and was diagnosed with chronic active EBV infection-associated T/NK-cell lymphoproliferative disorder (CAEBV/TNK-LPD). She developed systemic EBV+ CTL at age 47 years during the 2.5-year clinical course of CAEBV/TNK-LPD, despite multiagent chemotherapy and allogeneic stem cell transplantation. Afterward, she had a rapidly deteriorating clinical course and died at age 48 years. The immunophenotype of the EBV+ CTL was consistently a CD3, CD8, and cytotoxic molecule-positive type with the same clonality in polymerase chain reaction analysis of T-cell receptor-γ chain gene rearrangement. This is the first reported case of EBV+ CTL preceding the clinical presentation of CAEBV/TNK-LPD. The present case was unique in suggesting a close relationship between EBV+ CTL and chronic active EBV infection. © 2013.

Recognition and killing of brain tumor stem-like initiating cells by CD8+ cytolytic T cells.

PubMed

Brown, Christine E; Starr, Renate; Martinez, Catalina; Aguilar, Brenda; D'Apuzzo, Massimo; Todorov, Ivan; Shih, Chu-Chih; Badie, Behnam; Hudecek, Michael; Riddell, Stanley R; Jensen, Michael C

2009-12-01

Solid tumors contain a subset of stem-like cells that are resistant to the cytotoxic effects of chemotherapy/radiotherapy, but their susceptibility to cytolytic T lymphocyte (CTL) effector mechanisms has not been well characterized. Using a panel of early-passage human brain tumor stem/initiating cell (BTSC) lines derived from high-grade gliomas, we show that BTSCs are subject to immunologic recognition and elimination by CD8(+) CTLs. Compared with serum-differentiated CD133(low) tumor cells and established glioma cell lines, BTSCs are equivalent with respect to expression levels of HLA class I and ICAM-1, similar in their ability to trigger degranulation and cytokine synthesis by antigen-specific CTLs, and equally susceptible to perforin-dependent CTL-mediated cytolysis. BTSCs are also competent in the processing and presentation of antigens as evidenced by the killing of these cells by CTL when antigen is endogenously expressed. Moreover, we show that CTLs can eliminate all BTSCs with tumor-initiating activity in an antigen-specific manner in vivo. Current models predict that curative therapies for many cancers will require the elimination of the stem/initiating population, and these studies lay the foundation for developing immunotherapeutic approaches to eradicate this tumor population.

Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function

PubMed Central

Shahid, Aniqa; Olvera, Alex; Anmole, Gursev; Kuang, Xiaomei T.; Cotton, Laura A.; Plana, Montserrat; Brander, Christian; Brockman, Mark A.

2015-01-01

ABSTRACT HLA-B*13 is associated with superior in vivo HIV-1 viremia control. Protection is thought to be mediated by sustained targeting of key cytotoxic T lymphocyte (CTL) epitopes and viral fitness costs of CTL escape in Gag although additional factors may contribute. We assessed the impact of 10 published B*13-associated polymorphisms in Gag, Pol, and Nef, in 23 biologically relevant combinations, on HIV-1 replication capacity and Nef-mediated reduction of cell surface CD4 and HLA class I expression. Mutations were engineered into HIV-1NL4.3, and replication capacity was measured using a green fluorescent protein (GFP) reporter T cell line. Nef-mediated CD4 and HLA-A*02 downregulation was assessed by flow cytometry, and T cell recognition of infected target cells was measured via coculture with an HIV-specific luciferase reporter cell line. When tested individually, only Gag-I147L and Gag-I437L incurred replicative costs (5% and 17%, respectively), consistent with prior reports. The Gag-I437L-mediated replication defect was rescued to wild-type levels by the adjacent K436R mutation. A novel B*13 epitope, comprising 8 residues and terminating at Gag147, was identified in p24Gag (GQMVHQAIGag140–147). No other single or combination Gag, Pol, or Nef mutant impaired viral replication. Single Nef mutations did not affect CD4 or HLA downregulation; however, the Nef double mutant E24Q-Q107R showed 40% impairment in HLA downregulation with no evidence of Nef stability defects. Moreover, target cells infected with HIV-1-NefE24Q-Q107R were recognized better by HIV-specific T cells than those infected with HIV-1NL4.3 or single Nef mutants. Our results indicate that CTL escape in Gag and Nef can be functionally costly and suggest that these effects may contribute to long-term HIV-1 control by HLA-B*13. IMPORTANCE Protective effects of HLA-B*13 on HIV-1 disease progression are mediated in part by fitness costs of CTL escape mutations in conserved Gag epitopes, but other mechanisms remain incompletely known. We extend our knowledge of the impact of B*13-driven escape on HIV-1 replication by identifying Gag-K436R as a compensatory mutation for the fitness-costly Gag-I437L. We also identify Gag-I147L, the most rapidly and commonly selected B*13-driven substitution in HIV-1, as a putative C-terminal anchor residue mutation in a novel B*13 epitope. Most notably, we identify a novel escape-driven fitness defect: B*13-driven substitutions E24Q and Q107R in Nef, when present together, substantially impair this protein's ability to downregulate HLA class I. This, in turn, increases the visibility of infected cells to HIV-specific T cells. Our results suggest that B*13-associated escape mutations impair HIV-1 replication by two distinct mechanisms, that is, by reducing Gag fitness and dampening Nef immune evasion function. PMID:26355081

Immediate-Early Transactivator Rta of Epstein-Barr Virus (EBV) Shows Multiple Epitopes Recognized by EBV-Specific Cytotoxic T Lymphocytes

PubMed Central

Pepperl, Sandra; Benninger-Döring, Gerlinde; Modrow, Susanne; Wolf, Hans; Jilg, Wolfgang

1998-01-01

We analyzed the immediate-early transactivator Rta of Epstein-Barr virus (EBV) for its role as a target for specific cytotoxic T lymphocytes (CTL). Panels of overlapping peptides covering the entire amino acid sequence of Rta were synthesized and used to induce and analyze specific CTL responses in EBV-positive donors. Using peptide-pulsed target cells, we found nine different CTL epitopes that are distributed over the entire protein sequence. One epitope restricted by HLA-A24 could be mapped to the decameric sequence DYCNVLNKEF between amino acid positions 28 and 37 of the Rta protein. A second epitope could be assigned to the same region of Rta (residues 25 to 39) and was shown to be restricted by HLA-B18. Another, minimal epitope could be mapped to the nonameric sequence ATIGTAMYK between amino acid positions 134 and 142; this peptide was restricted by HLA-A11. Another four epitopes were proven to be restricted by HLA-A2, -A3, -B61, and -Cw4 and were located between Rta residues 225 and 239, 145 and 159, 529 and 543, and 393 and 407, respectively. For two other epitopes, only the location within the Rta protein is known so far (residues 121 to 135 and 441 to 455); their exact HLA restriction patterns have not yet been identified. Using target cells infected with recombinant vaccinia virus containing the gene for Rta, we showed that six of eight Rta-specific CTL lines recognized the corresponding peptides also after endogenous processing. These data suggest that Rta comprises an important target for EBV-specific cellular cytotoxicity. Together with recent findings of other immediate-early and early proteins also acting as CTL targets, they reveal the role of proteins of the lytic cycle in the immune recognition of EBV-infected cells. PMID:9765404

HIV-1 dynamics revisited: biphasic decay by cytotoxic T lymphocyte killing?

PubMed Central

Arnaout, R A; Nowak, M A; Wodarz, D

2000-01-01

The biphasic decay of blood viraemia in patients being treated for human immunodeficiency virus type 1 (HIV-1) infection has been explained as the decay of two distinct populations of cells: the rapid death of productively infected cells followed by the much slower elimination of a second population the identity of which remains unknown. Here we advance an alternative explanation based on the immune response against a single population of infected cells. We show that the biphasic decay can be explained simply, without invoking multiple compartments: viral load falls quickly while cytotoxic T lymphocytes (CTL) are still abundant, and more slowly as CTL disappear. We propose a method to test this idea, and develop a framework that is readily applicable to treatment of other infections. PMID:10972131

T Cell Receptor Vβ Staining Identifies the Malignant Clone in Adult T cell Leukemia and Reveals Killing of Leukemia Cells by Autologous CD8+ T cells

PubMed Central

Witkover, Aviva; Tanaka, Yuetsu; Fields, Paul; Bangham, Charles R. M.

2016-01-01

There is growing evidence that CD8+ cytotoxic T lymphocyte (CTL) responses can contribute to long-term remission of many malignancies. The etiological agent of adult T-cell leukemia/lymphoma (ATL), human T lymphotropic virus type-1 (HTLV-1), contains highly immunogenic CTL epitopes, but ATL patients typically have low frequencies of cytokine-producing HTLV-1-specific CD8+ cells in the circulation. It remains unclear whether patients with ATL possess CTLs that can kill the malignant HTLV-1 infected clone. Here we used flow cytometric staining of TCRVβ and cell adhesion molecule-1 (CADM1) to identify monoclonal populations of HTLV-1-infected T cells in the peripheral blood of patients with ATL. Thus, we quantified the rate of CD8+-mediated killing of the putative malignant clone in ex vivo blood samples. We observed that CD8+ cells from ATL patients were unable to lyse autologous ATL clones when tested directly ex vivo. However, short in vitro culture restored the ability of CD8+ cells to kill ex vivo ATL clones in some donors. The capacity of CD8+ cells to lyse HTLV-1 infected cells which expressed the viral sense strand gene products was significantly enhanced after in vitro culture, and donors with an ATL clone that expressed the HTLV-1 Tax gene were most likely to make a detectable lytic CD8+ response to the ATL cells. We conclude that some patients with ATL possess functional tumour-specific CTLs which could be exploited to contribute to control of the disease. PMID:27893842

Viral CTL escape mutants are generated in lymph nodes and subsequently become fixed in plasma and rectal mucosa during acute SIV infection of macaques.

PubMed

Vanderford, Thomas H; Bleckwehl, Chelsea; Engram, Jessica C; Dunham, Richard M; Klatt, Nichole R; Feinberg, Mark B; Garber, David A; Betts, Michael R; Silvestri, Guido

2011-05-01

SIV(mac239) infection of rhesus macaques (RMs) results in AIDS despite the generation of a strong antiviral cytotoxic T lymphocyte (CTL) response, possibly due to the emergence of viral escape mutants that prevent recognition of infected cells by CTLs. To determine the anatomic origin of these SIV mutants, we longitudinally assessed the presence of CTL escape variants in two MamuA*01-restricted immunodominant epitopes (Tat-SL8 and Gag-CM9) in the plasma, PBMCs, lymph nodes (LN), and rectal biopsies (RB) of fifteen SIV(mac239)-infected RMs. As expected, Gag-CM9 did not exhibit signs of escape before day 84 post infection. In contrast, Tat-SL8 escape mutants were apparent in all tissues by day 14 post infection. Interestingly LNs and plasma exhibited the highest level of escape at day 14 and day 28 post infection, respectively, with the rate of escape in the RB remaining lower throughout the acute infection. The possibility that CTL escape occurs in LNs before RBs is confirmed by the observation that the specific mutants found at high frequency in LNs at day 14 post infection became dominant at day 28 post infection in plasma, PBMC, and RB. Finally, the frequency of escape mutants in plasma at day 28 post infection correlated strongly with the level Tat-SL8-specific CD8 T cells in the LN and PBMC at day 14 post infection. These results indicate that LNs represent the primary source of CTL escape mutants during the acute phase of SIV(mac239) infection, suggesting that LNs are the main anatomic sites of virus replication and/or the tissues in which CTL pressure is most effective in selecting SIV escape variants.

Viral CTL Escape Mutants Are Generated in Lymph Nodes and Subsequently Become Fixed in Plasma and Rectal Mucosa during Acute SIV Infection of Macaques

PubMed Central

Vanderford, Thomas H.; Bleckwehl, Chelsea; Engram, Jessica C.; Dunham, Richard M.; Klatt, Nichole R.; Feinberg, Mark B.; Garber, David A.; Betts, Michael R.; Silvestri, Guido

2011-01-01

SIVmac239 infection of rhesus macaques (RMs) results in AIDS despite the generation of a strong antiviral cytotoxic T lymphocyte (CTL) response, possibly due to the emergence of viral escape mutants that prevent recognition of infected cells by CTLs. To determine the anatomic origin of these SIV mutants, we longitudinally assessed the presence of CTL escape variants in two MamuA*01-restricted immunodominant epitopes (Tat-SL8 and Gag-CM9) in the plasma, PBMCs, lymph nodes (LN), and rectal biopsies (RB) of fifteen SIVmac239-infected RMs. As expected, Gag-CM9 did not exhibit signs of escape before day 84 post infection. In contrast, Tat-SL8 escape mutants were apparent in all tissues by day 14 post infection. Interestingly LNs and plasma exhibited the highest level of escape at day 14 and day 28 post infection, respectively, with the rate of escape in the RB remaining lower throughout the acute infection. The possibility that CTL escape occurs in LNs before RBs is confirmed by the observation that the specific mutants found at high frequency in LNs at day 14 post infection became dominant at day 28 post infection in plasma, PBMC, and RB. Finally, the frequency of escape mutants in plasma at day 28 post infection correlated strongly with the level Tat-SL8-specific CD8 T cells in the LN and PBMC at day 14 post infection. These results indicate that LNs represent the primary source of CTL escape mutants during the acute phase of SIVmac239 infection, suggesting that LNs are the main anatomic sites of virus replication and/or the tissues in which CTL pressure is most effective in selecting SIV escape variants. PMID:21625590

Adoptive transfer of Epstein-Barr virus-specific cytotoxic T-lymphocytes for the treatment of angiocentric lymphomas.

PubMed

Cho, Hyun-Il; Hong, Young Seon; Lee, Myung Ah; Kim, Eun-Kyung; Yoon, Sung-Hee; Kim, Chun-Choo; Kim, Tai-Gyu

2006-01-01

Angiocentric lymphoma, known as natural killer (NK)/T-cell non-Hodgkin's lymphoma, has been reported to be associated with the Epstein-Barr virus (EBV). We performed adoptive transfer of EBV-specific polyclonal T-cell lines in 3 patients with extranodal NK/T-cell lymphoma, nasal type, and evaluated the treatment for safety, immunologic reconstitution, and clinical outcomes. The tissue samples collected from the 3 patients were confirmed by polymerase chain reaction analysis to be EBV positive. In the cases of the first and second patients, EBV-transformed B-lymphoblastoid cell lines (LCLs) and T-cell lines were generated from peripheral lymphocytes of HLA-matched sibling donors. The third patient's T-cell lines were induced with autologous lymphocytes. Polyclonal T-cell infusion was carried out after high-dose radiotherapy because active relapsed disease remained in all of the patients. The first patient received 4 weekly infusions of 2 3 10(7) cells/m(2), and the second and third patients underwent treatment with 2 cycles of infusions of the same dosage. All T-cell lines showed >60% NK activity, cytotoxic T-lymphocyte (CTL) responses of >40% against autologous LCLs, and no CTL activity against patient-derived lymphoblasts. The level of cytotoxicity increased substantially in all patients after cell infusion. The 2 patients who received T-cell therapy twice had stabilized disease for more than 3 years. These safe treatments exhibited no severe inflammatory response, and no serious toxicity developed during T-cell therapy. Our findings demonstrate that adoptively transferred cells may provide reconstitution of EBV-specific CTL responses in patients with active relapsed angiocentric lymphoma. These results provide a rationale for the immunotherapy of angiocentric lymphoma.

T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2- restricted and melanocyte-lineage-specific CTL clone

PubMed Central

1993-01-01

HLA-A2+ melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA- A2+ melanoma patient and selected for T cell receptor (TCR)-dependent, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing. Despite absence of oligoclonality in fresh TIL and PBL, as well as in T cells of day 28 MLTC (day of cloning), sequence analysis of TCR alpha and beta chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a V alpha 2.1 gene segment associated with either V beta 13.2, 14, or w22. Clones A81 (V alpha 2.1/J alpha IGRJ alpha 04/C alpha and V beta 14/D beta 1/J beta 1.2/C beta 1) and A21 (V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lytic patterns, but both were HLA-A2 restricted and lysed only HLA-A2+ melanomas and normal melanocytes, thus indicating recognition of two distinct HLA-A2-associated and tissue-related Ags. Finally, by the inverse PCR technique, the specific TCR beta chain (V beta 2.1/D beta 1/J beta 1.1/C beta 1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V beta 2 sequences expressed in the fresh tumor sample and in the purified TIL, respectively, but < 0.19% of V beta 2+ sequences expressed in PBL. These results are consistent with the hypothesis that a clonal expansion/accumulation of a melanocyte-lineage-specific and HLA-A2-restricted T cell clone occurred in vivo at the site of tumor growth. PMID:8376931

Feasibility of Cancer Immunotherapy with WT1 Peptide Vaccination for Solid and Hematological Malignancies in Children.

PubMed

Sawada, Akihisa; Inoue, Masami; Kondo, Osamu; Yamada-Nakata, Kayo; Ishihara, Takashi; Kuwae, Yuko; Nishikawa, Masanori; Ammori, Yasuhiro; Tsuboi, Akihiro; Oji, Yusuke; Koyama-Sato, Maho; Oka, Yoshihiro; Yasui, Masahiro; Sugiyama, Haruo; Kawa, Keisei

2016-02-01

Advances in cancer immunotherapy in the pediatric field are needed in order to improve the prognosis of children with malignancies. We conducted a prospective phase I/II study of WT1 peptide vaccination for children with relapsed or refractory malignancies. The main eligibility criteria were affected tissues or leukemic cells expressing the WT1 gene, and patients (and donors for allogeneic hematopoietic stem cell transplantation) having HLA-A*24:02. Vaccination using the WT1 peptide (CYTWNQMNL), which was modified for higher affinity to this HLA-type molecule with the adjuvant Montanide ISA51, was performed weekly 12 times. Twenty-six patients were enrolled and 13 (50.0%) completed the vaccination 12 times. Evidence for the induction of WT1-specific cytotoxic T-lymphocyte (CTL) responses without severe systemic side effects was obtained. Two out of 12 patients with bulky disease exhibited a transient clinical effect (one mixed response and one stable disease), three out of six patients with minimal residual disease achieved transient molecular remission, and five out of eight patients without a detectable level of the molecular marker, but with a high risk of relapse, had the best outcome of long-term continuous complete remission. WT1 vaccination is a safe immunotherapy and induced WT1-specific CTL responses in children; however, as a single agent, vaccination only provided patients in remission, but with a high risk of relapse, with "long-term benefits" in the context of its use for relapse prevention. WT1 peptide-based treatments in combination with other modalities, such as anti-tumor drugs or immunomodulating agents, need to be planned. © 2015 Wiley Periodicals, Inc.

The IDO1 selective inhibitor epacadostat enhances dendritic cell immunogenicity and lytic ability of tumor antigen-specific T cells.

PubMed

Jochems, Caroline; Fantini, Massimo; Fernando, Romaine I; Kwilas, Anna R; Donahue, Renee N; Lepone, Lauren M; Grenga, Italia; Kim, Young-Seung; Brechbiel, Martin W; Gulley, James L; Madan, Ravi A; Heery, Christopher R; Hodge, James W; Newton, Robert; Schlom, Jeffrey; Tsang, Kwong Y

2016-06-21

Epacadostat is a novel inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1) that suppresses systemic tryptophan catabolism and is currently being evaluated in ongoing clinical trials. We investigated the effects of epacadostat on (a) human dendritic cells (DCs) with respect to maturation and ability to activate human tumor antigen-specific cytotoxic T-cell (CTL) lines, and subsequent T-cell lysis of tumor cells, (b) human regulatory T cells (Tregs), and (c) human peripheral blood mononuclear cells (PBMCs) in vitro. Simultaneous treatment with epacadostat and IFN-γ plus lipopolysaccharide (LPS) did not change the phenotype of matured human DCs, and as expected decreased the tryptophan breakdown and kynurenine production. Peptide-specific T-cell lines stimulated with DCs pulsed with peptide produced significantly more IFN-γ, TNFα, GM-CSF and IL-8 if the DCs were treated with epacadostat. These T cells also displayed higher levels of tumor cell lysis on a per cell basis. Epacadostat also significantly decreased Treg proliferation induced by IDO production from IFN-γ plus LPS matured human DCs, although the Treg phenotype did not change. Multicolor flow cytometry was performed on human PBMCs treated with epacadostat; analysis of 123 discrete immune cell subsets revealed no changes in major immune cell types, an increase in activated CD83+ conventional DCs, and a decrease in immature activated Tim3+ NK cells. These studies show for the first time several effects of epacadostat on human DCs, and subsequent effects on CTL and Tregs, and provide a rationale as to how epacadostat could potentially increase the efficacy of immunotherapeutics, including cancer vaccines.

Direct Coal -to-Liquids (CTL) for Jet Fuel Using Biomass-Derived Solvents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chauhan, Satya P.; Garbark, Daniel B.; Taha, Rachid

Battelle has demonstrated a novel and potentially breakthrough technology for a direct coal-to-liquids (CTL) process for producing jet fuel using biomass-derived coal solvents (bio-solvents). The Battelle process offers a significant reduction in capital and operating costs and a substantial reduction in greenhouse gas (GHG) emissions, without requiring carbon capture and storage (CCS). The results of the project are the advancement of three steps of the hybrid coal/biomass-to-jet fuel process to the technology readiness level (TRL) of 5. The project objectives were achieved over two phases. In Phase 1, all three major process steps were explored and refined at bench-scale, including:more » (1) biomass conversion to high hydrogen-donor bio-solvent; (2) coal dissolution in biomass-derived bio-solvent, without requiring molecular H 2, to produce a synthetic crude (syncrude); and (3) two-stage catalytic hydrotreating/hydrogenation of syncrude to jet fuel and other distillates. In Phase 2, all three subsystems of the CTL process were scaled up to a pre-pilot scale, and an economic analysis was carried out. A total of over 40 bio-solvents were identified and prepared. The most unique attribute of Battelle’s bio-solvents is their ability to provide much-needed hydrogen to liquefy coal and thus increase its hydrogen content so much that the resulting syncrude is liquid at room temperature. Based on the laboratory-scale testing with bituminous coals from Ohio and West Virginia, a total of 12 novel bio-solvent met the goal of greater than 80% coal solubility, with 8 bio-solvents being as good as or better than a well-known but expensive hydrogen-donor solvent, tetralin. The Battelle CTL process was then scaled up to 1 ton/day (1TPD) at a pre-pilot facility operated in Morgantown, WV. These tests were conducted, in part, to produce enough material for syncrude-upgrading testing. To convert the Battelle-CTL syncrude into a form suitable as a blending stock for jet turbine fuel, a two-step catalytic upgrading process was developed at laboratory scale and then demonstrated at pre-pilot scale facility in Pittsburg, PA. Several drums of distillate products were produced, which were then distilled into unblended (neat) synthetic jet fuel and diesel products for a detailed characterization. Based on a detailed characterization of the synthetic jet fuel, a 20% synthetic, 80% commercial jet fuel blend was prepared, which met all specifications. An analysis of the synthetic diesel product showed that it has the promise of being a drop-in fuel as super-low (less than 15 ppm)-sulfur diesel fuel. A detailed economic analysis showed that the Battelle liquefaction process is economical at between 1000 metric tons/day (MT/day) and 2000 MT/day. The unit capital cost for Battelle CTL process for making jet fuel is 50K USD/daily bbl compared to 151K USD/daily bbl for indirect CTL, based on 2011 dollars. The jet-fuel selling cost at the refinery, including a 12% capital cost factor (which included profit), for the Battelle CTL process is 61USD/bbl (1.45 USD/gallon). This is competitive with crude oil price of 48 USD/bbl. At the same time, the GHG emissions of 3.56 MT CO 2/MT fuel were lower than the GHG emissions of 3.79 MT CO 2/MTfuel for petroleum-based fuels and 7.77 MT CO 2/MT fuel for indirect CTL. Thus, the use of bio-solvents completely eliminates the need for carbon capture in the case of Battelle CTL process. The superior economics and low GHG emissions for the Battelle CTL process has thus sparked worldwide interest and some potential commercialization opportunities are emerging.« less

Structural and functional characterization of peptide-{beta}{sub 2}m fused HLA-A2/MART1{sub 27-35} complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen Chuanlai; Chang Chienchung; Zhang Jianqiong

The uses of soluble HLA class I/peptide complexes to monitor antigen reactive T cells are often hampered by their low-yield and high-cost production. As an alternative strategy, the peptide-{beta}{sub 2}m fused, 2-component (2C) HLA class I/peptide complex has been developed, but its application is limited due to the lack of the comparison of its structural and functional characteristics with those of its conventional 3-component (3C) counterpart. In this study, we have demonstrated that the 2C and 3C HLA-A2/MART1{sub 27-35} complexes have a similar chromatographical profile and comparable stability, but the former has 2.5 times higher yield and significantly higher bindingmore » ability with HLA-A2/MART1{sub 27-35} complex-specific receptors than the latter. Furthermore, the 2C complex has a comparable ability to stimulate specific CTL proliferation, but appears to be more effective in eliciting the cytotoxicity of antigen-specific CTL, as compared to its 3C counterpart.« less

Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1).

PubMed

Wallace, Aaron; West, Kim; Rothman, Alan L; Ennis, Francis A; Lu, Shan; Wang, Shixia

2013-10-01

In the current study, immune responses induced by Gag DNA vaccines with different designs were evaluated in Balb/C mice. The results demonstrated that the DNA vaccine with the full length wild type gag gene (Wt-Gag) mainly produced Gag antigens intracellularly and induced a higher level of cell-mediated immune (CMI) responses, as measured by IFN-gamma ELISPOT, intracellular cytokine staining (ICS), and cytotoxic T lymphocytes (CTL) assays against a dominant CD8(+) T cell epitope (AMQMLKETI). In contrast, the addition of a tissue plasminogen activator (tPA) leader sequence significantly improved overall Gag protein expression/secretion and Gag-specific antibody responses; however, Gag-specific CMI responses were decreased. The mutation of zinc-finger motif changed Gag protein expression patterns and reduced the ability to generate both CMI and antibody responses against Gag. These findings indicate that the structure and post-translational processing of antigens expressed by DNA vaccines play a critical role in eliciting optimal antibody or CMI responses.

Development of Epstein-Barr virus-specific memory T cell receptor clonotypes in acute infectious mononucleosis

PubMed Central

1996-01-01

The importance of cytotoxic T lymphocytes (CTLs) in the immunosurveillance of Epstein-Barr virus (EBV)-infected B cells is firmly established, and the viral antigens of CTL recognition in latent infection are well defined. The epitopes targeted by CTLs during primary infection have not been identified, however, and there is only limited information about T cell receptor (TCR) selection. In the present report, we have monitored the development of memory TCR-beta clonotypes selected in response to natural EBV infection in a longitudinal study of an HLA-B8+ individual with acute infectious mononucleosis (IM). By stimulating peripheral blood lymphocytes with HLA-B8+ EBV-transformed B lymphoblastoid cells, the primary virus- specific CTL response was shown to include specificities for two HLA-B8- restricted antigenic determinants, FLRGRAYGL and QAKWRLQTL, which are encoded within the latent EBV nuclear antigen EBNA-3. TCR-beta sequence analysis of CTL clones specific for each epitope showed polyclonal TCR- beta repertoire selection, with structural restrictions on recognition that indicated antigen-driven selection. Furthermore, longitudinal repertoire analysis revealed long-term preservation of a multiclonal effector response throughout convalescence, with the reemergence of distinct memory T cell clonotypes sharing similar structural restrictions. Tracking the progression of specific TCR-beta clonotypes and antigen-specific TCR-V beta family gene expression in the peripheral repertoire ex vivo using semiquantitative PCR strongly suggested that selective TCR-beta expansions were present at the clonotype level, but not at the TCR-V beta family level. Overall, in this first analysis of antigen-specific TCR development in IM, a picture of polyclonal TCR stimulation is apparent. This diversity may be especially important in the establishment of an effective CTL control during acute EBV infection and in recovery from disease. PMID:8920869

Restoration of Viral Immunity in Immunodeficient Humans by the Adoptive Transfer of T Cell Clones

NASA Astrophysics Data System (ADS)

Riddell, Stanley R.; Watanabe, Kathe S.; Goodrich, James M.; Li, Cheng R.; Agha, Mounzer E.; Greenberg, Philip D.

1992-07-01

The adoptive transfer of antigen-specific T cells to establish immunity is an effective therapy for viral infections and tumors in animal models. The application of this approach to human disease would require the isolation and in vitro expansion of human antigen-specific T cells and evidence that such T cells persist and function in vivo after transfer. Cytomegalovirus-specific CD8^+ cytotoxic T cell (CTL) clones could be isolated from bone marrow donors, propagated in vitro, and adoptively transferred to immunodeficient bone marrow transplant recipients. No toxicity developed and the clones provided persistent reconstitution of CD8^+ cytomegalovirus-specific CTL responses.

Adeno-associated virus capsid antigen presentation is dependent on endosomal escape

PubMed Central

Li, Chengwen; He, Yi; Nicolson, Sarah; Hirsch, Matt; Weinberg, Marc S.; Zhang, Ping; Kafri, Tal; Samulski, R. Jude

2013-01-01

Adeno-associated virus (AAV) vectors are attractive for gene delivery-based therapeutics, but data from recent clinical trials have indicated that AAV capsids induce a cytotoxic T lymphocyte (CTL) response that eliminates transduced cells. In this study, we used traditional pharmacological agents and AAV mutants to elucidate the pathway of capsid cross-presentation in AAV-permissive cells. Endosomal acidification inhibitors blocked AAV2 antigen presentation by over 90%, while proteasome inhibitors completely abrogated antigen presentation. Using mutant viruses that are defective for nuclear entry, we observed a 90% decrease in capsid antigen presentation. Different antigen presentation efficiencies were achieved by selectively mutating virion nuclear localization signals. Low antigen presentation was demonstrated with basic region 1 (BR1) mutants, despite relatively high transduction efficiency, whereas there was no difference in antigen presentation between BR2 and BR3 mutants defective for transduction, as compared with wild-type AAV2. These results suggest that effective AAV2 capsid antigen presentation is dependent on AAV virion escape from the endosome/lysosome for antigen degradation by proteasomes, but is independent of nuclear uncoating. These results should facilitate the design of effective strategies to evade capsid-specific CTL-mediated elimination of AAV-transduced target cells in future clinical trials. PMID:23454772

Antigen sensitivity of CD22-specific chimeric T cell receptors is modulated by target epitope distance from the cell membrane

PubMed Central

James, Scott E.; Greenberg, Philip D.; Jensen, Michael C.; Lin, Yukang; Wang, Jinjuan; Till, Brian G.; Raubitschek, Andrew A.; Forman, Stephen J.; Press, Oliver W.

2008-01-01

We have targeted CD22 as a novel tumor-associated antigen for recognition by human CTL genetically modified to express chimeric T cell receptors (cTCR) recognizing this surface molecule. CD22-specifc cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR+ CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR+ CTL exhibited lower levels of maximum lysis and lower antigen sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of antigen engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope but constructed as a truncated CD22 molecule to approximate the length of a TCR:pMHC complex. The reduced sensitivity of CD22-specific cTCR+ CTL for antigen-induced triggering of effector functions has potential therapeutic applications, as such cells selectively lysed B cell lymphoma lines expressing high levels of CD22 but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength – and consequently antigen sensitivity – can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate antigen density. PMID:18453625

Induction of immunity to antigens expressed by recombinant adeno-associated virus depends on the route of administration.

PubMed

Brockstedt, D G; Podsakoff, G M; Fong, L; Kurtzman, G; Mueller-Ruchholtz, W; Engleman, E G

1999-07-01

Recombinant adeno-associated virus (rAAV) is a replication-defective parvovirus which is being explored as a vector for gene therapy because of its broad host range, excellent safety profile, and durable transgene expression in infected hosts. rAAV has also been reported by several groups to induce little or no immune response to its encoded transgene products. In this study we examined the immunogenicity of rAAV by studying the immune response of C57BL/6 mice to a single dose of rAAV-encoding ovalbumin (AAV-Ova) administered by a variety of routes. Mice injected with AAV-Ova intraperitoneally (ip), intravenously, or subcutaneously developed potent ovalbumin-specific cytotoxic T lymphocytes (CTL) as well as anti-ovalbumin antibodies and antibodies to AAV. In contrast, mice injected with AAV-Ova intramuscularly developed a humoral response to the virus and the transgene but minimal ovalbumin-specific CTLs. The induced CTL response after ip administration of AAV-Ova protected mice against a subsequent tumor challenge with an ovalbumin-transfected B16 melanoma cell line. Studies of the mechanism by which AAV-Ova induces CTL confirmed that the virus delivers the transgene product into the classical MHC class I pathway of antigen processing. Mice that previously had been exposed to rAAV vectors failed to develop ovalbumin-specific CTL following administration of AAV-Ova. Analysis of these mice revealed the presence of circulating anti-AAV antibodies that blocked rAAV transduction in vitro and inhibited CTL induction in vivo. These results suggest a possible role for rAAV in the immunotherapy of malignancies and viral infections, although induced antibody responses to AAV may limit its ability to be administered for repeated vaccinations. Copyright 1999 Academic Press.

Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes

PubMed Central

Dekimpe, Emily; Van Woensel, Matthias; Roobrouck, Valerie D.; Bullens, Dominique M.; Pinxteren, Jef; Verfaillie, Catherine M.; Van Gool, Stefaan W.

2016-01-01

MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8+ cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was—even after major histocompatibility complex class I upregulation—insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8−CD69+ T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Significance Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune therapy and have the advantage over mesenchymal stem cells (MSCs) of large-scale manufacturing and banking potential and thus prompt availability, it is important to understand how MAPCs interact with immune cells to validate their widespread therapeutic applicability. Cytotoxic immune effector cells play a crucial role in immune homeostasis and in the pathogenesis of some autoimmune diseases. This study assessed for the first time the in vitro influence of a clinical-grade human MAPC product (MultiStem) on the cytotoxic function of CD8+ T cells (CTLs) by evaluating the immunogenicity of MAPCs and the susceptibility of MAPCs toward CTL-mediated lysis and by analyzing the mechanism of MAPC-mediated modulation of CTL functionality. These results may represent a highly relevant contribution to the current knowledge and, in combination with the results of future phase II/III trials using MultiStem, could lead to an intriguing continuation of stem cell-based research for immunotherapy. PMID:27465071

Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes.

PubMed

Plessers, Jeroen; Dekimpe, Emily; Van Woensel, Matthias; Roobrouck, Valerie D; Bullens, Dominique M; Pinxteren, Jef; Verfaillie, Catherine M; Van Gool, Stefaan W

2016-12-01

: MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8 + cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was-even after major histocompatibility complex class I upregulation-insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8 - CD69 + T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune therapy and have the advantage over mesenchymal stem cells (MSCs) of large-scale manufacturing and banking potential and thus prompt availability, it is important to understand how MAPCs interact with immune cells to validate their widespread therapeutic applicability. Cytotoxic immune effector cells play a crucial role in immune homeostasis and in the pathogenesis of some autoimmune diseases. This study assessed for the first time the in vitro influence of a clinical-grade human MAPC product (MultiStem) on the cytotoxic function of CD8 + T cells (CTLs) by evaluating the immunogenicity of MAPCs and the susceptibility of MAPCs toward CTL-mediated lysis and by analyzing the mechanism of MAPC-mediated modulation of CTL functionality. These results may represent a highly relevant contribution to the current knowledge and, in combination with the results of future phase II/III trials using MultiStem, could lead to an intriguing continuation of stem cell-based research for immunotherapy. ©AlphaMed Press.

Application of a MRI based index to longitudinal atrophy change in Alzheimer disease, mild cognitive impairment and healthy older individuals in the AddNeuroMed cohort

PubMed Central

Aguilar, Carlos; Muehlboeck, J-Sebastian; Mecocci, Patrizia; Vellas, Bruno; Tsolaki, Magda; Kloszewska, Iwona; Soininen, Hilkka; Lovestone, Simon; Wahlund, Lars-Olof; Simmons, Andrew; Westman, Eric

2014-01-01

Cross sectional studies of patients at risk of developing Alzheimer disease (AD) have identified several brain regions known to be prone to degeneration suitable as biomarkers, including hippocampal, ventricular, and whole brain volume. The aim of this study was to longitudinally evaluate an index based on morphometric measures derived from MRI data that could be used for classification of AD and healthy control subjects, as well as prediction of conversion from mild cognitive impairment (MCI) to AD. Patients originated from the AddNeuroMed project at baseline (119 AD, 119 MCI, 110 controls (CTL)) and 1-year follow-up (62 AD, 73 MCI, 79 CTL). Data consisted of 3D T1-weighted MR images, demographics, MMSE, ADAS-Cog, CERAD and CDR scores, and APOE e4 status. We computed an index using a multivariate classification model (AD vs. CTL), using orthogonal partial least squares to latent structures (OPLS). Sensitivity, specificity and AUC were determined. Performance of the classifier (AD vs. CTL) was high at baseline (10-fold cross-validation, 84% sensitivity, 91% specificity, 0.93 AUC) and at 1-year follow-up (92% sensitivity, 74% specificity, 0.93 AUC). Predictions of conversion from MCI to AD were good at baseline (77% of MCI converters) and at follow-up (91% of MCI converters). MCI carriers of the APOE e4 allele manifested more atrophy and presented a faster cognitive decline when compared to non-carriers. The derived index demonstrated a steady increase in atrophy over time, yielding higher accuracy in prediction at the time of clinical conversion. Neuropsychological tests appeared less sensitive to changes over time. However, taking the average of the two time points yielded better correlation between the index and cognitive scores as opposed to using cross-sectional data only. Thus, multivariate classification seemed to detect patterns of AD changes before conversion from MCI to AD and including longitudinal information is of great importance. PMID:25071554

Resistance-based interval exercise acutely improves endothelial function in type 2 diabetes.

PubMed

Francois, Monique E; Durrer, Cody; Pistawka, Kevin J; Halperin, Frank A; Little, Jonathan P

2016-11-01

Different modes of exercise, disease, and training status can modify endothelial shear stress and result in distinct effects on endothelial function. To date, no study has examined the influence of type 2 diabetes (T2D) and training status on the acute endothelial response to different modes of interval exercise (INT). We examined the effect of a single session of resistance- and cardio-based INT compared with a time-matched control on endothelial function in 12 age-matched T2D participants, 12 untrained, and 11 trained adults (aged 56 ± 7 yr). Flow-mediated dilation (%FMD) of the brachial artery was assessed at baseline and immediately, 1, and 2 h after an acute bout of cardio interval (C-INT), resistance interval (R-INT), and seated control (CTL); these interventions were randomized and separated by >2 days. C-INT involved seven 1-min cycling intervals at 85% of peak power with 1-min recovery between. R-INT involved the same pattern of seven 1-min intervals using leg resistance exercises. Endothelial function (%FMD) was improved after R-INT in all groups (Condition × Time interaction, P < 0.01), an effect that was most robust in T2D where %FMD was higher immediately (+4.0 ± 2.8%), 1 h (+2.5 ± 2.5%), and 2 h (+1.9 ± 1.9%) after R-INT compared with CTL (P < 0.01 for all). C-INT improved %FMD in T2D at 1-h postexercise (+1.6 ± 2.2%, P = 0.03) compared with CTL. In conclusion, R-INT acutely improves endothelial function throughout the 2-h postexercise period in T2D patients. The long-term impact of resistance exercise performed in an interval pattern is warranted. Copyright © 2016 the American Physiological Society.

Resistance-based interval exercise acutely improves endothelial function in type 2 diabetes

PubMed Central

Francois, Monique E.; Durrer, Cody; Pistawka, Kevin J.; Halperin, Frank A.

2016-01-01

Different modes of exercise, disease, and training status can modify endothelial shear stress and result in distinct effects on endothelial function. To date, no study has examined the influence of type 2 diabetes (T2D) and training status on the acute endothelial response to different modes of interval exercise (INT). We examined the effect of a single session of resistance- and cardio-based INT compared with a time-matched control on endothelial function in 12 age-matched T2D participants, 12 untrained, and 11 trained adults (aged 56 ± 7 yr). Flow-mediated dilation (%FMD) of the brachial artery was assessed at baseline and immediately, 1, and 2 h after an acute bout of cardio interval (C-INT), resistance interval (R-INT), and seated control (CTL); these interventions were randomized and separated by >2 days. C-INT involved seven 1-min cycling intervals at 85% of peak power with 1-min recovery between. R-INT involved the same pattern of seven 1-min intervals using leg resistance exercises. Endothelial function (%FMD) was improved after R-INT in all groups (Condition × Time interaction, P < 0.01), an effect that was most robust in T2D where %FMD was higher immediately (+4.0 ± 2.8%), 1 h (+2.5 ± 2.5%), and 2 h (+1.9 ± 1.9%) after R-INT compared with CTL (P < 0.01 for all). C-INT improved %FMD in T2D at 1-h postexercise (+1.6 ± 2.2%, P = 0.03) compared with CTL. In conclusion, R-INT acutely improves endothelial function throughout the 2-h postexercise period in T2D patients. The long-term impact of resistance exercise performed in an interval pattern is warranted. PMID:27638878

Studies of Cell-Mediated Immunity Against Immune Disorders Using Synthetic Peptides and Rotating Bioreactor System

NASA Technical Reports Server (NTRS)

Sastry, Jagannadha K.

1997-01-01

Our proposed experiments included: (1) immunzing mice with synthetic peptides; (2) preparing spleen and lymph node cells; (3) growing them under conventional conditions as well as in the rotatory vessel in appropriate medium reconstituting with synthetic peptides and/or cytokines as needed; and (4) comparing at regular time intervals the specific CTL activity as well as helper T-cell activity (in terms of both proliferative responses and cytokine production) using established procedures in my laboratory. We further proposed that once we demonstrated the merit of rotatory vessel technology to achieve desired results, these studies would be expanded to include immune cells from non-human primates (rhesus monkeys and chimpanzees) and also humans. We conducted a number of experiments to determine CTL induction by the synthetic peptides corresponding to antigenic proteins in HIV and HPV in different mouse strains that express MHC haplotypes H-2b or H-2d. We immunized mice with 100 ug of the synthetic peptide, suspended in sterile water, and emulsified in CFA (1:1). The immune lymph node cells obtained after 7 days were restimulated by culturing in T25 flask, HARV-10, or STLV-50, in the presence of the peptide at 20 ug/ml. The results from the 5'Cr-release assay consistently revealed complete abrogation of CTL activity of cells grown in the bioreactors (both HARV and STLV), while significant antigen-specific CTL activity was observed with cells cultured in tissue culture flasks. Thus, overall the data we generated in this study proved the usefulness of the NASA-developed developed technology for understanding the known immune deficiency during space travel. Additionally, this ex vivo microgravity technology since it mimics effectively the in vivo situation, it is also useful in understanding immune disorders in general. Thus, our proposed studies in TMC-NASA contract round II application benefit from data generated in this TMC-NASA contract round I study.

Activation of antigen-specific cytotoxic T lymphocytes by beta 2-microglobulin or TAP1 gene disruption and the introduction of recipient-matched MHC class I gene in allogeneic embryonic stem cell-derived dendritic cells.

PubMed

Matsunaga, Yusuke; Fukuma, Daiki; Hirata, Shinya; Fukushima, Satoshi; Haruta, Miwa; Ikeda, Tokunori; Negishi, Izumi; Nishimura, Yasuharu; Senju, Satoru

2008-11-01

A method for the genetic modification of dendritic cells (DC) was previously established based on the in vitro differentiation of embryonic stem (ES) cells to DC (ES-DC). The unavailability of human ES cells genetically identical to the patients will be a problem in the future clinical application of this technology. This study attempted to establish a strategy to overcome this issue. The TAP1 or beta(2)-microglobulin (beta(2)m) gene was disrupted in 129 (H-2(b))-derived ES cells and then expression vectors for the H-2K(d) or beta(2)m-linked form of K(d) (beta2m-K(d)) were introduced, thus resulting in two types of genetically engineered ES-DC, TAP1(-/-)/K(d) ES-DC and beta(2)m(-/-)/beta(2)m-K(d) ES-DC. As intended, both of the transfectant ES-DC expressed K(d) but not the intrinsic H-2(b) haplotype-derived MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) and TAP1(-/-)/K(d) ES-DC were not recognized by pre-activated H-2(b)-reactive CTL and did not prime H-2(b) reactive CTL in vitro or in vivo. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC and TAP1(-/-)/K(d) ES-DC had a survival advantage in comparison to beta(2)m(+/-)/beta(2)m-K(d) ES-DC and TAP1(+/+)/K(d) ES-DC, when transferred into BALB/c mice. K(d)-restricted RSV-M2-derived peptide-loaded ES-DC could prime the epitope-specific CTL upon injection into the BALB/c mice, irrespective of the cell surface expression of intrinsic H-2(b) haplotype-encoded MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC were significantly more efficient in eliciting immunity against RSV M2 protein-expressing tumor cells than beta(2)m(+/-)/beta(2)m-K(d) ES-DC. The modification of the beta(2)m or TAP gene may therefore be an effective strategy to resolve the problem of HLA class I allele mismatch between human ES or induced pluripotent stem cells and the recipients to be treated.

Cytotoxic T lymphocytes to Ebola Zaire virus are induced in mice by immunization with liposomes containing lipid A.

PubMed

Rao, M; Matyas, G R; Grieder, F; Anderson, K; Jahrling, P B; Alving, C R

1999-08-06

An eight amino acid sequence (TELRTFSI) present in the carboxy terminal end (aa 577-584) of membrane-anchored GP, the major structural protein of Ebola virus, was identified as an H-2k-specific murine cytotoxic T cell epitope. Cytotoxic T lymphocytes (CTLs) to this epitope were induced by immunizing B10.BR mice intravenously with either irradiated Ebola virus or with irradiated Ebola virus encapsulated in liposomes containing lipid A. The CTL response induced by irradiated Ebola virus could not be sustained after the second round of in vitro stimulation of immune splenocytes with the peptide, unless the irradiated virus was encapsulated in liposomes containing lipid A. The identification of an Ebola GP-specific CTL epitope and the requirement of liposomal lipid A for CTL memory recall responses could prove to be a promising approach for developing a vaccine against Ebola virus infection.

In vitro Peptide Immunization ofTargetTax Protein HumanT-Cell Leukemia Virus Type 1 – Specific CD4+ Helper T Lymphocytes

PubMed Central

Kobayashi, Hiroya; Ngato, Toshihiro; Sato, Keisuke; Aoki, Naoko; Kimura, Shoji; Tanaka, Yuetsu; Aizawa, Hitoshi; Tateno, Masatoshi; Celis, Esteban

2006-01-01

Purpose Adult T-cell leukemia/lymphoma induced by human T-cell leukemia virus type 1 (HTLV-1) is usually a fatal lymphoproliferative malignant disease. HTLV-1 Tax protein plays a critical role in HTLV-1-associated leukemogenesis and is an attractive target for vaccine development. Although HTLV-1Tax is the most dominant antigen for HTLV-1-specific CD8+ CTLs in HTLV-1-infected individuals, few epitopes recognized by CD4+ helper T lymphocytes in HTLV-1Tax protein have been described.The aim of the present study was to study T-helper-cell responses to HTLV-1 Tax and to identify naturally processed MHC class II – restricted epitopes that could be used for vaccine development. Experimental Design An MHC class II binding peptide algorithm was used to predict potential T-helper cell epitope peptides from HTLV-1 Tax. We assessed the ability of the corresponding peptides to elicit helper T-cell responses by in vitro vaccination of purified CD4+ T lymphocytes. Results Peptides Tax191–205 and Tax305–319 were effective in inducingT-helper-cell responses. Although Tax191–205 was restricted by the HLA-DR1 and DR9 alleles, responses to Tax305–319 were restricted by either DR15 or DQ9. Both these epitopes were found to be naturally processed by HTLV-1+ T-cell lymphoma cells and by autologous antigen-presenting cells that were pulsed with HTLV-1Tax+ tumor lysates. Notably, the two newly identified helper T-cell epitopes are found to lie proximal to known CTL epitopes, which will facilitate the development of prophylactic peptide – based vaccine capable of inducing simultaneous CTL andT-helper responses. Conclusion Our data suggest that HTLV-1 Tax protein could serve as tumor-associated antigen for CD4+ helper T cells and that the present epitopes might be used for T-cell-based immunotherapy against tumors expressing HTLV-1. PMID:16778109

Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames

PubMed Central

1996-01-01

An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within introns. We have used a series of frameshifts (FS) introduced into the Influenza A/PR/8 /34 nucleoprotein (NP) gene to confirm the previous in vitro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presentation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of translation. By introducing additional mutations to the construct exhibiting the most potent presentation, we have identified initiation codon readthrough (termed scanthrough here, where the scanning ribosome bypasses the conventional initiation codon, initiating translation further downstream) as the likely mechanism of epitope production. Further mutational analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a major source of processing substrate in our system. These findings suggest (i) that the full array of self- and pathogen-derived epitopes available during thymic selection and infection has not been fully appreciated and (ii) that cryptic epitope expression should be considered when the specificity of a CTL response cannot be identified or in therapeutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules. PMID:8879204

Soil compaction associated with cut-to-length and whole-tree harvesting of a coniferous forest

Treesearch

Sang-Kyun Han; Han Han-Sup; Deborah Page-Dumroese; Leonard R. Johnson

2009-01-01

The degree and extent of soil compaction, which may reduce productivity of forest soils, is believed to vary by the type of harvesting system, and a field-based study was conducted to compare soil compaction from cut-to-length (CTL) and whole-tree (WT) harvesting operations. The CTL harvesting system used less area to transport logs to the landings than did the WT...

Induction of human dendritic cell maturation using transfection with RNA encoding a dominant positive toll-like receptor 4.

PubMed

Cisco, Robin M; Abdel-Wahab, Zeinab; Dannull, Jens; Nair, Smita; Tyler, Douglas S; Gilboa, Eli; Vieweg, Johannes; Daaka, Yehia; Pruitt, Scott K

2004-06-01

Maturation of dendritic cells (DC) is critical for the induction of Ag-specific immunity. Ag-loaded DC matured with LPS, which mediates its effects by binding to Toll-like receptor 4 (TLR4), induce Ag-specific CTL in vitro and in vivo in animal models. However, clinical use of LPS is limited due to potential toxicity. Therefore, we sought to mimic the maturation-inducing effects of LPS on DC by stimulating TLR4-mediated signaling in the absence of exogenous LPS. We developed a constitutively active TLR4 (caTLR4) and demonstrated that transfection of human DC with RNA encoding caTLR4 led to IL-12 and TNF-alpha secretion. Transfection with caTLR4 RNA also induced a mature DC phenotype. Functionally, transfection of DC with caTLR4 RNA enhanced allostimulation of CD4(+) T cells. DC transfected with RNA encoding the MART (Melan-A/MART-1) melanoma Ag were then used to stimulate T cells in vitro. Cotransfection of these DC with caTLR4 RNA enhanced the generation of MART-specific CTL. This CTL activity was superior to that seen when DC maturation was induced using either LPS or a standard mixture of cytokines (TNF-alpha, IL-6, IL-1beta, and PGE(2)). We conclude that transfection of DC with RNA encoding a functional signaling protein, such as caTLR4, may provide a new tool for studying TLR signaling in DC and may be a promising approach for the induction of DC maturation for tumor immunotherapy.

Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

PubMed

Chahroudi, A; Silvestri, G; Feinberg, M B

2003-10-01

Cytotoxic T lymphocytes (CTLs) play a major role in the immune response against viruses and other intracellular pathogens. In addition, CTLs are implicated in the control of tumor cells in certain settings. Accurate measures of CTL function are of critical importance to study the pathogenesis of infectious diseases and to evaluate the efficacy of new vaccines and immunotherapies. To this end, we have recently developed a flow cytometry-based CTL (FCC) assay that measures the CTL-induced caspase activation within target cells using cell permeable fluorogenic caspase substrates. This novel assay reliably detects, by flow cytometry or fluorescence/confocal microscopy, antigen-specific CTLs in a wide variety of human and murine systems, and is safer and more informative than the standard 51Cr-release assay. In addition, the flow cytometric CTL (FCC) assay provides an alternative method that is often more sensitive and physiologically informative when compared to previously described FCC assays, as it measures a biological indicator of apoptosis within the target cell. The FCC assay may thus represent a useful tool to further understand the molecular and cellular mechanisms that underlie CTL-mediated killing during tumorigenesis or following infection with viruses or other intracellular pathogens.

Reexamining the role of choline transporter-like (Ctlp) proteins in choline transport.

PubMed

Zufferey, Rachel; Santiago, Teresa C; Brachet, Valerie; Ben Mamoun, Choukri

2004-02-01

In Saccharomyces cerevisiae, choline enters the cell via a single high-affinity transporter, Hnmlp. hnm1delta cells lacking HNM1 gene are viable. However, they are unable to transport choline suggesting that no additional active choline transporters are present in this organism. A complementation study of a choline auxotrophic mutant, ctrl-ise (hnm1-ise), using a cDNA library from Torpedo marmorata electric lobe identified a membrane protein named Torpedo marmorata choline transporter-like, tCtl1p. tCtllp was proposed to mediate a high-affinity choline transport (O'Regan et al., 1999, Proc. Natl. Acad. Sci.). Homologs of tCtl1p have been identified in other organisms, including yeast (Pns1p, YOR161c) and are postulated to function as choline transporters. Here we provide several lines of evidence indicating that Ctlp proteins are not involved in choline transport. Loss of PNS1 has no effect on choline transport and overexpression of either PNS1 or tCTL1 does not restore choline uptake activity of choline transport-defective mutants. The data presented here call into question the role of proteins of the CTL family in choline transport and suggest that the mechanism by which tCTL1 complements hnm1-ise mutant is independent of its ability to transport choline.

Defining the expression hierarchy of latent T-cell epitopes in Epstein-Barr virus infection with TCR-like antibodies

PubMed Central

Sim, Adrian Chong Nyi; Too, Chien Tei; Oo, Min Zin; Lai, Junyun; Eio, Michelle Yating; Song, Zhenying; Srinivasan, Nalini; Tan, Diane Ai Lin; Pang, Shyue Wei; Gan, Shu Uin; Lee, Kok Onn; Loh, Thomas Kwok Seng; Chen, Jianzhu; Chan, Soh Ha; MacAry, Paul Anthony

2013-01-01

Epstein-Barr virus (EBV) is a gamma herpesvirus that causes a life-long latent infection in human hosts. The latent gene products LMP1, LMP2A and EBNA1 are expressed by EBV-associated tumors and peptide epitopes derived from these can be targeted by CD8 Cytotoxic T-Lymphocyte (CTL) lines. Whilst CTL-based methodologies can be utilized to infer the presence of specific latent epitopes, they do not allow a direct visualization or quantitation of these epitopes. Here, we describe the characterization of three TCR-like monoclonal antibodies (mAbs) targeting the latent epitopes LMP1125–133, LMP2A426–434 or EBNA1562–570 in association with HLA-A0201. These are employed to map the expression hierarchy of endogenously generated EBV epitopes. The dominance of EBNA1562–570 in association with HLA-A0201 was consistently observed in cell lines and EBV-associated tumor biopsies. These data highlight the discordance between MHC-epitope density and frequencies of associated CTL with implications for cell-based immunotherapies and/or vaccines for EBV-associated disease. PMID:24240815

In vivo induction of a high-avidity, high-frequency cytotoxic T-lymphocyte response is associated with antiviral protective immunity.

PubMed

Sedlik, C; Dadaglio, G; Saron, M F; Deriaud, E; Rojas, M; Casal, S I; Leclerc, C

2000-07-01

Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8(+) cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8(+) class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8(+) T-cell epitopes, bound to 1-microm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503-7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4(+) T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies.

In Vivo Induction of a High-Avidity, High-Frequency Cytotoxic T-Lymphocyte Response Is Associated with Antiviral Protective Immunity†

PubMed Central

Sedlik, C.; Dadaglio, G.; Saron, M. F.; Deriaud, E.; Rojas, M.; Casal, S. I.; Leclerc, C.

2000-01-01

Many approaches are currently being developed to deliver exogenous antigen into the major histocompatibility complex class I-restricted antigen pathway, leading to in vivo priming of CD8+ cytotoxic T cells. One attractive possibility consists of targeting the antigen to phagocytic or macropinocytic antigen-presenting cells. In this study, we demonstrate that strong CD8+ class I-restricted cytotoxic responses are induced upon intraperitoneal immunization of mice with different peptides, characterized as CD8+ T-cell epitopes, bound to 1-μm synthetic latex microspheres and injected in the absence of adjuvant. The cytotoxic response induced against a lymphocytic choriomeningitis virus (LCMV) peptide linked to these microspheres was compared to the cytotoxic T-lymphocyte (CTL) response obtained upon immunization with the nonreplicative porcine parvovirus-like particles (PPV:VLP) carrying the same peptide (PPV:VLP-LCMV) previously described (C. Sedlik, M. F. Saron, J. Sarraseca, I. Casal, and C. Leclerc, Proc. Natl. Acad. Sci. USA 94:7503–7508, 1997). We show that the induction of specific CTL activity by peptides bound to microspheres requires CD4+ T-cell help in contrast to the CTL response obtained with the peptide delivered by viral pseudoparticles. Furthermore, PPV:VLP are 100-fold more efficient than microspheres in generating a strong CTL response characterized by a high frequency of specific T cells of high avidity. Moreover, PPV:VLP-LCMV are able to protect mice against a lethal LCMV challenge whereas microspheres carrying the LCMV epitope fail to confer such protection. This study demonstrates the crucial involvement of the frequency and avidity of CTLs in conferring antiviral protective immunity and highlights the importance of considering these parameters when developing new vaccine strategies. PMID:10846055

Bioactivity and Safety of IL13Rα2-Redirected Chimeric Antigen Receptor CD8+ T Cells in Patients with Recurrent Glioblastoma.

PubMed

Brown, Christine E; Badie, Behnam; Barish, Michael E; Weng, Lihong; Ostberg, Julie R; Chang, Wen-Chung; Naranjo, Araceli; Starr, Renate; Wagner, Jamie; Wright, Christine; Zhai, Yubo; Bading, James R; Ressler, Julie A; Portnow, Jana; D'Apuzzo, Massimo; Forman, Stephen J; Jensen, Michael C

2015-09-15

A first-in-human pilot safety and feasibility trial evaluating chimeric antigen receptor (CAR)-engineered, autologous primary human CD8(+) cytotoxic T lymphocytes (CTL) targeting IL13Rα2 for the treatment of recurrent glioblastoma (GBM). Three patients with recurrent GBM were treated with IL13(E13Y)-zetakine CD8(+) CTL targeting IL13Rα2. Patients received up to 12 local infusions at a maximum dose of 10(8) CAR-engineered T cells via a catheter/reservoir system. We demonstrate the feasibility of manufacturing sufficient numbers of autologous CTL clones expressing an IL13(E13Y)-zetakine CAR for redirected HLA-independent IL13Rα2-specific effector function for a cohort of patients diagnosed with GBM. Intracranial delivery of the IL13-zetakine(+) CTL clones into the resection cavity of 3 patients with recurrent disease was well-tolerated, with manageable temporary brain inflammation. Following infusion of IL13-zetakine(+) CTLs, evidence for transient anti-glioma responses was observed in 2 of the patients. Analysis of tumor tissue from 1 patient before and after T-cell therapy suggested reduced overall IL13Rα2 expression within the tumor following treatment. MRI analysis of another patient indicated an increase in tumor necrotic volume at the site of IL13-zetakine(+) T-cell administration. These findings provide promising first-in-human clinical experience for intracranial administration of IL13Rα2-specific CAR T cells for the treatment of GBM, establishing a foundation on which future refinements of adoptive CAR T-cell therapies can be applied. ©2015 American Association for Cancer Research.

A New Approach to Simulate Groundwater Table Dynamics and Its Validation in China

NASA Astrophysics Data System (ADS)

Lv, M.; Lu, H.; Dan, L.; Yang, K.

2017-12-01

The groundwater has very important role in hydrology-climate-human activity interaction. But the groundwater table dynamics currently is not well simulated in global-scale land surface models. Meanwhile, almost all groundwater schemes are adopting a specific yield method to estimate groundwater table, in which how to determine the proper specific yield value remains a big challenge. In this study, we developed a Soil Moisture Correlation (SMC) method to simulate groundwater table dynamics. We coupled SMC with a hydrological model (named as NEW) and compared it with the original model in which a specific yield method is used (named as CTL). Both NEW and CTL were tested in Tangnaihai Subbasin of Yellow River and Jialingjiang Subbasin along Yangtze River, where underground water is less impacted by human activities. The simulated discharges by NEW and CTL are compared against gauge observations. The comparison results reveal that after calibration both models are able to reproduce the discharge well. However, there is no parameter needed to be calibrated for SMC. It indicates that SMC method is more efficient and easy-to-use than the specific yield method. Since there is no direct groundwater table observation in these two basins, simulated groundwater table were compared with a global data set provided by Fan et al. (2013). Both NEW and CTL estimate lower depths than Fan does. Moreover, when comparing the variation of terrestrial water storage (TWS) derived from NEW with that observed by GRACE, good agreements were confirmed. It demonstrated that SMC method is able to reproduce groundwater level dynamics reliably.

KIR and HLA Genotypes Implicated in Reduced Killer Lymphocytes Immunity Are Associated with Vogt-Koyanagi-Harada Disease

PubMed Central

Levinson, Ralph D.; Yung, Madeline; Meguro, Akira; Ashouri, Elham; Yu, Fei; Mizuki, Nobuhisa; Ohno, Shigeaki

2016-01-01

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are killer lymphocytes that provide defense against viral infections and tumor transformation. Analogous to that of CTL, interactions of killer-cell immunoglobulin-like receptors (KIR) with specific human leukocyte antigen (HLA) class I ligands calibrate NK cell education and response. Gene families encoding KIRs and HLA ligands are located on different chromosomes, and feature variation in the number and type of genes. The independent segregation of KIR and HLA genes results in variable KIR-HLA interactions in individuals, which may impact disease susceptibility. We tested whether KIR-HLA combinations are associated with Vogt-Koyanagi-Harada (VKH) disease, a bilateral granulomatous panuveitis that has strong association with HLA-DR4. We present a case control study of 196 VKH patients and 209 controls from a highly homogeneous native population of Japan. KIR and HLA class I genes were typed using oligonucleotide hybridization method and analyzed using two-tailed Fisher’s exact probabilities. The incidence of Bx-KIR genotypes was decreased in VKH patients (odds ratio [OR] 0.58, P = 0.007), due primarily to a decrease in centromeric B-KIR motif and its associated KIRs 2DS2, 2DL2, 2DS3, and 2DL5B. HLA-B22, implicated in poor immune response, was increased in VKH (OR = 4.25, P = 0.0001). HLA-Bw4, the ligand for KIR3DL1, was decreased in VKH (OR = 0.59, P = 0.01). The KIR-HLA combinations 2DL2+C1/C2 and 3DL1+Bw4, which function in NK education, were also decreased in VKH (OR = 0.49, P = 0.012; OR = 0.59, P = 0.013). Genotypes missing these two inhibitory KIR-HLA combinations in addition to missing activating KIRs 2DS2 and 2DS3 were more common in VKH (OR = 1.90, P = 0.002). These results suggest that synergistic hyporesponsiveness of NK cells (due to poor NK education along with missing of activating KIRs) and CTL (due to HLA-B22 restriction) fail to mount an effective immune response against viral-infection that may trigger VKH pathogenesis in genetically susceptible individuals, such as HLA-DR4 carriers. PMID:27490240

Established human papillomavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides.

PubMed

Zwaveling, Sander; Ferreira Mota, Sandra C; Nouta, Jan; Johnson, Mark; Lipford, Grayson B; Offringa, Rienk; van der Burg, Sjoerd H; Melief, Cornelis J M

2002-07-01

Peptide-based vaccines aimed at the induction of effective T cell responses against established cancers have so far only met with limited clinical success and clearly need to be improved. In a preclinical model of human papillomavirus (HPV)16-induced cervical cancer we show that prime-boost vaccinations with the HPV16-derived 35 amino-acid long peptide E7(43-77), containing both a CTL epitope and a Th epitope, resulted in the induction of far more robust E7-specific CD8(+) T cell responses than vaccinations with the minimal CTL epitope only. We demonstrate that two distinct mechanisms are responsible for this effect. First, vaccinations with the long peptide lead to the generation of E7-specific CD4(+) Th cells. The level of the induced E7-specific CD8(+) T cell response proved to be dependent on the interactions of these Th cells with professional APC. Second, we demonstrate that vaccination with the long peptide and dendritic cell-activating agents resulted in a superior induction of E7-specific CD8(+) T cells, even when T cell help was excluded. This suggests that, due to its size, the long peptide was preferably endocytosed, processed, and presented by professional APCs. Moreover, the efficacy of this superior HPV-specific T cell induction was demonstrated in therapeutic prime-boost vaccinations in which the long peptide admixed with the dendritic cell-activating adjuvant oligodeoxynucleotide-CpG resulted in the eradication of large, established HPV16-expressing tumors. Because the vaccine types used in this study are easy to prepare under good manufacturing practice conditions and are safe to administer to humans, these data provide important information for future clinical trials.

A Subdominant CD8+ Cytotoxic T Lymphocyte (CTL) Epitope from the Plasmodium yoelii Circumsporozoite Protein Induces CTLs That Eliminate Infected Hepatocytes from Culture

PubMed Central

Franke, Eileen D.; Sette, Alessandro; Sacci, John; Southwood, Scott; Corradin, Giampietro; Hoffman, Stephen L.

2000-01-01

Previous studies indicated that the Plasmodium yoelii circumsporozoite protein (PyCSP) 57–70 region elicits T cells capable of eliminating infected hepatocytes in vitro. Herein, we report that the PyCSP58–67 sequence contains an H-2d binding motif, which binds purified Kd molecules in vitro with low affinity (3,267 nM) and encodes an H-2d-restricted cytotoxic T lymphocyte (CTL) epitope. Immunization of BALB/c mice with three doses of a multiple antigen peptide (MAP) construct containing four branches of amino acids 57 to 70 linked to a lysine-glycine core [MAP4(PyCSP57–70)] and Lipofectin as the adjuvant induced both T-cell proliferation and a peptide-specific CTL response that was PyCSP59–67 specific, H-2d restricted, and CD8+ T cell dependent. Immunization with either DNA encoding the PyCSP or irradiated sporozoites demonstrated that this CTL epitope is subdominant since it is not recognized in the context of whole CSP immunization. The biological relevance of this CTL response was underlined by the demonstration that it could mediate genetically restricted, CD8+- and nitric-oxide-dependent elimination of infected hepatocytes in vitro, as well as partial protection of BALB/c mice against sporozoite challenge. These findings indicate that subdominant epitopes with low major histocompatibility complex affinity can be used to engineer epitope-based vaccines and have implications for the selection of epitopes for subunit-based vaccines. PMID:10816491

Granzyme B ELISPOT assay to measure influenza-specific cellular immunity.

PubMed

Salk, Hannah M; Haralambieva, Iana H; Ovsyannikova, Inna G; Goergen, Krista M; Poland, Gregory A

2013-12-15

The immunogenicity and efficacy of influenza vaccination are markedly lower in the elderly. Granzyme B (GrzB), quantified in fresh cell lysates, has been suggested to be a marker of cytotoxic T lymphocyte (CTL) response and a predictor of influenza illness among vaccinated older individuals. We have developed an influenza-specific GrzB ELISPOT assay using cryopreserved PBMCs. This method was tested on 106 healthy older subjects (ages 50-74) at baseline (Day 0) and three additional time points post-vaccination (Day 3, Day 28, Day 75) with influenza A/H1N1-containing vaccine. No significant difference was seen in GrzB response between any of the time points, although influenza-specific GrzB response appears to be elevated at all post-vaccination time points. There was no correlation between GrzB response and hemagglutination inhibition (HAI) titers, indicating no relationship between the cytolytic activity and humoral antibody levels in this cohort. Additionally, a significant negative correlation between GrzB response and age was observed. These results reveal a reduction in influenza-specific GrzB response as one ages. In conclusion, we have developed and optimized an influenza-specific ELISPOT assay for use with frozen cells to quantify the CTL-specific serine protease GrzB, as a measure of cellular immunity after influenza vaccination. © 2013.

Generation of CTL responses against pancreatic cancer in vitro using dendritic cells co-transfected with MUC4 and survivin RNA.

PubMed

Chen, Jiang; Guo, Xiao-Zhong; Li, Hong-Yu; Liu, Xu; Ren, Li-Nan; Wang, Di; Zhao, Jia-Jun

2013-09-23

Pancreatic cancer (PC) is one of the most devastating human malignancies without effective therapies. Tumor vaccine based on RNA-transfected dendritic cells (DCs) has emerged as an alternative therapeutic approach for a variety of human cancers including advanced PC. In the present study we compared the cytotoxic T lymphocyte (CTL) responses against PC cells in vitro, which were induced by DCs co-transfected with two mRNAs of tumor associated-antigens (TAA) MUC4 and survivin, versus DCs transfected with a single mRNA encoding either MUC4 or survivin. DCs co-transfected with two TAA mRNAs were found to induce stronger CTL responses against PC target cells in vitro, compared with the DCs transfected with a single mRNA. Moreover, the antigen-specific CTL responses were MHC class I-restricted. These results provide an experimental foundation for further clinical investigations of DC vaccines encoding multiple TAA epitopes for metastatic PC. Copyright © 2013 Elsevier Ltd. All rights reserved.

Molecular and functional characterization of choline transporter in the human trophoblastic cell line JEG-3 cells.

PubMed

Yara, M; Iwao, B; Hara, N; Yamanaka, T; Uchino, H; Inazu, M

2015-06-01

Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine (PC), the methyl donor betaine and the neurotransmitter acetylcholine (ACh), which is involved in several vital biological functions that play key roles in fetal development. In this study, we examined the molecular and functional characteristics of choline uptake in the human trophoblastic cell line JEG-3. We examined [(3)H]choline uptake in the human trophoblastic cell line JEG-3. The expression of CTL1 and CTL2 was evaluated by quantitative real-time PCR, western blotting and immunocytochemistry. We demonstrated that JEG-3 cells take up [(3)H] choline by a saturable process that is mediated by a Na(+)-independent and pH-dependent transport system. The cells have two different [(3)H] choline transport systems, high- and low-affinity, with Km values of 28.4 ± 5.0 μM and 210.6 ± 55.1 μM, respectively. Cationic compounds and hemicholinium-3 (HC-3) inhibited choline uptake. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in JEG-3 cells and were localized to the plasma membrane. The present results suggest that choline is mainly transported via a high-affinity choline transport system (CTL1) and a low-affinity choline transport system (CTL2) in human trophoblastic JEG-3 cells. These transporters play an important role in the growth of the fetus. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ventilation Increases with Lower Extremity Venous Occlusion in Young Adults

PubMed Central

Keller-Ross, Manda L.; Cowl, Andrielle L.; Cross, Troy; Johnson, Bruce D.; Olson, Thomas P.

2015-01-01

Introduction Venous distention via sub-systolic occlusion of the lower limbs may augment ventilation via stimulation of group III/IV afferent neurons. Purpose The purpose of this study was to examine the ventilatory response to graded lower extremity venous occlusion during exercise in healthy adults. Methods Nineteen adults (9 men, 25±5 yr) completed two visits. Visit 1: a maximal cycle ergometry exercise test. Visit 2 included a 30% peak workload cycle exercise with randomized inflations of bilateral thigh pressure tourniquets to 20, 40, 60, 80, 100 mmHg for 2 min each, separated by 2 min of deflation. Three min of cycling occurred prior to cuffing (CTL). Expired minute ventilation (VE), whole body gas exchange, rating of perceived exertion and dyspnea were measured during each session. Results VE increased significantly from the control condition (exercise only, control, CTL) to each occlusion pressure (p<0.05) with the greatest increase at 100 mmHg (CTL to 100 mmHg: 31.5±6.6 to 40.1±10.7 L/min). Respiratory rate (RR) increased as well (CTL to 100 mmHg: 24.8±6.0 to 30.9±11.5 breaths/min, p<0.05, condition effect) with no change in tidal volume (p>0.05). Tidal volume to inspiratory time (VT/TI) increased significantly from the CTL condition to each occlusion pressure (CTL to 100 mmHg: 1.5±0.3 to 1.8±0.4 L/min, p<0.05, all pressures). Dyspnea and RPE increased with all occlusion pressures from CTL exercise (p<0.05, all pressures). Conclusion Our findings suggest that mild-to-moderate venous occlusion of the lower extremity evokes a tachypneic breathing pattern which, in turn, augments VE and perceived breathing effort during exercise. PMID:26484951

Induced pluripotent stem cells: challenges and opportunities for cancer immunotherapy.

PubMed

Sachamitr, Patty; Hackett, Simon; Fairchild, Paul Jonathan

2014-01-01

Despite recent advances in cancer treatment over the past 30 years, therapeutic options remain limited and do not always offer a cure for malignancy. Given that tumor-associated antigens (TAA) are, by definition, self-proteins, the need to productively engage autoreactive T cells remains at the heart of strategies for cancer immunotherapy. These have traditionally focused on the administration of autologous monocyte-derived dendritic cells (moDC) pulsed with TAA, or the ex vivo expansion and adoptive transfer of tumor-infiltrating lymphocytes (TIL) as a source of TAA-specific cytotoxic T cells (CTL). Although such approaches have shown some efficacy, success has been limited by the poor capacity of moDC to cross present exogenous TAA to the CD8(+) T-cell repertoire and the potential for exhaustion of CTL expanded ex vivo. Recent advances in induced pluripotency offer opportunities to generate patient-specific stem cell lines with the potential to differentiate in vitro into cell types whose properties may help address these issues. Here, we review recent success in the differentiation of NK cells from human induced pluripotent stem (iPS) cells as well as minor subsets of dendritic cells (DCs) with therapeutic potential, including CD141(+)XCR1(+) DC, capable of cross presenting TAA to naïve CD8(+) T cells. Furthermore, we review recent progress in the use of TIL as the starting material for the derivation of iPSC lines, thereby capturing their antigen specificity in a self-renewing stem cell line, from which potentially unlimited numbers of naïve TAA-specific T cells may be differentiated, free of the risks of exhaustion.

Sequence conservation, HLA-E-Restricted peptide, and best-defined CTL/CD8+ epitopes in gag P24 (capsid) of HIV-1 subtype B

NASA Astrophysics Data System (ADS)

Prasetyo, Afiono Agung; Dharmawan, Ruben; Sari, Yulia; Sariyatun, Ratna

2017-02-01

Human immunodeficiency virus type 1 (HIV-1) remains a cause of global health problem. Continuous studies of HIV-1 genetic and immunological profiles are important to find strategies against the virus. This study aimed to conduct analysis of sequence conservation, HLA-E-restricted peptide, and best-defined CTL/CD8+ epitopes in p24 (capsid) of HIV-1 subtype B worldwide. The p24-coding sequences from 3,557 HIV subtype B isolates were aligned using MUSCLE and analysed. Some highly conserved regions (sequence conservation ≥95%) were observed. Two considerably long series of sequences with conservation of 100% was observed at base 349-356 and 550-557 of p24 (HXB2 numbering). The consensus from all aligned isolates was precisely the same as consensus B in the Los Alamos HIV Database. The HLA-E-restricted peptide in amino acid (aa) 14-22 of HIV-1 p24 (AISPRTLNA) was found in 55.9% (1,987/3,557) of HIV-1 subtype B worldwide. Forty-four best-defined CTL/CD8+ epitopes were observed, in which VKNWMTETL epitope (aa 181-189 of p24) restricted by B*4801 was the most frequent, as found in 94.9% of isolates. The results of this study would contribute information about HIV-1 subtype B and benefits for further works willing to develop diagnostic and therapeutic strategies against the virus.

Graded Alternating-Time Temporal Logic

NASA Astrophysics Data System (ADS)

Faella, Marco; Napoli, Margherita; Parente, Mimmo

Graded modalities enrich the universal and existential quantifiers with the capability to express the concept of at least k or all but k, for a non-negative integer k. Recently, temporal logics such as μ-calculus and Computational Tree Logic, Ctl, augmented with graded modalities have received attention from the scientific community, both from a theoretical side and from an applicative perspective. Both μ-calculus and Ctl naturally apply as specification languages for closed systems: in this paper, we add graded modalities to the Alternating-time Temporal Logic (Atl) introduced by Alur et al., to study how these modalities may affect specification languages for open systems.

Variation at Extra-epitopic Amino Acid Residues Influences Suppression of Influenza Virus Replication by M158-66 Epitope-Specific CD8+ T Lymphocytes.

PubMed

van de Sandt, Carolien E; Pronk, Mark R; van Baalen, Carel A; Fouchier, Ron A M; Rimmelzwaan, Guus F

2018-06-01

Influenza virus-specific CD8 + T lymphocytes (CTLs) contribute to clearance of influenza virus infections and reduce disease severity. Variation at amino acid residues located in or outside CTL epitopes has been shown to affect viral recognition by virus-specific CTLs. In the present study, we investigated the effect of naturally occurring variation at residues outside the conserved immunodominant and HLA*0201-restricted M1 58-66 epitope, located in the influenza virus M1 protein, on the extent of virus replication in the presence of CTLs specific for the epitope. To this end, we used isogenic viruses with an M1 gene segment derived from either an avian or a human influenza virus, HLA-transgenic human epithelial cells, human T cell clones specific for the M1 58-66 epitope or a control epitope, and a novel, purposely developed in vitro system to coculture influenza virus-infected cells with T cells. We found that the M gene segment of a human influenza A/H3N2 virus afforded the virus the capacity to replicate better in the presence of M1 58-66 -specific CTLs than the M gene segment of avian viruses. These findings are in concordance with previously observed differential CTL activation, caused by variation at extra-epitopic residues, and may reflect an immune adaptation strategy of human influenza viruses that allows them to cope with potent CTL immunity to the M1 58-66 epitope in HLA-A*0201-positive individuals, resulting in increased virus replication and shedding and possibly increasing disease severity. IMPORTANCE Influenza viruses are among the leading causes of acute respiratory tract infections. CD8 + T lymphocytes display a high degree of cross-reactivity with influenza A viruses of various subtypes and are considered an important correlate of protection. Unraveling viral immune evasion strategies and identifying signs of immune adaptation are important for defining the role of CD8 + T lymphocytes in affording protection more accurately. Improving our insight into the interaction between influenza viruses and virus-specific CD8 + T lymphocyte immunity may help to advance our understanding of influenza virus epidemiology, aid in risk assessment of potentially pandemic influenza virus strains, and benefit the design of vaccines that induce more broadly protective immunity. Copyright © 2018 American Society for Microbiology.

Studies of Cell-Mediated Immunity Against Immune Disorders Using Synthetic Peptides and Rotating Bioreactor System

NASA Technical Reports Server (NTRS)

Sastry, Jagannadha K.

1998-01-01

We conducted a series of experiments using mouse immune-precursor cells, and observed that bioreactor culturing results in the loss of antigen-specific cytotoxic T lymphocyte (CTL) function. The reason for the abrogation of CTL function is microgravity conditions in the bioreactor, but not the antigen per se or its MHC restriction. Similarly, we observed that allostimulation of human PBMC in the bioreactor, but not in the T flask, resulted in the blunting of both allo-CTL function and the NK activity, indicating that the microgravity-associated functional defects are not unique to the mouse system. These results provide further confirmation to the microgravity-associated immune dysfunction, and constitute ground-based confirmatory data for those related to space-travel.

Nectin-like 4 Complexes with Choline Transporter-like Protein-1 and Regulates Schwann Cell Choline Homeostasis and Lipid Biogenesis in Vitro*

PubMed Central

Heffernan, Corey; Jain, Mohit R.; Liu, Tong; Kim, Hyosung; Barretto, Kevin; Li, Hong; Maurel, Patrice

2017-01-01

Nectin-like 4 (NECL4, CADM4) is a Schwann cell-specific cell adhesion molecule that promotes axo-glial interactions. In vitro and in vivo studies have shown that NECL4 is necessary for proper peripheral nerve myelination. However, the molecular mechanisms that are regulated by NECL4 and affect peripheral myelination currently remain unclear. We used an in vitro approach to begin identifying some of the mechanisms that could explain NECL4 function. Using mass spectrometry and Western blotting techniques, we have identified choline transporter-like 1 (CTL1) as a putative complexing partner with NECL4. We show that intracellular choline levels are significantly elevated in NECL4-deficient Schwann cells. The analysis of extracellular d9-choline uptake revealed a deficit in the amount of d9-choline found inside NECL4-deficient Schwann cells, suggestive of either reduced transport capabilities or increased metabolization of transported choline. An extensive lipidomic screen of choline derivatives showed that total phosphatidylcholine and phosphatidylinositol (but not diacylglycerol or sphingomyelin) are significantly elevated in NECL4-deficient Schwann cells, particularly specific subspecies of phosphatidylcholine carrying very long polyunsaturated fatty acid chains. Finally, CTL1-deficient Schwann cells are significantly impaired in their ability to myelinate neurites in vitro. To our knowledge, this is the first demonstration of a bona fide cell adhesion molecule, NECL4, regulating choline homeostasis and lipid biogenesis. Phosphatidylcholines are major myelin phospholipids, and several phosphorylated phosphatidylinositol species are known to regulate key aspects of peripheral myelination. Furthermore, the biophysical properties imparted to plasma membranes are regulated by fatty acid chain profiles. Therefore, it will be important to translate these in vitro observations to in vivo studies of NECL4 and CTL1-deficient mice. PMID:28119456

Cognitive Performances Are Selectively Enhanced during Chronic Caloric Restriction or Resveratrol Supplementation in a Primate

PubMed Central

Marchal, Julia; Picq, Jean-Luc; Aujard, Fabienne

2011-01-01

Effects of an 18-month treatment with a moderate, chronic caloric restriction (CR) or an oral supplementation with resveratrol (RSV), a potential CR mimetic, on cognitive and motor performances were studied in non-human primates, grey mouse lemurs (Microcebus murinus). Thirty-three adult male mouse lemurs were assigned to three different groups: a control (CTL) group fed ad libitum, a CR group fed 70% of the CTL caloric intake, and an RSV group (RSV supplementation of 200 mg.kg−1.day−1) fed ad libitum. Three different cognitive tests, two motor tests, one emotional test and an analysis of cortisol level were performed in each group. Compared to CTL animals, CR or RSV animals did not show any change in motor performances evaluated by rotarod and jump tests, but an increase in spontaneous locomotor activity was observed in both groups. Working memory was improved by both treatments in the spontaneous alternation task. Despite a trend for CR group, only RSV supplementation increased spatial memory performances in the circular platform task. Finally, none of these treatments induced additional stress to the animals as reflected by similar results in the open field test and cortisol analyses compared to CTL animals. The present data provided the earliest evidence for a beneficial effect of CR or RSV supplementation on specific cognitive functions in a primate. Taken together, these results suggest that RSV could be a good candidate to mimic long-term CR effects and support the growing evidences that nutritional interventions can have beneficial effects on brain functions even in adults. PMID:21304942

Intravital imaging of CTLs killing islet cells in diabetic mice

PubMed Central

Coppieters, Ken; Amirian, Natalie; von Herrath, Matthias

2011-01-01

Type 1 diabetes (T1D) is caused by autoimmune destruction of the insulin-producing β cells in the pancreatic islets, which are essentially mini-organs embedded in exocrine tissue. CTLs are considered to have a predominant role in the autoimmune destruction underlying T1D. Visualization of CTL-mediated killing of β cells would provide new insight into the pathogenesis of T1D, but has been technically challenging to achieve. Here, we report our use of intravital 2-photon imaging in mice to visualize the dynamic behavior of a virally expanded, diabetogenic CTL population in the pancreas at cellular resolution. Following vascular arrest and extravasation, CTLs adopted a random motility pattern throughout the compact exocrine tissue and displayed unimpeded yet nonlinear migration between anatomically nearby islets. Upon antigen encounter within islets, a confined motility pattern was acquired that allowed the CTLs to scan the target cell surface. A minority of infiltrating CTLs subsequently arrested at the β cell junction, while duration of stable CTL–target cell contact was on the order of hours. Slow-rate killing occurred in the sustained local presence of substantial numbers of effector cells. Collectively, these data portray the kinetics of CTL homing to and between antigenic target sites as a stochastic process at the sub-organ level and argue against a dominant influence of chemotactic gradients. PMID:22133877

Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing

PubMed Central

Gameiro, Sofia R.; Jammed, Momodou L.; Wattenberg, Max M.; Tsang, Kwong Y.; Ferrone, Soldano; Hodge, James W.

2014-01-01

Radiation therapy (RT) is used for local tumor control through direct killing of tumor cells. Radiation-induced cell death can trigger tumor antigen-specific immune responses, but these are often noncurative. Radiation has been demonstrated to induce immunogenic modulation (IM) in various tumor types by altering the biology of surviving cells to render them more susceptible to T cell-mediated killing. Little is known about the mechanism(s) underlying IM elicited by sub-lethal radiation dosing. We have examined the molecular and immunogenic consequences of radiation exposure in breast, lung, and prostate human carcinoma cells. Radiation induced secretion of ATP and HMGB1 in both dying and surviving tumor cells. In vitro and in vivo tumor irradiation induced significant upregulation of multiple components of the antigen-processing machinery and calreticulin cell-surface expression. Augmented CTL lysis specific for several tumor-associated antigens was largely dictated by the presence of calreticulin on the surface of tumor cells and constituted an adaptive response to endoplasmic reticulum stress, mediated by activation of the unfolded protein response. This study provides evidence that radiation induces a continuum of immunogenic alterations in tumor biology, from immunogenic modulation to immunogenic cell death. We also expand the concept of immunogenic modulation, where surviving tumor cells recovering from radiation-induced endoplasmic reticulum stress become more sensitive to CTL killing. These observations offer a rationale for the combined use of radiation with immunotherapy, including for patients failing RT alone. PMID:24480782

Antigen Cross-Presentation of Immune Complexes

PubMed Central

Platzer, Barbara; Stout, Madeleine; Fiebiger, Edda

2014-01-01

The ability of dendritic cells (DCs) to cross-present tumor antigens has long been a focus of interest to physicians, as well as basic scientists, that aim to establish efficient cell-based cancer immune therapy. A prerequisite for exploiting this pathway for therapeutic purposes is a better understanding of the mechanisms that underlie the induction of tumor-specific cytotoxic T-lymphocyte (CTL) responses when initiated by DCs via cross-presentation. The ability of humans DC to perform cross-presentation is of utmost interest, as this cell type is a main target for cell-based immunotherapy in humans. The outcome of a cross-presentation event is guided by the nature of the antigen, the form of antigen uptake, and the subpopulation of DCs that performs presentation. Generally, CD8α+ DCs are considered to be the most potent cross-presenting DCs. This paradigm, however, only applies to soluble antigens. During adaptive immune responses, immune complexes form when antibodies interact with their specific epitopes on soluble antigens. Immunoglobulin G (IgG) immune complexes target Fc-gamma receptors on DCs to shuttle exogenous antigens efficiently into the cross-presentation pathway. This receptor-mediated cross-presentation pathway is a well-described route for the induction of strong CD8+ T cell responses. IgG-mediated cross-presentation is intriguing because it permits the CD8− DCs, which are commonly considered to be weak cross-presenters, to efficiently cross-present. Engaging multiple DC subtypes for cross-presentation might be a superior strategy to boost CTL responses in vivo. We here summarize our current understanding of how DCs use IgG-complexed antigens for the efficient induction of CTL responses. Because of its importance for human cell therapy, we also review the recent advances in the characterization of cross-presentation properties of human DC subsets. PMID:24744762

Identification of novel HLA-A(*)0201-restricted CTL epitopes from Pokemon.

PubMed

Yuan, Bangqing; Zhao, Lin; Xian, Ronghua; Zhao, Gang

2012-01-01

Pokemon is a member of the POK family of transcriptional repressors and aberrant overexpressed in various human cancers. Therefore, the related peptide epitopes derived from Pokemon is essential for the development of specific immunotherapy of malignant tumors. In this study, we predicted and identified HLA-A(*)0201-restricted cytotoxic T lymphocyte (CTL) epitopes derived from Pokemon with computer-based epitope prediction, peptide-binding assay and testing of the induced CTLs toward different kinds of carcinoma cells. The results demonstrated that effectors induced by peptides of Pokemon containing residues 32-40, 61-69, 87-95, and 319-327 could specifically secrete IFN-γ and lyse tumor cell lines of Pokemon-positive and HLA-A2-matched. The results suggest that Pokemon32, Pokemon61, Pokemon87, and Pokemon319 peptides are novel HLA-A(*)0201-restricted restricted CTL epitopes, and could be utilized in the cancer immunotherapy against a broad spectrum of tumors. Copyright © 2012 Elsevier Inc. All rights reserved.

Mild aerobic training with blood flow restriction increases the hypertrophy index and MuSK in both slow and fast muscles of old rats: Role of PGC-1α.

PubMed

Bahreinipour, Mohammad-Ali; Joukar, Siyavash; Hovanloo, Fariborz; Najafipour, Hamid; Naderi, Vida; Rajiamirhasani, Alireza; Esmaeili-Mahani, Saeed

2018-06-01

Existing evidence emphasize the role of mitochondrial dysfunction in sarcopenia which is revealed as loss of skeletal muscle mass and neuromuscular junction remodeling. We assessed the effect of low-intensity aerobic training along with blood flow restriction on muscle hypertrophy index, muscle-specific kinase (MuSK), a pivotal protein of the neuromuscular junction and Peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) in aged male rats. Animals groups were control (CTL), sham (Sh), leg blood flow restriction (BFR), exercise (Ex), sham + exercise (Sh + Ex), and BFR plus exercise (BFR + Ex) groups. The exercise groups were trained with low intensity exercise for 10 weeks. 48 h after the last training session, animals were sacrificed under anesthesia. Soleus and EDL muscles were isolated, hypertrophy index was estimated and MuSK and PGC-1α were measured by western blot method. Hypertrophy index enhanced in soleus and Extensor digitorum longus (EDL) muscles of BFR + Ex group (P < 0.01 versus CTL and Sh groups, and P < 0.001 versus other groups). The MuSK protein of soleus and EDL muscles increased in BFR + Ex group (P < 0.01 and P < 0.001, respectively) in comparison with CTL and Sh groups. In BFR + Ex group, the PGC-1α protein increased in both soleus and EDL (P < 0.001 compared to other groups). Also the PGC-1α of soleus muscle was higher in Ex and Sh + Ex groups versus CTL and Sh groups (P < 0.05). Findings suggest that low endurance exercise plus BFR improves the MuSK and hypertrophy index of both slow and fast muscles of elderly rats probably through the rise of PGC-1α expression. Copyright © 2018. Published by Elsevier Inc.

Activation of cellular immunity and marked inhibition of liver cancer in a mouse model following gene therapy and tumor expression of GM-SCF, IL-21, and Rae-1.

PubMed

Cheng, Mingrong; Zhi, Kangkang; Gao, Xiaoyan; He, Bing; Li, Yingchun; Han, Jiang; Zhang, Zhiping; Wu, Yan

2013-12-18

Cancer is both a systemic and a genetic disease. The pathogenesis of cancer might be related to dampened immunity. Host immunity recognizes nascent malignant cells - a process referred to as immune surveillance. Augmenting immune surveillance and suppressing immune escape are crucial in tumor immunotherapy. A recombinant plasmid capable of co-expressing granulocyte-macrophage colony- stimulating factor (GM-SCF), interleukin-21 (IL-21), and retinoic acid early transcription factor-1 (Rae-1) was constructed, and its effects determined in a mouse model of subcutaneous liver cancer. Serum specimens were assayed for IL-2 and INF-γ by ELISA. Liver cancer specimens were isolated for Rae-1 expression by RT-PCR and Western blot, and splenocytes were analyzed by flow cytometry. The recombinant plasmid inhibited the growth of liver cancer and prolonged survival of tumor-loaded mice. Activation of host immunity might have contributed to this effect by promoting increased numbers and cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) following expression of GM-SCF, IL-21, and Rae-1. By contrast, the frequency of regulatory T cells was decreased, Consequently, activated CTL and NK cells enhanced their secretion of INF-γ, which promoted cytotoxicity of NK cells and CTL. Moreover, active CTL showed dramatic secretion of IL-2, which stimulates CTL. The recombinant expression plasmid also augmented Rae-1 expression by liver cancer cells. Rae-1 receptor expressing CTL and NK cells removed liver cancer. The recombinant expression plasmid inhibited liver cancer by a mechanism that involved activation of cell-mediated immunity and Rae-1 in liver cancer.

MUC1-specific cytotoxic T lymphocytes eradicate tumors when adoptively transferred in vivo.

PubMed

Mukherjee, P; Ginardi, A R; Tinder, T L; Sterner, C J; Gendler, S J

2001-03-01

We have reported previously that MUC1 transgenic mice with spontaneous tumors of the pancreas (designated MET) naturally develop MHC class I-restricted, MUC1-specific CTLs as tumors progress (P. Mukherjee et al., J. Immunol., 165: 3451-3460, 2000). From these MET mice, we have isolated, expanded, and cloned naturally occurring MUC1-specific CTLs in vitro. In this report, we show that the CTL line is predominantly CD8+ T cells and expresses T-cell receptor Vbeta chains 5.1/5.2, 11, 13, and 2 and Valpha chains 2, 8.3, 3.2, and 11.1/11.2. These CTLs recognize several epitopes on the MUC1 tandem repeat with highest affinity to APGSTAPPA. The CTL clone, on the other hand, is 100% CD8+ cells and expresses a single Vbeta chain of 5.1/5.2 and Valpha2. It recognizes only the H-2Db class I-restricted epitope of MUC1, APGSTAPPA. When adoptively transferred, the CTLs were effective in eradicating MUC1-expressing injected tumor cells including mammary gland cells (C57mg) and B16 melanomas. These results suggest that MUC1-specific CTLs are capable of possibly preventing, or at least substantially delaying, MUC1-expressing tumor formation. To our knowledge, this is the first evidence that demonstrates that the naturally occurring MUC1-specific CTLs isolated from one tumor model has antitumor effects on other MUC1-expressing tumors in vivo. Therefore, our data confirm that MUC1 is an important tumor rejection antigen and can serve as a target for immunotherapy.

CXCL9 and CXCL10 expression are critical for control of genital herpes simplex virus type 2 infection through mobilization of HSV-specific CTL and NK cells to the nervous system1

PubMed Central

Thapa, Manoj; Welner, Robert S.; Pelayo, Rosana; Carr, Daniel J.J.

2007-01-01

CXCL9 and CXCL10 mediate the recruitment of T lymphocytes and NK cells known to be important in viral surveillance. The relevance of CXCL10 in comparison to CXCL9 in response to genital HSV-2 infection was determined using mice deficient in CXCL9 (CXCL9−/−) and CXCL10 (CXCL10−/−) along with wild type (WT) C57BL/6 mice. An increased sensitivity to infection was found in CXCL10−/− mice in comparison to CXCL9−/− or WT mice as determined by detection of HSV-2 in the central nervous system (CNS) at day 3 post infection. However, by day 7 post infection both CXCL9−/− & CXCL10−/−mice possessed significantly higher viral titers in the CNS in comparison to WT mice consistent with mortality (18–35%) of these mice within the first 7 days after infection. Even though CXCL9−/− and CXCL10−/− mice expressed elevated levels of CCL2, CCL3, CCL5, and CXCL1 in the spinal cord in comparison to WT mice, there was a reduction in NK cell and virus-specific CD8+ T cell mobilization to this tissue suggesting CXCL9 and CXCL10 are critical for recruitment of these effector cells to the spinal cord following genital HSV-2 infection. Moreover, leukocytes from the spinal cord but not draining lymph nodes or spleens of infected CXCL9−/− or CXCL10−/− mice displayed reduced CTL activity in comparison to effector cells from WT mice. Thus, the absence of CXCL9 or CXCL10 expression significantly alters the ability of the host to control genital HSV-2 infection through the mobilization of effector cells to sites of infection. PMID:18178850

Strong and multi-antigen specific immunity by hepatitis B core antigen (HBcAg)-based vaccines in a murine model of chronic hepatitis B: HBcAg is a candidate for a therapeutic vaccine against hepatitis B virus.

PubMed

Akbar, Sheikh Mohammad Fazle; Chen, Shiyi; Al-Mahtab, Mamun; Abe, Masanori; Hiasa, Yoichi; Onji, Morikazu

2012-10-01

Experimental evidence suggests that hepatitis B core antigen (HBcAg)-specific cytotoxic T lymphocytes (CTL) are essential for the control of hepatitis B virus (HBV) replication and prevention of liver damage in patients with chronic hepatitis B (CHB). However, most immune therapeutic approaches in CHB patients have been accomplished with hepatitis B surface antigen (HBsAg)-based prophylactic vaccines with unsatisfactory clinical outcomes. In this study, we prepared HBsAg-pulsed dendritic cells (DC) and HBcAg-pulsed DC by culturing spleen DC from HBV transgenic mice (HBV TM) and evaluated the immunomodulatory capabilities of these antigens, which may serve as a better therapy for CHB. The kinetics of HBsAg, antibody levels against HBsAg (anti-HBs), proliferation of HBsAg- and HBcAg-specific lymphocytes, production of antigen-specific CTL, and activation of endogenous DC were compared between HBV TM vaccinated with either HBsAg- or HBcAg-pulsed DC. Vaccination with HBsAg-pulsed DC induced HBsAg-specific immunity, but failed to induce HBcAg-specific immunity in HBV TM. However, immunization of HBV TM with HBcAg-pulsed DC resulted in: (1) HBsAg negativity, (2) production of anti-HBs, and (3) development of HBsAg- and HBcAg-specific T cells and CTL in the spleen and the liver. Additionally, significantly higher levels of activated endogenous DC were detected in HBV TM immunized with HBcAg-pulsed DC compared to HBsAg-pulsed DC (p<0.05). The capacity of HBcAg to modulate both HBsAg- and HBcAg-specific immunity in HBV TM, and activation of endogenous DC in HBV TM without inducing liver damage suggests that HBcAg should be an integral component of the therapeutic vaccine against CHB. Copyright © 2012 Elsevier B.V. All rights reserved.

CD8+ T cell recognition of an endogenously processed epitope is regulated primarily by residues within the epitope

PubMed Central

1992-01-01

Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides associated with cell surface class I major histocompatibility complex (MHC) molecules. This association presumably occurs between newly synthesized class I MHC molecules and peptide fragments in a pre-Golgi compartment. Little is known about the factors that regulate the formation of these antigenic peptide fragments within the cell. To examine the role of residues within a core epitope and in the flanking sequences for the generation and presentation of the newly synthesized peptide fragment recognized by CD8+ CTL, we have mutagenized the coding sequence for the CTL epitope spanning residues 202-221 in the influenza A/Japan/57 hemagglutinin (HA). In this study over 60 substitution mutations in the epitope were tested for their effects on target cell sensitization using a cytoplasmic viral expression system. The HA202- 221 site contains two overlapping subsites defined by CTL clones 11-1 and 40-2. Mutations in HA residues 204-213 or residues 210-219 often abolished target cell lysis by CTL clones 11-1 and 40-2, respectively. Although residues outside the core epitope did not usually affect the ability to be lysed by CTL clones, substitution of a Gly residue for Val-214 abolished lysis by clone 11-1. These data suggest that residues within a site that affect MHC binding and T cell receptor recognition appear to play the predominant role in dictating the formation of the antigenic complex recognized by CD8+ CTL, and therefore the antigenicity of the protein antigen presented to CD8+ T cells. Most alterations in residues flanking the endogenously expressed epitope do not appreciably affect the generation and recognition of the site. PMID:1383384

Polymer nanoparticles for cross-presentation of exogenous antigens and enhanced cytotoxic T-lymphocyte immune response

PubMed Central

Song, Chanyoung; Noh, Young-Woock; Lim, Yong Taik

2016-01-01

Effective induction of an antigen-specific cytotoxic T lymphocyte (CTL) immune response is one of the key goals of cancer immunotherapy. We report the design and fabrication of polyethylenimine (PEI)-coated polymer nanoparticles (NPs) as efficient antigen-delivery carriers that can induce antigen cross-presentation and a strong CTL response. After synthesis of poly(d,l-lactide-co-glycolide) (PLGA) NPs containing ovalbumin (OVA) by the double-emulsion solvent-evaporation method, cationic-charged PLGA NPs were generated by coating them with PEI. In a methyl tetrazolium salt assay, no discernible cytotoxic effect of PEI-coated PLGA (OVA) NPs was observed. The capacity and mechanism of PEI-coated PLGA (OVA) NPs for antigen delivery and cross-presentation on dendritic cells (DCs) were determined by fluorescence microscopy and flow cytometry. PEI-coated PLGA (OVA) NPs were internalized efficiently via phagocytosis or macropinocytosis in DCs and induced efficient cross-presentation of the antigen on MHC class I molecules via both endosome escape and a lysosomal processing mechanism. The DCs treated with PEI-coated PLGA (OVA) NPs induced a release of IL-2 cytokine from OVA-specific CD8-OVA1.3 T cells more efficiently than DCs treated with PLGA (OVA) NPs. Therefore, the PEI-coated PLGA (OVA) NPs can induce antigen cross-presentation and are expected to be used for induction of a strong CTL immune response and for efficient anticancer immunotherapy. PMID:27540289

Evolutionarily Conserved Epitopes on Human Immunodeficiency Virus Type 1 (HIV-1) and Feline Immunodeficiency Virus Reverse Transcriptases Detected by HIV-1-Infected Subjects

PubMed Central

Sanou, Missa P.; Roff, Shannon R.; Mennella, Antony; Sleasman, John W.; Rathore, Mobeen H.; Levy, Jay A.

2013-01-01

Anti-human immunodeficiency virus (HIV) cytotoxic T lymphocyte (CTL)-associated epitopes, evolutionarily conserved on both HIV type 1 (HIV-1) and feline immunodeficiency virus (FIV) reverse transcriptases (RT), were identified using gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and carboxyfluorescein diacetate succinimide ester (CFSE) proliferation assays followed by CTL-associated cytotoxin analysis. The peripheral blood mononuclear cells (PBMC) or T cells from HIV-1-seropositive (HIV+) subjects were stimulated with overlapping RT peptide pools. The PBMC from the HIV+ subjects had more robust IFN-γ responses to the HIV-1 peptide pools than to the FIV peptide pools, except for peptide-pool F3. In contrast, much higher and more frequent CD8+ T-cell proliferation responses were observed with the FIV peptide pools than with the HIV peptide pools. HIV-1-seronegative subjects had no proliferation or IFN-γ responses to the HIV and FIV peptide pools. A total of 24% (40 of 166) of the IFN-γ responses to HIV pools and 43% (23 of 53) of the CD8+ T-cell proliferation responses also correlated to responses to their counterpart FIV pools. Thus, more evolutionarily conserved functional epitopes were identified by T-cell proliferation than by IFN-γ responses. In the HIV+ subjects, peptide-pool F3, but not the HIV H3 counterpart, induced the most IFN-γ and proliferation responses. These reactions to peptide-pool F3 were highly reproducible and persisted over the 1 to 2 years of testing. All five individual peptides and epitopes of peptide-pool F3 induced IFN-γ and/or proliferation responses in addition to inducing CTL-associated cytotoxin responses (perforin, granzyme A, granzyme B). The epitopes inducing polyfunctional T-cell activities were highly conserved among human, simian, feline, and ungulate lentiviruses, which indicated that these epitopes are evolutionarily conserved. These results suggest that FIV peptides could be used in an HIV-1 vaccine. PMID:23824804

Development of contextual teaching and learning based science module for junior high school for increasing creativity of students

NASA Astrophysics Data System (ADS)

Kurniasari, H.; Sukarmin; Sarwanto

2018-03-01

The purpose of this research are to analyze the the properness of contextual teaching and learning (CTL)-based science module for Junior High School for increasing students’ creativity and using CTL-based science module to increase students’ learning creativity. Development of CTL-based science module for Junior High School is Research and Development (R&D) using 4D Model consist of 4 steps: define, design, develop, and disseminate. Module is validated by 3 expert validators (Material, media, and language experts), 2 reviewer and 1 peer reviewer. . Based on the results of data analysis, it can be concluded that: the results of the validation, the average score of CTL-based science module is 88.28%, the value exceeded the value of the cut off score of 87.5%, so the media declared eligible for the study. Research shows that the gain creativity class that uses CTL-based science module has a gain of 0.72. Based on the results of the study showed that CTL-based science module effectively promotes creativity of students

Leishmania mexicana Gp63 cDNA Using Gene Gun Induced Higher Immunity to L. mexicana Infection Compared to Soluble Leishmania Antigen in BALB/C

PubMed Central

Rezvan, H; Rees, R; Ali, SA

2011-01-01

Background Leishmaniasis is a worldwide disease prevalent in tropical and sub tropical countries. Many attempts have been made and different strategies have been approached to develop a potent vaccine against Leishmania. DNA immunisation is a method, which is shown to be effective in Leishmania vaccination. Leishmania Soluble Antigen (SLA) has also recently been used Leishmania vaccination. Methods The immunity generated by SLA and L. mexicana gp63 cDNA was compared in groups of 6 mice, which were statistically analysed by student t- test with the P-value of 0.05. SLA was administered by two different methods; intramuscular injection and injection of dendritic cells (DCs) loaded with SLA. L. mexicana gp63 cDNA was administered by the gene gun. Results Immunisation of BALB/c mice with L. mexicana gp63 resulted in high levels of Th1-type immune response and cytotoxic T lymphocytes (CTL) activity, which were accompanied with protection induced by the immunisation against L. mexicana infection. In contrast, administration of SLA, produced a mixed Th1/Th2-type immune responses as well as a high level of CTL activity but did not protect mice from the infection. Conclusion The results indicate higher protection by DNA immunisation using L. mexicana gp63 cDNA compared to SLA, which is accompanied by a high level of Th1 immune response. However, the CTL activity does not necessarily correlate with the protection induced by the vaccine. Also, gene gun immunisation is a potential approach in Leishmania vaccination. These findings would be helpful in opening new windows in Leishmania vaccine research. PMID:22347315

Neuroprotective intervention by interferon-γ blockade prevents CD8+ T cell–mediated dendrite and synapse loss

PubMed Central

Kreutzfeldt, Mario; Bergthaler, Andreas; Fernandez, Marylise; Brück, Wolfgang; Steinbach, Karin; Vorm, Mariann; Coras, Roland; Blümcke, Ingmar; Bonilla, Weldy V.; Fleige, Anne; Forman, Ruth; Müller, Werner; Becher, Burkhard; Misgeld, Thomas; Kerschensteiner, Martin; Pinschewer, Daniel D.

2013-01-01

Neurons are postmitotic and thus irreplaceable cells of the central nervous system (CNS). Accordingly, CNS inflammation with resulting neuronal damage can have devastating consequences. We investigated molecular mediators and structural consequences of CD8+ T lymphocyte (CTL) attack on neurons in vivo. In a viral encephalitis model in mice, disease depended on CTL-derived interferon-γ (IFN-γ) and neuronal IFN-γ signaling. Downstream STAT1 phosphorylation and nuclear translocation in neurons were associated with dendrite and synapse loss (deafferentation). Analogous molecular and structural alterations were also found in human Rasmussen encephalitis, a CTL-mediated human autoimmune disorder of the CNS. Importantly, therapeutic intervention by IFN-γ blocking antibody prevented neuronal deafferentation and clinical disease without reducing CTL responses or CNS infiltration. These findings identify neuronal IFN-γ signaling as a novel target for neuroprotective interventions in CTL-mediated CNS disease. PMID:23999498

Cytotoxic T lymphocyte response to peptide vaccination predicts survival in stage III colorectal cancer.

PubMed

Kawamura, Junichiro; Sugiura, Fumiaki; Sukegawa, Yasushi; Yoshioka, Yasumasa; Hida, Jin-Ichi; Hazama, Shoichi; Okuno, Kiyotaka

2018-02-23

We previously reported a phase I clinical trial of a peptide vaccine ring finger protein 43 (RNF43) and 34-kDa translocase of the outer mitochondrial membrane (TOMM34) combined with uracil-tegafur (UFT)/LV for patients with metastatic colorectal cancer (CRC), and demonstrated the safety and immunological responsiveness of this combination therapy. In this study, we evaluated vaccination-induced immune responses to clarify the survival benefit of the combination therapy as adjuvant treatment. We enrolled 44 patients initially in an HLA-masked fashion. After the disclosure of HLA, 28 patients were in the HLA-A*2402-matched and 16 were in the unmatched group. In the HLA-matched group, 14 patients had positive CTL responses specific for the RNF43 and/or TOMM34 peptides after 2 cycles of treatment and 9 had negative responses; in the HLA-unmatched group, 10 CTL responses were positive and 2 negative. In the HLA-matched group, 3-year relapse-free survival (RFS) was significantly better in the positive CTL subgroup than in the negative-response subgroup. Patients with negative vaccination-induced CTL responses showed a significant trend towards shorter RFS than those with positive responses. Moreover, in the HLA-unmatched group, the positive CTL response subgroup showed an equally good 3-year RFS as in the HLA-matched group. In conclusion, vaccination-induced CTL response to peptide vaccination could predict survival in the adjuvant setting for stage III CRC. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Transmission of HIV-1 CTL Escape Variants Provides HLA-Mismatched Recipients with a Survival Advantage

PubMed Central

Chopera, Denis R.; Woodman, Zenda; Mlisana, Koleka; Mlotshwa, Mandla; Martin, Darren P.; Seoighe, Cathal; Treurnicht, Florette; de Rosa, Debra Assis; Hide, Winston; Karim, Salim Abdool; Gray, Clive M.; Williamson, Carolyn

2008-01-01

One of the most important genetic factors known to affect the rate of disease progression in HIV-infected individuals is the genotype at the Class I Human Leukocyte Antigen (HLA) locus, which determines the HIV peptides targeted by cytotoxic T-lymphocytes (CTLs). Individuals with HLA-B*57 or B*5801 alleles, for example, target functionally important parts of the Gag protein. Mutants that escape these CTL responses may have lower fitness than the wild-type and can be associated with slower disease progression. Transmission of the escape variant to individuals without these HLA alleles is associated with rapid reversion to wild-type. However, the question of whether infection with an escape mutant offers an advantage to newly infected hosts has not been addressed. Here we investigate the relationship between the genotypes of transmitted viruses and prognostic markers of disease progression and show that infection with HLA-B*57/B*5801 escape mutants is associated with lower viral load and higher CD4+ counts. PMID:18369479

Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawano, Masaaki; Morikawa, Katsuma; Suda, Tatsuya

Virus-like particles (VLPs) are a promising vaccine platform due to the safety and efficiency. However, it is still unclear whether polyomavirus-based VLPs are useful for this purpose. Here, we attempted to evaluate the potential of polyomavirus VLPs for the antiviral vaccine using simian virus 40 (SV40). We constructed chimeric SV40-VLPs carrying an HLA-A{sup ⁎}02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A{sup ⁎}02:01-transgenic mice were then immunized with the chimeric SV40-VLPs. The chimeric SV40-VLPs effectively induced influenza-specific CTLs and heterosubtypic protection against influenza A viruses without the need of adjuvants. Because DNase I treatment of the chimericmore » SV40-VLPs did not disrupt CTL induction, the intrinsic adjuvant property may not result from DNA contaminants in the VLP preparation. In addition, immunization with the chimeric SV40-VLPs generated long-lasting memory CTLs. We here propose that the chimeric SV40-VLPs harboring an epitope may be a promising CTL-based vaccine platform with self-adjuvant properties. - Highlights: • We constructed chimeric SV40-VLPs carrying an influenza virus-derived CTL epitope. • Chimeric SV40-VLPs induce influenza-specific CTLs in mice without adjuvants. • Chimeric SV40-VLPs induce heterosubtypic protection against influenza A viruses. • Chimeric SV40-VLPs induce long-lasting memory CTLs. • Chimeric SV40-VLPs is a promising vaccine platform with self-adjuvant properties.« less

Time course expression of Foxo transcription factors in skeletal muscle following corticosteroid administration

PubMed Central

Cho, John E.; Fournier, Mario; Da, Xiaoyu

2010-01-01

Increased expression of forkhead box O (Foxo) transcription factors were reported in cultured myotubes and mouse limb muscle with corticosteroid (CS) treatment. We previously reported that administration of CS to rats resulted in muscle fiber atrophy only by day 7. The aim of this study, therefore, was to evaluate the time-course changes in the expression of Foxo transcription factors and muscle-specific ubiquitin E3 ligases in rat limb muscle following CS administration. Triamcinolone (TRI; 1 mg · kg−1 · day−1 im) was administered for 1, 3, or 7 days. Control (CTL) rats were given saline. Muscle mRNA was analyzed by real-time RT-PCR. Compared with CTL, body weights of TRI-treated animals decreased by 3, 12, and 21% at days 1, 3, and 7, respectively. Muscle IGF-1 mRNA levels decreased by 33, 65, and 58% at days 1, 3, and 7 in TRI-treated rats compared with CTL. Levels of phosphorylated Akt were 28, 50, and 36% lower in TRI animals at these time points. Foxo1 mRNA increased progressively by 1.2-, 1.4-, and 2.5-fold at days 1, 3, and 7 in TRI animals. Similar changes were noted in the expression of Foxo3a mRNA (1.3-, 1.4-, and 2.6-fold increments). By contrast, Foxo4 mRNA was not significantly changed in TRI animals. With TRI, muscle atrophy F box/Atrogin-1 increased by 1.8-, 4.1-, and 7.5-fold at days 1, 3, and 7 compared with CTL rats. By contrast, muscle RING finger 1 increased only from day 7 (2.7-fold). Gradual reduction in IGF-I expression with TRI over the time series paralleled that of Akt. These findings are consistent with a progressive stimulus to muscle protein degradation and the need to process/remove disassembled muscle proteins via the ubiquitin-proteasome system. Elucidating the dynamic catabolic responses to CS challenge is important in understanding the mechanisms underlying muscle atrophy and therapeutic measures to offset this. PMID:19850732

Tumor Expression of CD200 Inhibits IL-10 Production by Tumor-Associated Myeloid Cells and Prevents Tumor Immune Evasion of CTL Therapy

PubMed Central

Wang, Lixin; Liu, Jin-Qing; Talebian, Fatemeh; El-Omrani, Hani Y.; Khattabi, Mazin; Yu, Li; Bai, Xue-Feng

2010-01-01

CD200 is a cell-surface glycoprotein that functions through interaction with the CD200 receptor (CD200R) on myeloid lineage cells to regulate myeloid cell functions. Expression of CD200 has been implicated in multiple types of human cancer, however the impact of tumor expression of CD200 on tumor immunity remains poorly understood. To evaluate this issue, we generated CD200-positive mouse plasmacytoma J558 and mastocytoma P815 cells. We found that established CD200-positive tumors were often completely rejected by adoptively transferred CTL without tumor recurrence; in contrast, CD200-negative tumors were initially rejected by adoptively transferred CTL but the majority of tumors recurred. Tumor expression of CD200 significantly inhibited suppressive activity and IL-10 production by tumor-associated myeloid cells (TAMC), and as a result, more CTL accumulated in the tumor and exhibited a greater capacity to produce IFN-γ in CD200-positive tumors than in CD200-negative tumors. Neutralization of IL-10 significantly inhibited the suppressor activity of TAMC, and IL-10-deficiency allowed TAMC to kill cancer cells and their antigenic variants, which prevented tumor recurrence during CTL therapy. Thus, tumor expression of CD200 prevents tumor recurrence via inhibiting IL-10 production by TAMC. PMID:20662098

Functional analysis of choline transporters in rheumatoid arthritis synovial fibroblasts.

PubMed

Seki, Masayuki; Kawai, Yuiko; Ishii, Chikanao; Yamanaka, Tsuyoshi; Odawara, Masato; Inazu, Masato

2017-11-01

In this study, we examined the functional characteristics of choline uptake and sought to identify the transporters in rheumatoid arthritis synovial fibroblasts (RASFs). The expression of choline transporters was evaluated by quantitative real-time PCR, western blotting, and immunocytochemistry. Time course, Na + -dependency, and kinetics of [ 3 H]choline uptake were investigated. Effects of cationic drugs on the uptake of [ 3 H]choline, cell viability, and caspase-3/7 activity were also examined. Finally, we investigated the influence of choline uptake inhibitor, hemicholinium-3 (HC-3), and choline deficiency on cell viability and caspase-3/7 activity. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in RASFs and were localized to the plasma membrane. [ 3 H]Choline uptake occurred via a Na + -independent and pH-dependent transport system. The cells have two different [ 3 H]choline transport systems, high- and low-affinity. Various organic cations, HC-3 and choline deficiency inhibited both [ 3 H]choline uptake and cell viability, and enhanced the caspase-3/7 activity. The functional inhibition of choline transporters could promote apoptotic cell death. In RASFs, [ 3 H]choline uptake was significantly increased compared with that in OASFs without a change in gene expression. These results suggest that CTL1 (high-affinity) and CTL2 (low-affinity) are highly expressed in RASFs and choline may be transported by a choline/H +  antiport system. Identification of this CTL1- and CTL2-mediated choline transport system should provide a potential new target for RA therapy.

Comparative effects of two multi-enzyme combinations and a Bacillus probiotic on growth performance, digestibility of energy and nutrients, disappearance of non-starch polysaccharides, and gut microflora in broiler chickens.

PubMed

Wealleans, A L; Walsh, M C; Romero, L F; Ravindran, V

2017-12-01

The efficacy of two exogenous enzyme combinations and a multi-strain Bacillus probiotic (DFM) on the growth performance, nutrient digestibility, disappearance of non-starch polysaccharides (NSP) and gut microbial composition was investigated in broilers. One-day old Ross 308 chicks were assigned to 36 pens with 22 birds/pen and 6 pens/treatment (Experiment 1) or 36 cages with 8 birds/cage and 6 cages/treatment (Experiment 2). Treatment additives were added to nutritionally complete corn/soy based starter (d 1 to 21) and finisher (d 22 to 42) diets. Treatments included 1) a control diet containing 500 FTU/kg phytase (CTL), 2) CTL + xylanase (2,000 U/kg) and amylase (200 U/kg; XA), 3) CTL+XA + protease (4000 U/g; XAP), 4) CTL+DFM (150,000 cfu/g of 3 strains of Bacillus spp), 5) CTL+DFM+XA, and 6) CTL+DFM+XAP. Supplementation with DFM increased BW, BWG, and FI compared with the CTL (P < 0.05); XAP, but not XA, resulted in increased final BW, BWG and FI compared to the control (P < 0.05). XA and XAP improved apparent ileal digestibility (AID) of starch and fat on d 22 to 42 with XAP improving AMEn (by ∼82 kcal) compared with CTL birds (P < 0.01). DFM+XAP improved apparent ileal digestible energy (AIDE), AID of fat and starch on d 22 to 42, and additionally had a greater than additive effect on AIDE and AMEn. Supplementation with DFM+XAP reduced the ileal and total tract flow of insoluble arabinose and additionally total tract flow of soluble and insoluble xylose and total galactose (P < 0.05); similar effects of XA+DFM were not seen or were lower in magnitude, suggesting that the protease component plays an important role in increasing the availability of NSP for hydrolysis. Supplementation with DFM alone did not affect gut bacterial populations, but XA and XAP reduced numbers of Campylobacter species (by > 2.5 log cfu/g; P < 0.001) and Bacteroides (P < 0.02) in the cecum compared with CTL birds. © The Author 2017. Published by Oxford University Press on behalf of Poultry Science Association.

Comparative effects of two multi-enzyme combinations and a Bacillus probiotic on growth performance, digestibility of energy and nutrients, disappearance of non-starch polysaccharides, and gut microflora in broiler chickens

PubMed Central

Walsh, M C; Romero, L F; Ravindran, V

2017-01-01

Abstract The efficacy of two exogenous enzyme combinations and a multi-strain Bacillus probiotic (DFM) on the growth performance, nutrient digestibility, disappearance of non-starch polysaccharides (NSP) and gut microbial composition was investigated in broilers. One-day old Ross 308 chicks were assigned to 36 pens with 22 birds/pen and 6 pens/treatment (Experiment 1) or 36 cages with 8 birds/cage and 6 cages/treatment (Experiment 2). Treatment additives were added to nutritionally complete corn/soy based starter (d 1 to 21) and finisher (d 22 to 42) diets. Treatments included 1) a control diet containing 500 FTU/kg phytase (CTL), 2) CTL + xylanase (2,000 U/kg) and amylase (200 U/kg; XA), 3) CTL+XA + protease (4000 U/g; XAP), 4) CTL+DFM (150,000 cfu/g of 3 strains of Bacillus spp), 5) CTL+DFM+XA, and 6) CTL+DFM+XAP. Supplementation with DFM increased BW, BWG, and FI compared with the CTL (P < 0.05); XAP, but not XA, resulted in increased final BW, BWG and FI compared to the control (P < 0.05). XA and XAP improved apparent ileal digestibility (AID) of starch and fat on d 22 to 42 with XAP improving AMEn (by ∼82 kcal) compared with CTL birds (P < 0.01). DFM+XAP improved apparent ileal digestible energy (AIDE), AID of fat and starch on d 22 to 42, and additionally had a greater than additive effect on AIDE and AMEn. Supplementation with DFM+XAP reduced the ileal and total tract flow of insoluble arabinose and additionally total tract flow of soluble and insoluble xylose and total galactose (P < 0.05); similar effects of XA+DFM were not seen or were lower in magnitude, suggesting that the protease component plays an important role in increasing the availability of NSP for hydrolysis. Supplementation with DFM alone did not affect gut bacterial populations, but XA and XAP reduced numbers of Campylobacter species (by > 2.5 log cfu/g; P < 0.001) and Bacteroides (P < 0.02) in the cecum compared with CTL birds. PMID:29053809

Immune mechanisms in organ allograft rejection. V. Pivotal role of the cytotoxic-suppressor T cell subset in the rejection of heart grafts bearing isolated class I disparities in the inbred rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lowry, R.P.; Forbes, R.D.; Blackburn, J.H.

1985-11-01

The cellular requirements for rejection of heart grafts bearing isolated major histocompatibility complex (MHC) subregion RT1A-encoded class I disparities was assessed by adoptive transfer. Sublethally irradiated (780 rads) (PVG X WF)F1 recipients of irradiated PVG-RT1r1 heart grafts were selectively reconstituted with spleen cells from syngeneic donors previously sensitized with two sequential PVG-RT1r1 skin grafts. PVG-RT1r1 heart grafts were rejected acutely in recipients reconstituted with 10 X 10(6) unfractionated immune spleen cells or inocula (5 X 10(6) cells) depleted of SIg+ cells, but additional depletion of cytotoxic T cells and their precursors resulted in marked prolongation of graft survival. Reducing themore » reconstituting inocula from 4 X 10(6) to 2.5 X 10(6) spleen cells prolonged graft survival to that observed in unreconstituted recipients. Additional studies were performed to define the immunologic basis for prolonged survival of PVG-RT1r1 heart grafts in homozygous PVG recipients. Although lymphoid cells of naive PVG failed to proliferate on coculture with irradiated PVG-RT1r1, bulk cultures yielding but weak and variable CTL generation, lymphoid cells from specifically sensitized PVG proliferated and generated greater cytotoxic T lymphocyte (CTL) activity under identical conditions, strongly suggesting, therefore, that prolonged heart graft survival in this strain combination is related to low CTL precursor frequency.« less

Expanding specificity of class 1 restricted CD8+ T cells for viral epitopes following multiple inoculations of swine with a human adenivorus vectored foot-and-mouth disease virus (FMDV) vaccine

USDA-ARS?s Scientific Manuscript database

The immune response to the highly acute foot-and-mouth disease virus (FMDV) is routinely reported as a measure of serum antibody. However, a critical effector function of immune responses combating viral infection of mammals is the cytotoxic T lymphocyte (CTL) response, mediated by virus specific ...

Official Guard and Reserve Manpower Strengths and Statistics. FY 2000 Summary

DTIC Science & Technology

2000-01-01

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Modulation of dendritic cell maturation and function by the Tax protein of human T cell leukemia virus type 1

PubMed Central

Jain, Pooja; Ahuja, Jaya; Khan, Zafar K.; Shimizu, Saori; Meucci, Olimpia; Jennings, Stephen R.; Wigdahl, Brian

2009-01-01

Human T cell leukemia virus type 1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is characterized by the generation of an intense CTL cell response directed against the viral transactivator protein Tax. In addition, patients diagnosed with HAM/TSP exhibit rapid activation and maturation of dendritic cells (DC), likely contributing to the robust, Tax-specific CTL response. In this study, extracellular Tax has been shown to induce maturation and functional alterations in human monocyte-derived DC, critical observations being confirmed in freshly isolated myeloid DC. Tax was shown to promote the production of proinflammatory cytokines and chemokines involved in the DC activation process in a dose- and time-dependent manner. Furthermore, Tax induced the expression of DC activation (CD40, CD80, and CD86) and maturation (CD83) markers and enhanced the T cell proliferation capability of DC. Heat inactivation of Tax resulted in abrogation of these effects, indicating a requirement for the native structure of Tax, which was found to bind efficiently to the DC membrane and was internalized within a few hours, suggesting that extracellular Tax may possess an intracellular mechanism of action subsequent to entry. Finally, inhibitors of cellular signaling pathways, NF-κB, protein kinase, tyrosine kinase, and phospholipase C, were shown to inhibit Tax-mediated DC activation. This is the first study reporting the immunomodulatory effects of extracellular Tax in the DC compartment. These results suggest that DC, once exposed to Tax by uptake from the extracellular environment, can undergo activation, providing constant antigen presentation and costimulation to T cells, leading to the intense T cell proliferation and inflammatory responses underlying HAM/TSP. PMID:17442856

Recombinant measles viruses expressing respiratory syncytial virus proteins induced virus-specific CTL responses in cotton rats.

PubMed

Yamaji, Yoshiaki; Nakayama, Tetsuo

2014-07-31

Respiratory syncytial virus (RSV) is a common cause of serious lower respiratory tract illnesses in infants. Natural infections with RSV provide limited protection against reinfection because of inefficient immunological responses that do not induce long-term memory. RSV natural infection has been shown to induce unbalanced immune response. The effective clearance of RSV is known to require the induction of a balanced Th1/Th2 immune response, which involves the induction of cytotoxic T lymphocytes (CTL). In our previous study, recombinant AIK-C measles vaccine strains MVAIK/RSV/F and MVAIK/RSV/G were developed, which expressed the RSV fusion (F) protein or glycoprotein (G). These recombinant viruses elicited antibody responses against RSV in cotton rats, and no infectious virus was recovered, but small amounts of infiltration of inflammatory cells were observed in the lungs following RSV challenge. In the present study, recombinant AIK-C measles vaccine strains MVAIK/RSV/M2-1 and MVAIK/RSV/NP were developed, expressing RSV M2-1 or Nucleoprotein (NP), respectively. These viruses exhibited temperature-sensitivity (ts), which was derived from AIK-C, and expressed respective RSV antigens. The intramuscular inoculation of cotton rats with the recombinant measles virus led to the induction of CD8(+) IFN-γ(+) cells. No infectious virus was recovered from a lung homogenate following the challenge. A Histological examination of the lungs revealed a significant reduction in inflammatory reactions without alveolar damage. These results support the recombinant measles viruses being effective vaccine candidates against RSV that induce RSV-specific CTL responses with or without the development of an antibody response. Copyright © 2014 Elsevier Ltd. All rights reserved.

Peptide-independent Recognition by Alloreactive Cytotoxic T Lymphocytes (CTL)

PubMed Central

Smith, Pamela A.; Brunmark, Anders; Jackson, Michael R.; Potter, Terry A.

1997-01-01

We have isolated several H-2Kb–alloreactive cytotoxic T cell clones and analyzed their reactivity for several forms of H-2Kb. These cytotoxic T lymphocytes (CTL) were elicited by priming with a skin graft followed by in vitro stimulation using stimulator cells that express an H-2Kb molecule unable to bind CD8. In contrast to most alloreactive T cells, these CTL were able to recognize H-2Kb on the surface of the antigen processing defective cell lines RMA-S and T2. Furthermore, this reactivity was not increased by the addition of an extract containing peptides from C57BL/6 (H-2b) spleen cells, nor was the reactivity decreased by treating the target cells with acid to remove peptides bound to MHC molecules. The CTL were also capable of recognizing targets expressing the mutant H-2Kbm8 molecule. These findings suggested that the clones recognized determinants on H-2Kb that were independent of peptide. Further evidence for this hypothesis was provided by experiments in which H-2Kb produced in Drosophila melanogaster cells and immobilized on the surface of a tissue culture plate was able to stimulate hybridomas derived from these alloreactive T cells. Precursor frequency analysis demonstrated that skin graft priming, whether with skin expressing the wild-type or the mutant H-2Kb molecule, is a strong stimulus to elicit peptide-independent CTL. Moreover, reconstitution experiments demonstrated that the peptide-independent CTL clones were capable of mediating rapid and complete rejection of H-2–incompatible skin grafts. These findings provide evidence that not all allorecognition is peptide dependent. PMID:9091576

Naïve CD8 T cell activation by liver bone marrow-derived cells leads to a "neglected" IL-2low Bimhigh phenotype, poor CTL function and cell death.

PubMed

Holz, Lauren E; Benseler, Volker; Vo, Michelle; McGuffog, Claire; Van Rooijen, Nico; McCaughan, Geoffrey W; Bowen, David G; Bertolino, Patrick

2012-10-01

The occurrence of primary CD8 T cell activation within the liver, unique among the non-lymphoid organs, is now well accepted. However, the outcome of intrahepatic T cell activation remains controversial. We have previously reported that activation initiated by hepatocytes results in a tolerogenic phenotype characterized by low expression of CD25 and IL-2, poor cytotoxic T lymphocyte (CTL) function, and excessive expression of the pro-apoptotic protein Bim. To investigate whether this phenotype was due to activation in the absence of co-stimulation, we generated bone marrow (bm) radiation chimeras in which adoptively transferred naïve transgenic CD8 T cells were activated in the presence of co-stimulation by liver bm-derived cells. Despite expressing pro-inflammatory cytokines, high levels of CD25 and CD54, donor T cells activated by liver bm-derived cells did not produce detectable IL-2 and displayed poor CTL function, suggesting incomplete acquisition of effector function. Simultaneously, these cells expressed high levels of Bim and died by neglect. Transfer of Bim-deficient T cells resulted in increased T cell numbers. These results imply that expression of CD25 and CD54 is co-stimulation dependent and distinguishes T cell activated by hepatocytes and liver bm-derived cells. In contrast, low expression of IL-2, poor CTL function and excess Bim production represent a more universal phenotype defining T cells undergoing primary activation by both types of hepatic antigen presenting cells (APC). These results have important implications for transplantation, in which all liver antigen presenting cells contribute to activation of T cells specific for the allograft. Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Pitching performance and longevity after revision ulnar collateral ligament reconstruction in Major League Baseball pitchers.

PubMed

Marshall, Nathan E; Keller, Robert A; Lynch, Jonathan R; Bey, Michael J; Moutzouros, Vasilios

2015-05-01

Medial ulnar collateral ligament (UCL) reconstruction is a common procedure performed on professional pitchers in Major League Baseball (MLB). Although a great deal is known about primary reconstruction, much less is known about revision reconstruction. The purpose of this study was to evaluate statistical performance, return to play, and career longevity in MLB pitchers after revision UCL surgery, with the hypothesis that pitching performance and career longevity will decline after revision surgery. Cohort study; Level of evidence, 3. A total of 33 MLB pitchers who underwent revision UCL reconstruction surgery (UCL-R group) were identified and compared with 33 age- and position-matched controls (CTL group). Return to play, total years played, and statistical performance were evaluated and compared with controls. After revision surgery, 65.5% of UCL-R pitchers returned to the MLB level. On average, the UCL-R pitchers played 0.8 years less in the majors (P<.01) than did the control pitchers. The UCL-R pitchers who returned to the MLB level had a similar earned run average (UCL-R: 4.88, CTL: 4.76, P=.82) and walks/hits per innings pitched (UCL-R: 1.58, CTL: 1.44, P=.22) compared with the control pitchers. There were significant declines, however, in terms of innings pitched (UCL-R: 36.95, CTL: 75.00, P<.01), walks per 9 innings (UCL-R: 4.75, CTL: 3.49, P<.01), and wins (UCL-R: 1.88, CTL: 4.10, P<.01) as well as nonsignificant declines in wins above replacement (UCL-R: 0.25, CTL: 0.62, P=.06) and runs above replacement (UCL-R: 3.26, CTL: 6.91, P=.07). MLB pitchers who undergo UCL-R have a low rate of return to MLB play and have shortened careers after return. Pitchers who returned to the MLB level maintained performance in several statistics such as earned run average and walks/hits per innings pitched; however, pitchers returned with a significantly decreased workload. © 2015 The Author(s).

HIV: current opinion in escapology.

PubMed

Klenerman, Paul; Wu, Ying; Phillips, Rodney

2002-08-01

Much recent work strongly supports the hypothesis that CD8(+) T lymphocytes (CTLs) exert important immune control over HIV and so are a major selective force in its evolution. We analyse this host-pathogen interplay and focus on new data that describe the overall 'effectiveness' of CTL responses (strength, spread, specificity and 'stamina') and the mechanisms by which HIV may evade this suppressive activity. CTLs directed against HIV recognise very large numbers of distinct epitopes across the genome, are largely functional, turn over rapidly, and possess a phenotype that is distinct from CD8(+) lymphocytes specific for other viruses. Mutation of HIV epitopes that alters or abolishes CTL recognition altogether appears to be the most important immune escape mechanism, as the variation that HIV generates defies the limits of the T cell repertoire. However, this immune evasion is still only well-studied in a few patients. The rules that govern immune escape, and the ultimate limits of CTL capacity to deal with the variant epitopes that currently circulate, are not understood. This information will determine the feasibility of current vaccine approaches that, so far, make no provision for the enormous antigenic plasticity of HIV.

Coal-to-liquids bill introduced in the Senate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buchsbaum, L.

2006-06-15

Of immense importance to the coal industry is the announcement, on 7 June 2006 by US Senators Barack Obama (D-IL) and Jim Bunning (R-KY) of S.3325, the 'Coal-to-Liquid Fund Promotion Act of 2006'. This legislation creates tax incentives for coal-to-liquids (CTL) technologies and construction of CTL plants. If passed, this will create the infrastructure needed to make CTL a viable energy resource throughout America. The article gives comment and background to this proposed legislation. Illinois Basin coal is well suited for CTL because of its high Btu content. If Sasol constructs a proposed plant in Illinois it would increase coalmore » production in the state by 10 mt. 1 fig.« less

CSF neurofilament proteins as diagnostic and prognostic biomarkers for amyotrophic lateral sclerosis.

PubMed

Rossi, Daniela; Volanti, Paolo; Brambilla, Liliana; Colletti, Tiziana; Spataro, Rossella; La Bella, Vincenzo

2018-03-01

Elevated cerebrospinal fluid (CSF), Neurofilament Light (NF-L) and phosphorylated Heavy (pNF-H) chain levels have been found in Amyotrophic Lateral Sclerosis (ALS), with studies reporting a correlation of both neurofilaments (NFs) with the disease progression. Here, we measured NF-L and pNF-H concentrations in the CSF of ALS patients from a single tertiary Center and investigated their relationship with disease-related variables. A total of 190 ALS patients (Bulbar, 29.9%; Spinal, 70.1%; M/F = 1.53) and 130 controls with mixed neurological diseases were recruited. Demographic and clinical variables were recorded, and ΔFS was used to rate the disease progression. Controls were divided into two cohorts: (1) patients with non-inflammatory neurological diseases (CTL-1); (2) patients with acute/subacute inflammatory diseases and tumors, expected to lead to significant axonal and tissue damage (CTL-2). For each patient and control, CSF was taken at the time of the diagnostic work-up and stored following the published guidelines. CSF NF-L and pNF-H were assayed with commercially available ELISA-based methods. Standard curves (from independent ELISA kits) were highly reproducible for both NFs, with a coefficient of variation < 20%. We found that CSF NF-L and pNF-H levels in ALS were significantly increased when compared to CTL-1 (NF-L: ALS, 4.7 ng/ml vs CTL-1, 0.61 ng/ml, p < 0.001; pNF-H: ALS, 1.7 ng/ml vs CTL-1, 0.03 ng/ml, p < 0.0001), but not to CTL-2. Analysis of different clinical and prognostic variables disclosed meaningful correlations with both NF-L and pNF-H levels. Our results, from a relatively large ALS cohort, confirm that CSF NF-L and pNF-H represent valuable diagnostic and prognostic biomarkers in ALS.

Cobalt germanide nanostructure formation and memory characteristic enhancement in silicon oxide films

NASA Astrophysics Data System (ADS)

Joo, Beom Soo; Kim, Hyunseung; Jang, Seunghun; Han, Dongwoo; Han, Moonsup

2018-08-01

We investigated nano-floating gate memory having a charge trap layer (CTL) composed of cobalt germanide nanostructure (ns-CoGe). A tunneling oxide layer; a CTL containing Co, Ge, and Si; and a blocking oxide layer were sequentially deposited on a p-type silicon substrate by RF magnetron sputtering and low-pressure chemical vapor deposition. We optimized the CTL formation conditions by rapid thermal annealing at a somewhat low temperature (about 830 °C) by considering the differences in Gibbs free energy and chemical enthalpy among the components. To characterize the charge storage properties, capacitance-voltage (C-V) measurements were performed. Further, we used X-ray photoelectron spectroscopy for chemical analysis of the CTL. In this work, we not only report that the C-V measurement shows a remarkable opening of the memory window for the ns-CoGe compared with those of nanostructures composed of Co or Ge alone, but also clarify that the improvement in the memory characteristics originates in the nanostructure formation, which consists mainly of Co-Ge bonds. We expect ns-CoGe to be a strong candidate for fabrication of next-generation memory devices.

CTL-Tumor Cell Interaction: The Generation of Molecular Probes of Monitoring the HLA-A*0201-HER-2/neu Peptide Complex

DTIC Science & Technology

2005-03-01

anti- HLA -A2, -A24, -A28 mAb CR11-351 (13,14); anti- HLA -A2, -A28 mAb KS-1 (14); anti- HLA -B7, - B27 , -Bw42, -Bw54, -Bw55, -Bw56, -Bw67, -Bw73 mAb KS-4 (15...AD Award Number: W81XWH-04-1-0372 TITLE: CTL-Tumor Cell Interaction: The Generation of Molecular Probes of Monitoring the HLA -A*0201-HER-2/neu...AND SUBTITLE 5. FUNDING NUMBERS CTL-Tumor Cell Interaction: The Generation of Molecular W81XWH-04-1-0372 Probes of Monitoring the HLA -A*0201-HER-2/neu

The roles of convective entrainment in spatial distributions and temporal variations of precipitation over tropical oceans

NASA Astrophysics Data System (ADS)

Hirota, N.; Takayabu, Y. N.; Watanabe, M.; Kimoto, M.; Chikira, M.

2013-12-01

This study shows that a proper treatment of convective entrainment is essential in determining spatial distributions and temporal variations of precipitation by numerical experiments. They have performed and compared four experiments with different entrainment characteristics: a control (Ctl), no entrainment (NoEnt), original Arakawa Schubert (AS), and AS with simple empirical suppression of convection (ASRH). The fractional entrainment rate of AS and ASRH are constant for each cloud type and are very small near cloud base compared to Ctl, in which half of buoyancy-generated energy is consumed by the entrainment. Ctl well reproduces the spatial and temporal variations, whereas NoEnt and AS, which are very similar to each other, significantly underestimated the variations with the so-called the double ITCZ problem. The enhanced variations in Ctl are due to the larger entrainment that strengthens the coupling of convection and free tropospheric humidity. Time variations are also more realistic in Ctl; mid-height convection moistens mid-troposphere and large precipitation events occur after sufficient moisture is available. In contrast, deep convection is more frequent but with smaller precipitation amount in NoEnt and AS. ASRH shows smaller spatial but excessive temporal variations suggesting that its empirical suppression condition is too simple and a more sophisticated formulation is required for more realistic precipitation variations. This study was supported by the Ministry of Education, Culture, Sports, Science and Technology (GRENE), and by the Ministry of the Environment (2A-1201), Japan.

Immunization of Mice by BCG Formulated HCV Core Protein Elicited Higher Th1-Oriented Responses Compared to Pluronic-F127 Copolymer

PubMed Central

Yazdanian, Maryam; Memarnejadian, Arash; Mahdavi, Mehdi; Sadat, Seyed Mehdi; Motevali, Fatemeh; Vahabpour, Rouhollah; Khanahmad, Hossein; Siadat, Seyed Davar; Aghasadeghi, Mohammad Reza; Roohvand, Farzin

2013-01-01

Background A supreme vaccine for Hepatitis C virus (HCV) infection should elicit strong Th1-oriented cellular responses. In the absence of a Th1-specific adjuvant, immunizations by protein antigens generally induce Th2-type and weak cellular responses. Objectives To evaluate the adjuvant effect of BCG in comparison with nonionic copolymer-Pluronic F127 (F127) as a classic adjuvant in the formulation of HCV core protein (HCVcp) as a candidate vaccine for induction of Th1 immune responses. Materials and Methods Expression of N-terminally His-Tagged HCVcp (1-122) by pIVEX2.4a-core vector harboring the corresponding gene under the control of arabinose-inducible (araBAD) promoter was achieved in BL21-AI strain of E.coli and purified through application of nitrilotriacetic acid (Ni-NTA) chromatography. Mice were immunized subcutaneously (s.c.) in base of the tail with 100 μl of immunogen (F127+HCVcp or BCG+HCVcp; 5 μgHCVcp/mouse/dose) or control formulations (PBS, BCG, F127) at weeks 0, 3, 6. Total and subtypes of IgG, as well as cellular immune responses (Proliferation, In vivo CTL and IFN-γ/IL-4 ELISpot assays against a strong and dominant H2-d restricted, CD8+-epitopic peptide, core 39-48; RRGPRLGVRA of HCVcp) were compared in each group of immunized animals. Results Expression and purification of core protein around the expected size (21 kDa) was confirmed by Western blotting. The HCVcp + BCG vaccinated mice showed significantly higher lymphocyte proliferation and IFN-γ production but lower levels of cell lysis (45% versus 62% in CTL assay) than the HCVcp+F127 immunized animals. “Besides, total anti-core IgG and IgG1 levels were significantly higher in HCVcp + F127 immunized mice as compared to HCVcp + BCG vaccinated animals, indicating relatively higher efficacy of F127 for the stimulation of humoral and Th2-oriented immune responses”. Conclusions Results showed that HCVcp + BCG induced a moderate CTL and mixed Th1/Th2 immune responses with higher levels of cell proliferation and IFN-γ secretion, indicating that BCG may have a better outcome when formulated in HCVcp-based subunit vaccines. PMID:24348641

Cellular kinetics of CTL019 in relapsed/refractory B-cell acute lymphoblastic leukemia and chronic lymphocytic leukemia

PubMed Central

Maude, Shannon L.; Porter, David L.; Frey, Noelle; Wood, Patricia; Han, Xia; Waldron, Edward; Chakraborty, Abhijit; Awasthi, Rakesh; Levine, Bruce L.; Melenhorst, J. Joseph; Grupp, Stephan A.; June, Carl H.; Lacey, Simon F.

2017-01-01

Tisagenlecleucel (CTL019) is an investigational immunotherapy that involves reprogramming a patient’s own T cells with a transgene encoding a chimeric antigen receptor to identify and eliminate CD19-expressing cells. We previously reported that CTL019 achieved impressive clinical efficacy in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), including the expansion and persistence of CTL019 cells, which correlates with response to therapy. Here, we performed formal cellular kinetic analyses of CTL019 in a larger cohort of 103 patients treated with CTL019 in 2 different diseases (ALL and CLL). CTL019 was measured in peripheral blood and bone marrow, using quantitative polymerase chain reaction and flow cytometry. CTL019 levels in peripheral blood typically peaked at 10 to 14 days postinfusion and then declined slowly over time. Patients with complete response (CR)/CR with incomplete count recovery had higher levels of CTL019 in peripheral blood, with greater maximal concentration and area under the curve values compared with nonresponding patients (P < .0001 for each). CTL019 transgene levels were measurable up to 780 days in peripheral blood. CTL019 trafficking and persistence were observed in bone marrow and cerebrospinal fluid. CTL019 expansion correlated with severity of cytokine release syndrome (CRS) and preinfusion tumor burden in pediatric ALL. The results described here are the first detailed formal presentation of cellular kinetics across 2 diseases and highlight the importance of the application of in vivo cellular kinetic analyses to characterize clinical efficacy and CRS severity associated with CTL019 therapy. PMID:28935694

Two patients with Hermansky Pudlak syndrome type 2 and novel mutations in AP3B1

PubMed Central

Wenham, Matt; Grieve, Samantha; Cummins, Michelle; Jones, Matthew L.; Booth, Sarah; Kilner, Rachel; Ancliff, Philip J.; Griffiths, Gillian M.; Mumford, Andrew D.

2010-01-01

Hermansky Pudlak syndrome type 2 (HPS2) is a rare disorder associated with mutations in the Adaptor Protein 3 (AP-3) complex, which is involved in sorting transmembrane proteins to lysosomes and related organelles. We now report 2 unrelated subjects with HPS2 who show a characteristic clinical phenotype of oculocutaneous albinism, platelet and T-lymphocyte dysfunction and neutropenia. The subjects were homozygous for different deletions within AP3B1 (g.del180242-180866, c.del153-156), which encodes the AP-3β3A subunit, resulting in frame shifts and introduction of nonsense substitutions (p.E693fsX13, p.E52fsX11). In the subject with p.E693fsX13, this resulted in expression of a truncated variant β3A protein. Cytotoxic T-lymphocyte (CTL) clones from both study subjects showed increased cell-surface expression of CD63 and reduced cytotoxicity. Platelets showed impaired aggregation and reduced uptake of 3H-serotonin. These findings are consistent with CTL granule and platelet dense granule defects, respectively. This report extends the clinical and laboratory description of HPS2. PMID:19679886

A Marked Reduction in Priming of Cytotoxic CD8+ T Cells Mediated by Stress-Induced Glucocorticoids Involves Multiple Deficiencies in Cross-Presentation by Dendritic Cells

PubMed Central

Hunzeker, John T.; Elftman, Michael D.; Mellinger, Jennifer C.; Princiotta, Michael F.; Bonneau, Robert H.; Truckenmiller, Mary E.; Norbury, Christopher C.

2013-01-01

Protracted psychological stress elevates circulating glucocorticoids, which can suppress CD8+ T cell-mediated immunity, but the mechanisms are incompletely understood. Dendritic cells (DCs), required for initiating CTL responses, are vulnerable to stress/corticosterone, which can contribute to diminished CTL responses. Cross-priming of CD8+ T cells by DCs is required for initiating CTL responses against many intracellular pathogens that do not infect DCs. We examined the effects of stress/corticosterone on MHC class I (MHC I) cross-presentation and priming and show that stress/corticosterone-exposed DCs have a reduced ability to cross-present OVA and activate MHC I-OVA257–264-specific T cells. Using a murine model of psychological stress and OVA-loaded β2-microglobulin knockout “donor” cells that cannot present Ag, DCs from stressed mice induced markedly less Ag-specific CTL proliferation in a glucocorticoid receptor-dependent manner, and endogenous in vivo T cell cytolytic activity generated by cross-presented Ag was greatly diminished. These deficits in cross-presentation/priming were not due to altered Ag donation, Ag uptake (phagocytosis, receptor-mediated endocytosis, or fluid-phase uptake), or costimulatory molecule expression by DCs. However, proteasome activity in corticosterone-treated DCs or splenic DCs from stressed mice was partially suppressed, which limits formation of antigenic peptide-MHC I complexes. In addition, the lymphoid tissue-resident CD11b−CD24+CD8α+ DC subset, which carries out cross-presentation/priming, was preferentially depleted in stressed mice. At the same time, CD11b−CD24+CD8α− DC precursors were increased, suggesting a block in development of CD8α+ DCs. Therefore, glucocorticoid-induced changes in both the cellular composition of the immune system and intracellular protein degradation contribute to impaired CTL priming in stressed mice. PMID:21098225

CD4 T cell-mediated masking effects of the immunogenicity of tumor-associated antigens are qualitatively and quantitatively different depending on the individual antigens.

PubMed

Okano, Shinji; Matsumoto, Yoshihiro; Yoshiya, Shohei; Yamashita, Yo-ichi; Harimoto, Norifumi; Ikegami, Toru; Shirabe, Ken; Harada, Mamoru; Yoshikai, Yasunobu; Maehara, Yoshihiko

2013-01-01

The use of cancer immunotherapy as part of multidisciplinary therapies for cancer is a promising strategy for the cure of advanced cancer patients. In cancer immunotherapy, the effective priming of tumor-associated antigen (TAA)-specific CD8+ T cells is essential, and therefore, the appropriate selection of the best peptide for targeting the cancer is a most important concern. One criticism in the selection of a TAA is the immunogenicity of the TAA, the vaccination of which effectively elicits clinical responses. However, the critical basic immunological factors that affect the differences in the immunogenicity of TAAs remain to be elucidated. Here we found that CD4 T-cell responses suppressed the immunogenicity of the concomitant TAA in a murine melanoma model in which intratumoral activated dendritic therapy (ITADT) was used for treatment of the established cancer, and we observed that the antitumor effects were largely dependent on the CD8 T-cell response. CD4 T-cell depletion simply enhanced the tyrosinase-related protein (TRP)-2(180-188) peptide-specific cytotoxic T-cell (CTL) responses, and CD4 T-cell depletion provided immunogenicity for mgp100(25-33) peptide, to which a CTL response could not be detected at all in CD4 T-cell-intact mice in the early therapeutic phase. Further, the mgp100(25-33) peptide-specific CTL response again became undetectable after the recovery of CD4 T cells in previously CD4-depleted, tumor-eradicated mice, whereas the TRP-2(180-188) peptide-specific CTL response was still much stronger in CD4-depleted mice than in CD4-intact mice. These findings suggest that the CD4 T cell-mediated masking effects of the immunogenicity of tumor-associated antigens are qualitatively and quantitatively different depending on the individual antigens.

Nectin-like 4 Complexes with Choline Transporter-like Protein-1 and Regulates Schwann Cell Choline Homeostasis and Lipid Biogenesis in Vitro.

PubMed

Heffernan, Corey; Jain, Mohit R; Liu, Tong; Kim, Hyosung; Barretto, Kevin; Li, Hong; Maurel, Patrice

2017-03-17

Nectin-like 4 (NECL4, CADM4) is a Schwann cell-specific cell adhesion molecule that promotes axo-glial interactions. In vitro and in vivo studies have shown that NECL4 is necessary for proper peripheral nerve myelination. However, the molecular mechanisms that are regulated by NECL4 and affect peripheral myelination currently remain unclear. We used an in vitro approach to begin identifying some of the mechanisms that could explain NECL4 function. Using mass spectrometry and Western blotting techniques, we have identified choline transporter-like 1 (CTL1) as a putative complexing partner with NECL4. We show that intracellular choline levels are significantly elevated in NECL4-deficient Schwann cells. The analysis of extracellular d 9 -choline uptake revealed a deficit in the amount of d 9 -choline found inside NECL4-deficient Schwann cells, suggestive of either reduced transport capabilities or increased metabolization of transported choline. An extensive lipidomic screen of choline derivatives showed that total phosphatidylcholine and phosphatidylinositol (but not diacylglycerol or sphingomyelin) are significantly elevated in NECL4-deficient Schwann cells, particularly specific subspecies of phosphatidylcholine carrying very long polyunsaturated fatty acid chains. Finally, CTL1-deficient Schwann cells are significantly impaired in their ability to myelinate neurites in vitro To our knowledge, this is the first demonstration of a bona fide cell adhesion molecule, NECL4, regulating choline homeostasis and lipid biogenesis. Phosphatidylcholines are major myelin phospholipids, and several phosphorylated phosphatidylinositol species are known to regulate key aspects of peripheral myelination. Furthermore, the biophysical properties imparted to plasma membranes are regulated by fatty acid chain profiles. Therefore, it will be important to translate these in vitro observations to in vivo studies of NECL4 and CTL1-deficient mice. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

HLA class I expression predicts prognosis and therapeutic benefits from tyrosine kinase inhibitors in metastatic renal-cell carcinoma patients.

PubMed

Wang, Jiajun; Liu, Li; Qu, Yang; Xi, Wei; Xia, Yu; Bai, Qi; Xiong, Ying; Long, Qilai; Xu, Jiejie; Guo, Jianming

2018-01-01

Classical HLA class I antigen is highly involved in antigen presentation and adaptive immune response against tumor. In this study, we explored its predictive value for treatment response and survival in metastatic renal-cell carcinoma (mRCC) patients. A TKI cohort of 111 mRCC patients treated with sunitinib or sorafenib and a non-TKI cohort of 160 mRCC patients treated with interleukin-2 or interferon-α-based immunotherapy at a single institution were retrospectively enrolled. HLA class I expression and cytotoxic T lymphocyte (CTL) density was assessed by immunohistochemistry on tissue microarrays. Association between HLA class I and CTL was also assessed in the TCGA KIRC cohort. In the TKI cohort, down-regulated HLA class I was associated with lower objective response rate of TKI therapy (P = 0.004), shorter overall survival (OS) (P = 0.001), and shorter progression free survival (PFS) (P < 0.001). Multivariate Cox regression model defined HLA expression as an independent prognostic factor for both OS [hazard ratio 1.687 (95% CI 1.045-2.724), P = 0.032] and PFS [hazard ratio 2.139 (95% CI 1.376-3.326), P = 0.001]. In the non-TKI cohort, HLA class I was not significantly associated with survival. HLA class I expression was associated with CTL infiltration and function, and its prognostic value was more predominant in CTL high-density tumors (P < 0.001) rather than CTL low-density tumors (P = 0.294). Classical HLA class I expression can serve as a potential predictive biomarker for TKI therapy in mRCC patients. Its predictive value was restricted in CTL high-density tumors. However, further external validations and functional investigations are still required.

D-Cateslytin: a new antifungal agent for the treatment of oral Candida albicans associated infections.

PubMed

Dartevelle, Pauline; Ehlinger, Claire; Zaet, Abdurraouf; Boehler, Christian; Rabineau, Morgane; Westermann, Benoit; Strub, Jean-Marc; Cianferani, Sarah; Haïkel, Youssef; Metz-Boutigue, Marie-Hélène; Marban, Céline

2018-06-18

The excessive use of antifungal agents, compounded by the shortage of new drugs being introduced into the market, is causing the accumulation of multi-resistance phenotypes in many fungal strains. Consequently, new alternative molecules to conventional antifungal agents are urgently needed to prevent the emergence of fungal resistance. In this context, Cateslytin (Ctl), a natural peptide derived from the processing of Chromogranin A, has already been described as an effective antimicrobial agent against several pathogens including Candida albicans. In the present study, we compared the antimicrobial activity of two conformations of Ctl, L-Ctl and D-Ctl against Candida albicans. Our results show that both D-Ctl and L-Ctl were potent and safe antifungal agents. However, in contrast to L-Ctl, D-Ctl was not degraded by proteases secreted by Candida albicans and was also stable in saliva. Using video microscopy, we also demonstrated that D-Ctl can rapidly enter C. albicans, but is unable to spread within a yeast colony unless from a mother cell to a daughter cell during cellular division. Besides, we revealed that the antifungal activity of D-Ctl could be synergized by voriconazole, an antifungal of reference in the treatment of Candida albicans related infections. In conclusion, D-Ctl can be considered as an effective, safe and stable antifungal and could be used alone or in a combination therapy with voriconazole to treat Candida albicans related diseases including oral candidosis.

Membrane transport mechanisms of choline in human intestinal epithelial LS180 cells.

PubMed

Horie, Asuka; Ishida, Kazuya; Watanabe, Yuri; Shibata, Kaito; Hashimoto, Yukiya

2014-12-01

The aim of the present study was to investigate the membrane transport mechanisms of choline using human intestinal epithelial LS180 cells. The mRNA of choline transporter-like proteins (CTLs) was expressed significantly in LS180 cells, and the rank order was CTL1 > CTL4 > CTL3 > CTL2 > CTL5. In contrast, the mRNA expression of other choline transporters, organic cation transporter (OCT) 1, OCT2 and high-affinity choline transporter 1 (CHT1), was considerably lower in LS180 cells. Five mm unlabelled choline, hemicolinium-3 and guanidine, but not tetraethylammonium, inhibited the cellular uptake of 100 µm choline in LS180 cells. The uptake of choline into LS180 cells was virtually Na(+)-independent. The uptake of choline was significantly decreased by acidification of the extracellular pH; however, it was not increased by alkalization of the extracellular pH. In addition, both acidification and alkalization of intracellular pH decreased the uptake of choline, indicating that the choline uptake in LS180 cells is not stimulated by the outward H(+) gradient. On the other hand, the uptake of choline was decreased by membrane depolarization along with increasing extracellular K(+) concentration. In addition, the Na(+)-independent uptake of choline was saturable, and the Km value was estimated to be 108 µm. These findings suggest that the uptake of choline into LS180 cells is membrane potential-dependent, but not outward H(+) gradient-dependent. Copyright © 2014 John Wiley & Sons, Ltd.

[Anti-metastatic effect of vascular endothelial growth factor receptor 2 extracellular domain gene-modified dendritic cell vaccination in murine model with experimental pulmonary metastasis].

PubMed

Pan, Jian-ping; Weng, Yue-song; Wu, Qian-qian

2006-09-01

To investigate the anti-metastatic effect of vascular endothelial growth factor receptor 2 extracellular domain gene-modified dendritic cell (DC-sVEGFR-2) vaccination. Dendritic cells (DC) were electroporated with pcDNA3. 1/sVEGFR-2 plasmid DNA. Expression of sVEGFR-2 was determined by ELISA. For immunization, C57BL/6 mice were intravenously injected three times with 1 x 10(5) cells per mouse of DC, pcDNA3. 1-transfected DC (DC-vector) , DC-sVEGFR-2, or 100 microl of PBS at 7-day intervals. At 10 days after the last immunization, the immunized mice were subjected to assessment of cytotoxic T lymphocyte ( CTL) response to VEGFR-2, alginate bead analysis of tumor cell-induced angiogenesis, and observation of the anti-metastatic effect in B16 melanoma metastasis model. CTL activity was determined by a standard 4-h 51Cr release assay against VEGFR-2 + vascular endothelial cell line H5V, 3LL cells stably transfected with pcDNA3. 1/sVEGFR-2 (3LL,-sVEGFR-2), and VEGFR-2- cell lines EL-4 and 3LL. Monoclonal antibodies GK1.5 anti-CD4 and 2.43 anti-CD8 were used to deplete in vivo CD4 + T cells and CD8' T cells, respectively. DC-sVEGFR-2 could effectively express sVEGFR-2, whereas DC-vector and DC could not. Immunization of mice with DC-sVEGFR-2 significantly induce CTL activity against VEGFR-2 + cell lines H5V and 3LL-sVEGFR-2, however, no significant CTL activity was observed when VEGFR-2- syngeneic cell lines EL-4 and 3LL. were used as target cells, implying this CTL activity was VEGFR-2 specific. Alginate bead analysis of in vivo neoangiogenesis showed that the inhibition reached 50% in mice vaccinated with DC-sVEGFR-2 compared with mice vaccinated with DC, DC-vector or PBS. Anti-metastatic experiment showed that profound reduction in pulmonary metastases was found in mice immunized with DC-sVEGFR-2, while mice immunized with PBS, DC, DC-vector developed extensive pulmonary metastases. The number of tumor nodules on lung surface decreased by 81.9% in mice immunized with DC-sVEGFR-2 when compared with mice immunized with DC-vector (49.7+/-12.7 vs. 9.0+/-3.2). In vivo T cell subset depletion experiments showed that the anti-metastatic effect of DC-sVEGFR-2 vaccination was abrogated in CD8 + T cell-depleted but not in CD4+ T cell-depleted mice. Immunization of mice with DC-sVEGFR-2 could break self-tolerance and induce a significant CTL response to VEGFR-2, leading to profound inhibition of tumor-cell induced angiogenesis and metastasis. This anti-metastatic effect is mainly mediated by CD8+ T cells.

Oxadiazole-isopropylamides as Potent and Non-covalent Proteasome Inhibitors

PubMed Central

Ozcan, Sevil; Kazi, Aslamuzzaman; Marsilio, Frank; Fang, Bin; Guida, Wayne C.; Koomen, John; Lawrence, Harshani R.; Sebti, Saïd M.

2013-01-01

Screening of the 50,000 ChemBridge compound library led to the identification of the oxadiazole-isopropylamide 1 (PI-1833) which inhibited CT-L activity (IC50 0.60 μM) with little effects on the other 2 major proteasome proteolytic activities, T-L and PGPH-L. LC/MS-MS and dialysis show that 1 is a non-covalent and rapidly reversible CT-L inhibitor. Focused library synthesis provided 11ad (PI-1840) with CT-L activity (IC50 27 nM). Detailed SAR studies indicate that the amide moiety and the 2 phenyl rings are sensitive toward modifications. Hydrophobic residues, such as propyl or butyl, in the para-position (not ortho or meta) of the A-ring and a meta-pyridyl group as B-ring significantly improve activity. Compound 11ad (IC50 0.37 μM) is more potent than 1 (IC50 3.5 μM) at inhibiting CT-L activity in intact MDA-MB-468 human breast cancer cells and inhibiting their survival. The activity of 11ad warrants further pre-clinical investigation of this class as non-covalent proteasome inhibitors. PMID:23547706

An antigen receptor-driven, interleukin 2-independent pathway for proliferation of murine cytolytic T lymphocyte clones

PubMed Central

1986-01-01

Proliferation of T lymphocytes can be induced by IL-2, either through an autocrine pathway in which the responding cell produces its own IL-2 or through an exocrine pathway in which IL-2 secreted by Th stimulates proliferation of IL-2-dependent CTL. However, proliferation of at least some CTL clones, such as CTL L3 and CTL dB45, also can be induced by stimulation of the antigen receptor in the absence of IL-2. Stimulation of these cloned CTL with T cell-depleted allogeneic spleen cells, allogeneic tumor cells, or immobilized mAb reactive with the T cell antigen receptor (TCR) induced thymidine incorporation, entry into cell cycle, and secretion of macrophage activating factor, but these stimuli did not induce the secretion of IL-2. Several observations indicated that such proliferation of cloned CTL induced by stimulation of the TCR was independent of IL-2; IL-2 could not be detected in supernatants from stimulated CTL cells. mAbs reactive with the murine IL-2-R efficiently blocked IL-2-mediated thymidine incorporation in cloned CTL and Th, but had no inhibitory effect on TCR-driven thymidine incorporation in the CTL clones. TCR-driven thymidine incorporation in cloned Th L2 cells was profoundly inhibited by these antibodies, indicating the operation of an IL-2-mediated autocrine pathway for proliferation in this cloned Th. When antibodies to the TCR were used to stimulate cloned CTL and Th, IFN-gamma mRNA was easily shown in the cloned CTL and Th. Although IL-2 mRNA could be detected in the cloned Th, it was never observed in the cloned CTL. These findings provide evidence for the existence of a TCR-mediated, IL-2-independent pathway for induction of cellular proliferation in cloned murine CTL. PMID:3486939

Antibodies attenuate the capacity of dendritic cells to stimulate HIV-specific cytotoxic T lymphocytes

PubMed Central

Posch, Wilfried; Cardinaud, Sylvain; Hamimi, Chiraz; Fletcher, Adam; Mühlbacher, Annelies; Loacker, Klaus; Eichberger, Paul; Dierich, Manfred P.; Pancino, Gianfranco; Lass-Flörl, Cornelia; Moris, Arnaud; Saez-Cirion, Asier; Wilflingseder, Doris

2014-01-01

Background Control of HIV is suggested to depend on potent effector functions of the virus-specific CD8+ T-cell response. Antigen opsonization can modulate the capture of antigen, its presentation, and the priming of specific CD8+ T-cell responses. Objective We have previously shown that opsonization of retroviruses acts as an endogenous adjuvant for dendritic cell (DC)–mediated induction of specific cytotoxic T lymphocytes (CTLs). However, in some HIV-positive subjects, high levels of antibodies and low levels of complement fragments coat the HIV surface. Methods Therefore we analyzed the effect of IgG opsonization on the antigen-presenting capacity of DCs by using CD8+ T-cell proliferation assays after repeated prime boosting, by measuring the antiviral activity against HIV-infected autologous CD4+ T cells, and by determining IFN-γ secretion from HIV-specific CTL clones. Results We find that DCs exposed to IgG-opsonized HIV significantly decreased the HIV-specific CD8+ T-cell response compared with the earlier described efficient CD8+ T-cell activation induced by DCs loaded with complement-opsonized HIV. DCs exposed to HIV bearing high surface IgG levels after incubation in plasma from HIV-infected subjects acted as weak stimulators for HIV-specific CTL clones. In contrast, HIV opsonized with plasma from patients exhibiting high complement and low IgG deposition on the viral surface favored significantly higher activation of HIV-specific CD8+ T-cell clones. Conclusion Our ex vivo and in vitro observations provide the first evidence that IgG opsonization of HIV is associated with a decreased CTL-stimulatory capacity of DCs. PMID:23063584

Inventory of File sref_nmb.t03z.pgrb221.ctl.grib2

Science.gov Websites

006 10 m above ground UGRD analysis U-Component of Wind [m/s] ENS=low-res ctl 007 10 m above ground VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl 008 surface WEASD analysis Water Equivalent of -Component of Wind [m/s] ENS=low-res ctl 021 250 mb VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl

Inventory of File sref_nmb.t03z.pgrb132.ctl.grib2

Science.gov Websites

006 10 m above ground UGRD analysis U-Component of Wind [m/s] ENS=low-res ctl 007 10 m above ground VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl 008 surface WEASD analysis Water Equivalent of -Component of Wind [m/s] ENS=low-res ctl 021 250 mb VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl

Inventory of File sref_nmb.t03z.pgrb243.ctl.grib2

Science.gov Websites

006 10 m above ground UGRD analysis U-Component of Wind [m/s] ENS=low-res ctl 007 10 m above ground VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl 008 surface WEASD analysis Water Equivalent of -Component of Wind [m/s] ENS=low-res ctl 021 250 mb VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl

Inventory of File sref_nmb.t03z.pgrb216.ctl.grib2

Science.gov Websites

006 10 m above ground UGRD analysis U-Component of Wind [m/s] ENS=low-res ctl 007 10 m above ground VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl 008 surface WEASD analysis Water Equivalent of -Component of Wind [m/s] ENS=low-res ctl 021 250 mb VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl

Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants.

PubMed

Kawano, Masaaki; Morikawa, Katsuma; Suda, Tatsuya; Ohno, Naohito; Matsushita, Sho; Akatsuka, Toshitaka; Handa, Hiroshi; Matsui, Masanori

2014-01-05

Virus-like particles (VLPs) are a promising vaccine platform due to the safety and efficiency. However, it is still unclear whether polyomavirus-based VLPs are useful for this purpose. Here, we attempted to evaluate the potential of polyomavirus VLPs for the antiviral vaccine using simian virus 40 (SV40). We constructed chimeric SV40-VLPs carrying an HLA-A*02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A*02:01-transgenic mice were then immunized with the chimeric SV40-VLPs. The chimeric SV40-VLPs effectively induced influenza-specific CTLs and heterosubtypic protection against influenza A viruses without the need of adjuvants. Because DNase I treatment of the chimeric SV40-VLPs did not disrupt CTL induction, the intrinsic adjuvant property may not result from DNA contaminants in the VLP preparation. In addition, immunization with the chimeric SV40-VLPs generated long-lasting memory CTLs. We here propose that the chimeric SV40-VLPs harboring an epitope may be a promising CTL-based vaccine platform with self-adjuvant properties. © 2013 Elsevier Inc. All rights reserved.

An attenuated herpes simplex virus type 1 (HSV1) encoding the HIV-1 Tat protein protects mice from a deadly mucosal HSV1 challenge.

PubMed

Sicurella, Mariaconcetta; Nicoli, Francesco; Gallerani, Eleonora; Volpi, Ilaria; Berto, Elena; Finessi, Valentina; Destro, Federica; Manservigi, Roberto; Cafaro, Aurelio; Ensoli, Barbara; Caputo, Antonella; Gavioli, Riccardo; Marconi, Peggy C

2014-01-01

Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and dissemination.

An Attenuated Herpes Simplex Virus Type 1 (HSV1) Encoding the HIV-1 Tat Protein Protects Mice from a Deadly Mucosal HSV1 Challenge

PubMed Central

Sicurella, Mariaconcetta; Nicoli, Francesco; Gallerani, Eleonora; Volpi, Ilaria; Berto, Elena; Finessi, Valentina; Destro, Federica; Manservigi, Roberto; Cafaro, Aurelio; Ensoli, Barbara; Caputo, Antonella; Gavioli, Riccardo; Marconi, Peggy C.

2014-01-01

Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and dissemination. PMID:25033084

Effects of purified lignin and mannan oligosaccharides on intestinal integrity and microbial populations in the ceca and litter of broiler chickens.

PubMed

Baurhoo, B; Phillip, L; Ruiz-Feria, C A

2007-06-01

A study was conducted to evaluate lignin and mannan oligosaccharides as potential alternatives to antibiotic growth promoters in broilers. Dietary treatments included an antibiotic-free diet (CTL-), a positive control (CTL+, 11 mg/kg of virginiamycin), and an antibiotic-free diet containing BioMos (MOS, 0.2% to 21 d and 0.1% thereafter) or Alcell lignin at 1.25% (LL) or 2.5% (HL) of the diet. Each treatment was randomly assigned to 4 floor pen replicates (40 birds each). Body weight and feed conversion were recorded weekly throughout 42 d. Jejunum histology was analyzed at d 14, 28, and 42. At d 28 and 42, cecal contents were assayed for Escherichia coli, Salmonella, lactobacilli, and bifidobacteria, and the litter was analyzed for E. coli and Salmonella. Birds fed the CTL- diet were heavier (P<0.05) than those fed the other dietary treatments, but feed conversion was not affected by dietary treatments. Birds fed MOS and LL had increased jejunum villi height and a higher number of goblet cells per villus (P<0.05) when compared with those fed the CTL+ diet. At d 42, birds fed MOS, LL, or HL had greater lactobacilli numbers than those fed the CTL+ diet. Compared with the CTL+ diet, the MOS diet increased the populations of bifidobacteria (P<0.05) in the ceca. Litter E. coli load was lower in birds fed MOS (P<0.05) than in birds fed the CTL+ diet but comparable to that of birds fed the LL or HL diet. Broiler performance was similar in birds fed antibiotics or antibiotic-free diets containing either MOS or lignin. However, birds fed MOS and LL had a comparative advantage over birds fed antibiotics as evidenced by an increased population of beneficial bacteria in the ceca, increased villi height and number of goblet cells in the jejunum, and lower population of E. coli in the litter.

A high selective cataluminescence sensor for the determination of tetrahydrofuran vapor

NASA Astrophysics Data System (ADS)

Cao, Xiaoan; Dai, Huimei; Chen, Suilin; Zeng, Jiayi; Zhang, Keke; Sun, Yan

2013-02-01

A novel tetrahydrofuran (THF) vapor sensor was designed based on the cataluminescence (CTL) of THF on nanosized γ-Al2O3/MgO (mol ratio = 1.5:1). SEM and XRD were applied for its characterization. We found that the CTL was strongly produced when THF vapor flowed through a nanosized Al-Mg mixed-metal oxide surface, while the CTL was weakly generated when THF vapor flowed through a single nanosized γ-Al2O3 or MgO surface. Quantitative analysis was performed at an optimal temperature of 279 °C, a wavelength of 460 nm and a flow rate of 360 mL min-1. The linear range of the CTL intensity versus concentrations of THF vapor was 1.0-3000 mL m-3 with a detection limit of 0.67 mL m-3. No (or only very low) interference was observed by formaldehyde, methanol, ethanol, benzene, toluene, ethyl acetate, ammonia, cyclohexane, chloroform, glycol armour ether, glycol ether, isopropyl ether and n-butyl ether or acetic acid. Since the response of the sensor was rapid and the system was easy to handle, we believe that the sensor has great potential for real-world use.

Functional analysis of [methyl-(3)H]choline uptake in glioblastoma cells: Influence of anti-cancer and central nervous system drugs.

PubMed

Taguchi, Chiaki; Inazu, Masato; Saiki, Iwao; Yara, Miki; Hara, Naomi; Yamanaka, Tsuyoshi; Uchino, Hiroyuki

2014-04-01

Positron emission tomography (PET) and PET/computed tomography (PET-CT) studies with (11)C- or (18)F-labeled choline derivatives are used for PET imaging in glioblastoma patients. However, the nature of the choline transport system in glioblastoma is poorly understood. In this study, we performed a functional characterization of [methyl-(3)H]choline uptake and sought to identify the transporters that mediate choline uptake in the human glioblastoma cell lines A-172 and U-251MG. In addition, we examined the influence of anti-cancer drugs and central nervous system drugs on the transport of [methyl-(3)H]choline. High- and low-affinity choline transport systems were present in A-172 cells, U-251MG cells and astrocytes, and these were Na(+)-independent and pH-dependent. Cell viability in A-172 cells was not affected by choline deficiency. However, cell viability in U-251MG cells was significantly inhibited by choline deficiency. Both A-172 and U-251MG cells have two different choline transporters, choline transporter-like protein 1 (CTL1) and CTL2. In A-172 cells, CTL1 is predominantly expressed, whereas in U-251MG cells, CTL2 is predominantly expressed. Treatment with anti-cancer drugs such as cisplatin, etoposide and vincristine influenced [methyl-(3)H]choline uptake in U-251MG cells, but not A-172 cells. Central nervous system drugs such as imipramine, fluvoxamine, paroxetine, reboxetine, citalopram and donepezil did not affect cell viability or [methyl-(3)H]choline uptake. The data presented here suggest that CTL1 and CTL2 are functionally expressed in A-172 and U-251MG cells and are responsible for [methyl-(3)H]choline uptake that relies on a directed H(+) gradient as a driving force. Furthermore, while anti-cancer drugs altered [methyl-(3)H]choline uptake, central nervous system drugs did not affect [methyl-(3)H]choline uptake. Copyright © 2014 Elsevier Inc. All rights reserved.

Low Dose Decitabine Treatment Induces CD80 Expression in Cancer Cells and Stimulates Tumor Specific Cytotoxic T Lymphocyte Responses

PubMed Central

Zhou, Ji-Hao; Yao, Yu-Shi; Li, Yong-Hui; Xu, Yi-Han; Li, Jing-Xin; Gao, Xiao-Ning; Zhou, Min-Hang; Jiang, Meng-Meng; Gao, Li; Ding, Yi; Lu, Xue-Chun; Shi, Jin-Long; Luo, Xu-Feng; Wang, Jia; Wang, Li-Li; Qu, Chunfeng; Bai, Xue-Feng; Yu, Li

2013-01-01

Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8+, but not CD4+ T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy. PMID:23671644

Low dose decitabine treatment induces CD80 expression in cancer cells and stimulates tumor specific cytotoxic T lymphocyte responses.

PubMed

Wang, Li-Xin; Mei, Zhen-Yang; Zhou, Ji-Hao; Yao, Yu-Shi; Li, Yong-Hui; Xu, Yi-Han; Li, Jing-Xin; Gao, Xiao-Ning; Zhou, Min-Hang; Jiang, Meng-Meng; Gao, Li; Ding, Yi; Lu, Xue-Chun; Shi, Jin-Long; Luo, Xu-Feng; Wang, Jia; Wang, Li-Li; Qu, Chunfeng; Bai, Xue-Feng; Yu, Li

2013-01-01

Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8(+), but not CD4(+) T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy.

Optimal Waist-to-Height Ratio Values for Cardiometabolic Risk Screening in an Ethnically Diverse Sample of South African Urban and Rural School Boys and Girls

PubMed Central

Matsha, Tandi E.; Kengne, Andre-Pascal; Yako, Yandiswa Y.; Hon, Gloudina M.; Hassan, Mogamat S.; Erasmus, Rajiv T.

2013-01-01

Background The proposed waist-to-height ratio (WHtR) cut-off of 0.5 is less optimal for cardiometabolic risk screening in children in many settings. The purpose of this study was to determine the optimal WHtR for children from South Africa, and investigate variations by gender, ethnicity and residence in the achieved value. Methods Metabolic syndrome (MetS) components were measured in 1272 randomly selected learners, aged 10–16 years, comprising of 446 black Africans, 696 mixed-ancestry and 130 Caucasians. The Youden’s index and the closest-top-left (CTL) point approaches were used to derive WHtR cut-offs for diagnosing any two MetS components, excluding the waist circumference. Results The two approaches yielded similar cut-off in girls, 0.465 (sensitivity 50.0, specificity 69.5), but two different values in boys, 0.455 (42.9, 88.4) and 0.425 (60.3, 67.7) based on the Youden’s index and the CTL point, respectively. Furthermore, WHtR cut-off values derived differed substantially amongst the regions and ethnic groups investigated, whereby the highest cut-off was observed in semi-rural and white children, respectively, Youden’s index0.505 (31.6, 87.1) and CTL point 0.475 (44.4, 75.9). Conclusion The WHtR cut-off of 0.5 is less accurate for screening cardiovascular risk in South African children. The optimal value in this setting is likely gender and ethnicity-specific and sensitive to urbanization. PMID:23967160

Clostridium perfringens enterotoxin is a superantigen reactive with human T cell receptors V beta 6.9 and V beta 22

PubMed Central

1992-01-01

Candidate superantigens were screened for their ability to induce lysis of human histocompatibility leukocyte antigen class II-positive targets by human CD8+ influenza-specific cytotoxic T cell (CTL) lines. Clostridium perfringens enterotoxin (CPET) induced major histocompatibility complex unrestricted killing by some but not all CTL lines. Using "anchored" polymerase chain reactions, CPET was shown to selectively stimulate peripheral blood lymphocytes bearing T cell receptor V beta 6.9 and V beta 22 in five healthy donors. V beta 24, V beta 21, V beta 18, V beta 5, and V beta 6.1-5 appeared to be weakly stimulated. Antigen processing was not required for CPET to induce proliferation. Like the staphylococcal enterotoxins, CPET is a major cause of food poisoning. These data suggest that superantigenic and enterotoxigenic properties may be closely linked. PMID:1512551

Adult Autism Subthreshold Spectrum (AdAS Spectrum): Validation of a questionnaire investigating subthreshold autism spectrum.

PubMed

Dell'Osso, L; Gesi, C; Massimetti, E; Cremone, I M; Barbuti, M; Maccariello, G; Moroni, I; Barlati, S; Castellini, G; Luciano, M; Bossini, L; Rocchetti, M; Signorelli, M; Aguglia, E; Fagiolini, A; Politi, P; Ricca, V; Vita, A; Carmassi, C; Maj, M

2017-02-01

Increasing literature has shown the usefulness of a dimensional approach to autism. The present study aimed to determine the psychometric properties of the Adult Autism Subthreshold Spectrum (AdAS Spectrum), a new questionnaire specifically tailored to assess subthreshold forms of autism spectrum disorder (ASD) in adulthood. 102 adults endorsing at least one DSM-5 symptom criterion for ASD (ASDc), 143 adults diagnosed with a feeding and eating disorder (FED), and 160 subjects with no mental disorders (CTL), were recruited from 7 Italian University Departments of Psychiatry and administered the following: SCID-5, Autism-Spectrum Quotient (AQ), Ritvo Autism and Asperger Diagnostic Scale 14-item version (RAADS-14), and AdAS Spectrum. The AdAS Spectrum demonstrated excellent internal consistency for the total score (Kuder-Richardson's coefficient=.964) as well as for five out of seven domains (all coefficients>.80) and sound test-retest reliability (ICC=.976). The total and domain AdAS Spectrum scores showed a moderate to strong (>.50) positive correlation with one another and with the AQ and RAADS-14 total scores. ASDc subjects reported significantly higher AdAS Spectrum total scores than both FED (p<.001) and CTL (p<.001), and significantly higher scores on the Childhood/adolescence, Verbal communication, Empathy, Inflexibility and adherence to routine, and Restricted interests and rumination domains (all p<.001) than FED, while on all domains compared to CTL. CTL displayed significantly lower total and domain scores than FED (all p<.001). A significant effect of gender emerged for the Hyper- and hyporeactivity to sensory input domain, with women showing higher scores than men (p=.003). A Diagnosis* Gender interaction was also found for the Verbal communication (p=.019) and Empathy (p=.023) domains. When splitting the ASDc in subjects with one symptom criterion (ASD 1 ) and those with a ASD, and the FED in subjects with no ASD symptom criteria (FED 0 ) and those with one ASD symptom criterion (FED 1 ) , a gradient of severity in AdAS Spectrum scores from CTL subjects to ASD patients, across FED 0 , ASD 1 , FED 1 was shown. The AdAS Spectrum showed excellent internal consistency and test-retest reliability and strong convergent validity with alternative dimensional measures of ASD. The questionnaire performed differently among the three diagnostic groups and enlightened some significant effects of gender in the expression of autistic traits. Copyright © 2016 Elsevier Inc. All rights reserved.

In vitro reactivity of allospecific cytotoxic T lymphocytes does not explain the taboo phenomenon.

PubMed

Stobbe, I; van der Meer-Prins, E; Smits, J M; Doxiadis, I I; Claas, F H

1999-12-01

Matching for human leucocyte antigens (HLA) is important for graft survival in kidney transplantation. Nevertheless, most patients receive a kidney graft with multiple HLA mismatches. Some of these mismatches seem to be more harmful than others. By studying the effect of single HLA mismatches in the context of the patients' own HLA, we have previously identified donor/recipient combinations with a significantly higher incidence of early graft failure, the so-called taboo combinations. In the present study we investigated whether a higher cytotoxic T lymphocyte (CTL) response towards taboo mismatches may be involved in this phenomenon. CTL reactivity was determined both in taboo and control combinations by in vitro CTL precursor assays, using peripheral blood mononuclear cells and proximal tubular epithelial cells as target cells. Inhibition studies with CD8-antibody as well as Cyclosporin A were performed to identify high avidity and primed CTLs. Furthermore, in committed CTLp assays indirect recognition of the taboo mismatch was tested using synthetic peptides. The CTL precursor frequencies in taboo combinations were always lower than the CTL precursor frequencies in control combinations. No difference in avidity and activation status of the CTLs could be detected when taboo combinations were compared with the controls. In the committed CTLp assays no reactivity towards any of the synthetic peptides was observed. The significantly poorer graft survival of taboo combinations cannot be explained by a higher number of donor-specific CTLs. Furthermore, the avidity or activation status of these CTLs does not provide a clue to the taboo phenomenon.

CRTAM determines the CD4+ cytotoxic T lymphocyte lineage

PubMed Central

Takeuchi, Arata; Badr, Mohamed El Sherif Gadelhaq; Miyauchi, Kosuke; Ishihara, Chitose; Onishi, Reiko; Guo, Zijin; Sasaki, Yoshiteru; Ike, Hiroshi; Takumi, Akiko; Tsuji, Noriko M.; Murakami, Yoshinori; Katakai, Tomoya; Kubo, Masato

2016-01-01

Naive T cells differentiate into various effector T cells, including CD4+ helper T cell subsets and CD8+ cytotoxic T cells (CTL). Although cytotoxic CD4+ T cells (CD4+CTL) also develop from naive T cells, the mechanism of development is elusive. We found that a small fraction of CD4+ T cells that express class I–restricted T cell–associated molecule (CRTAM) upon activation possesses the characteristics of both CD4+ and CD8+ T cells. CRTAM+ CD4+ T cells secrete IFN-γ, express CTL-related genes, such as eomesodermin (Eomes), Granzyme B, and perforin, after cultivation, and exhibit cytotoxic function, suggesting that CRTAM+ T cells are the precursor of CD4+CTL. Indeed, ectopic expression of CRTAM in T cells induced the production of IFN-γ, expression of CTL-related genes, and cytotoxic activity. The induction of CD4+CTL and IFN-γ production requires CRTAM-mediated intracellular signaling. CRTAM+ T cells traffic to mucosal tissues and inflammatory sites and developed into CD4+CTL, which are involved in mediating protection against infection as well as inducing inflammatory response, depending on the circumstances, through IFN-γ secretion and cytotoxic activity. These results reveal that CRTAM is critical to instruct the differentiation of CD4+CTL through the induction of Eomes and CTL-related gene. PMID:26694968

Aberrant apoptotic machinery confers melanoma dual resistance to BRAFV600E inhibitor and immune effector cells: immunosensitization by a histone deacetylase inhibitor

PubMed Central

Jazirehi, Ali R; Nazarian, Ramin; Torres-Collado, Antoni Xavier; Economou, James S

2014-01-01

BRAFV600E-inhibitors (BRAFi; e.g., vemurafenib) and modern immune-based therapies such as PD-1/PD-L1 and CTLA-4 checkpoints blockade and adoptive cell transfer (ACT) have significantly improved the care of melanoma patients. Having these two effective (BRAFi and immunotherapy) therapies raises the question whether there is a rational biological basis for using them in combination. We developed an in vitro model to determine whether tumor resistance mechanisms to a small molecule inhibitor of a driver oncogene, and to cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-delivered apoptotic death signals were exclusive or intersecting. We generated melanoma sublines resistant to BRAFi vemurafenib and to CTL recognizing the MART-1 melanoma antigen. Vemurafenib-resistant (VemR) sublines were cross-resistant to MART CTL and NK cells indicating that a common apoptotic pathway governing tumor response to both modalities was disrupted. Pretreatment of VemR melanomas with a histone deacetylase inhibitor (HDACi) restored sensitivity to MART CTL and NK apoptosis by skewing the apoptotic gene programs towards a proapoptotic phenotype. Our in vitro findings suggest that during the course of acquisition of BRAFi resistance, melanomas develop cross-resistance to CTL- and NK-killing. Further, aberrant apoptotic pathways, amenable by an FDA-approved chromatin remodeling drug, regulate tumor resistance mechanisms to immune effector cells. These results may provide rational molecular basis for further investigations to combine these therapies clinically. PMID:24660121

Respondents' answers to community attitudinal surveys represent impressions of soundscapes and not merely reactions to the physical noise.

PubMed

Schomer, Paul; Mestre, Vincent; Schulte-Fortkamp, Brigitte; Boyle, James

2013-07-01

Noise dose as a sole independent variable accounts only for less than half of the variance in community response data. Non-acoustic factors, such as attitude to the noise source identified by Job [(1988). J. Acoust. Soc. Am. 83(3), 991-1001] and others, reduce the unexplained variance. This paper suggests that non-acoustic factors reflect the context in which the sonic environment is perceived; hence, these factors constitute the judgments of a soundscape. Fidell et al. [(2011). J. Acoust. Soc. Am. 130(2), 791-806] show that a "community tolerance level" (CTL) is a one-number, community-specific, independent variable that represents the aggregate influence on annoyance judgments of all non-acoustic influences. Taken together these findings suggest that: (i) CTL is a one-number quantification of a soundscape, and (ii) a soundscape can be quantified by a single, numeric variable. It follows, if one can predict or calculate the single numeric quantification of a soundscape, that number will be the CTL. These results can only be true for a soundscape judged on the basis of annoyance. Virtually no data exist for which the respondents even had the possibility to rate the sonic environment positively, i.e., pleasing, so the application of CTL to soundscapes judged positively is not clear.

Intranasal delivery of recombinant parvovirus-like particles elicits cytotoxic T-cell and neutralizing antibody responses.

PubMed

Sedlik, C; Dridi, A; Deriaud, E; Saron, M F; Rueda, P; Sarraseca, J; Casal, J I; Leclerc, C

1999-04-01

We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4(+) and CD8(+) T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8(+) T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8(+) T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8(+) T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration.

Intranasal Delivery of Recombinant Parvovirus-Like Particles Elicits Cytotoxic T-Cell and Neutralizing Antibody Responses

PubMed Central

Sedlik, C.; Dridi, A.; Deriaud, E.; Saron, M. F.; Rueda, P.; Sarraseca, J.; Casal, J. I.; Leclerc, C.

1999-01-01

We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4+ and CD8+ T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8+ T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8+ T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8+ T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration. PMID:10074120

Photochemical Internalization of Peptide Antigens Provides a Novel Strategy to Realize Therapeutic Cancer Vaccination

PubMed Central

Haug, Markus; Brede, Gaute; Håkerud, Monika; Nedberg, Anne Grete; Gederaas, Odrun A.; Flo, Trude H.; Edwards, Victoria T.; Selbo, Pål K.; Høgset, Anders; Halaas, Øyvind

2018-01-01

Effective priming and activation of tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) is crucial for realizing the potential of therapeutic cancer vaccination. This requires cytosolic antigens that feed into the MHC class I presentation pathway, which is not efficiently achieved with most current vaccination technologies. Photochemical internalization (PCI) provides an emerging technology to route endocytosed material to the cytosol of cells, based on light-induced disruption of endosomal membranes using a photosensitizing compound. Here, we investigated the potential of PCI as a novel, minimally invasive, and well-tolerated vaccination technology to induce priming of cancer-specific CTL responses to peptide antigens. We show that PCI effectively promotes delivery of peptide antigens to the cytosol of antigen-presenting cells (APCs) in vitro. This resulted in a 30-fold increase in MHC class I/peptide complex formation and surface presentation, and a subsequent 30- to 100-fold more efficient activation of antigen-specific CTLs compared to using the peptide alone. The effect was found to be highly dependent on the dose of the PCI treatment, where optimal doses promoted maturation of immature dendritic cells, thus also providing an adjuvant effect. The effect of PCI was confirmed in vivo by the successful induction of antigen-specific CTL responses to cancer antigens in C57BL/6 mice following intradermal peptide vaccination using PCI technology. We thus show new and strong evidence that PCI technology holds great potential as a novel strategy for improving the outcome of peptide vaccines aimed at triggering cancer-specific CD8+ CTL responses. PMID:29670624

Uptake of Biotin by Chlamydia Spp. through the Use of a Bacterial Transporter (BioY) and a Host-Cell Transporter (SMVT)

PubMed Central

Fisher, Derek J.; Fernández, Reinaldo E.; Adams, Nancy E.; Maurelli, Anthony T.

2012-01-01

Chlamydia spp. are obligate intracellular Gram-negative bacterial pathogens that cause disease in humans and animals. Minor variations in metabolic capacity between species have been causally linked to host and tissue tropisms. Analysis of the highly conserved genomes of Chlamydia spp. reveals divergence in the metabolism of the essential vitamin biotin with genes for either synthesis (bioF_2ADB) and/or transport (bioY). Streptavidin blotting confirmed the presence of a single biotinylated protein in Chlamydia. As a first step in unraveling the need for divergent biotin acquisition strategies, we examined BioY (CTL0613) from C. trachomatis 434/Bu which is annotated as an S component of the type II energy coupling-factor transporters (ECF). Type II ECFs are typically composed of a transport specific component (S) and a chromosomally unlinked energy module (AT). Intriguingly, Chlamydia lack recognizable AT modules. Using 3H-biotin and recombinant E. coli expressing CTL0613, we demonstrated that biotin was transported with high affinity (a property of Type II ECFs previously shown to require an AT module) and capacity (apparent K(m) of 3.35 nM and V(max) of 55.1 pmol×min−1×mg−1). Since Chlamydia reside in a host derived membrane vacuole, termed an inclusion, we also sought a mechanism for transport of biotin from the cell cytoplasm into the inclusion vacuole. Immunofluorescence microscopy revealed that the mammalian sodium multivitamin transporter (SMVT), which transports lipoic acid, biotin, and pantothenic acid into cells, localizes to the inclusion. Since Chlamydia also are auxotrophic for lipoic and pantothenic acids, SMVT may be subverted by Chlamydia to move multiple essential compounds into the inclusion where BioY and another transporter(s) would be present to facilitate transport into the bacterium. Collectively, our data validates the first BioY from a pathogenic organism and describes a two-step mechanism by which Chlamydia transport biotin from the host cell into the bacterial cytoplasm. PMID:23029384

L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy.

PubMed

Mobley, Christopher Brooks; Fox, Carlton D; Ferguson, Brian S; Amin, Rajesh H; Dalbo, Vincent J; Baier, Shawn; Rathmacher, John A; Wilson, Jacob M; Roberts, Michael D

2014-01-01

The purpose of this study was to examine if L-leucine (Leu), β-hydroxy-β-methylbutyrate (HMB), or creatine monohydrate (Crea) prevented potential atrophic effects of myostatin (MSTN) on differentiated C2C12 myotubes. After four days of differentiation, myotubes were treated with MSTN (10 ng/ml) for two additional days and four treatment groups were studied: 1) 3x per day 10 mM Leu, 2) 3x per day 10 mM HMB, 3) 3x per day 10 mM Crea, 4) DM only. Myotubes treated with DM without MSTN were analyzed as the control condition (DM/CTL). Following treatment, cells were analyzed for total protein, DNA content, RNA content, muscle protein synthesis (MPS, SUnSET method), and fiber diameter. Separate batch treatments were analyzed for mRNA expression patterns of myostatin-related genes (Akirin-1/Mighty, Notch-1, Ski, MyoD) as well as atrogenes (MuRF-1, and MAFbx/Atrogin-1). MSTN decreased fiber diameter approximately 30% compared to DM/CTL myotubes (p < 0.001). Leu, HMB and Crea prevented MSTN-induced atrophy. MSTN did not decrease MPS levels compared to DM/CTL myotubes, but MSTN treatment decreased the mRNA expression of Akirin-1/Mighty by 27% (p < 0.001) and MyoD by 26% (p < 0.01) compared to DM/CTL myotubes. shRNA experiments confirmed that Mighty mRNA knockdown reduced myotube size, linking MSTN treatment to atrophy independent of MPS. Remarkably, MSTN + Leu and MSTN + HMB myotubes had similar Akirin-1/Mighty and MyoD mRNA levels compared to DM/CTL myotubes. Furthermore, MSTN + Crea myotubes exhibited a 36% (p < 0.05) and 86% (p < 0.001) increase in Akirin-1/Mighty mRNA compared to DM/CTL and MSTN-only treated myotubes, respectively. Leu, HMB and Crea may reduce MSTN-induced muscle fiber atrophy by influencing Akirin-1/Mighty mRNA expression patterns. Future studies are needed to examine if Leu, HMB and Crea independently or synergistically affect Akirin-1/Mighty expression, and how Akirin-1/Mighty expression mechanistically relates to skeletal muscle hypertrophy in vivo.

L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy

PubMed Central

2014-01-01

Background The purpose of this study was to examine if L-leucine (Leu), β-hydroxy-β-methylbutyrate (HMB), or creatine monohydrate (Crea) prevented potential atrophic effects of myostatin (MSTN) on differentiated C2C12 myotubes. Methods After four days of differentiation, myotubes were treated with MSTN (10 ng/ml) for two additional days and four treatment groups were studied: 1) 3x per day 10 mM Leu, 2) 3x per day 10 mM HMB, 3) 3x per day 10 mM Crea, 4) DM only. Myotubes treated with DM without MSTN were analyzed as the control condition (DM/CTL). Following treatment, cells were analyzed for total protein, DNA content, RNA content, muscle protein synthesis (MPS, SUnSET method), and fiber diameter. Separate batch treatments were analyzed for mRNA expression patterns of myostatin-related genes (Akirin-1/Mighty, Notch-1, Ski, MyoD) as well as atrogenes (MuRF-1, and MAFbx/Atrogin-1). Results MSTN decreased fiber diameter approximately 30% compared to DM/CTL myotubes (p < 0.001). Leu, HMB and Crea prevented MSTN-induced atrophy. MSTN did not decrease MPS levels compared to DM/CTL myotubes, but MSTN treatment decreased the mRNA expression of Akirin-1/Mighty by 27% (p < 0.001) and MyoD by 26% (p < 0.01) compared to DM/CTL myotubes. shRNA experiments confirmed that Mighty mRNA knockdown reduced myotube size, linking MSTN treatment to atrophy independent of MPS. Remarkably, MSTN + Leu and MSTN + HMB myotubes had similar Akirin-1/Mighty and MyoD mRNA levels compared to DM/CTL myotubes. Furthermore, MSTN + Crea myotubes exhibited a 36% (p < 0.05) and 86% (p < 0.001) increase in Akirin-1/Mighty mRNA compared to DM/CTL and MSTN-only treated myotubes, respectively. Conclusions Leu, HMB and Crea may reduce MSTN-induced muscle fiber atrophy by influencing Akirin-1/Mighty mRNA expression patterns. Future studies are needed to examine if Leu, HMB and Crea independently or synergistically affect Akirin-1/Mighty expression, and how Akirin-1/Mighty expression mechanistically relates to skeletal muscle hypertrophy in vivo. PMID:25132809

Th cells promote CTL survival and memory via acquired pMHC-I and endogenous IL-2 and CD40L signaling and by modulating apoptosis-controlling pathways.

PubMed

Umeshappa, Channakeshava Sokke; Xie, Yufeng; Xu, Shulin; Nanjundappa, Roopa Hebbandi; Freywald, Andrew; Deng, Yulin; Ma, Hong; Xiang, Jim

2013-01-01

Involvement of CD4(+) helper T (Th) cells is crucial for CD8(+) cytotoxic T lymphocyte (CTL)-mediated immunity. However, CD4(+) Th's signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4(+) Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4(+) T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2K(b)/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4(+) Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4(+) Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7) molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4(+) Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy.

Th Cells Promote CTL Survival and Memory via Acquired pMHC-I and Endogenous IL-2 and CD40L Signaling and by Modulating Apoptosis-Controlling Pathways

PubMed Central

Umeshappa, Channakeshava Sokke; Xie, Yufeng; Xu, Shulin; Nanjundappa, Roopa Hebbandi; Freywald, Andrew; Deng, Yulin; Ma, Hong; Xiang, Jim

2013-01-01

Involvement of CD4+ helper T (Th) cells is crucial for CD8+ cytotoxic T lymphocyte (CTL)-mediated immunity. However, CD4+ Th’s signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4+ Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4+ T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2Kb/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4+ Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4+ Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7) molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4+ Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy. PMID:23785406

Acute high-intensity interval exercise reduces the postprandial glucose response and prevalence of hyperglycaemia in patients with type 2 diabetes.

PubMed

Gillen, J B; Little, J P; Punthakee, Z; Tarnopolsky, M A; Riddell, M C; Gibala, M J

2012-06-01

High-volume endurance exercise (END) improves glycaemic control in type 2 diabetes (T2D) but many individuals cite 'lack of time' as a barrier to regular participation. High-intensity interval training (HIT) is a time-efficient method to induce physiological adaptations similar to END, but little is known regarding the effect of HIT in T2D. Using continuous glucose monitoring (CGM), we examined the 24-h blood glucose response to one session of HIT consisting of 10 × 60 s cycling efforts at ~90% maximal heart rate, interspersed with 60 s rest. Seven adults with T2D underwent CGM for 24-h on two occasions under standard dietary conditions: following acute HIT and on a non-exercise control day (CTL). HIT reduced hyperglycaemia measured as proportion of time spent above 10 mmol/l (HIT: 4.5 ± 4.4 vs. CTL: 15.2 ± 12.3%, p = 0.04). Postprandial hyperglycaemia, measured as the sum of post-meal areas under the glucose curve, was also lower after HIT vs. CTL (728 ± 331 vs. 1142 ± 556 mmol/l·9 h, p = 0.01). These findings highlight the potential for HIT to improve glycaemic control in T2D. © 2012 Blackwell Publishing Ltd.

Interleukin-6 and vascular endothelial growth factor release by renal cell carcinoma cells impedes lymphocyte-dendritic cell cross-talk.

PubMed

Cabillic, F; Bouet-Toussaint, F; Toutirais, O; Rioux-Leclercq, N; Fergelot, P; de la Pintière, C Thomas; Genetet, N; Patard, J-J; Catros-Quemener, V

2006-12-01

Anti-tumour T cell response requires antigen presentation via efficient immunological synapse between antigen presenting cells, e.g. dendritic cells (DC), and specific T cells in an adapted Th1 cytokine context. Nine renal cell carcinoma (RCC) primary culture cells were used as sources of tumour antigens which were loaded on DC (DC-Tu) for autologous T cell activation assays. Cytotoxic activity of lymphocytes stimulated with DC-Tu was evaluated against autologous tumour cells. Assays were performed with 75 grays irradiated tumour cells (Tu irr) and with hydrogen peroxide +/- heat shock (Tu H(2)O(2) +/- HS) treated cells. DC-Tu irr failed to enhance cytotoxic activity of autologous lymphocytes in seven of 13 assays. In all these defective assays, irradiated tumour cells displayed high interleukin (IL)-6 and vascular endothelial growth factor (VEGF) release. Conversely, when tumour cells released low IL-6 levels (n = 4), DC-Tu irr efficiently enhanced CTL activity. When assays were performed with the same RCC cells treated with H(2)O(2) + HS, DC-Tu stimulation resulted in improved CTL activity. H(2)O(2) + HS treatment induced post-apoptotic cell necrosis of tumour cells, totally abrogated their cytokine release [IL-6, VEGF, transforming growth factor (TGF)-beta1] and induced HSP70 expression. Taken together, data show that reduction in IL-6 and VEGF release in the environment of the tumour concomitantly to tumour cell HSP expression favours induction of a stronger anti-tumour CTL response.

Functional overreaching: the key to peak performance during the taper?

PubMed

Aubry, Anaël; Hausswirth, Christophe; Louis, Julien; Coutts, Aaron J; LE Meur, Yann

2014-09-01

The purpose of this study is to examine whether performance supercompensation during taper is maximized in endurance athletes after experiencing overreaching during an overload training (OT) period. Thirty-three trained male triathletes were assigned to either OT (n = 23) or normal training groups (n = 10, CTL) during 8 wk. Cycling performance and maximal oxygen uptake (V˙O2max) were measured after 1 wk of moderate training, a 3-wk period of OT, and then each week during 4-wk taper. Eleven of the 23 subjects from the OT group were diagnosed as functionally overreached (F-OR) after the overload period (decreased performance with concomitant high perceived fatigue), whereas the 12 other subjects were only acutely fatigued (AF) (no decrease in performance). According to qualitative statistical analysis, the AF group demonstrated a small to large greater peak performance supercompensation than the F-OR group (2.6% ± 1.1%) and the CTL group (2.6% ± 1.6%). V˙O2max increased significantly from baseline at peak performance only in the CTL and AF groups. Of the peak performances, 60%, 83%, and 73% occurred within the two first weeks of taper in CTL, AF, and OR, respectively. Ten cases of infection were reported during the study with higher prevalence in F-OR (70%) than that in AF (20%) and CTL (10%). This study showed that 1) greater gains in performance and V˙O2max can be achieved when higher training load is prescribed before the taper but not in the presence of F-OR; 2) peak performance is not delayed during taper when heavy training loads are completed immediately prior; and 3) F-OR provides higher risk for training maladaptation, including increased infection risks.

Exercise Increases and Browns Muscle Lipid in High-Fat Diet-Fed Mice.

PubMed

Morton, Tiffany L; Galior, Kornelia; McGrath, Cody; Wu, Xin; Uzer, Gunes; Uzer, Guniz Bas; Sen, Buer; Xie, Zhihui; Tyson, David; Rubin, Janet; Styner, Maya

2016-01-01

Muscle lipid increases with high-fat feeding and diabetes. In trained athletes, increased muscle lipid is not associated with insulin resistance, a phenomenon known as the athlete's paradox. To understand if exercise altered the phenotype of muscle lipid, female C57BL/6 mice fed CTL or high-fat diet (HFD for 6 or 18 weeks) were further divided into sedentary or exercising groups (CTL-E or HFD-E) with voluntary access to running wheels for the last 6 weeks of experiments, running 6 h/night. Diet did not affect running time or distance. HFD mice weighed more than CTL after 18 weeks (p < 0.01). Quadriceps muscle TG was increased in running animals and in sedentary mice fed HFD for 18 weeks (p < 0.05). In exercised animals, markers of fat, Plin1, aP2, FSP27, and Fasn, were increased significantly in HFD groups. Ucp1 and Pgc1a, markers for brown fat, increased with exercise in the setting of high fat feeding. Fndc5, which encodes irisin, and CytC were sensitive to exercise regardless of diet. Plin5 was increased with HFD and unaffected by exercise; the respiratory exchange ratio was 15% lower in the 18-week HFD group compared with CTL (p < 0.001) and 10% lower in 18 weeks HFD-E compared with CTL-E (p < 0.001). Increased Ucp1 and Pgc1a in exercised muscle of running mice suggests that a beige/brown fat phenotype develops, which differs from the fat phenotype that induces insulin resistance in high fat feeding. This suggests that increased muscle lipid may develop a "brown" phenotype in the setting of endurance exercise training, a shift that is further promoted by HFD.

Development of a rectal sexually transmitted infection (STI) Model in Rhesus macaques using Chlamydia trachomatis serovars E and L2.

PubMed

Henning, Tara R; Morris, Monica; Ellis, Shanon; Kelley, Kristen; Phillips, Christi; Ritter, Jana; Jones, Tara; Nachamkin, Eli; Chen, Cheng Y; Hong, Jaeyoung; Kang, Joseph; Patton, Dorothy; McNicholl, Janet; Papp, John; Kersh, Ellen N

2017-10-01

Rectal STI coinfection models enhance the understanding of rectal HIV transmission risk factors. Rhesus macaques (n=9) were exposed to one of three rectal Chlamydia trachomatis (CT) challenges: C. trachomatis L 2 (CT-L 2 ); C. trachomatis serovar E (CT-E), followed by CT-L 2 ; or CT-E, treatment/clearance, then CT-L 2 . Infections were monitored by PCR. Weekly blood and rectal secretion/lavage samples were collected for cytokine analyzes and/or epithelial sloughing, occult, and overt blood determinations. Chlamydial infections were successfully established in each animal, with varying degrees of persistence. Mucosal IL-1beta was upregulated in animals consecutively infected with CT-E then CT-L 2 (P=.05). Epithelial sloughing was also significantly increased post-infection in this group (P=.0003). This study demonstrates successful rectal infection of rhesus macaques with CT-E and CT-L 2 and describes measures of assessing rectal inflammation and pathology. Different infection strategies yield varying inflammatory and pathologic outcomes, providing well-described models for future SIV/SHIV susceptibility studies. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Toll-like receptor 9 agonist enhances anti-tumor immunity and inhibits tumor-associated immunosuppressive cells numbers in a mouse cervical cancer model following recombinant lipoprotein therapy.

PubMed

Chang, Li-Sheng; Leng, Chih-Hsiang; Yeh, Yi-Chen; Wu, Chiao-Chieh; Chen, Hsin-Wei; Huang, Hai-Mei; Liu, Shih-Jen

2014-03-19

Although cytotoxic T lymphocytes (CTLs) play a major role in eradicating cancer cells during immunotherapy, the cancer-associated immunosuppressive microenvironment often limits the success of such therapies. Therefore, the simultaneous induction of cancer-specific CTLs and reversal of the immunosuppressive tumor microenvironment may be more effectively achieved through a single therapeutic vaccine. A recombinant lipoprotein with intrinsic Toll-like receptor 2 (TLR2) agonist activity containing a mutant form of E7 (E7m) and a bacterial lipid moiety (rlipo-E7m) has been demonstrated to induce robust CTL responses against small tumors. This treatment in combination with other TLR agonists is able to eliminate large tumors. Mouse bone marrow-derived dendritic cells (DCs) were employed to determine the synergistic production of pro-inflammatory cytokines upon combination of rlipo-E7m and other TLR agonists. Antigen-specific CTL responses were investigated using immunospots or in vivo cytolytic assays after immunization in mice. Mice bearing various tumor sizes were used to evaluate the anti-tumor effects of the formulation. Specific subpopulations of immunosuppressive cells in the tumor infiltrate were quantitatively determined by flow cytometry. We demonstrate that a TLR9 agonist (unmethylated CpG oligodeoxynucleotide, CpG ODN) enhances CTL responses and eradicates large tumors when combined with rlipo-E7m. Moreover, combined treatment with rlipo-E7m and CpG ODN effectively increases tumor infiltration by CTLs and reduces the numbers of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) in the tumor microenvironment. These findings suggest that the dramatic anti-tumor effects of the recombinant lipoprotein together with CpG ODN may reflect the amplification of CTL responses and the repression of the immunosuppressive environment. This promising approach could be applied for the development of additional therapeutic cancer vaccines.

ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis.

PubMed

Silva-Sánchez, Aarón; Meza-Pérez, Selene; Flores-Langarica, Adriana; Donis-Maturano, Luis; Estrada-García, Iris; Calderón-Amador, Juana; Hernández-Pando, Rogelio; Idoyaga, Juliana; Steinman, Ralph M; Flores-Romo, Leopoldo

2015-01-01

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen's naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205+ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4+ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ+ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205+ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.

ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis

PubMed Central

Silva-Sánchez, Aarón; Meza-Pérez, Selene; Flores-Langarica, Adriana; Donis-Maturano, Luis; Estrada-García, Iris; Calderón-Amador, Juana; Hernández-Pando, Rogelio; Idoyaga, Juliana; Flores-Romo, Leopoldo

2015-01-01

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen’s naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205+ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4+ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ+ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205+ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions. PMID:25915045

A novel HLA-A*0201 restricted peptide derived from cathepsin G is an effective immunotherapeutic target in acute myeloid leukemia.

PubMed

Zhang, Mao; Sukhumalchandra, Pariya; Enyenihi, Atim A; St John, Lisa S; Hunsucker, Sally A; Mittendorf, Elizabeth A; Sergeeva, Anna; Ruisaard, Kathryn; Al-Atrache, Zein; Ropp, Patricia A; Jakher, Haroon; Rodriguez-Cruz, Tania; Lizee, Gregory; Clise-Dwyer, Karen; Lu, Sijie; Molldrem, Jeffrey J; Glish, Gary L; Armistead, Paul M; Alatrash, Gheath

2013-01-01

Immunotherapy targeting aberrantly expressed leukemia-associated antigens has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy. We used Immune Epitope Database and in vitro binding assays to identify immunogenic epitopes derived from CG. Flow cytometry, immunoblotting, and confocal microscopy were used to characterize the expression and processing of CG in AML patient samples, leukemia stem cells, and normal neutrophils. Cytotoxicity assays determined the susceptibility of AML to CG-specific cytotoxic T lymphocytes (CTL). Dextramer staining and cytokine flow cytometry were conducted to characterize the immune response to CG in patients. CG was highly expressed and ubiquitinated in AML blasts, and was localized outside granules in compartments that facilitate antigen presentation. We identified five HLA-A*0201 binding nonameric peptides (CG1-CG5) derived from CG, and showed immunogenicity of the highest HLA-A*0201 binding peptide, CG1. We showed killing of primary AML by CG1-CTL, but not normal bone marrow. Blocking HLA-A*0201 abrogated CG1-CTL-mediated cytotoxicity, further confirming HLA-A*0201-dependent killing. Finally, we showed functional CG1-CTLs in peripheral blood from AML patients following allogeneic stem cell transplantation. CG is aberrantly expressed and processed in AML and is a novel immunotherapeutic target that warrants further development.

STRESS-INDUCED REDISTRIBUTION OF IMMUNE CELLS - FROM BARRACKS TO BOULEVARDS TO BATTLEFIELDS: A TALE OF THREE HORMONES - CURT RICHTER AWARD WINNER

PubMed Central

Dhabhar, Firdaus S.; Malarkey, William B.; Neri, Eric; McEwen, Bruce S.

2012-01-01

Background The surveillance and effector functions of the immune system are critically dependent on the appropriate distribution of immune cells in the body. An acute or short-term stress response induces a rapid and significant redistribution of immune cells among different body compartments. Stress-induced leukocyte redistribution may be a fundamental survival response that directs leukocyte subpopulations to specific target organs during stress, and significantly enhances the speed, efficacy and regulation of an immune response. Immune responses are generally enhanced in compartments (e.g., skin) that are enriched with leukocytes, and suppressed in compartments that are depleted of leukocytes during/following stress. The experiments described here were designed to elucidate the: 1) Time-course, trajectory, and subpopulation-specificity of stress-induced mobilization and trafficking of blood leukocytes. 2) Individual and combined actions of the principal stress hormones, norepinephrine (NE), epinephrine (EPI), and corticosterone (CORT), in mediating mobilization or trafficking of specific leukocyte subpopulations. 3) Effects of stress/stress hormones on adhesion molecule, L-selectin (CD62L), expression by each subpopulation to assess its adhesion / functional / maturation status. Methods Male Sprague Dawley rats were stressed (short-term restraint, 2–120 min), or adrenalectomized and injected with vehicle (VEH), NE, EPI, CORT, or their combinations, and blood was collected for measurement of hormones and flow cytometric quantification of leukocyte subpopulations. Results Acute stress induced an early increase/mobilization of neutrophils, lymphocytes, helper T cells (Th), cytolytic T cells (CTL), and B cells into the blood, followed by a decrease/trafficking of all cell types out of the blood, except neutrophil numbers that continued to increase. CD62L expression was increased on neutrophils, decreased on Th, CTL, and natural killer (NK) cells, and showed a biphasic decrease on monocytes & B cells, suggesting that CD62L is involved in mediating the redistribution effects of stress. Additionally, we observed significant differences in the direction, magnitude, and subpopulation specificity of the effects of each hormone: NE increased leukocyte numbers, most notably CD62L−/+ neutrophils and CD62L− B cells. EPI increased monocyte and neutrophil numbers, most notably CD62L−/+ neutrophils and CD62L− monocytes, but decreased lymphocyte numbers with CD62L−/+ CTL and CD62L+ B cells being especially sensitive. CORT decreased monocyte, lymphocyte, Th, CTL, and B cell numbers with CD62L− and CD62L+ cells being equally affected. Thus, naïve (CD62L+) vs. memory (CD62L−) T cells, classical (CD62L+) vs. non-classical (CD62L−) monocytes, and similarly distinct functional subsets of other leukocyte populations are differentially mobilized into the blood and trafficked to tissues by stress hormones. Conclusion Stress hormones orchestrate a large-scale redistribution of immune cells in the body. NE and EPI mobilize immune cells into the bloodstream, and EPI and CORT induce traffic out of the blood possibly to tissue surveillance pathways, lymphoid tissues, and sites of ongoing or de novo immune activation. Immune cell subpopulations appear to show differential sensitivities and redistribution responses to each hormone depending on the type of leukocyte (neutrophil, monocyte or lymphocyte) and its maturation/functional characteristics (e.g., resident or inflammatory monocyte, naïve or central/effector memory T cell). Thus, stress hormones could be administered simultaneously or sequentially to induce specific leukocyte subpopulations to be mobilized into the blood, or to traffic from blood to tissues. Stress hormone-mediated changes in immune cell distribution could be clinically harnessed to: 1) Direct leukocytes to sites of vaccination, wound healing, infection, or cancer and thereby enhance protective immunity. 2) Reduce leukocyte traffic to sites of inflammatory/autoimmune reactions. 3) Sequester immune cells in relatively protected compartments to minimize exposure to cytotoxic treatments like radiation or localized chemotherapy. 4) Measure biological resistance/sensitivity to stress hormones in vivo. In keeping with the guidelines for Richter Award manuscripts, in addition to original data we also present a model and synthesis of findings in the context of the literature on the effects of short-term stress on immune cell distribution and function. PMID:22727761

[Immune response induced by HIV DNA vaccine combined with recombinant adeno-associated virus].

PubMed

Liu, Yan-zheng; Zhou, Ling; Wang, Qi; Ye, Shu-qing; Li, Hong-xia; Zeng, Yi

2004-09-01

HIV-1 DNA vaccine and recombinant adeno-associated virus (rAAV) expressing gagV3 gene of HIV-1 subtype B were constructed and BALB/c mice were immunized by vaccination regimen consisting of consecutive priming with DNA vaccine and boosting with rAAV vaccine; the CTL and antibody response were detected and compared with those induced by DNA vaccine or rAAV vaccine separately. HIV-1 subtype B gagV3 gene was inserted into the polyclonal site of plasmid pCI-neo, DNA vaccine pCI-gagV3 was thereby constructed; pCI-gagV3 was transfected into p815 cells, G-418-resistant cells were obtained through screening transfected cells with G418, the expression of HIV-1 antigen in G-418-resistant cells was detected by EIA; BALB/c mice were immunized with pCI-gagV3 and the immune response was tested; BALB/c mouse immunized with pCI-gagV3 and combined with rAAV expressing the same gagV3 genes were tested for antibody level in sera by EIA method and cytotoxicity response by LDH method. pCI-gagV3 could express HIV-1 gene in p815 cells; pCI-gagV3 could induce HIV-1 specific humoral and cell-mediated immune response in BALB/c mice. The HIV-1 specific antibody level was 1/20; when the ratio of effector cells: target cells was 50:1, the average specific cytotoxicity was 41.7%; there was no evident increase in the antibody level induced by pCI-gagV3 combined with rAAV, but there was increase in CTL response, the average specific cytotoxicity was 61.3% when effector cells: target cells ratio was 50:1. HIV-1 specific cytotoxicity in BALB/c mice can be increased by immunization of BALB/c mice with DNA vaccine combined with rAAV vaccine.

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus and in vitro properties.

PubMed

Anderson, J; Byrne, J A; Schreiber, R; Patterson, S; Oldstone, M B

1985-02-01

We have generated lymphocytic choriomeningitis virus-specific, H-2-restricted cytotoxic thymus-derived lymphocyte (CTL) clones. By using these reagents in several in vitro assays with infected target cells, we show that CTLs by themselves prevent the release of infectious virus into culture fluids and significantly lower the titers of infectious virus previously produced. This ability of cloned CTLs is not influenced by monensin. However, monensin does abrogate the ability of CTLs from spleens of mice primed 6 to 8 days previously with virus to kill virus-infected syngeneic targets. When tested for the participation of lymphokines in this system, the CTLs proliferate when reacted with syngeneic lymphocytic choriomeningitis virus-infected macrophages but fail to make interleukin-2. These CTLs make gamma interferon when reacted with syngeneic virus-infected targets. However, the production of interferon does not directly correlate with CTL-mediated killing. The number of H-2K and D molecules expressed on the target cell surface is not altered during the course of lymphocytic choriomeningitis virus infection. Electron microscopy shows finger-like projections of the CTL clone thrust into the infected cell and lesions bearing an internal diameter of approximately 15 nm in those membranes, illustrating the lytic process.

Epstein-Barr virus-transformed B-cells as efficient antigen presenting cells to propagate Aspergillus-specific cytotoxic T-lymphocytes.

PubMed

Ramadan, Gamal

2008-01-01

To overcome the cytotoxic T-lymphocytes (CTL) expansion limitations imposed by the lack of sufficient dendritic cells (DC) alternative sources of autologous antigen presenting cells (APC) such as Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (BLCL), which are easy to establish in vitro, have been considered and studied in the present work. Non-adherent peripheral blood mononuclear cells of three healthy donors were repeatedly primed with autologous Aspergillus fumigatus commercial culture-filtrate antigen-pulsed fast monocyte-derived DC (Aspf-CFA-DC) alone, Aspf-CFA-pulsed BLCL (Aspf-CFA-BLCL) alone or Aspf-CFA-BLCL after one, two, or three primings with Aspf-CFA-DC (1DC/BLCL, 2DC/BLCL or 3DCIBLCL; respectively). After 5th priming, lines generated by Aspf-CFA-BLCL only showed strong/weak lytic activity for EBV/Aspf; respectively. Aspf-specific lytic activity in all donors was increased by increasing the number of primings with Aspf-CFA-DC before switching to Aspf-CFA-BLCL (18.20 +/- 1.65% versus 35.67 +/- 1.02% and 40.03 +/- 1.41% in bulk cultures generated by 1DC/BLCL versus 2DC/BLCL and 3DC/BLCL, respectively). Bulk cultures generated by Aspf-CFA-BLCL after at least two primings with Aspf-CFA-DC showed approximately the same Aspf-specific lytic activity, effector cell phenotype, expansion level and percentage expression of IFN-gamma, CD69 and CD107a without any significant differences (p > 0.05) as standard bulk cultures generated by only Aspf-CFA-DC. Thus, this study explored the use of a combined DC/BLCL protocol to establish/propagate Aspf-specific CTL for adoptive immunotherapy to prevent or treat invasive pulmonary aspergillosis.

Immunotherapy of murine retrovirus-induced acquired immunodeficiency by CD4 T regulatory cell depletion and PD-1 blockade.

PubMed

Li, Wen; Green, William R

2011-12-01

LP-BM5 retrovirus induces a complex disease featuring an acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice, such as C57BL/6 (B6). CD4 T helper effector cells are required for MAIDS induction and progression of viral pathogenesis. CD8 T cells are not needed for viral pathogenesis, but rather, are essential for protection from disease in resistant strains, such as BALB/c. We have discovered an immunodominant cytolytic T lymphocyte (CTL) epitope encoded in a previously unrecognized LP-BM5 retroviral alternative (+1 nucleotide [nt]) gag translational open reading frame. CTLs specific for this cryptic gag epitope are the basis of protection from LP-BM5-induced immunodeficiency in BALB/c mice, and the inability of B6 mice to mount an anti-gag CTL response appears critical to the initiation and progression of LP-BM5-induced MAIDS. However, uninfected B6 mice primed by LP-BM5-induced tumors can generate CTL responses to an LP-BM5 retrovirus infection-associated epitope(s) that is especially prevalent on such MAIDS tumor cells, indicating the potential to mount a protective CD8 T-cell response. Here, we utilized this LP-BM5 retrovirus-induced disease system to test whether modulation of normal immune down-regulatory mechanisms can alter retroviral pathogenesis. Thus, following in vivo depletion of CD4 T regulatory (Treg) cells and/or selective interruption of PD-1 negative signaling in the CD8 T-cell compartment, retroviral pathogenesis was significantly decreased, with the combined treatment of CD4 Treg cell depletion and PD-1 blockade working in a synergistic fashion to substantially reduce the induction of MAIDS.

Long-term control of HIV-1 in hemophiliacs carrying slow-progressing allele HLA-B*5101.

PubMed

Kawashima, Yuka; Kuse, Nozomi; Gatanaga, Hiroyuki; Naruto, Takuya; Fujiwara, Mamoru; Dohki, Sachi; Akahoshi, Tomohiro; Maenaka, Katsumi; Goulder, Philip; Oka, Shinichi; Takiguchi, Masafumi

2010-07-01

HLA-B*51 alleles are reported to be associated with slow disease progression to AIDS, but the mechanism underlying this association is still unclear. In the present study, we analyzed the effect of HLA-B*5101 on clinical outcome for Japanese hemophiliacs who had been infected with HIV-1 before 1985 and had been recruited in 1998 for this study. HLA-B*5101(+) hemophiliacs exhibited significantly slow progression. The analysis of HLA-B*5101-restricted HIV-1-specific cytotoxic T-lymphocyte (CTL) responses to 4 HLA-B*-restricted epitopes in 10 antiretroviral-therapy (ART)-free HLA-B*5101(+) hemophiliacs showed that the frequency of Pol283-8-specific CD8(+) T cells was inversely correlated with the viral load, whereas the frequencies of CD8(+) T cells specific for 3 other epitopes were positively correlated with the viral load. The HLA-B*5101(+) hemophiliacs whose HIV-1 replication had been controlled for approximately 25 years had HIV-1 possessing the wild-type Pol283-8 sequence or the Pol283-8V mutant, which does not critically affect T-cell recognition, whereas other HLA-B*5101(+) hemophiliacs had HIV-1 with escape mutations in this epitope. The results suggest that the control of HIV-1 over approximately 25 years in HLA-B*5101-positive hemophiliacs is associated with a Pol283-8-specific CD8(+) T-cell response and that lack of control of HIV-1 is associated with the appearance of Pol283-8-specific escape mutants.

Protective specific immunity induced by doxorubicin plus TNF-alpha combination treatment of EL4 lymphoma-bearing C57BL/6 mice.

PubMed

Ehrke, M J; Verstovsek, S; Maccubbin, D L; Ujházy, P; Zaleskis, G; Berleth, E; Mihich, E

2000-07-01

The therapeutic efficacy of a single (day 8), moderate dose (4 mg/kg, i.v.) of doxorubicin (DOX, Adriamycin) combined with recombinant human TNF-alpha (3 different doses and 5 different schedules, i.v.) was evaluated in C57BL/6 mice bearing an implant (s.c.) of the DOX-sensitive, TNF-alpha-resistant EL4 lymphoma. In parallel to monitoring survival, the levels of several host anti-tumor cytolytic effector functions of splenocytes and thymocytes were evaluated throughout the treatment period and in long-term survivors (LTS). DOX treatment alone resulted in a moderate (approx. 20%) increase in life span but no cures. TNF-alpha alone, at any tested dose or schedule, had little or no positive effect on survival. The combinations of DOX and TNF-alpha were only slightly better than DOX alone with respect to the time to death of mice that died (approx. 29% increase); however, each of the combinations involving 1,000 U TNF-alpha/injection produced a fraction (20% to 80%) of LTS. The host defense activities examined included those of splenic and thymic cytolytic T lymphocytes (CTL) and lymphokine-activated killer cells as well as splenic tumoricidal macrophages. Although most activities were modulated by tumor growth and/or treatment, only CTL responsiveness appeared to correlate with survival. CTL activity in the treated groups with LTS was significantly higher than in control groups late in the treatment period. Finally, ex vivo analyses of splenocytes and thymocytes together with the rejection of implanted tumor at 17 months established that LTS displayed specific long-term immune memory. Copyright 2000 Wiley-Liss, Inc.

Mitochondrial DNA copy numbers in pyramidal neurons are decreased and mitochondrial biogenesis transcriptome signaling is disrupted in Alzheimer's disease hippocampi.

PubMed

Rice, Ann C; Keeney, Paula M; Algarzae, Norah K; Ladd, Amy C; Thomas, Ravindar R; Bennett, James P

2014-01-01

Alzheimer's disease (AD) is the major cause of adult-onset dementia and is characterized in its pre-diagnostic stage by reduced cerebral cortical glucose metabolism and in later stages by reduced cortical oxygen uptake, implying reduced mitochondrial respiration. Using quantitative PCR we determined the mitochondrial DNA (mtDNA) gene copy numbers from multiple groups of 15 or 20 pyramidal neurons, GFAP(+) astrocytes and dentate granule neurons isolated using laser capture microdissection, and the relative expression of mitochondrial biogenesis (mitobiogenesis) genes in hippocampi from 10 AD and 9 control (CTL) cases. AD pyramidal but not dentate granule neurons had significantly reduced mtDNA copy numbers compared to CTL neurons. Pyramidal neuron mtDNA copy numbers in CTL, but not AD, positively correlated with cDNA levels of multiple mitobiogenesis genes. In CTL, but not in AD, hippocampal cDNA levels of PGC1α were positively correlated with multiple downstream mitobiogenesis factors. Mitochondrial DNA copy numbers in pyramidal neurons did not correlate with hippocampal Aβ1-42 levels. After 48 h exposure of H9 human neural stem cells to the neurotoxic fragment Aβ25-35, mtDNA copy numbers were not significantly altered. In summary, AD postmortem hippocampal pyramidal neurons have reduced mtDNA copy numbers. Mitochondrial biogenesis pathway signaling relationships are disrupted in AD, but are mostly preserved in CTL. Our findings implicate complex alterations of mitochondria-host cell relationships in AD.

HIV-I Nef inhibitors: a novel class of HIV-specific immune adjuvants in support of a cure.

PubMed

Dekaban, Gregory A; Dikeakos, Jimmy D

2017-09-12

The success of many current vaccines relies on a formulation that incorporates an immune activating adjuvant. This will hold true for the design of a successful therapeutic HIV vaccine targeted at controlling reactivated virus following cessation of combined antiretroviral therapy (cART). The HIV accessory protein Nef functions by interfering with HIV antigen presentation through the major histocompatibility complex I (MHC-I) pathway thereby suppressing CD8 + cytotoxic T cell (CTL)-mediated killing of HIV infected cells. Thus, this important impediment to HIV vaccine success must be circumvented. This review covers our current knowledge of Nef inhibitors that may serve as immune adjuvants that will specifically restore and enhance CTL-mediated killing of reactivated HIV infected cells as part of an overall vaccine strategy to affect a cure for HIV infection.

Students’ Creativity: Problem Posing in Structured Situation

NASA Astrophysics Data System (ADS)

Amalina, I. K.; Amirudin, M.; Budiarto, M. T.

2018-01-01

This is a qualitative research concerning on students’ creativity on problem posing task. The study aimed at describing the students’ creative thinking ability to pose the mathematics problem in structured situations with varied condition of given problems. In order to find out the students’ creative thinking ability, an analysis of mathematics problem posing test based on fluency, novelty, and flexibility and interview was applied for categorizing students’ responses on that task. The data analysis used the quality of problem posing and categorized in 4 level of creativity. The results revealed from 29 secondary students grade 8, a student in CTL (Creative Thinking Level) 1 met the fluency. A student in CTL 2 met the novelty, while a student in CTL 3 met both fluency and novelty and no one in CTL 4. These results are affected by students’ mathematical experience. The findings of this study highlight that student’s problem posing creativity are dependent on their experience in mathematics learning and from the point of view of which students start to pose problem.

A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes.

PubMed

Schaft, Niels; Dörrie, Jan; Müller, Ina; Beck, Verena; Baumann, Stefanie; Schunder, Tanja; Kämpgen, Eckhart; Schuler, Gerold

2006-09-01

Effective T cell receptor (TCR) transfer until now required stable retroviral transduction. However, retroviral transduction poses the threat of irreversible genetic manipulation of autologous cells. We, therefore, used optimized RNA transfection for transient manipulation. The transfection efficiency, using EGFP RNA, was >90%. The electroporation of primary T cells, isolated from blood, with TCR-coding RNA resulted in functional cytotoxic T lymphocytes (CTLs) (>60% killing at an effector to target ratio of 20:1) with the same HLA-A2/gp100-specificity as the parental CTL clone. The TCR-transfected T cells specifically recognized peptide-pulsed T2 cells, or dendritic cells electroporated with gp100-coding RNA, in an IFNgamma-secretion assay and retained this ability, even after cryopreservation, over 3 days. Most importantly, we show here for the first time that the electroporated T cells also displayed cytotoxicity, and specifically lysed peptide-loaded T2 cells and HLA-A2+/gp100+ melanoma cells over a period of at least 72 h. Peptide-titration studies showed that the lytic efficiency of the RNA-transfected T cells was similar to that of retrovirally transduced T cells, and approximated that of the parental CTL clone. Functional TCR transfer by RNA electroporation is now possible without the disadvantages of retroviral transduction, and forms a new strategy for the immunotherapy of cancer.

Dynamics of an HIV-1 infection model with cell mediated immunity

NASA Astrophysics Data System (ADS)

Yu, Pei; Huang, Jianing; Jiang, Jiao

2014-10-01

In this paper, we study the dynamics of an improved mathematical model on HIV-1 virus with cell mediated immunity. This new 5-dimensional model is based on the combination of a basic 3-dimensional HIV-1 model and a 4-dimensional immunity response model, which more realistically describes dynamics between the uninfected cells, infected cells, virus, the CTL response cells and CTL effector cells. Our 5-dimensional model may be reduced to the 4-dimensional model by applying a quasi-steady state assumption on the variable of virus. However, it is shown in this paper that virus is necessary to be involved in the modeling, and that a quasi-steady state assumption should be applied carefully, which may miss some important dynamical behavior of the system. Detailed bifurcation analysis is given to show that the system has three equilibrium solutions, namely the infection-free equilibrium, the infectious equilibrium without CTL, and the infectious equilibrium with CTL, and a series of bifurcations including two transcritical bifurcations and one or two possible Hopf bifurcations occur from these three equilibria as the basic reproduction number is varied. The mathematical methods applied in this paper include characteristic equations, Routh-Hurwitz condition, fluctuation lemma, Lyapunov function and computation of normal forms. Numerical simulation is also presented to demonstrate the applicability of the theoretical predictions.

Recombinant parvovirus-like particles as an antigen carrier: A novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells

PubMed Central

Sedlik, C.; Saron, M.-F.; Sarraseca, J.; Casal, I.; Leclerc, C.

1997-01-01

To develop a strategy that promotes efficient antiviral immunity, hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus VP2 capsid protein carrying a CD8+ T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated- or virus-infected-target cells. This CD8+ class I-restricted cytotoxic activity persisted in vivo for at least 9 months. Furthermore, the hybrid parvovirus-like particles were able to induce a complete protection of mice against a lethal lymphocytic choriomeningitis virus infection. To our knowledge, this study represents the first demonstration that hybrid nonreplicative VLP carrying a single viral CTL epitope can induce protection against a viral lethal challenge, in the absence of any adjuvant. These recombinant particles containing a single type of protein are easily produced by the baculovirus expression system and, therefore, represent a promising and safe strategy to induce strong CTL responses for the elimination of virus-infected cells. PMID:9207121

Axioms for Obligation and Robustness with Temporal Logic

NASA Astrophysics Data System (ADS)

French, Tim; McCabe-Dansted, John C.; Reynolds, Mark

RoCTL* was proposed to model and specify the robustness of reactive systems. RoCTL* extended CTL* with the addition of Obligatory and Robustly operators, which quantify over failure-free paths and paths with one more failure respectively. This paper gives an axiomatisation for all the operators of RoCTL* with the exception of the Until operator; this fragment is able to express similar contrary-to-duty obligations to the full RoCTL* logic. We call this formal system NORA, and give a completeness proof. We also consider the fragments of the language containing only path quantifiers (but where variables are dependent on histories). We examine semantic properties and potential axiomatisations for these fragments.

Elicitation of anti-Sendai virus cytotoxic T lymphocytes by viral and H-2 antigens incorporated into the same lipid bilayer by membrane fusion and by reconstitution into liposomes.

PubMed

Hale, A H; Lyles, D S; Fan, D P

1980-02-01

We have investigated the minimal molecular requirements for elicitation of anti-Sendai virus cytotoxic T lymphocytes (CTL), and the minimal molecular requirements for the recognition and lysis processes associated with anti-Sendai virus CTL-target cell interactions. This report demonstrates a) that the hemagglutinin-neuraminidase and/or fusion glycoproteins of Sendai virus can elicit anti-Sendai virus CTL and b) that these glycoproteins and H-2 antigens must be within the same membrane lipid bilayer for effective elicitation of anti-Sendai-virus CTL and for effective recognition and lysis of target cells by anti-Sendai virus CTL.

The B-Cell Follicle in HIV Infection: Barrier to a Cure.

PubMed

Bronnimann, Matthew P; Skinner, Pamela J; Connick, Elizabeth

2018-01-01

The majority of HIV replication occurs in secondary lymphoid organs (SLOs) such as the spleen, lymph nodes, and gut-associated lymphoid tissue. Within SLOs, HIV RNA + cells are concentrated in the B-cell follicle during chronic untreated infection, and emerging data suggest that they are a major source of replication in treated disease as well. The concentration of HIV RNA + cells in the B-cell follicle is mediated by several factors. Follicular CD4 + T-cell subsets including T-follicular helper cells and T-follicular regulatory cells are significantly more permissive to HIV than extrafollicular subsets. The B cell follicle also contains a large reservoir of extracellular HIV virions, which accumulate on the surface of follicular dendritic cells (FDCs) in germinal centers. FDC-bound HIV virions remain infectious even in the presence of neutralizing antibodies and can persist for months or even years. Moreover, the B-cell follicle is semi-immune privileged from CTL control. Frequencies of HIV- and SIV-specific CTL are lower in B-cell follicles compared to extrafollicular regions as the majority of CTL do not express the follicular homing receptor CXCR5. Additionally, CTL in the B-cell follicle may be less functional than extrafollicular CTL as many exhibit the recently described CD8 T follicular regulatory phenotype. Other factors may also contribute to the follicular concentration of HIV RNA + cells. Notably, the contribution of NK cells and γδ T cells to control and/or persistence of HIV RNA + cells in secondary lymphoid tissue remains poorly characterized. As HIV research moves increasingly toward the development of cure strategies, a greater understanding of the barriers to control of HIV infection in B-cell follicles is critical. Although no strategy has as of yet proven to be effective, a range of novel therapies to address these barriers are currently being investigated including genetically engineered CTL or chimeric antigen receptor T cells that express the follicular homing molecule CXCR5, treatment with IL-15 or an IL-15 superagonist, use of bispecific antibodies to harness the killing power of the follicular CD8 + T cell population, and disruption of the follicle through treatments such as rituximab.

Concrete material characterization reinforced concrete tank structure Multi-Function Waste Tank Facility

NASA Astrophysics Data System (ADS)

Winkel, B. V.

1995-03-01

The purpose of this report is to document the Multi-Function Waste Tank Facility (MWTF) Project position on the concrete mechanical properties needed to perform design/analysis calculations for the MWTF secondary concrete structure. This report provides a position on MWTF concrete properties for the Title 1 and Title 2 calculations. The scope of the report is limited to mechanical properties and does not include the thermophysical properties of concrete needed to perform heat transfer calculations. In the 1970's, a comprehensive series of tests were performed at Construction Technology Laboratories (CTL) on two different Hanford concrete mix designs. Statistical correlations of the CTL data were later generated by Pacific Northwest Laboratories (PNL). These test results and property correlations have been utilized in various design/analysis efforts of Hanford waste tanks. However, due to changes in the concrete design mix and the lower range of MWTF operating temperatures, plus uncertainties in the CTL data and PNL correlations, it was prudent to evaluate the CTL data base and PNL correlations, relative to the MWTF application, and develop a defendable position. The CTL test program for Hanford concrete involved two different mix designs: a 3 kip/sq in mix and a 4.5 kip/sq in mix. The proposed 28-day design strength for the MWTF tanks is 5 kip/sq in. In addition to this design strength difference, there are also differences between the CTL and MWTF mix design details. Also of interest, are the appropriate application of the MWTF concrete properties in performing calculations demonstrating ACI Code compliance. Mix design details and ACI Code issues are addressed in Sections 3.0 and 5.0, respectively. The CTL test program and PNL data correlations focused on a temperature range of 250 to 450 F. The temperature range of interest for the MWTF tank concrete application is 70 to 200 F.

Laparoendoscopic single-site surgery versus standard laparoscopic simple nephrectomy: a prospective randomized study.

PubMed

Tugcu, Volkan; Ilbey, Yusuf Ozlem; Mutlu, Bircan; Tasci, Ali Ihsan

2010-08-01

Laparoendoscopic single-site surgery (LESS), an attempt to further enhance the cosmetic benefits of minimally invasive surgery while minimizing the potential morbidity associated with multiple incisions, has been developed recently. Our aim was to compare LESS simple nephrectomy (LESS-SN) and conventional transperitoneal laparoscopic simple nephrectomy (CTL-SN). In this randomized study that was conducted between December 2008 and September 2009, 27 patients who needed simple nephrectomy were randomized to either LESS-SN or CTL-SN. All procedures in both groups were performed by the first author, who is experienced in laparoscopic surgery. Patient characteristics, perioperative details, and time to return to work were recorded. Postoperative evaluation of pain and use of analgesic medication were recorded. There was no difference in median operative time (117.5 vs 114 min, P = 0.52), blood loss (50.71 vs 47.15 mL, P = 0.60), transfusion rates (0% for both), and hospitalization time (2.07 vs 2.11 days, P = 0.74) between the LESS-SN and CTL-SN groups. Time to return to normal activities was shorter in the LESS-SN group compared with the CTL-SN group (10.7 vs 13.5 days, P = 0.001). Both the visual analogue scale and the postoperative use of analgesics were significantly lower during postoperative days 1, 2, and 3 in patients who underwent LESS-SN, compared with patients who underwent CTL-SN. There were no intraoperative or postoperative complications in both groups. Compared with CTL-SN, LESS-SN was more expensive, but all patients undergoing LESS-SN were very pleased with the cosmetic outcome (no visible scars). The early experience described in this study suggests that LESS-SN is a safe and effective alternative to CTL-SN that provides surgeons with a minimally invasive surgical option and the ability to hide the surgical incision within the umbilicus; however, a larger series is necessary to confirm these findings and to determine if there are any benefits in pain, recovery, or cosmesis.

Hard truths: facing the hard truths about energy. Topic Paper No. 18: Coal to liquids and gas

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

2007-07-18

The report presents the issues associated with and the potential of coal to liquids (CTL) and coal to gas (CTG) technologies. The other important outcome from this report is to view and understand the inputs and assumptions from various publications and the range of production estimates from CTG and CTL technology. The examination of the publications demonstrates a large uncertainty for CTL, due to various assumptions from petroleum price to technological abilities. Key assumptions are left unexamined, such as product transportation, labor, equipment availability, and environmental risk. Overall, the published CTL production estimates are small in the total global petroleummore » market perspective; even in the most optimistic scenario the volume from CTL amounts to only 20% of the U.S. petroleum market in the Southern States Energy Board (SSEB) report. The National Coal Council (NCC) saw a 10% market share, whereas the various Energy Information Administration (EIA) scenarios saw 0% to 6% of the U.S market share. The NCC and SSEB both mentioned the added benefit of using the CO{sub 2} for enhanced oil recovery (EOR). It begins by introducing the process, giving a detailed technological understanding, and then outlining each issue with each report from coal availability to oil price assumptions. The incremental gains from CTL and other technology areas, such as oil shale, could have a significant impact on U.S. energy cost and foreign dependency. The use of coal allows the added benefit of relying on a resource that is domestically more plentiful than petroleum, but this reliance must be carefully balanced with the economics of developing the resource, since CTL facilities can cost more than $1 billion per 10,000 days of production, which implicates the competitiveness of the U.S. economy within the global economy. 33 refs.« less

A Two-Component DNA-Prime/Protein-Boost Vaccination Strategy for Eliciting Long-Term, Protective T Cell Immunity against Trypanosoma cruzi

PubMed Central

Gupta, Shivali; Garg, Nisha J.

2015-01-01

In this study, we evaluated the long-term efficacy of a two-component subunit vaccine against Trypanosoma cruzi infection. C57BL/6 mice were immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach and challenged with T. cruzi at 120 or 180 days post-vaccination (dpv). We examined whether vaccine-primed T cell immunity was capable of rapid expansion and intercepting the infecting T. cruzi. Our data showed that D/P vaccine elicited CD4+ (30-38%) and CD8+ (22-42%) T cells maintained an effector phenotype up to 180 dpv, and were capable of responding to antigenic stimulus or challenge infection by a rapid expansion (CD8>CD4) with type 1 cytokine (IFNγ+ and TFNα+) production and cytolytic T lymphocyte (CTL) activity. Subsequently, challenge infection at 120 or 180 dpv, resulted in 2-3-fold lower parasite burden in vaccinated mice than was noted in unvaccinated/infected mice. Co-delivery of IL-12- and GMCSF-encoding expression plasmids provided no significant benefits in enhancing the anti-parasite efficacy of the vaccine-induced T cell immunity. Booster immunization (bi) with recombinant TcG2/TcG4 proteins 3-months after primary vaccine enhanced the protective efficacy, evidenced by an enhanced expansion (1.2-2.8-fold increase) of parasite-specific, type 1 CD4+ and CD8+ T cells and a potent CTL response capable of providing significantly improved (3-4.5-fold) control of infecting T. cruzi. Further, CD8+T cells in vaccinated/bi mice were predominantly of central memory phenotype, and capable of responding to challenge infection 4-6-months post bi by a rapid expansion to a poly-functional effector phenotype, and providing a 1.5-2.3-fold reduction in tissue parasite replication. We conclude that the TcG2/TcG4 D/P vaccine provided long-term anti-T. cruzi T cell immunity, and bi would be an effective strategy to maintain or enhance the vaccine-induced protective immunity against T. cruzi infection and Chagas disease. PMID:25951312

Global stability for an HIV-1 infection model with Beddington-DeAngelis incidence rate and CTL immune response

NASA Astrophysics Data System (ADS)

Lv, Cuifang; Huang, Lihong; Yuan, Zhaohui

2014-01-01

In this paper, an HIV-1 infection model with Beddington-DeAngelis incidence rate and CTL immune response is investigated. One main feature of this model is that an eclipse stage for the infected cells is included and a portion of these cells is reverted to uninfected cells. We derive the basic reproduction number R1 and the immune response reproduction number R2 for the HIV-1 infection model. By constructing Lyapunov functions, the global stabilities for the equilibria have been analyzed.

Pathogen recognition of a novel C-type lectin from Marsupenaeus japonicus reveals the divergent sugar-binding specificity of QAP motif.

PubMed

Alenton, Rod Russel R; Koiwai, Keiichiro; Miyaguchi, Kohei; Kondo, Hidehiro; Hirono, Ikuo

2017-04-04

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins known to assist the innate immune system as pattern recognition receptors (PRRs). The binding specificity of CTLs lies in the motif of their carbohydrate recognition domain (CRD), the tripeptide motifs EPN and QPD bind to mannose and galactose, respectively. However, variants of these motifs were discovered including a QAP sequence reported in shrimp believed to have the same carbohydrate specificity as QPD. Here, we characterized a novel C-type lectin (MjGCTL) possessing a CRD with a QAP motif. The recombinant MjGCTL has a calcium-dependent agglutinating capability against both Gram-negative and Gram-positive bacteria, and its sugar specificity did not involve either mannose or galactose. In an encapsulation assay, agarose beads coated with rMjGCTL were immediately encapsulated from 0 h followed by melanization at 4 h post-incubation with hemocytes. These results confirm that MjGCTL functions as a classical CTL. The structure of QAP motif and carbohydrate-specificity of rMjGCTL was found to be different to both EPN and QPD, suggesting that QAP is a new motif. Furthermore, MjGCTL acts as a PRR binding to hemocytes to activate their adherent state and initiate encapsulation.

Pathogen recognition of a novel C-type lectin from Marsupenaeus japonicus reveals the divergent sugar-binding specificity of QAP motif

PubMed Central

Alenton, Rod Russel R.; Koiwai, Keiichiro; Miyaguchi, Kohei; Kondo, Hidehiro; Hirono, Ikuo

2017-01-01

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins known to assist the innate immune system as pattern recognition receptors (PRRs). The binding specificity of CTLs lies in the motif of their carbohydrate recognition domain (CRD), the tripeptide motifs EPN and QPD bind to mannose and galactose, respectively. However, variants of these motifs were discovered including a QAP sequence reported in shrimp believed to have the same carbohydrate specificity as QPD. Here, we characterized a novel C-type lectin (MjGCTL) possessing a CRD with a QAP motif. The recombinant MjGCTL has a calcium-dependent agglutinating capability against both Gram-negative and Gram-positive bacteria, and its sugar specificity did not involve either mannose or galactose. In an encapsulation assay, agarose beads coated with rMjGCTL were immediately encapsulated from 0 h followed by melanization at 4 h post-incubation with hemocytes. These results confirm that MjGCTL functions as a classical CTL. The structure of QAP motif and carbohydrate-specificity of rMjGCTL was found to be different to both EPN and QPD, suggesting that QAP is a new motif. Furthermore, MjGCTL acts as a PRR binding to hemocytes to activate their adherent state and initiate encapsulation. PMID:28374848

Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

PubMed Central

Mouchacca, Pierre; Schmitt-Verhulst, Anne-Marie; Boyer, Claude

2013-01-01

To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells’ plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo. PMID:23840635

Inventory of File sref_nmb.t03z.pgrb212.ctl.grib2

Science.gov Websites

10 m above ground UGRD 3 hour fcst U-Component of Wind [m/s] ENS=low-res ctl 004 10 m above ground VGRD 3 hour fcst V-Component of Wind [m/s] ENS=low-res ctl 005 mean sea level PRMSL 3 hour fcst 250 mb UGRD 3 hour fcst U-Component of Wind [m/s] ENS=low-res ctl 014 500 mb UGRD 3 hour fcst U

Identification of Novel Saccharomyces cerevisiae Proteins with Nuclear Export Activity: Cell Cycle-Regulated Transcription Factor Ace2p Shows Cell Cycle-Independent Nucleocytoplasmic Shuttling

PubMed Central

Jensen, Torben Heick; Neville, Megan; Rain, Jean Christophe; McCarthy, Terri; Legrain, Pierre; Rosbash, Michael

2000-01-01

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiae proteins not previously known to have nuclear export activity. These proteins are the 5′ RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G1-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G1 but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner. PMID:11027275

Adolescent social instability stress increases aggression in a food competition task in adult male Long-Evans rats.

PubMed

Cumming, Mark J; Thompson, Madison A; McCormick, Cheryl M

2014-11-01

Adolescent social instability stress (SS; daily 1 hr isolation + new cage partners postnatal days 30-45; thereafter with original cage partner, also in the SS condition) and control (CTL) rats competed for access to a preferred food in five sessions against their cage partner. In the first session, SS pairs displayed more aggression (face whacks, p = .02; rear attacks, p = .03), were less likely to relinquish access to the food voluntarily (p = .03), spent more time at the feeder than CTL pairs (p = .06), but did not differ in latency to access the feeder (p = .41). Pairs were considered in dominant-submissive relationships (DSR) if one rat spent significantly more time at the feeder than the other; 8 of 12 SS and 8 of 12 CTL pairs displayed DSRs (remaining: no-DSR). Aggression increased from the 1st to 5th session (p < .001), was greater in no-DSR than DSR pairs (p = .04; consistent with the proposed function of DSRs to be the reduction of aggression in groups), and was higher in SS than CTL pairs (p = .05). Because the increased aggression of SS compared with CTL pairs did not result in a significant increase in their time at the feeder, the increased aggression may be considered maladaptive, and may reflect an increased motivation for food reward. These results add to evidence that SS in adolescence modifies the adult social repertoire of rats and highlight the importance of adolescent social experiences for adult behavior. © 2014 Wiley Periodicals, Inc.

Biofuel feedstock and blended coproducts compared with deoiled corn distillers grains in feedlot diets: Effects on cattle growth performance, apparent total tract nutrient digestibility, and carcass characteristics.

PubMed

Opheim, T L; Campanili, P R B; Lemos, B J M; Ovinge, L A; Baggerman, J O; McCuistion, K C; Galyean, M L; Sarturi, J O; Trojan, S J

2016-01-01

Crossbred steers (British × Continental; = 192; initial BW 391 ± 28 kg) were used to evaluate the effects of feeding ethanol coproducts on feedlot cattle growth performance, apparent nutrient digestibility, and carcass characteristics. Steers were blocked by initial BW and assigned randomly to 1 of 6 dietary treatments within block. Treatments (replicated in 8 pens with 4 steers/pen) included 1) control, steam-flaked corn-based diet (CTL), 2) corn dried distillers grains with solubles (DGS; DRY-C), 3) deoiled corn dried DGS (DRY-CLF), 4) blended 50/50 corn/sorghum dried DGS (DRY-C/S), 5) sorghum dried DGS (DRY-S), and 6) sorghum wet DGS (WET-S). Inclusion of DGS was 25% (DM basis). The DGS diets were isonitrogenous, CTL was formulated for 13.5% CP, and all diets were balanced for ether extract. Final shrunk BW, ADG, and DMI did not differ among CTL and DGS treatments ( ≥ 0.19). Overall G:F did not differ from CTL for DRY-C, DRY-CLF, or WET-S ( ≥ 0.12); however, G:F was 9.6% less for DRY-S compared with CTL ( < 0.01) and tended ( = 0.09) to be less for DRY-C/S than CTL. For grain source, ADG and G:F were less for DRY-S vs. DRY-C ( < 0.05), but blending DRY-C/S tended ( = 0.07) to increase ADG and increased ( = 0.05) carcass-adjusted G:F vs. DRY-S. For WET-S, final BW and ADG were greater ( < 0.05), and G:F tended ( = 0.06) to be greater than for DRY-S. There was no difference in ADG, DMI, or G:F of steers fed DRY-C vs. DRY-CLF ( ≥ 0.35). Apparent DM and OM digestibility did not differ for CTL, DRY-C, DRY-CLF, and WET-S ( ≥ 0.30) but were lower for DRY-C/S and DRY-S ( < 0.05). Nutrient digestibility was lower for DRY-S vs. DRY-C ( < 0.01), but apparent digestibility of OM, DM, NDF, ADF, CP, ether extract, and starch were increased ( < 0.01) for DRY-C/S vs. DRY-S. Although starch digestibility did not differ between DRY-S and WET-S ( 0.18), digestibility of other measured nutrients was greater for WET-S vs. DRY-S ( < 0.01). Ether extract digestibility was greater for DRY-CLF vs. DRY-C ( < 0.05). Carcass weight, dressing percent, and marbling score did not differ between CTL and DGS diets ( ≥ 0.23). For DRY-S, HCW was lower than for DRY-C ( = 0.02); however, compared with DRY-S, HCW tended to be greater for DRY-C/S ( = 0.10) and WET-S ( = 0.07). At a moderately high (25% DM) inclusion, blending C/S or feeding WET-S resulted in cattle growth performance and carcass characteristics similar to those of CTL and corn-based coproducts.

Transcutaneous immunization with a novel imiquimod nanoemulsion induces superior T cell responses and virus protection.

PubMed

Lopez, Pamela Aranda; Denny, Mark; Hartmann, Ann-Kathrin; Alflen, Astrid; Probst, Hans Christian; von Stebut, Esther; Tenzer, Stefan; Schild, Hansjörg; Stassen, Michael; Langguth, Peter; Radsak, Markus P

2017-09-01

Transcutaneous immunization (TCI) is a novel vaccination strategy utilizing the skin associated lymphatic tissue to induce immune responses. TCI using a cytotoxic T lymphocyte (CTL) epitope and the Toll-like receptor 7 (TLR7) agonist imiquimod mounts strong CTL responses by activation and maturation of skin-derived dendritic cells (DCs) and their migration to lymph nodes. However, TCI based on the commercial formulation Aldara only induces transient CTL responses that needs further improvement for the induction of durable therapeutic immune responses. Therefore we aimed to develop a novel imiquimod solid nanoemulsion (IMI-Sol) for TCI with superior vaccination properties suited to induce high quality T cell responses for enhanced protection against infections. TCI was performed by applying a MHC class I or II restricted epitope along with IMI-Sol or Aldara (each containing 5% Imiquimod) on the shaved dorsum of C57BL/6, IL-1R, Myd88, Tlr7 or Ccr7 deficient mice. T cell responses as well as DC migration upon TCI were subsequently analyzed by flow cytometry. To determine in vivo efficacy of TCI induced immune responses, CTL responses and frequency of peptide specific T cells were evaluated on day 8 or 35 post vaccination and protection in a lymphocytic choriomeningitis virus (LCMV) infection model was assessed. TCI with the imiquimod formulation IMI-Sol displayed equal skin penetration of imiquimod compared to Aldara, but elicited superior CD8 + as well as CD4 + T cell responses. The induction of T-cell responses induced by IMI-Sol TCI was dependent on the TLR7/MyD88 pathway and independent of IL-1R. IMI-Sol TCI activated skin-derived DCs in skin-draining lymph nodes more efficiently compared to Aldara leading to enhanced protection in a LCMV infection model. Our data demonstrate that IMI-Sol TCI can overcome current limitations of previous imiquimod based TCI approaches opening new perspectives for transcutaneous vaccination strategies and allowing the use of this enhanced cutaneous drug-delivery system to be tailored for the improved prevention and treatment of infectious diseases and cancers. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Complex antigen presentation pathway for an HLA-A*0201-restricted epitope from Chikungunya 6K protein

PubMed Central

Lemonnier, François A.; Esteban, Mariano

2017-01-01

Background The adaptive cytotoxic T lymphocyte (CTL)-mediated immune response is critical for clearance of many viral infections. These CTL recognize naturally processed short viral antigenic peptides bound to human leukocyte antigen (HLA) class I molecules on the surface of infected cells. This specific recognition allows the killing of virus-infected cells. The T cell immune T cell response to Chikungunya virus (CHIKV), a mosquito-borne Alphavirus of the Togaviridae family responsible for severe musculoskeletal disorders, has not been fully defined; nonetheless, the importance of HLA class I-restricted immune response in this virus has been hypothesized. Methodology/Principal findings By infection of HLA-A*0201-transgenic mice with a recombinant vaccinia virus that encodes the CHIKV structural polyprotein (rVACV-CHIKV), we identified the first human T cell epitopes from CHIKV. These three novel 6K transmembrane protein-derived epitopes are presented by the common HLA class I molecule, HLA-A*0201. One of these epitopes is processed and presented via a complex pathway that involves proteases from different subcellular locations. Specific chemical inhibitors blocked these events in rVACV-CHIKV-infected cells. Conclusions/Significance Our data have implications not only for the identification of novel Alphavirus and Togaviridae antiviral CTL responses, but also for analyzing presentation of antigen from viruses of different families and orders that use host proteinases to generate their mature envelope proteins. PMID:29084215

Complex antigen presentation pathway for an HLA-A*0201-restricted epitope from Chikungunya 6K protein.

PubMed

Lorente, Elena; Barriga, Alejandro; García-Arriaza, Juan; Lemonnier, François A; Esteban, Mariano; López, Daniel

2017-10-01

The adaptive cytotoxic T lymphocyte (CTL)-mediated immune response is critical for clearance of many viral infections. These CTL recognize naturally processed short viral antigenic peptides bound to human leukocyte antigen (HLA) class I molecules on the surface of infected cells. This specific recognition allows the killing of virus-infected cells. The T cell immune T cell response to Chikungunya virus (CHIKV), a mosquito-borne Alphavirus of the Togaviridae family responsible for severe musculoskeletal disorders, has not been fully defined; nonetheless, the importance of HLA class I-restricted immune response in this virus has been hypothesized. By infection of HLA-A*0201-transgenic mice with a recombinant vaccinia virus that encodes the CHIKV structural polyprotein (rVACV-CHIKV), we identified the first human T cell epitopes from CHIKV. These three novel 6K transmembrane protein-derived epitopes are presented by the common HLA class I molecule, HLA-A*0201. One of these epitopes is processed and presented via a complex pathway that involves proteases from different subcellular locations. Specific chemical inhibitors blocked these events in rVACV-CHIKV-infected cells. Our data have implications not only for the identification of novel Alphavirus and Togaviridae antiviral CTL responses, but also for analyzing presentation of antigen from viruses of different families and orders that use host proteinases to generate their mature envelope proteins.

Activin receptor-like kinase5 inhibition suppresses mouse melanoma by ubiquitin degradation of Smad4, thereby derepressing eomesodermin in cytotoxic T lymphocytes

PubMed Central

Yoon, Jeong-Hwan; Jung, Su Myung; Park, Seok Hee; Kato, Mitsuyasu; Yamashita, Tadashi; Lee, In-Kyu; Sudo, Katsuko; Nakae, Susumu; Han, Jin Soo; Kim, Ok-Hee; Oh, Byung-Chul; Sumida, Takayuki; Kuroda, Masahiko; Ju, Ji-Hyeon; Jung, Kyeong Cheon; Park, Seong Hoe; Kim, Dae-Kee; Mamura, Mizuko

2013-01-01

Varieties of transforming growth factor-β (TGF-β) antagonists have been developed to intervene with excessive TGF-β signalling activity in cancer. Activin receptor-like kinase5 (ALK5) inhibitors antagonize TGF-β signalling by blocking TGF-β receptor-activated Smad (R-Smad) phosphorylation. Here we report the novel mechanisms how ALK5 inhibitors exert a therapeutic effect on a mouse B16 melanoma model. Oral treatment with a novel ALK5 inhibitor, EW-7197 (2.5 mg/kg daily) or a representative ALK5 inhibitor, LY-2157299 (75 mg/kg bid) suppressed the progression of melanoma with enhanced cytotoxic T-lymphocyte (CTL) responses. Notably, ALK5 inhibitors not only blocked R-Smad phosphorylation, but also induced ubiquitin-mediated degradation of the common Smad, Smad4 mainly in CD8+ T cells in melanoma-bearing mice. Accordingly, T-cell-specific deletion of Smad4 was sufficient to suppress the progression of melanoma. We further identified eomesodermin (Eomes), the T-box transcription factor regulating CTL functions, as a specific target repressed by TGF-β via Smad4 and Smad3 in CD8+ T cells. Thus, ALK5 inhibition enhances anti-melanoma CTL responses through ubiquitin-mediated degradation of Smad4 in addition to the direct inhibitory effect on R-Smad phosphorylation. PMID:24127404

Activated natural killer cell-mediated immunity is required for the inhibition of tumor metastasis by dendritic cell vaccination.

PubMed

Kim, Aeyung; Noh, Young-Woock; Kim, Kwang Dong; Jang, Yong-Suk; Choe, Yong-Kyung; Lim, Jong-Seok

2004-10-31

Immunization with dendritic cells (DCs) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL), which is responsible for tumor protection and regression. In this study, we examined whether DCs pulsed with necrotic tumor lysates can efficiently prevent malignant melanoma tumor cell metastasis to the lung. DCs derived from mouse bone marrow were found to produce remarkably elevated levels of IL-12 after being pulsed with the tumor lysates. Moreover, immunization with these DCs induced CTL activation and protected mice from metastasis development by intravenously inoculated tumor cells. In addition, these DCs activated NK cells in vitro in a contact-dependent manner, and induced NK activities in vivo. Furthermore, NK cell depletion before DC vaccination significantly reduced the tumor-specific CTL activity, IFN-gamma production, and IFN-gamma- inducible gene expression, and eventually interfered with the antitumor effect of tumor-pulsed DCs. Finally, similar findings with respect to NK cell dependency were obtained in the C57BL/ 6J-bg/bg mice, which have severe deficiency in cytolytic activity of NK cells. These data suggest that the antitumor effect elicited by DC vaccination, at least in a B16 melanoma model, requires the participation of both cytolytic NK and CD8(+) T cells. The findings of this study would provide important data for the effective design of DC vaccines for cancer immunotherapy.

Stimulating CTL Towards HER2/neu Overexpressing Breast Cancer

DTIC Science & Technology

1998-10-01

296. 9. Jardetzky, T.S., W.S. Lane, R.A. Robinson, D.R. Madden, and D.C. Wiley, Identification of self peptides bound to purified HLA - B27 . Nature...neu (HN654-662) has been shown to bind to HLA -A2.1 and stimulate cytotoxic T lymphocytes (CTL) that lyse primary tumors from ovarian or breast cancer...demonstrating HN654-662 is an extremely poor HLA -A2.1 binding peptide. In a novel approach, we will make use of biophysical techniques that have recently

Granulocyte-macrophage colony-stimulating factor induces the differentiation of murine erythroleukaemia cells into dendritic cells.

PubMed Central

Cao, X; Zhao, Y; Yu, Y; Wang, Y; Zhang, M; Zhang, W; Wang, J

1998-01-01

Dendritic cells (DC) are professional antigen-presenting cells (APC) within the immune system and antigen-pulsed DC can be used as an effective vaccine for active immunotherapy of cancer. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the generation of DC. We previously showed that GM-CSF can induce murine erythroleukaemia cells (FBL-3) to differentiate into monocyte-like cells. To develop a new vaccinating method to stimulate the host immune response to leukaemia, we further investigate whether FBL-3 cells induced by GM-CSF can differentiate into DC in the present study. After being treated with GM-CSF, FBL-3 cells expressed high levels of 33D1 and NLDC-145, which are the specific markers of DC. The expression of MHC-II, B7-1, B7-2 and vascular cell adhesion molecule-1 (VCAM-1) was up-regulated markedly; the typical morphology of DC were also observed by electron microscopy. Functionally, the GM-CSF-induced FBL-3 cells could apparently stimulate the proliferation of naive allogeneic and autologous T lymphocytes and induce the generation of specific CTL more efficiently than the wild-type FBL-3 cells. Mice immunized with GM-CSF-induced FBL-3 cells could resist the subsequent challenge with the wild-type FBL-3 cells. Collectively, these data indicate that GM-CSF differentiates murine erythroleukaemia cells into DC phenotypically, morphologically and functionally. FBL-3-derived DC can be used as a new type of vaccine. Our results may have important implications for the immunotherapy of leukaemia. Images Figure 3 Figure 4 PMID:9767469

Accumulation of Pol Mutations Selected by HLA-B*52:01-C*12:02 Protective Haplotype-Restricted Cytotoxic T Lymphocytes Causes Low Plasma Viral Load Due to Low Viral Fitness of Mutant Viruses

PubMed Central

Murakoshi, Hayato; Koyanagi, Madoka; Chikata, Takayuki; Rahman, Mohammad Arif; Kuse, Nozomi; Sakai, Keiko; Gatanaga, Hiroyuki; Oka, Shinichi

2016-01-01

ABSTRACT HLA-B*52:01-C*12:02, which is the most abundant haplotype in Japan, has a protective effect on disease progression in HIV-1-infected Japanese individuals, whereas HLA-B*57 and -B*27 protective alleles are very rare in Japan. A previous study on HLA-associated polymorphisms demonstrated that the number of HLA-B*52:01-associated mutations at four Pol positions was inversely correlated with plasma viral load (pVL) in HLA-B*52:01-negative individuals, suggesting that the transmission of HIV-1 with these mutations could modulate the pVL in the population. However, it remains unknown whether these mutations were selected by HLA-B*52:01-restricted CTLs and also reduced viral fitness. In this study, we identified two HLA-B*52:01-restricted and one HLA-C*12:02-restricted novel cytotoxic T-lymphocyte (CTL) epitopes in Pol. Analysis using CTLs specific for these three epitopes demonstrated that these CTLs failed to recognize mutant epitopes or more weakly recognized cells infected with mutant viruses than wild-type virus, supporting the idea that these mutations were selected by the HLA-B*52:01- or HLA-C*12:02-restricted T cells. We further showed that these mutations reduced viral fitness, although the effect of each mutation was weak. The present study demonstrated that the accumulation of these Pol mutations selected by HLA-B*52:01- or HLA-C*12:02-restricted CTLs impaired viral replication capacity and thus reduced the pVL. The fitness cost imposed by the mutations partially accounted for the effect of the HLA-B*52:01-C*12:02 haplotype on clinical outcome, together with the effect of HLA-B*52:01-restricted CTLs on viral replication, which had been previously demonstrated. IMPORTANCE Numerous population-based studies identified HLA-associated HIV-1 mutations to predict HIV-1 escape mutations from cytotoxic T lymphocytes (CTLs). However, the majority of these HLA-associated mutations have not been identified as CTL escape mutations. Our previous population-based study showed that five HLA-B*52:01-associated mutations at four Pol positions were inversely correlated with the plasma viral load in HLA-B*52:01-negative Japanese individuals. In the present study, we demonstrated that these mutations were indeed selected by CTLs specific for novel B*52:01- and C*12:02-restricted epitopes and that the accumulation of these mutations reduced the viral fitness in vitro. This study elucidated the mechanism by which the accumulation of these CTL escape mutations contributed to the protective effect of the HLA-B*52:01-HLA-C*12:02 haplotype on disease progression in HIV-1-infected Japanese individuals. PMID:27903797

Accumulation of Pol Mutations Selected by HLA-B*52:01-C*12:02 Protective Haplotype-Restricted Cytotoxic T Lymphocytes Causes Low Plasma Viral Load Due to Low Viral Fitness of Mutant Viruses.

PubMed

Murakoshi, Hayato; Koyanagi, Madoka; Chikata, Takayuki; Rahman, Mohammad Arif; Kuse, Nozomi; Sakai, Keiko; Gatanaga, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

2017-02-15

HLA-B*52:01-C*12:02, which is the most abundant haplotype in Japan, has a protective effect on disease progression in HIV-1-infected Japanese individuals, whereas HLA-B*57 and -B*27 protective alleles are very rare in Japan. A previous study on HLA-associated polymorphisms demonstrated that the number of HLA-B*52:01-associated mutations at four Pol positions was inversely correlated with plasma viral load (pVL) in HLA-B*52:01-negative individuals, suggesting that the transmission of HIV-1 with these mutations could modulate the pVL in the population. However, it remains unknown whether these mutations were selected by HLA-B*52:01-restricted CTLs and also reduced viral fitness. In this study, we identified two HLA-B*52:01-restricted and one HLA-C*12:02-restricted novel cytotoxic T-lymphocyte (CTL) epitopes in Pol. Analysis using CTLs specific for these three epitopes demonstrated that these CTLs failed to recognize mutant epitopes or more weakly recognized cells infected with mutant viruses than wild-type virus, supporting the idea that these mutations were selected by the HLA-B*52:01- or HLA-C*12:02-restricted T cells. We further showed that these mutations reduced viral fitness, although the effect of each mutation was weak. The present study demonstrated that the accumulation of these Pol mutations selected by HLA-B*52:01- or HLA-C*12:02-restricted CTLs impaired viral replication capacity and thus reduced the pVL. The fitness cost imposed by the mutations partially accounted for the effect of the HLA-B*52:01-C*12:02 haplotype on clinical outcome, together with the effect of HLA-B*52:01-restricted CTLs on viral replication, which had been previously demonstrated. Numerous population-based studies identified HLA-associated HIV-1 mutations to predict HIV-1 escape mutations from cytotoxic T lymphocytes (CTLs). However, the majority of these HLA-associated mutations have not been identified as CTL escape mutations. Our previous population-based study showed that five HLA-B*52:01-associated mutations at four Pol positions were inversely correlated with the plasma viral load in HLA-B*52:01-negative Japanese individuals. In the present study, we demonstrated that these mutations were indeed selected by CTLs specific for novel B*52:01- and C*12:02-restricted epitopes and that the accumulation of these mutations reduced the viral fitness in vitro This study elucidated the mechanism by which the accumulation of these CTL escape mutations contributed to the protective effect of the HLA-B*52:01-HLA-C*12:02 haplotype on disease progression in HIV-1-infected Japanese individuals. Copyright © 2017 American Society for Microbiology.

Natural killer cells facilitate PRAME-specific T-cell reactivity against neuroblastoma

PubMed Central

Spel, Lotte; Boelens, Jaap-Jan; van der Steen, Dirk M.; Blokland, Nina J.G.; van Noesel, Max M.; Molenaar, Jan J.; Heemskerk, Mirjam H.M.

2015-01-01

Neuroblastoma is the most common solid tumor in children with an estimated 5-year progression free survival of 20–40% in stage 4 disease. Neuroblastoma actively avoids recognition by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Although immunotherapy has gained traction for neuroblastoma treatment, these immune escape mechanisms restrain clinical results. Therefore, we aimed to improve neuroblastoma immunogenicity to further the development of antigen-specific immunotherapy against neuroblastoma. We found that neuroblastoma cells significantly increase surface expression of MHC I upon exposure to active NK cells which thereby readily sensitize neuroblastoma cells for recognition by CTLs. We show that oncoprotein PRAME serves as an immunodominant antigen for neuroblastoma as NK-modulated neuroblastoma cells are recognized by PRAMESLLQHLIGL/A2-specific CTL clones. Furthermore, NK cells induce MHC I upregulation in neuroblastoma through contact-dependent secretion of IFNγ. Our results demonstrate remarkable plasticity in the peptide/MHC I surface expression of neuroblastoma cells, which is reversed when neuroblastoma cells experience innate immune attack by sensitized NK cells. These findings support the exploration of NK cells as adjuvant therapy to enforce neuroblastoma-specific CTL responses. PMID:26452036

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

PubMed Central

Vereecke, Lars; Mc Guire, Conor; Sze, Mozes; Schuijs, Martijn J.; Willart, Monique; Itati Ibañez, Lorena; Hammad, Hamida; Lambrecht, Bart N.; Beyaert, Rudi; Saelens, Xavier; van Loo, Geert

2016-01-01

A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20AEC-KO mice during later stages of infection. When A20AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection. PMID:26815999

Cetuximab-activated natural killer (NK) and dendritic cells (DC) collaborate to trigger tumor antigen-specific T cell immunity in head and neck cancer patients

PubMed Central

Srivastava, Raghvendra M.; Lee, Steve C.; Filho, Pedro A. Andrade; Lord, Christopher A.; Jie, Hyun-bae; Davidson, H. Carter; López-Albaitero, Andrés; Gibson, Sandra P.; Gooding, William E.; Ferrone, Soldano; Ferris, Robert L.

2013-01-01

Purpose Tumor antigen (TA)-specific monoclonal antibodies (mAb) block oncogenic signaling and induce Fcγ receptor (FcγR)-mediated cytotoxicity. However, the role of CD8+ cytotoxic T lymphocyte (CTL) and FcγR in initiating innate and adaptive immune responses in mAb-treated human cancer patients is still emerging. Experimental Design FcγRIIIa codon 158 polymorphism was correlated with survival in 107 cetuximab-treated head and neck cancer (HNC) patients. Flow cytometry was performed to quantify EGFR-specific T cells in cetuximab-treated HNC patients. The effect of cetuximab on NK cell, dendritic cell (DC), and T cell activation was measured using IFN-γ release assays and flow cytometry. Results FcγR IIIa polymorphism did not predict clinical outcome in cetuximab-treated HNC patients, however elevated circulating EGFR -specific CD8+ 853-861 T cells were found in cetuximab-treated HNC patients (p<0.005). Cetuximab promoted EGFR-specific cellular immunity through the interaction of EGFR+ tumor cells and FcγRIIIa on NK cells, but not on the polymorphism per se. Cetuximab-activated NK cells induced IFN-γ dependent expression of DC maturation markers, antigen presentation machinery (APM) components such as TAP-1/2, and Th1 chemokines through NKG2D/MICA binding. Cetuximab initiated adaptive immune responses via NK-cell induced DC maturation, which enhanced cross-presentation to CTL specific for EGFR as well as another TA, MAGE-3. Conclusion Cetuximab-activated NK cells promote DC maturation and CD8+ T cell priming, leading to TA spreading and Th1 cytokine release through ‘NK-DC cross-talk.’ FcγRIIIa polymorphism did not predict clinical response to cetuximab, but was necessary for NK-DC interaction and mAb induced cross-presentation. EGFR-specific T cells in cetuximab treated HNC patients may contribute to clinical response. PMID:23444227

Predictors of social instability stress effects on social interaction and anxiety in adolescent male rats.

PubMed

Hodges, Travis E; Baumbach, Jennet L; McCormick, Cheryl M

2018-06-21

Adolescence is an important phase of development of social behaviors, which may be disrupted by the experience of stressors. We previously reported that exposure to social instability stress in adolescence (SS; postnatal day [PND] 30-45) in rats reduced social interactions with unfamiliar peers compared with non-stressed controls (CTL). In experiment 1, we replicated the effect of SS on social interaction and found that the pattern of neural activations based on Fos immunohistochemistry in brain regions during social interactions differed for SS and CTL rats. In experiment 2, we found that individual differences in novelty-seeking behavior on PND 30 and SS exposure were unique predictors of anxiety in the elevated plus maze on PND 46, and interacted to predict social interaction on PND 47; among high novelty-seeking rats, SS and CTL rats do not differ, whereas among low-novelty seeking rats, SS rats engaged in less social interaction than did CTL rats. Thus, high novelty-seeking may be a resilience factor against the effects of social stressors in adolescence. © 2018 Wiley Periodicals, Inc.

Metal-Free Cataluminescence Gas Sensor for Hydrogen Sulfide Based on Its Catalytic Oxidation on Silicon Carbide Nanocages.

PubMed

Wu, Liqian; Zhang, Lichun; Sun, Mingxia; Liu, Rui; Yu, Lingzhu; Lv, Yi

2017-12-19

Cataluminescence- (CTL-) based sensors are among the most attractive and effective tools for gas sensing, owing to their efficient selectivity, high sensitivity, and rapidity. As the sensing materials of CTL-based sensors, metal-based catalysts easily bring about high costs and environmental pollution of heavy metals. More importantly, the long-term stability of metal-based catalysts is usually rather poor. Metal-free catalysts have unique advantages such as environmental friendliness, low costs, and long-term stability, making them promising materials for CTL-based sensors. Herein, we report the fabrication of a CTL sensor based on a metal-free catalyst. F-doped cage-like SiC was synthesized by wet chemical etching. The as-prepared products showed a rapid, stable, highly selective, and sensitive cataluminescent response to H 2 S. The stability of the sensor was demonstrated to be fairly good for at least 15 days. After CTL tests, F-doped cage-like SiC retained its original morphology, structure, and chemical composition. In addition, to the best of our knowledge, this is the first report of a metal-free CTL sensor. Metal-free catalysts are environmentally friendly and low in cost and exhibit long-term stability, which could open a new avenue of CTL sensing.

Long-Term Control of HIV-1 in Hemophiliacs Carrying Slow-Progressing Allele HLA-B*5101▿ †

PubMed Central

Kawashima, Yuka; Kuse, Nozomi; Gatanaga, Hiroyuki; Naruto, Takuya; Fujiwara, Mamoru; Dohki, Sachi; Akahoshi, Tomohiro; Maenaka, Katsumi; Goulder, Philip; Oka, Shinichi; Takiguchi, Masafumi

2010-01-01

HLA-B*51 alleles are reported to be associated with slow disease progression to AIDS, but the mechanism underlying this association is still unclear. In the present study, we analyzed the effect of HLA-B*5101 on clinical outcome for Japanese hemophiliacs who had been infected with HIV-1 before 1985 and had been recruited in 1998 for this study. HLA-B*5101+ hemophiliacs exhibited significantly slow progression. The analysis of HLA-B*5101-restricted HIV-1-specific cytotoxic T-lymphocyte (CTL) responses to 4 HLA-B*-restricted epitopes in 10 antiretroviral-therapy (ART)-free HLA-B*5101+ hemophiliacs showed that the frequency of Pol283-8-specific CD8+ T cells was inversely correlated with the viral load, whereas the frequencies of CD8+ T cells specific for 3 other epitopes were positively correlated with the viral load. The HLA-B*5101+ hemophiliacs whose HIV-1 replication had been controlled for approximately 25 years had HIV-1 possessing the wild-type Pol283-8 sequence or the Pol283-8V mutant, which does not critically affect T-cell recognition, whereas other HLA-B*5101+ hemophiliacs had HIV-1 with escape mutations in this epitope. The results suggest that the control of HIV-1 over approximately 25 years in HLA-B*5101-positive hemophiliacs is associated with a Pol283-8-specific CD8+ T-cell response and that lack of control of HIV-1 is associated with the appearance of Pol283-8-specific escape mutants. PMID:20410273

Joint Mental Health Advisory Team 7 (J-MHAT 7) Operation Enduring Freedom 2010 Afghanistan (REDACTED)

DTIC Science & Technology

2011-02-22

level. 18 45% ctl 40% ’!:: Q) 35% ...., ·c 0 30% 0> c: 25% :.;::::; Q) Q) 20% ::!E ...., 15% c: Q) e 10% Q) (1... 5% 0% Figure 5.2.1...t::: (.) 60% 0 c: c.o Q)- 50% 0::: en ...... ctl 40% c: Q) Q)_J 30% ~ Q) 20% a. 10% 0% Figure 12.1.1: Representative Combat Experiences...maneuver units in the global war on terror. In part, this high demand may be due to our willingness through efforts such as the MHAT process to take a

BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes.

PubMed

Kessler, J H; Bres-Vloemans, S A; van Veelen, P A; de Ru, A; Huijbers, I J G; Camps, M; Mulder, A; Offringa, R; Drijfhout, J W; Leeksma, O C; Ossendorp, F; Melief, C J M

2006-10-01

For immunotherapy of residual disease in patients with Philadelphia-positive leukemias, the BCR-ABL fusion regions are attractive disease-specific T-cell targets. We analyzed these regions for the prevalence of cytotoxic T lymphocyte (CTL) epitopes by an advanced reverse immunology procedure. Seventeen novel BCR-ABL fusion peptides were identified to bind efficiently to the human lymphocyte antigen (HLA)-A68, HLA-B51, HLA-B61 or HLA-Cw4 HLA class I molecules. Comprehensive enzymatic digestion analysis showed that 10 out of the 28 HLA class I binding fusion peptides were efficiently excised after their C-terminus by the proteasome, which is an essential requirement for efficient cell surface expression. Therefore, these peptides are prime vaccine candidates. The other peptides either completely lacked C-terminal liberation or were only inefficiently excised by the proteasome, rendering them inappropriate or less suitable for inclusion in a vaccine. CTL raised against the properly processed HLA-B61 epitope AEALQRPVA from the BCR-ABL e1a2 fusion region, expressed in acute lymphoblastic leukemia (ALL), specifically recognized ALL tumor cells, proving cell surface presentation of this epitope, its applicability for immunotherapy and underlining the accuracy of our epitope identification strategy. Our study provides a reliable basis for the selection of optimal peptides to be included in immunotherapeutic BCR-ABL vaccines against leukemia.

Effects of Annatto (Bixa orellana L.) Seeds Powder on Physicochemical Properties, Antioxidant and Antimicrobial Activities of Pork Patties during Refrigerated Storage

PubMed Central

Cuong, Tran Van; Chin, Koo Bok

2016-01-01

This study investigated the effect of the powder produced by ball-milling the outer layer of annatto (Bixa orellana L.) seeds on the physicochemical properties as well as the antioxidant and antimicrobial activities of pork patties over 14 d of refrigerated storage (4±1℃). Five pork patty treatments were produced containing three different concentrations of annatto seeds, 0.1, 0.25 and 0.5% (ANT0.1, ANT0.25, ANT0.5), 0.1% ascorbic acid (AA0.1), and a control (CTL). Based on the results, annatto seed powder appeared to show antioxidant activity. The Hunter color values of pork patties were affected by the addition of annatto seed powder, which increased the redness and yellowness values, but decreased the lightness of the patties (p<0.05). To evaluate the antioxidative effects of annatto on pork patties, thiobarbituric acid reactive substances (TBARS) and peroxide values (POV) were analyzed over 14 d of refrigerated storage. Treatments containing annatto seed showed lower TBARS and POV than control (CTL) samples (p<0.05). The volatile basic nitrogen (VBN) of the pork patties containing annatto seeds were lower than that of CTL at the end of storage (p<0.05). Although no differences in total bacterial counts were observed between control and treated patties, those containing annatto seeds had lower microbial counts for Enterobacteriacease than CTL or AA 0.1%. Therefore, annatto seed powder might be a good source of natural antioxidants for the production of meat products. PMID:27621688

Plasmodium parasite as an effective hepatocellular carcinoma antigen glypican-3 delivery vector.

PubMed

Liu, Quan; Yang, Yijun; Tan, Xuefang; Tao, Zhu; Adah, Dickson; Yu, Songlin; Lu, Junnan; Zhao, Siting; Qin, Limei; Qin, Li; Chen, Xiaoping

2017-04-11

We have previously demonstrated that malaria parasite infection has an anti-tumor effect in a mouse model. This research aimed to investigate the possibility of using Plasmodium parasite as a novel vaccine vector for hepatocellular carcinoma (HCC) immunotherapy. We constructed a Plasmodium yoelii 17XNL strain (P.y) expressing murine glypican-3 (GPC3) protein (P.y-GPC3), and examined its therapeutic potency in a murine Hepa1-6-induced hepatoma model that highly expressed GPC3 protein. The prerequisites for invoking a CD8+ T cell response were assessed after P.y-based immunization, which included obviously increased concentrations of T helper cell type 1 (Th1)-associated cytokines, such as IL-2, IFN-γ and TNF-α, in serum and preferential expansion of the CD8α+ dendritic cell (DC) subset with higher expression of CD80 and CD86 molecules. Compared with uninfected and wild-type P.y-infected mice, a significant GPC3-specific cytotoxic T lymphocyte (CTL) response was detected in P.y-GPC3 vaccinated mice. Furthermore, P.y-GPC3-based vaccination dramatically inhibited Hepa1-6-induced tumor growth in the implanted HCC and prolonged the survival of tumor-bearing mice. We concluded that a Plasmodium-based vector is highly efficient in inducing tumor antigen-specific T cell-mediated immunity and protection against tumor cells. More broadly, this strategy supported our hypothesis that Plasmodium parasites, as novel therapeutic antigen vectors, may be applicable to tumor immunotherapy for patients with HCC.

Investigating Forest Soil Disturbance with Different Timber Harvesting Operations in South Korea

NASA Astrophysics Data System (ADS)

Im, Sangjun; Lee, Eunjai; Eu, Song; Han, Sang-Kyun

2017-04-01

Forest operation such as timber harvesting can influence to forest environment by displacing soil particles, compacting surface layers, and destroying soil structures. This results in increased surface runoff and associated soil erosion during rainy season, due to soil disturbance. The extent of soil disturbance depends on the skidding/yarding method, types of machine used, and soil types. In South Korea, cut-to-length (CTL) operation is traditionally used by excavator with grapple in most areas. Recently, whole-tree (WT) harvesting system by swing yarder has gained considerable attention as an alternative traditional extraction method. The objectives of this study were to describe the effects of two different harvesting methods (CTL and WT) on soil disturbance and soil physical properties. After the CTL observation, we found that severe disturbed soils and compacted area were more than WT. Rutting was influenced more than 50% of the deep disturbance classes by the uphill climbing and downhill extraction method, while exposing bare soil was most disturbance in WT operation. Soil physical properties were influenced considerably by the number of excavator passes and slash residual classes in both units. The results from the study would be useful for understanding soil disturbance influence by timber harvesting in Korea. But, more detailed observations are needed to accurately estimate erosion rates and sediment delivery associated with forest management and operation. Acknowledgements. This study was carried out with the support of 'R&D Program for Forestry Technology (Project No. S211316L020110)' provided by Korea Forest Service.

Interleukin-7 in Patients With Chronic Hepatitis B May Have Effect on T Follicular Helper Cells and Specific Cellular Immunity.

PubMed

Zhong, Hua; Xibing, Gu; Yaping, Dai; Zheng, Wang; Decai, Fu; Xiaoye, Guo; Hangyuan, Wu; Dong, Wang; Zhonghua, Lu

2016-09-01

In patients with chronic hepatitis B (CHB), the relation of interkeukin-7 (IL-7) to either the T follicular helper cells (Tfh cells) or to a specific cellular immune response is not clear. The present study aims to explore the possible relationship of IL-7 to Tfh cells and to hepatitis B virus (HBV)-specific cellular immune response in patients with CHB. Ninety-one adult patients with CHB were divided into groups A, B, and C, according to the patients' IL-7 levels (low, medium, and high). Tfh cells and HBV-specific cytotoxic T lymphocytes (CTLs) were detected with flow cytometry; IL-7 and IL-21 were determined with a double antibody sandwich enzyme-linked immunosorbent assay; and HBV DNA was determined by using a real-time fluorescent quantitative polymerase chain reaction. The results showed that the levels of IL-7, Tfh cells, IL-21, and HBV-specific CTLs of patients in group C were significantly higher than those of patients in group B, (P < 0.01 for each comparison) and that the levels of these four parameters of patients in group B were significantly higher than those of the patients in group A (P < 0.01 for each comparison). Meanwhile, the level of HBV DNA of the patients in group C was significantly lower than that of the patients in group B (P < 0.01), and that of the patients in group B was significantly lower than that of the patients in group A (P < 0.05). Multiple linear regression analyses showed that IL-7, Tfh cells, IL-21, and HBV-specific CTL might have effects on HBV DNA and that only the HBV-specific CTL had an independent effect on HBV DNA (P < 0.01). IL-7, Tfh cells, and IL-21 showed independent effects on HBV-specific CTL (P < 0.05, P < 0.01, and P < 0.01). This study suggests that the IL-7 level of CHB patients may be related to Tfh cells. In CHB patients, IL-7 possibly increases the level of Tfh cells and HBV-specific cellular immune responses and thereby reduces the HBV DNA level.

Chimeric Antigen Receptor–Modified T Cells for Acute Lymphoid Leukemia

PubMed Central

Barrett, David; Aplenc, Richard; Porter, David L.; Rheingold, Susan R.; Teachey, David T.; Chew, Anne; Hauck, Bernd; Wright, J. Fraser; Milone, Michael C.; Levine, Bruce L.; June, Carl H.

2014-01-01

Summary Chimeric antigen receptor–modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre–B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×106 to 1.2×107 CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce anti-leukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor–modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL. PMID:23527958

Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.

PubMed

Grupp, Stephan A; Kalos, Michael; Barrett, David; Aplenc, Richard; Porter, David L; Rheingold, Susan R; Teachey, David T; Chew, Anne; Hauck, Bernd; Wright, J Fraser; Milone, Michael C; Levine, Bruce L; June, Carl H

2013-04-18

Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.

Induction of protective anti-CTL epitope responses against HER-2-positive breast cancer based on multivalent T7 phage nanoparticles.

PubMed

Pouyanfard, Somayeh; Bamdad, Taravat; Hashemi, Hamidreza; Bandehpour, Mojgan; Kazemi, Bahram

2012-01-01

We report here the development of multivalent T7 bacteriophage nanoparticles displaying an immunodominant H-2k(d)-restricted CTL epitope derived from the rat HER2/neu oncoprotein. The immunotherapeutic potential of the chimeric T7 nanoparticles as anti-cancer vaccine was investigated in BALB/c mice in an implantable breast tumor model. The results showed that T7 phage nanoparticles confer a high immunogenicity to the HER-2-derived minimal CTL epitope, as shown by inducing robust CTL responses. Furthermore, the chimeric nanoparticles protected mice against HER-2-positive tumor challenge in both prophylactic and therapeutic setting. In conclusion, these results suggest that CTL epitope-carrying T7 phage nanoparticles might be a promising approach for development of T cell epitope-based cancer vaccines.

Induction of Protective Anti-CTL Epitope Responses against HER-2-Positive Breast Cancer Based on Multivalent T7 Phage Nanoparticles

PubMed Central

Pouyanfard, Somayeh; Bamdad, Taravat; Hashemi, Hamidreza; Bandehpour, Mojgan; Kazemi, Bahram

2012-01-01

We report here the development of multivalent T7 bacteriophage nanoparticles displaying an immunodominant H-2kd-restricted CTL epitope derived from the rat HER2/neu oncoprotein. The immunotherapeutic potential of the chimeric T7 nanoparticles as anti-cancer vaccine was investigated in BALB/c mice in an implantable breast tumor model. The results showed that T7 phage nanoparticles confer a high immunogenicity to the HER-2-derived minimal CTL epitope, as shown by inducing robust CTL responses. Furthermore, the chimeric nanoparticles protected mice against HER-2-positive tumor challenge in both prophylactic and therapeutic setting. In conclusion, these results suggest that CTL epitope-carrying T7 phage nanoparticles might be a promising approach for development of T cell epitope-based cancer vaccines. PMID:23166703

Pathology during acute infections: contributions of intracellular pathogens and the CTL response.

PubMed

Ganusov, Vitaly V; Antia, Rustom

2005-06-22

Previous work has shown how, in the case of cytotoxic T-lymphocyte (CTL) responses to persistent viral infections, pathology may arise as a consequence of cell destruction directly by the virus or indirectly due to the CTL response, leading to maximum pathology at intermediate efficacy of the immune response. We expand these studies to consider pathology arising during acute infections with intracellular pathogens controlled by the CTL response. We show that, in contrast to persistent infections, pathology during acute infections is minimized with increasing efficacy of the immune response. The implications of these results for vaccination are discussed.

Choline transport via choline transporter-like protein 1 in conditionally immortalized rat syncytiotrophoblast cell lines TR-TBT.

PubMed

Lee, N-Y; Choi, H-M; Kang, Y-S

2009-04-01

Choline is an essential nutrient for phospholipids and acetylcholine biosynthesis in normal development of fetus. In the present study, we investigated the functional characteristics of choline transport system and inhibitory effect of cationic drugs on choline transport in rat conditionally immortalized syncytiotrophoblast cell line (TR-TBT). Choline transport was weakly Na(+) dependent and significantly influenced by extracellular pH and by membrane depolarization. The transport process of choline is saturable with Michaelis-Menten constants (K(m)) of 68microM and 130microM in TR-TBT 18d-1 and TR-TBT 18d-2 respectively. Choline uptake in the cells was inhibited by unlabeled choline and hemicholinium-3 as well as various organic cations including guanidine, amiloride and acetylcholine. However, the prototypical organic cation tetraethylammonium and cimetidine showed very little inhibitory effect of choline uptake in TR-TBT cells. RT-PCR revealed that choline transporter-like protein 1 (CTL1) and organic cation transporter 2 (OCT2) are expressed in TR-TBT cells. The transport properties of choline in TR-TBT cells were similar or identical to that of CTL1 but not OCT2. CTL1 was also detected in human placenta. In addition, several cationic drugs such as diphenhydramine and verapamil competitively inhibited choline uptake in TR-TBT 18d-1 with K(i) of 115microM and 55microM, respectively. Our results suggest that choline transport system, which has intermediate affinity and weakly Na(+) dependent, in TR-TBT seems to occur through a CTL1 and this system may have relevance with the uptake of pharmacologically important organic cation drugs.

Effects of Mutations on Replicative Fitness and Major Histocompatibility Complex Class I Binding Affinity Are Among the Determinants Underlying Cytotoxic-T-Lymphocyte Escape of HIV-1 Gag Epitopes.

PubMed

Du, Yushen; Zhang, Tian-Hao; Dai, Lei; Zheng, Xiaojuan; Gorin, Aleksandr M; Oishi, John; Wu, Ting-Ting; Yoshizawa, Janice M; Li, Xinmin; Yang, Otto O; Martinez-Maza, Otoniel; Detels, Roger; Sun, Ren

2017-11-28

Certain "protective" major histocompatibility complex class I (MHC-I) alleles, such as B*57 and B*27, are associated with long-term control of HIV-1 in vivo mediated by the CD8 + cytotoxic-T-lymphocyte (CTL) response. However, the mechanism of such superior protection is not fully understood. Here we combined high-throughput fitness profiling of mutations in HIV-1 Gag, in silico prediction of MHC-peptide binding affinity, and analysis of intraperson virus evolution to systematically compare differences with respect to CTL escape mutations between epitopes targeted by protective MHC-I alleles and those targeted by nonprotective MHC-I alleles. We observed that the effects of mutations on both viral replication and MHC-I binding affinity are among the determinants of CTL escape. Mutations in Gag epitopes presented by protective MHC-I alleles are associated with significantly higher fitness cost and lower reductions in binding affinity with respect to MHC-I. A linear regression model accounting for the effect of mutations on both viral replicative capacity and MHC-I binding can explain the protective efficacy of MHC-I alleles. Finally, we found a consistent pattern in the evolution of Gag epitopes in long-term nonprogressors versus progressors. Overall, our results suggest that certain protective MHC-I alleles allow superior control of HIV-1 by targeting epitopes where mutations typically incur high fitness costs and small reductions in MHC-I binding affinity. IMPORTANCE Understanding the mechanism of viral control achieved in long-term nonprogressors with protective HLA alleles provides insights for developing functional cure of HIV infection. Through the characterization of CTL escape mutations in infected persons, previous researchers hypothesized that protective alleles target epitopes where escape mutations significantly reduce viral replicative capacity. However, these studies were usually limited to a few mutations observed in vivo Here we utilized our recently developed high-throughput fitness profiling method to quantitatively measure the fitness of mutations across the entirety of HIV-1 Gag. The data enabled us to integrate the results with in silico prediction of MHC-peptide binding affinity and analysis of intraperson virus evolution to systematically determine the differences in CTL escape mutations between epitopes targeted by protective HLA alleles and those targeted by nonprotective HLA alleles. We observed that the effects of Gag epitope mutations on HIV replicative fitness and MHC-I binding affinity are among the major determinants of CTL escape. Copyright © 2017 Du et al.

Specific depletion of Ly6C(hi) inflammatory monocytes prevents immunopathology in experimental cerebral malaria.

PubMed

Schumak, Beatrix; Klocke, Katrin; Kuepper, Janina M; Biswas, Aindrila; Djie-Maletz, Andrea; Limmer, Andreas; van Rooijen, Nico; Mack, Matthias; Hoerauf, Achim; Dunay, Ildiko Rita

2015-01-01

Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice leads to experimental cerebral malaria (ECM) that is commonly associated with serious T cell mediated damage. In other parasitic infection models, inflammatory monocytes have been shown to regulate Th1 responses but their role in ECM remains poorly defined, whereas neutrophils are reported to contribute to ECM immune pathology. Making use of the recent development of specific monoclonal antibodies (mAb), we depleted in vivo Ly6C(hi) inflammatory monocytes (by anti-CCR2), Ly6G+ neutrophils (by anti-Ly6G) or both cell types (by anti-Gr1) during infection with Ovalbumin-transgenic PbA parasites (PbTg). Notably, the application of anti-Gr1 or anti-CCR2 but not anti-Ly6G antibodies into PbTg-infected mice prevented ECM development. In addition, depletion of Ly6C(hi) inflammatory monocytes but not neutrophils led to decreased IFNγ levels and IFNγ+CD8+ T effector cells in the brain. Importantly, anti-CCR2 mAb injection did not prevent the generation of PbTg-specific T cell responses in the periphery, whereas anti-Gr1 mAb injection strongly diminished T cell frequencies and CTL responses. In conclusion, the specific depletion of Ly6C(hi) inflammatory monocytes attenuated brain inflammation and immune cell recruitment to the CNS, which prevented ECM following Plasmodium infection, pointing out a substantial role of Ly6C+ monocytes in ECM inflammatory processes.

Specific Depletion of Ly6Chi Inflammatory Monocytes Prevents Immunopathology in Experimental Cerebral Malaria

PubMed Central

Kuepper, Janina M.; Biswas, Aindrila; Djie-Maletz, Andrea; Limmer, Andreas; van Rooijen, Nico; Mack, Matthias; Hoerauf, Achim; Dunay, Ildiko Rita

2015-01-01

Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice leads to experimental cerebral malaria (ECM) that is commonly associated with serious T cell mediated damage. In other parasitic infection models, inflammatory monocytes have been shown to regulate Th1 responses but their role in ECM remains poorly defined, whereas neutrophils are reported to contribute to ECM immune pathology. Making use of the recent development of specific monoclonal antibodies (mAb), we depleted in vivo Ly6Chi inflammatory monocytes (by anti-CCR2), Ly6G+ neutrophils (by anti-Ly6G) or both cell types (by anti-Gr1) during infection with Ovalbumin-transgenic PbA parasites (PbTg). Notably, the application of anti-Gr1 or anti-CCR2 but not anti-Ly6G antibodies into PbTg-infected mice prevented ECM development. In addition, depletion of Ly6Chi inflammatory monocytes but not neutrophils led to decreased IFNγ levels and IFNγ+CD8+ T effector cells in the brain. Importantly, anti-CCR2 mAb injection did not prevent the generation of PbTg-specific T cell responses in the periphery, whereas anti-Gr1 mAb injection strongly diminished T cell frequencies and CTL responses. In conclusion, the specific depletion of Ly6Chi inflammatory monocytes attenuated brain inflammation and immune cell recruitment to the CNS, which prevented ECM following Plasmodium infection, pointing out a substantial role of Ly6C+ monocytes in ECM inflammatory processes. PMID:25884830

Nicotine Inhibits Memory CTL Programming

PubMed Central

Sun, Zhifeng; Smyth, Kendra; Garcia, Karla; Mattson, Elliot; Li, Lei; Xiao, Zhengguo

2013-01-01

Nicotine is the main tobacco component responsible for tobacco addiction and is used extensively in smoking and smoking cessation therapies. However, little is known about its effects on the immune system. We confirmed that multiple nicotinic receptors are expressed on mouse and human cytotoxic T lymphocytes (CTLs) and demonstrated that nicotinic receptors on mouse CTLs are regulated during activation. Acute nicotine presence during activation increases primary CTL expansion in vitro, but impairs in vivo expansion after transfer and subsequent memory CTL differentiation, which reduces protection against subsequent pathogen challenges. Furthermore, nicotine abolishes the regulatory effect of rapamycin on memory CTL programming, which can be attributed to the fact that rapamycin enhances expression of nicotinic receptors. Interestingly, naïve CTLs from chronic nicotine-treated mice have normal memory programming, which is impaired by nicotine during activation in vitro. In conclusion, simultaneous exposure to nicotine and antigen during CTL activation negatively affects memory development. PMID:23844169

Novel mucosal DNA-MVA HIV vaccination in which DNA-IL-12 plus cholera toxin B subunit (CTB) cooperates to enhance cellular systemic and mucosal genital tract immunity.

PubMed

Maeto, Cynthia; Rodríguez, Ana María; Holgado, María Pía; Falivene, Juliana; Gherardi, María Magdalena

2014-01-01

Induction of local antiviral immune responses at the mucosal portal surfaces where HIV-1 and other viral pathogens are usually first encountered remains a primary goal for most vaccines against mucosally acquired viral infections. Exploring mucosal immunization regimes in order to find optimal vector combinations and also appropriate mucosal adjuvants in the HIV vaccine development is decisive. In this study we analyzed the interaction of DNA-IL-12 and cholera toxin B subunit (CTB) after their mucosal administration in DNA prime/MVA boost intranasal regimes, defining the cooperation of both adjuvants to enhance immune responses against the HIV-1 Env antigen. Our results demonstrated that nasal mucosal DNA/MVA immunization schemes can be effectively improved by the co-delivery of DNA-IL-12 plus CTB inducing elevated HIV-specific CD8 responses in spleen and more importantly in genital tract and genito-rectal draining lymph nodes. Remarkably, these CTL responses were of superior quality showing higher avidity, polyfunctionality and a broader cytokine profile. After IL-12+CTB co-delivery, the cellular responses induced showed an enhanced breadth recognizing with higher efficiency Env peptides from different subtypes. Even more, an in vivo CTL cytolytic assay demonstrated the higher specific CD8 T-cell performance after the IL-12+CTB immunization showing in an indirect manner its potential protective capacity. Improvements observed were maintained during the memory phase where we found higher proportions of specific central memory and T memory stem-like cells T-cell subpopulations. Together, our data show that DNA-IL-12 plus CTB can be effectively employed acting as mucosal adjuvants during DNA prime/MVA boost intranasal vaccinations, enhancing magnitude and quality of HIV-specific systemic and mucosal immune responses.

Protective Role of Cytotoxic T Lymphocytes in Filovirus Hemorrhagic Fever

PubMed Central

Warfield, Kelly Lyn; Olinger, Gene Garrard

2011-01-01

Infection with many emerging viruses, such as the hemorrhagic fever disease caused by the filoviruses, Marburg (MARV), and Ebola virus (EBOV), leaves the host with a short timeframe in which to mouse a protective immune response. In lethal cases, uncontrolled viral replication and virus-induced immune dysregulation are too severe to overcome, and mortality is generally associated with a lack of notable immune responses. Vaccination studies in animals have demonstrated an association of IgG and neutralizing antibody responses against the protective glycoprotein antigen with survival from lethal challenge. More recently, studies in animal models of filovirus hemorrhagic fever have established that induction of a strong filovirus-specific cytotoxic T lymphocyte (CTL) response can facilitate complete viral clearance. In this review, we describe assays used to discover CTL responses after vaccination or live filovirus infection in both animal models and human clinical trials. Unfortunately, little data regarding CTL responses have been collected from infected human survivors, primarily due to the low frequency of disease and the inability to perform these studies in the field. Advancements in assays and technologies may allow these studies to occur during future outbreaks. PMID:22253531

Cytotoxic T lymphocytes and CD4 epitope mutations in the pre-core/core region of hepatitis B virus in chronic hepatitis B carriers in Northeast Iran.

PubMed

Zhand, Sareh; Tabarraei, Alijan; Nazari, Amineh; Moradi, Abdolvahab

2017-07-01

Hepatitis B virus (HBV) is vulnerable to many various mutations. Those within epitopes recognized by sensitized T cells may influence the re-emergence of the virus. This study was designed to investigate the mutation in immune epitope regions of HBV pre-core/core among chronic HBV patients of Golestan province, Northeast Iran. In 120 chronic HBV carriers, HBV DNA was extracted from blood plasma samples and PCR was done using specific primers. Direct sequencing and alignment of the pre-core/core region were applied using reference sequence from Gene Bank database (Accession Number AB033559). The study showed 27 inferred amino acid substitutions, 9 of which (33.3%) were in CD4 and 2 (7.4%) in cytotoxic T lymphocytes' (CTL) epitopes and 16 other mutations (59.2%) were observed in other regions. CTL escape mutations were not commonly observed in pre-core/core sequences of chronic HBV carriers in the locale of study. It can be concluded that most of the inferred amino acid substitutions occur in different immune epitopes other than CTL and CD4.

You Are the Danger: Attenuated Insula Response in Methamphetamine Users During Aversive Interoceptive Decision-Making*

PubMed Central

Stewart, Jennifer L.; May, April C.; Poppa, Tasha; Davenport, Paul W.; Tapert, Susan F.; Paulus, Martin P.

2014-01-01

Background Drug dependent individuals often make drug-taking decisions when they do not feel well. Yet, few studies have examined the influence of an aversive state on decision-making related neural processing. Methods We investigate brain activation to decision-making during an aversive interoceptive challenge in methamphetamine users using functional magnetic resonance imaging (fMRI). Recently abstinent inpatients with methamphetamine use disorder (METH; n=20) and healthy comparison subjects (CTL; n=22) performed a two-choice prediction task at three fixed error rates (ER; 20%=reward, 50%=uncertainty, 80%=punishment) while anticipating and experiencing episodes of inspiratory breathing load during fMRI. Results METH exhibited higher trait anxiety in conjunction with lower anterior insula (AI) and inferior frontal gyrus (IFG) activation than CTL across trials. METH also showed lower posterior insula (PI) and anterior cingulate cortex (ACC) activation than CTL during breathing load independent of ER. For the crucial ER by interoception interaction, METH displayed lower ACC activation to punishment/loss than CTL during breathing load. Within METH, lower trait anxiety was linked to bilateral AI/IFG attenuation across trials. Conclusions AI/IFG attenuations in METH are suggestive of an executive functioning deficit, particularly in users with low anxiety, reflecting reduced resources allocated to choice selection. In contrast, PI/ACC reductions in METH appear specific to impairments in registering and evaluating interoceptive experiences. Taken together, inadequate activation of brain areas that are important for regulating when one does not feel well may be the neural basis for poor decision-making by METH. PMID:24993186

Stem-like tumor-initiating cells isolated from IL13Rα2 expressing gliomas are targeted and killed by IL13-zetakine-redirected T Cells.

PubMed

Brown, Christine E; Starr, Renate; Aguilar, Brenda; Shami, Andrew F; Martinez, Catalina; D'Apuzzo, Massimo; Barish, Michael E; Forman, Stephen J; Jensen, Michael C

2012-04-15

To evaluate IL13Rα2 as an immunotherapeutic target for eliminating glioma stem-like cancer initiating cells (GSC) of high-grade gliomas, with particular focus on the potential of genetically engineered IL13Rα2-specific primary human CD8(+) CTLs (IL13-zetakine(+) CTL) to target this therapeutically resistant glioma subpopulation. A panel of low-passage GSC tumor sphere (TS) and serum-differentiated glioma lines were expanded from patient glioblastoma specimens. These glioblastoma lines were evaluated for expression of IL13Rα2 and for susceptibility to IL13-zetakine(+) CTL-mediated killing in vitro and in vivo. We observed that although glioma IL13Rα2 expression varies between patients, for IL13Rα2(pos) cases this antigen was detected on both GSCs and more differentiated tumor cell populations. IL13-zetakine(+) CTL were capable of efficient recognition and killing of both IL13Rα2(pos) GSCs and IL13Rα2(pos) differentiated cells in vitro, as well as eliminating glioma-initiating activity in an orthotopic mouse tumor model. Furthermore, intracranial administration of IL13-zetakine(+) CTL displayed robust antitumor activity against established IL13Rα2(pos) GSC TS-initiated orthotopic tumors in mice. Within IL13Rα2 expressing high-grade gliomas, this receptor is expressed by GSCs and differentiated tumor populations, rendering both targetable by IL13-zetakine(+) CTLs. Thus, our results support the potential usefullness of IL13Rα2-directed immunotherapeutic approaches for eradicating therapeutically resistant GSC populations. ©2012 AACR.

Kinetics of HIV-1 CTL epitopes recognized by HLA I alleles in HIV-infected individuals at times near primary infection: the Provir/Latitude45 study.

PubMed

Papuchon, Jennifer; Pinson, Patricia; Guidicelli, Gwenda-Line; Bellecave, Pantxika; Thomas, Réjean; LeBlanc, Roger; Reigadas, Sandrine; Taupin, Jean-Luc; Baril, Jean Guy; Routy, Jean Pierre; Wainberg, Mark; Fleury, Hervé

2014-01-01

In patients responding successfully to ART, the next therapeutic step is viral cure. An interesting strategy is antiviral vaccination, particularly involving CD8 T cell epitopes. However, attempts at vaccination are dependent on the immunogenetic background of individuals. The Provir/Latitude 45 project aims to investigate which CTL epitopes in proviral HIV-1 will be recognized by the immune system when HLA alleles are taken into consideration. A prior study (Papuchon et al, PLoS ONE 2013) showed that chronically-infected patients under successful ART exhibited variations of proviral CTL epitopes compared to a reference viral strain (HXB2) and that a generic vaccine may not be efficient. Here, we investigated viral and/or proviral CTL epitopes at different time points in recently infected individuals of the Canadian primary HIV infection cohort and assessed the affinity of these epitopes for HLA alleles during the study period. An analysis of the results confirms that it is not possible to fully predict which epitopes will be recognized by the HLA alleles of the patients if the reference sequences and epitopes are taken as the basis of simulation. Epitopes may be seen to vary in circulating RNA and proviral DNA. Despite this confirmation, the overall variability of the epitopes was low in these patients who are temporally close to primary infection.

Kinetics of HIV-1 CTL Epitopes Recognized by HLA I Alleles in HIV-Infected Individuals at Times near Primary Infection: The Provir/Latitude45 Study

PubMed Central

Papuchon, Jennifer; Pinson, Patricia; Guidicelli, Gwenda-Line; Bellecave, Pantxika; Thomas, Réjean; LeBlanc, Roger; Reigadas, Sandrine; Taupin, Jean-Luc; Baril, Jean Guy; Routy, Jean Pierre; Wainberg, Mark; Fleury, Hervé

2014-01-01

In patients responding successfully to ART, the next therapeutic step is viral cure. An interesting strategy is antiviral vaccination, particularly involving CD8 T cell epitopes. However, attempts at vaccination are dependent on the immunogenetic background of individuals. The Provir/Latitude 45 project aims to investigate which CTL epitopes in proviral HIV-1 will be recognized by the immune system when HLA alleles are taken into consideration. A prior study (Papuchon et al, PLoS ONE 2013) showed that chronically-infected patients under successful ART exhibited variations of proviral CTL epitopes compared to a reference viral strain (HXB2) and that a generic vaccine may not be efficient. Here, we investigated viral and/or proviral CTL epitopes at different time points in recently infected individuals of the Canadian primary HIV infection cohort and assessed the affinity of these epitopes for HLA alleles during the study period. An analysis of the results confirms that it is not possible to fully predict which epitopes will be recognized by the HLA alleles of the patients if the reference sequences and epitopes are taken as the basis of simulation. Epitopes may be seen to vary in circulating RNA and proviral DNA. Despite this confirmation, the overall variability of the epitopes was low in these patients who are temporally close to primary infection. PMID:24964202

Inhibition of serine-peptidase activity enhances the generation of a survivin-derived HLA-A2-presented CTL epitope in colon-carcinoma cells.

PubMed

Preta, G; Marescotti, D; Fortini, C; Carcoforo, P; Castelli, C; Masucci, M; Gavioli, R

2008-12-01

Cytotoxic T lymphocytes eliminate tumor cells expressing antigenic peptides in the context of MHC-I molecules. Peptides are generated during protein degradation by the proteasome and resulting products, surviving cytosolic amino-peptidases activity, may be presented by MHC-I molecules. The MHC-I processing pathway is altered in a large number of malignancies and modulation of antigen generation is one strategy employed by cells to evade immune control. In this study we analyzed the generation and presentation of a survivin-derived CTL epitope in HLA-A2-positive colon-carcinoma cells. Although all cell lines expressed the anti-apoptotic protein survivin, some tumors were poorly recognized by ELTLGEFLKL (ELT)-specific CTL cultures. The expression of MHC-I or TAP molecules was similar in all cell lines suggesting that tumors not recognized by CTLs may present defects in the generation of the ELT-epitope which could be due either to lack of generation or to subsequent degradation of the epitope. The cells were analyzed for the expression and the activity of extra-proteasomal peptidases. A significant overexpression and higher activity of TPPII was observed in colon-carcinoma cells which are not killed by ELT-specific CTLs, suggesting a possible role of TPPII in the degradation of the ELT-epitope. To confirm the role of TPPII in the degradation of the ELT-peptide, we showed that treatment of colon-carcinoma cells with a TPPII inhibitor resulted in a dose-dependent increased sensitivity to ELT-specific CTLs. These results suggest that TPPII is involved in degradation of the ELT-peptide, and its overexpression may contribute to the immune escape of colon-carcinoma cells.

Engaging pre-service teachers to teach science contextually with scientific approach instructional video

NASA Astrophysics Data System (ADS)

Susantini, E.; Kurniasari, I.; Fauziah, A. N. M.; Prastowo, T.; Kholiq, A.; Rosdiana, L.

2018-01-01

Contextual teaching and learning (CTL) present new concepts in real experiences and situations, where students can find out the meaningful relationship between abstract ideas and practical applications. Implementation of CTL using scientific approach fosters teachers to find constructive ways of delivering and organizing science contents in science classroom settings. An instructional video for modelling by using a scientific approach in CTL was then developed. Questionnaires with open-ended questions were used to, asking whether modelling through instructional video could help them to teach science contextually with a scientific approach or not. Data for pre-service teachers’ views were analyzed descriptively. The aims of this research are to engage pre-service teachers in learning how to teach CTL and to show how their responses to learning and how to teach CTL using the video. The study showed that ten pre-service teachers in science department were involved, all observed through videos that demonstrated a combined material of CTL and scientific approach and completed worksheets to analyze the video contents. The results show that pre-service teachers could learn to teach contextual teaching and make use of scientific approach in science classroom settings with the help of model in the video.

High conservation level of CD8(+) T cell immunogenic regions within an unusual H1N2 human influenza variant.

PubMed

Komadina, Naomi; Quiñones-Parra, Sergio M; Kedzierska, Katherine; McCaw, James M; Kelso, Anne; Leder, Karin; McVernon, Jodie

2016-10-01

Current seasonal influenza vaccines require regular updates due to antigenic drift causing loss of effectiveness and therefore providing little or no protection against novel influenza A subtypes. Next generation vaccines capable of eliciting CD8(+) T cell (CTL) mediated cross-protective immunity may offer a long-term alternative strategy. However, measuring pre- and existing levels of CTL cross-protection in humans is confounded by differences in infection histories across individuals. During 2000-2003, H1N2 viruses circulated persistently in the human population for the first time and we hypothesized that the viral nucleoprotein (NP) contained novel CTL epitopes that may have contributed to the survival of the viruses. This study describes the immunogenic NP peptides of H1N1, H2N2, and H3N2 influenza viruses isolated from humans over the past century, 1918-2003, by comparing this historical dataset to reference NP peptides from H1N2 that circulated in humans during 2000-2003. Observed peptides sequences ranged from highly conserved (15%) to highly variable (12%), with variation unrelated to reported immunodominance. No unique NP peptides which were exclusive to the H1N2 viruses were noted. However, the virus had inherited the NP from a recently emerged H3N2 variant containing novel peptides, which may have assisted its persistence. Any advantage due to this novelty was subsequently lost with emergence of a newer H3N2 variant in 2003. Our approach has potential to provide insight into the population context in which influenza viruses emerge, and may help to inform immunogenic peptide selection for CTL-inducing influenza vaccines. J. Med. Virol. 88:1725-1732, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Advanced glycation end product (AGE) modified proteins in tears of diabetic patients.

PubMed

Zhao, Zhenjun; Liu, Jingfang; Shi, Bingyin; He, Shuixiang; Yao, Xiaoli; Willcox, Mark D P

2010-08-11

High glucose level in diabetic patients may lead to advanced glycation end product (AGE) modified proteins. This study investigated AGE modified proteins in tears and compared their levels in diabetic patients (DM) with non-diabetic controls (CTL). Basal tears were collected from DM with (DR) or without (DNR) retinopathy and CTL. Total AGE modified proteins were detected quantitatively by a dot immunobinding assay. The AGE modified proteins were separated in 1D- and 2D-SDS gels and detected by western-blotting. The individual AGE modified proteins were also compared between groups using densitometry. Compared with the CTL group, tear concentrations of AGE modified proteins were significantly elevated in DR and DNR groups. The concentration of AGE modified proteins in diabetic tears were positively correlated with AGE modified hemoglobin (HbA1c) and postprandial blood glucose level (PBG). Western blotting of AGE modified proteins from 1D-SDS gels showed several bands, the major one at around 60 kDa. The intensities of AGE modified protein bands were higher in DM tears than in CTL tears. Western blotting from 2D-SDS gels showed a strongly stained horizontal strip, which corresponded to the major band in 1D-SDS gels. Most of the other AGE modified protein species were within molecular weight of 30-60 kDa, PI 5.2-7.0. Densitometry analysis demonstrated several AGE modified proteins were elevated in DR or DNR tears. Total and some individual AGE modified proteins were elevated in DM tears. AGE modified proteins in tears may be used as biomarkers to diagnose diabetes and/or diabetic retinopathy.

Antitumor and immunomodulatory effects of weikangfu granule compound in tumor-bearing mice

PubMed Central

Nie, Xiaohua; Shi, Baojun; Ding, Yuting; Tao, Wenyi

2006-01-01

Background: Weikangfu granule compound (WKC) is a drug preparation based on a clinical prescription drug, Weikangfu-tang, which has been found to have therapeutic effects on gastric cancer. WKC comprises 7 components, including polysaccharides, saponin, flavonoids, and essential oil. Objective: The purpose of this study was to assess the antitumor and immunomodulatory effects of WKC in a tumor-bearing rodent model. Methods: Male and female Kuming mice weighing ∼20 g were subcutaneously implanted with sarcoma 180 (S180) tumor cells and randomly assigned to 1 of 5 treatment groups: oral WKC 175, 350, or 525 mg/kg·d, isotonic saline (negative control), or intraperitoneal cyclophosphamide 25 mg/kg·d (positive control). All treatments were administered daily for 10 days. After euthanization on day 11, the mice, tumors, and spleens were weighed. Lymphocyte proliferation and cytotoxic T lymphocyte (CTL) activity were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cellular viability assay method. Macrophage phagocytosis was identified using a yeast test. Results: Fifty mice were included in the study (10 mice were assigned to each group). The tumors of the mice administered WKC 175, 350, and 525 mg/kg·d were significantly regressed, as determined using MICs, compared with those in the negative-control group (P<0.05, P<0.01, and P<0.01, respectively), and the inhibitory rates were 30.43%, 46.72%, and 54.35%, respectively. Compared with those in the negative-control group, CTL activities and lymphocyte proliferations in the presence of concanavalin A were significantly greater in the WKC-treated groups at all doses (CTL activities: P<0.05, P<0.01, and P<0.01, respectively; lymphocyte proliferations: P<0.05, P<0.01, and P<0.01, respectively). In the groups receiving WKC 175, 350, and 525 mg/kg·d, the phagocytic rates were 1.5- to 2.0-fold those in the negative-control group (P<0.05, P<0.01, and P<0.01, respectively). In the groups receiving WKC 175, 350, and 525 mg/kg·d, the phagocytic indexes were 3.7- to 5.0-fold those in the negative-control group (all, P<0.01). In contrast, lymphocyte proliferation in the positive-control group was significantly less compared with that in the negative-control group (P<0.01), but no significant differences were found in CTL activities or macrophage phagocytosis between these 2 groups. Conclusion: The results of this study in a rodent model suggest that WKC exhibited antitumor and immunomodulatory activities in S180-bearing mice, and that WKC improved nonspecific and specific immune functions in mice, such as lymphocyte proliferation, CTL activity, and macrophage phagocytosis. PMID:24678090

Loss of DNAM-1 contributes to CD8+ T cell exhaustion in chronic HIV-1 infection

PubMed Central

Cella, Marina; Presti, Rachel; Vermi, William; Lavender, Kerry; Turnbull, Emma; Ochsenbauer-Jambor, Christina; Kappes, John C.; Ferrari, Guido; Kessels, Lisa; Williams, Ian; McMichael, Andrew J.; Haynes, Barton F.; Borrow, Persephone; Colonna, Marco

2011-01-01

Summary The hallmark of chronic viral infections is a progressive exhaustion of antigen specific CD8+ T cells that leads to persisting viral replication. It is generally believed that exhaustion is a consequence of the accumulation of multiple inhibitory receptors on CD8+ T cells that makes them dysfunctional. Here we show that during human chronic HIV-1 infection a CD8+ T cell positive costimulatory pathway mediated by DNAM-1 is also disrupted. Thus, DNAM-1 downregulation on CD8+ T cells aggravates the impairment of CTL effector function in chronic HIV-1 infection. PMID:20201043

Serine Proteases Enhance Immunogenic Antigen Presentation on Lung Cancer Cells

PubMed Central

Peters, Haley L.; Tripathi, Satyendra C.; Kerros, Celine; Katayama, Hiroyuki; Garber, Haven R.; St. John, Lisa S.; Federico, Lorenzo; Meraz, Ismail M.; Roth, Jack A.; Sepesi, Boris; Majidi, Mourad; Ruisaard, Kathryn; Clise-Dwyer, Karen; Roszik, Jason; Gibbons, Don L.; Heymach, John V.; Swisher, Stephen G.; Bernantchez, Chantale; Alatrash, Gheath; Hanash, Samir; Molldrem, Jeffrey J.

2017-01-01

Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these re-invigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAM were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTL from healthy donors that had been expanded against select peptides. Finally, CTL specific for serine proteases–induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. PMID:28254787

“Resting” CBF in the Epileptic Baboon: Correlation with Ketamine Dose and Interictal Epileptic Discharges

PubMed Central

Szabó, C. Ákos; Narayana, Shalini; Franklin, Crystal; Knape, Koyle D.; Davis, M. Duff; Fox, Peter T.; Leland, M. Michelle; Williams, Jeff T.

2011-01-01

Background Photosensitive epileptic (SZ) baboons demonstrate different cerebral blood flow (CBF) activation patterns from asymptomatic controls (CTL) during intermittent light stimulation (ILS). This study compares “resting” CBF between PS and CTL animals, and CBF correlations with ketamine dose and interictal epileptic discharges (IEDs) between PS and CTL animals. Methods Continuous intravenous ketamine was administered to eight PS and eight CTL baboons (matched for gender and weight), and maintained at subanesthetic doses (4.8–14.6 mg/kg/hr). Three resting H215O-PET studies were attempted in each animal (CTI/Siemens HR+ scanner). Images were acquired in 3D mode (63 contiguous slices, 2.4 mm thickness). PET images were co-registered with MRI images (3T Siemens Trio, T1-weighted 3D Turboflash sequence, TE/TR/TI = 3.04/2100/785 msec, flip angle=13 degrees). EEG was used to monitor depth of sedation and for quantification of IED rates. Regional CBF was compared between PS and CTL groups and correlations were analyzed for ketamine dose and IED rates. Results When subsets of animals of either group, receiving similar doses of ketamine were compared, PS animals demonstrated relative CBF increases in the occipital lobes and decreases in the frontal lobes. Correlation analyses with ketamine dose confirmed the frontal and occipital lobe changes in the PS animals. The negative correlations of CBF with ketamine dose and IED rate overlapped frontally. While frontal lobe CBF was also negatively correlated with IED rate, positive correlations were found in the parietal lobe. Conclusions “Resting” CBF differs between PS and CTL baboons. Correlation analyses of CBF and ketamine dose reveal that occipital lobe CBF increases and frontal lobe in PS animals are driven by ketamine. While frontal lobe CBF decreases may be related to ketamine’s propensity to activate IEDs, positive CBF correlations with IED rate suggest involvement of the parietal lobes in their generation. PMID:18801644

Defective activity of Epstein-Barr virus (EBV) specific cytotoxic T lymphocytes in children with chronic active EBV infection and in their parents.

PubMed

Fujieda, M; Wakiguchi, H; Hisakawa, H; Kubota, H; Kurashige, T

1993-10-01

Antibodies of Epstein-Barr virus (EBV), EBV-specific cytotoxic T lymphocyte (EBVCTL) activity and the lymphocyte subset of CTL were examined in 13 Japanese children with chronic active EBV infection (CAEBV) and their parents (eight fathers and 10 mothers). Anti-virus-capsid antigen (VCA)-IgG antibody titers ranged from 1:640 to 1:5120 in the patients with CAEBV and from 1:40 to 1:640 in the parents. While anti-VCA-IgM antibody was detected in three patients, anti-VCA-IgA antibody in five and anti-early-antigen (EA)-IgG antibody in 11, no antibody was detected in the parents except anti-EA antibody, which was positive in the mothers of cases 5 and 13 (1:10 and 1:40). Anti-EBV-associated nuclear antigen (EBNA) antibody was < or = 1:10 in six out of 13 patients with CAEBV and in 10 out of 18 parents tested. Epstein-Barr virus activity was significantly lower (P < 0.005) both in the children with CAEBV and in their parents than in seropositive age-matched controls. Proportions of a CTL subset (CD8+ CD11- lymphocytes) in the patients with CAEBV were significantly higher (P < 0.005) than in controls, while those in the parents were at the same level as in controls. Defective EBVCTL activity and anti-EBNA-antibody responses were frequently observed both in children with CAEBV and in their parents, which may suggest that the abnormal immune response to EBV may be based on a familial disorder, though no familial involvement has been reported in Japanese children with CAEBV.

Dietary milled flaxseed and flaxseed oil improve N-3 fatty acid status and do not affect glycemic control in individuals with well-controlled type 2 diabetes.

PubMed

Taylor, Carla G; Noto, Amy D; Stringer, Danielle M; Froese, Sylvia; Malcolmson, Linda

2010-02-01

To determine the effects of dietary consumption of milled flaxseed or flaxseed oil on glycemic control, n-3 fatty acid status, anthropometrics, and adipokines in individuals with type 2 diabetes. Thirty-four participants were randomized into a parallel, controlled trial. The participants were adults with type 2 diabetes (age 52.4 +/- 1.5 years, body mass index 32.4 +/- 1.0 kg/m(2), n = 17 men and 17 women). Participants consumed a selection of bakery products containing no flax (control group [CTL], n = 9), milled flaxseed (FXS, n = 13; 32 g/d), or flaxseed oil (FXO, n = 12; 13 g/d) daily for 12 weeks. The FXS and FXO groups received equivalent amounts of alpha-linolenic acid (ALA; 7.4 g/day). The primary outcome measures were fasting plasma hemoglobin A(1c), glucose, insulin, and phospholipid fatty acid composition. The secondary outcome measures were fasting circulating leptin and adiponectin, as well as body weight, body mass index, and waist circumference. Dietary intake assessment and calculations for homeostasis model assessment for insulin resistance and quantified insulin sensitivity check were also completed. The FXS and FXO groups had increases in plasma phospholipid n-3 fatty acids (ALA, eicosapentaenoic acid [EPA], or decosapentaenoic acid [DPA], but not docosahexaenoic acid), and the FXO group had more EPA and DPA in plasma phospholipids compared to the FXS group. All groups had similar caloric intakes; however, the CTL group experienced a 4% weight gain compared to baseline (p < 0.05), while both flax groups had constant body weights during the study period. All other parameters, including glycemic control, were unchanged by dietary treatment. Milled FXS and FXO intake does not affect glycemic control in adults with well-controlled type 2 diabetes. Possible prevention of weight gain by flax consumption warrants further investigation.

Reappraisal of nodal Epstein-Barr Virus-negative cytotoxic T-cell lymphoma: identification of indolent CD5+ diseases.

PubMed

Yamashita, Daisuke; Shimada, Kazuyuki; Takata, Katsuyoshi; Miyata-Takata, Tomoko; Kohno, Kei; Satou, Akira; Sakakibara, Ayako; Nakamura, Shigeo; Asano, Naoko; Kato, Seiichi

2018-05-29

Nodal cytotoxic molecule (CM)-positive peripheral T-cell lymphoma (CTL) has recently been recognized as a clinicopathologically distinct disease. To further characterize this disease, here we compared 58 patients with Epstein-Barr virus (EBV)-negative CTL to 48 patients with EBV-positive CTL. The two groups did not differ in histopathology, T-cell receptor (TCR) expression or rearrangement incidences, or survival curves. However, patients with EBV-negative CTL less frequently showed hepatic involvement (P = 0.007), B symptoms (P = 0.020), hemophagocytosis (P = 0.024), and detectable CD4 (P = 0.002) and CD5 (P = 0.009). Univariate and multivariate analyses identified three factors that independently predicted favorable survival, onset age <60 years (P = 0.002), CD5 expression (P = 0.002), and mixed morphology (P = 0.013), TCRαβ was not an independent predictor (P = 0.30), but was strongly linked with long survivorship among patients younger than 60 years old. A prognostic model incorporating these factors worked well for prognostic delineation, independently of the International Prognostic Index (P = 0.007 vs. P = 0.082) and Prognostic Index for PTCL (P = 0.020 vs. P = 0.15). Moreover, this constellation of findings indicated two nodal indolent diseases: CD5 + TCRαβ (n = 13), and CD5 + NK-cell type lacking TCR expression or clonal TCRγ rearrangement (n = 4). The survival curves for these two groups were significantly superior to others (n = 29, P < 0.001). These diseases appear to be unique in their indolent clinical behavior, and should be managed differently from other diseases. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Novel CTL epitopes identified through a Y. pestis proteome-wide analysis in the search for vaccine candidates against plague.

PubMed

Zvi, Anat; Rotem, Shahar; Zauberman, Ayelet; Elia, Uri; Aftalion, Moshe; Bar-Haim, Erez; Mamroud, Emanuelle; Cohen, Ofer

2017-10-20

The causative agent of Plague, Yersinia pestis, is a highly virulent pathogen and a potential bioweapon. Depending on the route of infection, two prevalent occurrences of the disease are known, bubonic and pneumonic. The latter has a high fatality rate. In the absence of a licensed vaccine, intense efforts to develop a safe and efficacious vaccine have been conducted, and humoral-driven subunit vaccines containing the F1 and LcrV antigens are currently under clinical trials. It is well known that a cellular immune response might have an essential additive value to immunity and protection against Y. pestis infection. Nevertheless, very few documented epitopes eliciting a protective T-cell response have been reported. Here, we present a combined high throughput computational and experimental effort towards identification of CD8 T-cell epitopes. All 4067 proteins of Y. pestis were analyzed with state-of-the-art recently developed prediction algorithms aimed at mapping potential MHC class I binders. A compilation of the results obtained from several prediction methods revealed a total of 238,000 peptide candidates, which necessitated downstream filtering criteria. Our previously established and proven approach for enrichment of true positive CTL epitopes, which relies on mapping clusters rich in tandem or overlapping predicted MHC binders ("hotspots"), was applied, as well as considerations of predicted binding affinity. A total of 1532 peptides were tested for their ability to elicit a specific T-cell response by following the production of IFNγ from splenocytes isolated from vaccinated mice. Altogether, the screen resulted in 178 positive responders (11.8%), all novel Y. pestis CTL epitopes. These epitopes span 113 Y. pestis proteins. Substantial enrichment of membrane-associated proteins was detected for epitopes selected from hotspots of predicted MHC binders. These results considerably expand the repertoire of known CTL epitopes in Y. pestis and pave the way to attest their protective potential, and hence their contribution to a future potent subunit vaccine. Copyright © 2017 Elsevier Ltd. All rights reserved.

Protective Role of Gamma Interferon during the Recall Response to Influenza Virus

PubMed Central

Bot, Adrian; Bot, Simona; Bona, Constantin A.

1998-01-01

During secondary immune responses to influenza virus, virus-specific T memory cells are a major source of gamma interferon (IFN-γ). We assessed the contribution of IFN-γ to heterologous protection against the A/WSN/33 (H1N1) virus of wild-type and IFN-γ−/− mice previously immunized with the A/HK/68 (H3N2) virus. The IFN-γ−/− mice displayed significantly reduced survival rates subsequent to a challenge with various doses of the A/WSN/33 virus. This was associated with an impaired ability of the IFN-γ−/− mice to completely clear the pulmonary virus by day 7 after the challenge, although significant reduction of the virus titers was noted. However, the IFN-γ−/− mice developed type A influenza virus cross-reactive cytotoxic T lymphocytes (CTLs) similar to the wild-type mice, as demonstrated by both cytotoxicity and a limiting-dilution assay for the estimation of CTL precursor frequency. The pulmonary recruitment of T cells in IFN-γ−/− mice was not dramatically affected, and the percentage of CD4+ and CD8+ T cells was similar to that of wild-type mice. The T cells from IFN-γ−/− mice did not display a significant switch toward a Th2 profile. Furthermore, the IFN-γ−/− mice retained the ability to mount significant titers of WSN and HK virus-specific hemagglutination-inhibiting antibodies. Together, these results are consistent with a protective role of IFN-γ during the heterologous response against influenza virus independently of the generation and local recruitment of cross-reactive CTLs. PMID:9658110

Model Checking Temporal Logic Formulas Using Sticker Automata

PubMed Central

Feng, Changwei; Wu, Huanmei

2017-01-01

As an important complex problem, the temporal logic model checking problem is still far from being fully resolved under the circumstance of DNA computing, especially Computation Tree Logic (CTL), Interval Temporal Logic (ITL), and Projection Temporal Logic (PTL), because there is still a lack of approaches for DNA model checking. To address this challenge, a model checking method is proposed for checking the basic formulas in the above three temporal logic types with DNA molecules. First, one-type single-stranded DNA molecules are employed to encode the Finite State Automaton (FSA) model of the given basic formula so that a sticker automaton is obtained. On the other hand, other single-stranded DNA molecules are employed to encode the given system model so that the input strings of the sticker automaton are obtained. Next, a series of biochemical reactions are conducted between the above two types of single-stranded DNA molecules. It can then be decided whether the system satisfies the formula or not. As a result, we have developed a DNA-based approach for checking all the basic formulas of CTL, ITL, and PTL. The simulated results demonstrate the effectiveness of the new method. PMID:29119114

An Oral DNA Vaccine Encoding Endoglin Eradicates Breast Tumors by Blocking Their Blood Supply

DTIC Science & Technology

2007-05-01

Marcantonio , Ph.D. Research Associate Dorothy...SB *pɘ.05 0 2 0 4 0 6 0 8 0 5 :1 1 0 :1 20 :1 E:T Ratio % S p e c i f i c L y s i s 100 101 102 103 104 FL2-Height Human Endoglin C o u n t s A B...Fig3. Cytotoxicity assay of breast cancer specific CTL. 0 5000 10000 15000 F l u o r e s c e i n l e v e l s / g HuEGEmpty Vector SB * *pɘ.005 B

Clonal predominance of CD8+ T cells in patients with unexplained neutropenia

PubMed Central

Wlodarski, Marcin Wojciech; Nearman, Zachary; Jiang, Ying; Lichtin, Alan; Maciejewski, Jaroslaw Pawel

2008-01-01

Objective T-cell-mediated autoimmunity may be involved in some cases of idiopathic neutropenia. We hypothesized that a precise T-cell receptor repertoire analysis may uncover cytotoxic T-cell (CTL) expansions that are less pronounced than those seen in T large granular lymphocyte leukemia (T-LGL), but are pathophysiologically analogous and thus can serve as markers of a T-cell-mediated process. Materials and Methods Using rational algorithms for T-cell receptor analysis and in vivo tracking of CTL responses previously established in our laboratory, we studied patients with unexplained chronic neutropenia (n = 20), T-LGL (n = 15), and healthy controls (n = 12). We further investigated the involvement of soluble inhibitory factors by coculture assays. To determine the level of immune activation, we studied interferon-g expression in CD8+cells using Taqman polymerase chain reaction. Results Fifteen expanded (immunodominant) CTL clones were detected in 12 of 20 patients. In comparison to LGL leukemia, these clones were less immunodominant, but clearly discernible from subclinical lymphoproliferations in controls. As a surrogate of cytotoxic activity, we found markedly increased production of interferon-γ in most of the neutropenia patients, irrespective of the presence of immunodominant CTL clones. Conclusions These results suggest that, while immunodominant CTL clones are detectable in a proportion of patients only, CTL-mediated pathophysiology may be a general mechanism operating in idiopathic neutropenia. Oligogoclonal CTL expansions in chronic neutropenia may indicate an ongoing autoimmune process, while highly polarized monoclonalities in a subset of neutropenic LGL patients may represent the “extreme” end of the clonal continuum. PMID:18279717

Evasion of adaptive immunity by HIV through the action of host APOBEC3G/F enzymes.

PubMed

Grant, Michael; Larijani, Mani

2017-09-12

APOBEC3G (A3G) and APOBEC3F (A3F) are DNA-mutating enzymes expressed in T cells, dendritic cells and macrophages. A3G/F have been considered innate immune host factors, based on reports that they lethally mutate the HIV genome in vitro. In vivo, A3G/F effectiveness is limited by viral proteins, entrapment in inactive complexes and filtration of mutations during viral life cycle. We hypothesized that the impact of sub-lethal A3G/F action could extend beyond the realm of innate immunity confined to the cytoplasm of infected cells. We measured recognition of wild type and A3G/F-mutated epitopes by cytotoxic T lymphocytes (CTL) from HIV-infected individuals and found that A3G/F-induced mutations overwhelmingly diminished CTL recognition of HIV peptides, in a human histocompatibility-linked leukocyte antigen (HLA)-dependent manner. Furthermore, we found corresponding enrichment of A3G/F-favored motifs in CTL epitope-encoding sequences within the HIV genome. These findings illustrate that A3G/F-mediated mutations mediate immune evasion by HIV in vivo. Therefore, we suggest that vaccine strategies target T cell or antibody epitopes that are not poised for mutation into escape variants by A3G/F action.

Direct assessment of the role of NK cells in autoimmune diabetes.

PubMed

Shachner, M S; Markmann, J F; Bassiri, H; Kim, J I; Naji, A; Barker, C F

1992-06-01

Considerable indirect evidence implicates participation of natural killer cells (NK) in the pathogenesis of diabetes in BB rats. The most convincing evidence derives from studies showing that anti-CD8 antibody effectively prevents both primary disease onset and autoimmune damage to transplanted islets. However, anti-CD8 treatment depletes both NK and cytotoxic T cells (CTL) since both cell types express the CD8 marker. To study directly the role of NK in diabetic BB rats we used MCA 3.2.3, a monoclonal antibody which selectively depletes normal Lewis rats of NK cells but not CTL. A regimen of ip injected antibody achieved rapid reduction of NK cells in diabetic and nondiabetic BB rats by FACS analysis. NK cell activity remained low in rats treated weekly as evidenced by YAC tumor cell killing. We next studied the effect of NK depletion on disease incidence in diabetes-prone BB rats of which about one half are expected to develop diabetes. Onset and incidence of diabetes in 3.2.3-treated and control antibody-treated aged matched litter mates were equal. These studies suggest that NK cells are not necessary for autoimmune islet destruction in spontaneously diabetic BB rats and support a role for CTL in pathogenesis of the disease.

Effect of avocado/soybean unsaponifiables on osseointegration: a proof-of-principle preclinical in vivo study.

PubMed

de Oliveira, Guilherme José Pimentel Lopes; de Paula, Luiz Guilherme Freitas; Spin-Neto, Rubens; Stavropoulos, Andreas; Spolidório, Luis Carlos; Marcantonio, Elcio; Marcantonio, Rosemary Adriana Chiérici

2014-01-01

To evaluate the influence of administration of avocado/soybean unsaponifiables (ASU) on implant osseointegration in rat tibiae. Thirty rats were randomly assigned into one of three equal-sized groups: (1) ASU1: administration of ASU starting 7 days prior to implant placement; (2) ASU2: administration of ASU starting on the day of implant placement, and (3) CTL: administration of saline solution. In all animals, one titanium implant was placed in each tibia. All animals received ASU or saline solution by gavage daily until sacrifice 60 days postoperatively. Implant osseointegration and bone maturation were assessed by biomechanical and radiographic bone density analysis; descriptive histology; immunohistochemistry for bone morphogenetic protein 2 (BMP-2), transforming growth factor beta 1 (TGF-β1), and osteocalcin; and histomorphometric evaluation of bone-to-implant contact (BIC) and mineralized bone area fraction within the threads of the implant. ASU1 and ASU2 showed three times higher expression of BMP-2 and nine times higher expression of TGF-β1 compared with CTL (P < .05). Histomorphometric analysis, however, showed that both ASU1 and ASU2 groups presented significantly higher BIC values only in the cortical bone compartment when compared to CTL (P < .05). ASU consumption seems to exert only a subtle effect on implant osseointegration.

Phase 1 clinical study of cyclophilin B peptide vaccine for patients with lung cancer.

PubMed

Gohara, Rumi; Imai, Nobue; Rikimaru, Toru; Yamada, Akira; Hida, Naoya; Ichiki, Masao; Kawamoto, Mayumi; Matsunaga, Kazuko; Ashihara, Junko; Yano, Sayoko; Tamura, Mayumi; Ohkouchi, Shinya; Yamana, Hideaki; Oizumi, Kotaro; Itoh, Kyogo

2002-01-01

Cyclophilin B (CypB) possesses two antigenic epitopes (CypB(84-92) and CypB(91-99) ) recognized by HLA-A24-restricted and tumor-specific cytotoxic T lymphocytes (CTLs). To determine the safety of CypB-derived peptides and its ability to generate antitumor immune responses, patients with advanced lung cancer received subcutaneous vaccinations of these peptides or their modified peptides. All 16 patients were vaccinated with CypB(91-99) or its modified peptide, whereas only two patients were vaccinated with the modified CypB(84-92), as immediate-type hypersensitivity to CypB(84-92) or its modified peptide was observed in the remaining patients. No severe adverse events were associated with the vaccination. No significant increase in cellular responses to either peptides or tumor cells was observed in the postvaccination PBMCs by the conventional CTL assays in any patients tested. These results suggest that the vaccination of CypB(91-99) peptide was safe, but failed to induce objective immune responses at this regimen.

Dengue virus-specific human T cell clones. Serotype crossreactive proliferation, interferon gamma production, and cytotoxic activity

PubMed Central

1989-01-01

The severe complications of dengue virus infections, hemorrhagic manifestation and shock, are much more commonly observed during secondary infections caused by a different serotype of dengue virus than that which caused the primary infections. It has been speculated, therefore, that dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are caused by serotype crossreactive immunopathological mechanisms. We analyzed clones of dengue serotype crossreactive T lymphocytes derived from the PBMC of a donor who had been infected with dengue 3 virus. These PBMC responded best to dengue 3 antigen, but also responded to dengue 1, 2, and 4 antigens, in bulk culture proliferation assays. 12 dengue antigen-specific clones were established using a limiting dilution technique. All of the clones had CD3+ CD4+ CD8 phenotypes. Eight clones responded to dengue 1, 2, 3, and 4 antigens and are crossreactive, while four other clones responded predominantly to dengue 3 antigen. These results indicate that the serotype crossreactive dengue-specific T lymphocyte proliferation observed in bulk cultures reflects the crossreactive responses detected at the clonal level. Serotype crossreactive clones produced high titers of IFN- gamma after stimulation with dengue 3 antigens, and also produced IFN- gamma to lower levels after stimulation with dengue 1, 2, and 4 antigens. The crossreactive clones lysed autologous lymphoblastoid cell line (LCL) pulsed with dengue antigens, and the crossreactivity of CTL lysis by T cell clones was consistent with the crossreactivity observed in proliferation assays. Epidemiological studies have shown that secondary infections with dengue 2 virus cause DHF/DSS at a higher rate than the other serotypes. We hypothesized that the lysis of dengue virus-infected cells by CTL may lead to DHF/DSS; therefore, the clones were examined for cytotoxic activity against dengue 2 virus-infected LCL. All but one of the serotype crossreactive clones lysed dengue 2 virus-infected autologous LCL, and they did not lyse uninfected autologous LCL. The lysis of dengue antigen-pulsed or virus-infected LCL by the crossreactive CTL clones that we have examined is restricted by HLA DP or DQ antigens. These results indicate that primary dengue virus infections induce predominantly crossreactive memory CD4+ T lymphocytes. These crossreactive T lymphocytes proliferate and produce IFN-gamma after stimulation with a virus strain of another serotype, and demonstrate crossreactive cyotoxic activity against autologous cells infected with heterologous dengue viruses.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2475573

Minimizing aggression during mixing of gestating sows with supplementation of a tryptophan-enriched diet.

PubMed

Poletto, Rosangela; Kretzer, Fabiana C; Hötzel, Maria J

2014-06-10

Gestation stalls are criticized for its negative physical and psycho-physiological effects on sow welfare. Group housing benefits sow well-being and when planned properly can minimize aggression during mixing. This study aimed to evaluate the effect of short-term feeding of a TRP-enriched diet at a concentration of 220% the control (CTL) diet, on aggressiveness at mixing of sows at 4weeks of gestation. Treatment diets were fed for 7 consecutive days; from days 1 to 5 sows were housed in stalls, early in the morning on day 6 sows were grouped by parity and assessed until day 7. Eighteen pens with 4 sows each (n=72) of similar parity were assigned to CTL and TRP treatments. Sows' behaviors were recorded daily for 12h, from days 1 to 7. Inactive and active behaviors (alert, walking (pen), rooting, feeding, drinking, eliminating), stereotypic behaviors (bar biting and sham-chewing), and postures (standing, sitting, lying) were assessed by 10-minute scan sampling. Occurrence of agonistic interactions, number of actions such as bites, head knocks and pursuits and their sum per interaction were recorded for each pen using 2-h continuous behavioral observation, at days 6 and 7. Skin lesion scores were assessed from each sow at day 5 and at 48h post-mixing, using a sow body map subdivided into anterior, central and posterior body regions. A linear mixed model with day as repeated measure, stall or pen as experimental unit, tested the fixed effects of treatment, day, period within day, their interactions, and block by treatment interaction; stall (trt) or pen (trt) as appropriate was used as random effect. Blood concentration of TRP was higher on the mixing day in TRP-fed sows compared to baseline (76%) and CLT-fed sows at mixing (79%; P<0.05), while serotonin concentration did not differ between treatments (P>0.05). The TRP-enriched diet was effective in reducing sham-chewing in stall housed sows of parity 5-9 (P<0.05). In pens, TRP-fed sows spent more time rooting (TRP=28.0 vs. CTL=20.7±1.0%; P<0.05) and consequently less time lying down than CTL-fed sows (TRP=56.1 vs. CTL=65.1±2.0%; P<0.05). The total number of offensive actions per interaction was greater in the morning than afternoon for both days (P<0.05), but this was less evident in TRP-fed compared to CTL sows mainly on the morning following mixing (3.4 vs. 7.2±1.0, respectively; Trt∗period (day)=P<0.05). The average lesion score was lower in the anterior body region of TRP-fed compared to CTL sows (2.1 vs. 2.5±0.2; P<0.05), the most affected area during fights. The TRP-enriched diet reduced sow aggression while increasing behavioral activity, as evidenced by more time rooting and standing while sows had fewer offensive actions per agonistic interaction and lower skin lesion score 48h post-mixing. A TRP enriched diet provided to gestating sows for a short period prior to social mixing and continued for a short time after is an effective means of reducing aggression and improving the welfare of sows during group formation. Copyright © 2014 Elsevier Inc. All rights reserved.

Engineering parvovirus-like particles for the induction of B-cell, CD4(+) and CTL responses.

PubMed

Rueda, P; Martínez-Torrecuadrada, J L; Sarraseca, J; Sedlik, C; del Barrio, M; Hurtado, A; Leclerc, C; Casal, J I

1999-09-01

An antigen delivery system based on hybrid recombinant parvovirus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of porcine (PPV) or canine parvovirus (CPV) expressed in insect cells with the baculovirus system has been developed. PPV:VLPs containing a CD8(+) epitope from the LCMV nucleoprotein evoked a potent CTL response and were able to protect mice against a lethal infection with the virus. Also, PPV:VLPs containing the C3:T epitope from poliovirus elicited a CD4(+)3 log(10) units) against poliovirus. The possibility of combining different types of epitopes in different positions of a single particle to stimulate different branches of the immune system paves the way to the production of more potent vaccines in a simple and cheap way.

Radio-frequency coil selection for MR imaging of the carotid vessel wall

NASA Astrophysics Data System (ADS)

Mat Isa, S.; Shuaib, I. L.; Bauk, S.

2014-11-01

This aim of this study was to identify the radiofrequency coil that will produce optimum image quality for scanning the carotid vessel wall using magnetic resonance imaging. A comparative cross-sectional study was conducted using 10 volunteers. Each volunteer was scanned three times using a 1.5T Signa HDxt machine equipped with one of three different coils: a neurovascular array (NV) coil, an 8-channel CTL spine array coil, and a 3-inch surface coil. A qualitative image quality rating was assigned to each image. The images were also evaluated by measuring the signal to noise ratio (SNR) using Osirix 4.2.3 software. The noise was estimated from the mean intensities of the region of interest in the background of the images and the signal was measured in the muscle adjacent to the vessel wall. The SNRs of the three coils were compared using one-way ANOVA, with 104 images used for the data analysis. The mean image quality scores for the NV head coil, CTL coil, and 3-inch coil were 3.4, 3.33, and 1.67, respectively. In addition, the SNRs differed significantly (p < 0.05). The mean SNR for the 3-inch coil was significantly higher (56.21 ± 25.06) than those for the NV head coil (27.34 ± 15.47) and CTL coil (21.77 ± 13.14). The Bonferroni post-hoc test revealed that there was no significant difference between the NV head coil and the CTL coil (p = 0.21). The optimum SNR value was 20-27. These results indicate that the NV head coil and CTL coil can be used to evaluate the carotid arterial wall with optimum image quality and higher resolution. These coil can deliver fast and robust data to image the carotid vessel wall in vivo.

Exposure to severe urban air pollution influences cognitive outcomes, brain volume and systemic inflammation in clinically healthy children.

PubMed

Calderón-Garcidueñas, Lilian; Engle, Randall; Mora-Tiscareño, Antonieta; Styner, Martin; Gómez-Garza, Gilberto; Zhu, Hongtu; Jewells, Valerie; Torres-Jardón, Ricardo; Romero, Lina; Monroy-Acosta, Maria E; Bryant, Christopher; González-González, Luis Oscar; Medina-Cortina, Humberto; D'Angiulli, Amedeo

2011-12-01

Exposure to severe air pollution produces neuroinflammation and structural brain alterations in children. We tested whether patterns of brain growth, cognitive deficits and white matter hyperintensities (WMH) are associated with exposures to severe air pollution. Baseline and 1 year follow-up measurements of global and regional brain MRI volumes, cognitive abilities (Wechsler Intelligence Scale for Children-Revised, WISC-R), and serum inflammatory mediators were collected in 20 Mexico City (MC) children (10 with white matter hyperintensities, WMH(+), and 10 without, WMH(-)) and 10 matched controls (CTL) from a low polluted city. There were significant differences in white matter volumes between CTL and MC children - both WMH(+) and WMH(-) - in right parietal and bilateral temporal areas. Both WMH(-) and WMH(+) MC children showed progressive deficits, compared to CTL children, on the WISC-R Vocabulary and Digit Span subtests. The cognitive deficits in highly exposed children match the localization of the volumetric differences detected over the 1 year follow-up, since the deficits observed are consistent with impairment of parietal and temporal lobe functions. Regardless of the presence of prefrontal WMH, Mexico City children performed more poorly across a variety of cognitive tests, compared to CTL children, thus WMH(+) is likely only partially identifying underlying white matter pathology. Together these findings reveal that exposure to air pollution may perturb the trajectory of cerebral development and result in cognitive deficits during childhood. Copyright © 2011 Elsevier Inc. All rights reserved.

Characterization of CTL Recognized Epitopes on Human Breast Tumors

DTIC Science & Technology

1996-09-01

maturation and effector function of cellular immune cytotoxic effectors such as CTL (11). (c) The epitopes defined on tumor Ag are self-peptides of...have been reported to be expressed in breast and ovarian cancer cells (18), and they apparently function by maintaining the undifferentiated state...Body of the Report The purpose of the present work continues to be the characterization of the functional significance of the CTL epitopes as potential

Effect of Aerva lanata on cell-mediated immune responses and cytotoxic T-lymphocyte generation in normal and tumor-bearing mice.

PubMed

Siveen, K S; Kuttan, Girija

2012-01-01

Cell-mediated immunity offers protection against virus-infected cells and tumor cells, involves activation of natural killer (NK) cells, production of antigen-specific cytotoxic T-lymphocytes, and release of various cytokines in response to an antigen. Administration of an ethanolic extract of Aerva lanata was found to stimulate cell-mediated immunological responses in normal and tumor-bearing BALB/c mice. A significant enhancement in NK cell activity in both normal and tumor-bearing hosts was observed after administration of A. lanata. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were significantly enhanced as well in both sets of treated hosts. In addition, in vivo production of IL-2 and IFNg were each significantly enhanced by extract treatment. The stimulatory effect of A. lanata on cytotoxic T-lymphocyte (CTL) production was determined by Winn's neutralization assay using CTL-sensitive EL4 thymoma cells. A. lanata treatment caused a significant increase in CTL production in both in vivo and in vitro models, in each case as indicated by a significant increase in the life-spans of tumor-injected mice. Taken together, all of these results in the murine model indicate that administration of an ethanolic extract of A. lanata could enhance the cell-mediated anti-tumor response.

Regulatory effect of dietary intake of chromium propionate on the response of monocyte-derived macrophages from Holstein cows in mid lactation.

PubMed

Garcia, M; Qu, Y; Scholte, C M; O'Connor, D; Rounds, W; Moyes, K M

2017-08-01

Chromium (Cr) has been reported to enhance immune function and improve insulin sensitivity and performance in beef and dairy cattle. However, its effect on bovine macrophage inflammatory and metabolic response is unknown. The objective of this study was to characterize the effect of dietary Cr on the inflammatory and metabolic response of polarized macrophages ex vivo. Twelve primiparous and 16 multiparous healthy Holstein cows in mid lactation (143 ± 37 d in milk) were enrolled in this study. Cows were fed a common total mixed ration once per day that was top-dressed with 200 g of ground corn containing 1 of 2 dietary treatments: control (CTL, no Cr supplementation) or Cr propionate (CrP, 8 mg of Cr/cow per day) for 35 d. At d 1, 17, and 35 of treatment, blood monocytes were isolated and cultured to obtain 3 monocyte-derived macrophage (MDM) phenotypes: M0 (non-polarized), M1 (pro-inflammatory; IFN-γ polarized) and M2 (anti-inflammatory; IL-4 polarized). The experiment was set in a randomized complete block design. Neither dry matter intake nor milk yield was affected by treatment. Plasma concentrations of metabolites and the metabolic and inflammatory response of MDM in spent media were not affected by treatment. Neither the whole blood cell population nor the specific proportion of leukocytes was affected by the main effect of treatment. However, we did observe a trend for fewer circulating neutrophils in cows fed CrP than in cows fed CTL for 35 d, which may be partly attributable to a greater influx of neutrophils into peripheral tissues, a reduced pro-inflammatory response during disease, or both; this warrants future study. Expression of IGFI was increased in MDM-M0, and expression of CXCL11 tended to increase in MDM-M2 from cows fed CrP compared with cows fed CTL. Expression of SLC2A3 also tended to increase in MDM-M2 from cows fed CrP compared with cows fed CTL at 17 d. Our results suggest that CrP has minimal effect on the inflammatory and metabolic response of MDM for Holstein dairy cows in mid lactation. Future studies are warranted to evaluate the differential regulation of Cr on the inflammatory and metabolic response of leukocytes from dairy cows at different stages of lactation and parity. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens.

PubMed

Lokhandwala, Shehnaz; Waghela, Suryakant D; Bray, Jocelyn; Martin, Cameron L; Sangewar, Neha; Charendoff, Chloe; Shetti, Rashmi; Ashley, Clay; Chen, Chang-Hsin; Berghman, Luc R; Mwangi, Duncan; Dominowski, Paul J; Foss, Dennis L; Rai, Sharath; Vora, Shaunak; Gabbert, Lindsay; Burrage, Thomas G; Brake, David; Neilan, John; Mwangi, Waithaka

2016-11-01

The African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (CTLs) could be the key to complete protection. Hence, generation of an efficacious subunit vaccine depends on identification of CTL targets along with a suitable delivery method that will elicit effector CTLs capable of eliminating ASFV-infected host cells and confer long-term protection. To this end, we evaluated the safety and immunogenicity of an adenovirus-vectored ASFV (Ad-ASFV) multiantigen cocktail formulated in two different adjuvants and at two immunizing doses in swine. Immunization with the cocktail rapidly induced unprecedented ASFV antigen-specific antibody and cellular immune responses against all of the antigens. The robust antibody responses underwent rapid isotype switching within 1 week postpriming, steadily increased over a 2-month period, and underwent rapid recall upon boost. Importantly, the primed antibodies strongly recognized the parental ASFV (Georgia 2007/1) by indirect fluorescence antibody (IFA) assay and Western blotting. Significant antigen-specific gamma interferon-positive (IFN-γ + ) responses were detected postpriming and postboosting. Furthermore, this study is the first to demonstrate induction of ASFV antigen-specific CTL responses in commercial swine using Ad-ASFV multiantigens. The relevance of the induced immune responses in regard to protection needs to be evaluated in a challenge study. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens

PubMed Central

Waghela, Suryakant D.; Bray, Jocelyn; Martin, Cameron L.; Sangewar, Neha; Charendoff, Chloe; Shetti, Rashmi; Ashley, Clay; Chen, Chang-Hsin; Berghman, Luc R.; Mwangi, Duncan; Dominowski, Paul J.; Foss, Dennis L.; Rai, Sharath; Vora, Shaunak; Gabbert, Lindsay; Burrage, Thomas G.; Brake, David; Neilan, John

2016-01-01

The African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (CTLs) could be the key to complete protection. Hence, generation of an efficacious subunit vaccine depends on identification of CTL targets along with a suitable delivery method that will elicit effector CTLs capable of eliminating ASFV-infected host cells and confer long-term protection. To this end, we evaluated the safety and immunogenicity of an adenovirus-vectored ASFV (Ad-ASFV) multiantigen cocktail formulated in two different adjuvants and at two immunizing doses in swine. Immunization with the cocktail rapidly induced unprecedented ASFV antigen-specific antibody and cellular immune responses against all of the antigens. The robust antibody responses underwent rapid isotype switching within 1 week postpriming, steadily increased over a 2-month period, and underwent rapid recall upon boost. Importantly, the primed antibodies strongly recognized the parental ASFV (Georgia 2007/1) by indirect fluorescence antibody (IFA) assay and Western blotting. Significant antigen-specific gamma interferon-positive (IFN-γ+) responses were detected postpriming and postboosting. Furthermore, this study is the first to demonstrate induction of ASFV antigen-specific CTL responses in commercial swine using Ad-ASFV multiantigens. The relevance of the induced immune responses in regard to protection needs to be evaluated in a challenge study. PMID:27628166

The enkephalinergic nervous system and its immunomodulation on the developing immune system during the ontogenesis of oyster Crassostrea gigas.

PubMed

Liu, Zhaoqun; Zhou, Zhi; Wang, Lingling; Song, Xiaorui; Chen, Hao; Wang, Weilin; Liu, Rui; Wang, Mengqiang; Wang, Hao; Song, Linsheng

2015-08-01

Enkephalinergic neuroendocrine-immune regulatory system is one of the most important neuroendocrine-immune systems in both vertebrates and invertebrates for its significant role in the immune regulation. In the present study, the early onset of enkephalinergic nervous system and its immunomodulation on the developing immune system during the ontogenesis of oyster Crassostrea gigas were investigated to illustrate the function of neural regulation on the innate immune system in oyster larvae. [Met(5)]-enkephalin (Met-ENK) was firstly observed on the marginal of the dorsal half of D-hinged larvae. Six immune-related molecules, including four PRRs (CgCTL-1, CgCTL-2, CgCTL-4, CgNatterin-3) and two immune effectors (CgTNF-1 and CgEcSOD) were detected in the early developmental stages of trochophore, D-hinged and umbo larvae of oyster. After incubated with [Met(5)]-enkephalin, the mRNA expression level of all the PRRs changed significantly (p < 0.05). In trochophore larvae, the expression level of CgNatterin-3 decreased dramatically (p < 0.05) at 6 h, and the expression level of CgCTL-4 was significantly down-regulated at 3 h and 6 h (p < 0.05), respectively. In D-hinged and umbo larvae, only CgCTL-1 was significantly down-regulated and the differences were significant at 3 h and 6 h (p < 0.05), while the expression level of CgCTL-2 and CgCTL-4 increased significantly at 3 h after treatment (p < 0.05). Moreover, the expression levels of immune effectors were up-regulated significantly at 3 h and 6 h in trochophore larvae (p < 0.05). The expression level of CgTNF-1 in both blank and experiment groups was up-regulated but there was no significant difference in D-hinged larvae stage. On the contrary, the expression level of CgEcSOD in D-hinged larvae decreased dramatically at 3 h and 6 h after [Met(5)]-enkephalin incubation (p < 0.05). In umbo larvae, the expression level of CgTNF-1 and CgEcSOD in the experiment group increased significantly at 6 h after [Met(5)]-enkephalin treatment (p < 0.05), while no significant difference was found in the blank group. In addition, the anti-bacterial activities of the total protein extract from trochophore, D-hinged and umbo larvae increased significantly (p < 0.05) at both 3 h and 6 h after [Met(5)]-enkephalin incubation compared to that in the blank group, and PO activities of both D-hinged and umbo larvae total protein extract increased significantly (p < 0.05) while no significant difference was observed in trochophore larvae. The PO activities of the total protein extract in all the experiment groups decreased after the treatment with [Met(5)]-enkephalin for 6 h, but no significant difference was observed when compared to the blank group. Furthermore, after incubation for 6 h, the concentration of both CgTNF-1 and CgIL17-5 increased dramatically compared to that in the blank group (p < 0.05). These results together indicated that the enkephalinergic nervous system of oyster was firstly appeared in D-hinged larvae, while the primitive immune defense system existed in the region of prototroch in trochophore larvae and developed maturely after D-hinged larvae. The developing immune system could be regulated by the neurotransmitter [Met(5)]-enkephalin released by the neuroendocrine system in oyster C. gigas. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural and Environmental Characteristics of Extratropical Cyclones that Cause Tornado Outbreaks in the Warm Sector

NASA Astrophysics Data System (ADS)

Tochimoto, Eigo; Niino, Hiroshi

2016-04-01

The differences in structural and environmental characteristics of extratropical cyclones (hereafter, ECs) that cause tornado outbreaks and those that do not were examined through composite analyses of the newly-released Japanese reanalysis data (JRA-55) and idealized numerical experiments. ECs that developed in the United States in April and May between 1995 and 2012 are categorized into two groups: ECs accompanied by 15 or more tornadoes (hereafter, outbreak cyclones (OCs)) and ECs accompanied by 5 or less tornadoes (non-outbreak cyclones (NOCs)). 55 OCs and 41 NOCs that are of similar strength as OCs are selected in this study. The composite analyses show significant differences in convective environmental parameters between OCs and NOCs. For OCs, convective available potential energy (CAPE) and storm relative environmental helicity (SREH) are larger and the areas in which these parameters have significant values are wider in the warm sector. The larger CAPE in OCs is due to larger amount of low-level water vapor, while the larger SREH in OCs due to stronger southerly wind at low levels. A piecewise potential vorticity (PV) diagnostics (Davis and Emanuel, 1991) indicates that low- to mid-level PV anomalies mainly contribute to the difference in the low-level winds between OCs and NOCs. On the other hand, the low-level winds associated with upper-level PV anomalies are not the major contributor to the difference. The results of the idealized numerical experiments for OCs and NOCs (hereafter, referred to as OC-CTL and NOC-CTL, respectively) using WRF ver. 3.4 show that the characteristics of the low-level wind fields and SREH distributions for the simulated ECs in OC-CTL and NOC-CTL are similar to those for OCs and NOCs, respectively. In OC-CTL, SREH and low-level winds in the east-southeast region of the EC center is larger than those in NOC-CTL, respectively. It is suggested that these differences are due to the structures of jetstream. The structure of jetstream in OC-CTL has larger anticyclonic horizontal shear in the southern side of the jet axis than that in NOC-CTL. Larger horizontal anticyclonic shear of the jetstream in OC-CTL causes more meridionally-elongated structure of the EC, resulting stronger low-level winds and larger SREH in the southeast region of the cyclone center.

NK cells modulate the cytotoxic activity generated by Mycobacterium leprae-hsp65 in leprosy patients: role of IL-18 and IL-13

PubMed Central

DE LA BARRERA, S; FINIASZ, M; FINK, S; ILARREGUI, J; ALEMÁN, M; OLIVARES, L; FRANCO, M C; PIZZARIELLO, G; DEL CARMEN SASIAIN, M

2004-01-01

Protection against intracellular pathogens such as Mycobacterium leprae is critically dependent on the function of NK cells at early stages of the immune response and on Th1 cells at later stages. In the present report we evaluated the role of IL-18 and IL-13, two cytokines that can influence NK cell activity, in the generation of M. leprae-derived hsp65-cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) of leprosy patients. We demonstrated that IL-18 modulates hsp65-induced CTL generation and collaborates with IL-12 for this effect. In paucibacillary (PB) patients and normal controls (N) depletion of NK cells reduces the cytolytic activity. Under these conditions, IL-12 cannot up-regulate this CTL generation, while, in contrast, IL-18 increases the cytotoxic activity both in the presence or absence of NK cells. IL-13 down-regulates the hsp65-induced CTL generation and counteracts the positive effect of IL-18. The negative effect of IL-13 is observed in the early stages of the response, suggesting that this cytokine affects IFNγ production by NK cells. mRNA coding for IFNγ is induced by IL-18 and reduced in the presence of IL-13, when PBMC from N or PB patients are stimulated with hsp65. Neutralization of IL-13 in PBMC from multibacillary (MB) leprosy patients induces the production of IFNγ protein by lymphocytes. A modulatory role on the generation of hsp65 induced CTL is demonstrated for IL-18 and IL-13 and this effect takes place through the production of IFNγ. PMID:14678270

Light-intensity and high-intensity interval training improve cardiometabolic health in rats.

PubMed

Batacan, Romeo B; Duncan, Mitch J; Dalbo, Vincent J; Connolly, Kylie J; Fenning, Andrew S

2016-09-01

Physical activity has the potential to reduce cardiometabolic risk factors but evaluation of different intensities of physical activity and the mechanisms behind their health effects still need to be fully established. This study examined the effects of sedentary behaviour, light-intensity training, and high-intensity interval training on biometric indices, glucose and lipid metabolism, inflammatory and oxidative stress markers, and vascular and cardiac function in adult rats. Rats (12 weeks old) were randomly assigned to 1 of 4 groups: control (CTL; no exercise), sedentary (SED; no exercise and housed in small cages to reduce activity), light-intensity trained (LIT; four 30-min exercise bouts/day at 8 m/min separated by 2-h rest period, 5 days/week), and high-intensity interval trained (HIIT, four 2.5-min work bouts/day at 50 m/min separated by 3-min rest periods, 5 days/week). After 12 weeks of intervention, SED had greater visceral fat accumulation (p < 0.01) and slower cardiac conduction (p = 0.04) compared with the CTL group. LIT and HIIT demonstrated beneficial changes in body weight, visceral and epididymal fat weight, glucose regulation, low-density lipoprotein cholesterol, total cholesterol, and mesenteric vessel contractile response compared with the CTL group (p < 0.05). LIT had significant improvements in insulin sensitivity and cardiac conduction compared with the CTL and SED groups whilst HIIT had significant improvements in systolic blood pressure and endothelium-independent vasodilation to aorta and mesenteric artery compared with the CTL group (p < 0.05). LIT and HIIT induce health benefits by improving traditional cardiometabolic risk factors. LIT improves cardiac health while HIIT promotes improvements in vascular health.

Social instability stress in adolescent male rats reduces social interaction and social recognition performance and increases oxytocin receptor binding.

PubMed

Hodges, Travis E; Baumbach, Jennet L; Marcolin, Marina L; Bredewold, Remco; Veenema, Alexa H; McCormick, Cheryl M

2017-09-17

Social experiences in adolescence are essential for displaying context-appropriate social behaviors in adulthood. We previously found that adult male rats that underwent social instability stress (SS) in adolescence had reduced social interactions with unfamiliar peers compared with non-stressed controls (CTL). Here we determined whether SS altered social recognition and social reward and brain oxytocin and vasopressin receptor density in adolescence. We confirmed that SS rats spent less time interacting with unfamiliar peers than did CTL rats (p=0.006). Furthermore, CTL rats showed a preference for novel over familiar conspecifics in a social recognition test whereas SS rats did not, which may reflect reduced recognition, impaired memory, or reduced preference for novelty in SS rats. The reward value of social interactions was not affected by SS based on conditioned place preference tests and based on the greater time SS rats spent investigating stimulus rats than did CTL rats when the stimulus rat was behind wire mesh (p=0.03). Finally, oxytocin receptor binding density was higher in the dorsal lateral septum and nucleus accumbens shell in SS rats compared with CTL rats (p=0.02, p=0.01, respectively). No effect of SS was found for vasopressin 1a receptor binding density in any of the brain regions analyzed. We discuss the extent to which the differences in social behavior exhibited after social instability in adolescence involve changes in social salience and social competency, and the possibility that changes in oxytocin signaling in the brain underlie the differences in social behavior. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Dendritic cells loaded with HeLa-derived exosomes simulate an antitumor immune response.

PubMed

Ren, Guoping; Wang, Yanhong; Yuan, Shexia; Wang, Baolian

2018-05-01

The aim of the present study was to investigate the effect of loading dendritic cells (DCs) with HeLa-derived exosomes on cytotoxic T-lymphocyte (CTL) responses, and the cytotoxic effects of CTL responses on the HeLa cell line. Ultrafiltration centrifugation combined with sucrose density gradient ultracentrifugation was applied to isolate exosomes (HeLa-exo) from the supernatant of HeLa cells. Morphological features of HeLa-exo were identified by transmission electron microscopy (TEM), and the expression of cluster of differentiation (CD)63 was detected by western blotting. Next, monocytes were isolated from peripheral blood and cultured with the removal of adherent cells to induce DC proliferation. DCs were then phenotypically characterized by flow cytometry. Finally, MTT assays were performed to analyze the effects of DCs loaded with HeLa-exo on T cell proliferation and cytotoxicity assays to evaluate the effect of CTL responses on HeLa cells. TEM revealed that HeLa-exo exhibit typical cup-shaped morphology with a diameter range of 30-100 nm. It was also identified that the CD63 surface antigen is expressed on HeLa-exo. Furthermore, monocyte-derived DCs were able to express CD1a, suggesting that DC induction was a success. DCs exhibited hair-like protrusions and other typical dendritic cell morphology. Furthermore, DCs loaded with HeLa-exo could enhance CTL proliferation and the cytotoxic activity of CTLs compared with DCs without HeLa-exo (P<0.05). In conclusion, DCs loaded with HeLa-exo may promote T cell proliferation and induce CTL responses to inhibit the growth of cervical cancer cells in vitro .

Protective Efficacy of Plasmodium vivax Radiation-Attenuated Sporozoites in Colombian Volunteers: A Randomized Controlled Trial

PubMed Central

Arévalo-Herrera, Myriam; Vásquez-Jiménez, Juan M.; Lopez-Perez, Mary; Vallejo, Andrés F.; Amado-Garavito, Andrés B.; Céspedes, Nora; Castellanos, Angélica; Molina, Karen; Trejos, Johanna; Oñate, José; Epstein, Judith E.; Richie, Thomas L.; Herrera, Sócrates

2016-01-01

Background Immunizing human volunteers by mosquito bite with radiation-attenuated Plasmodium falciparum sporozoites (RAS) results in high-level protection against infection. Only two volunteers have been similarly immunized with P. vivax (Pv) RAS, and both were protected. A phase 2 controlled clinical trial was conducted to assess the safety and protective efficacy of PvRAS immunization. Methodology/Principal Findings A randomized, single-blinded trial was conducted. Duffy positive (Fy+; Pv susceptible) individuals were enrolled: 14 received bites from irradiated (150 ± 10 cGy) Pv-infected Anopheles mosquitoes (RAS) and 7 from non-irradiated non-infected mosquitoes (Ctl). An additional group of seven Fy- (Pv refractory) volunteers was immunized with bites from non-irradiated Pv-infected mosquitoes. A total of seven immunizations were carried out at mean intervals of nine weeks. Eight weeks after last immunization, a controlled human malaria infection (CHMI) with non-irradiated Pv-infected mosquitoes was performed. Nineteen volunteers completed seven immunizations (12 RAS, 2 Ctl, and 5 Fy-) and received a CHMI. Five of 12 (42%) RAS volunteers were protected (receiving a median of 434 infective bites) compared with 0/2 Ctl. None of the Fy- volunteers developed infection by the seventh immunization or after CHMI. All non-protected volunteers developed symptoms 8–13 days after CHMI with a mean pre-patent period of 12.8 days. No serious adverse events related to the immunizations were observed. Specific IgG1 anti-PvCS response was associated with protection. Conclusion Immunization with PvRAS was safe, immunogenic, and induced sterile immunity in 42% of the Fy+ volunteers. Moreover, Fy- volunteers were refractory to Pv malaria. Trial registration Identifier: NCT01082341. PMID:27760143

Phenylpyrazole insecticide fipronil induces male infertility in the estuarine meiobenthic crustacean Amphiascus tenuiremis.

PubMed

Cary, Tawnya L; Chandler, G Thomas; Volz, David C; Walse, Spencer S; Ferry, John L

2004-01-15

Copepods are the most abundant arthropods on earth and are often the most important secondary producers in estuarine/marine food webs. The new GABA (gamma-aminobutyric acid)-disrupting insecticide fipronil (FP) induces unique sex-specific reproductive dysfunction in male meiobenthic copepods, leading to trans-generational population depression at environmentally realistic concentrations (0.63 microg/L). Using a newly developed 96-well microplate lifecycle bioassay, more than 700 individual Stage-I juveniles were reared to adulthood in as short as 12 days in only 200 microL of control (CTL) or 0.63 microg-FP/L seawater solution. Individual virgin male: female pairs were then cross-mated for all possible combinations within and across rearing treatments and allowed to mate for an additional 12 days in CTL or 0.63 microg-FP/L solution. FP at 0.63 microg/L caused no significant lethality to any mating combinations but evoked 73% or 89% inhibition of reproduction when FP-reared males were mated with either a control- or FP-reared female in FP solution, respectively. In contrast, when CTL-reared males were mated with FP-reared females in FP solution, there was no difference in reproductive success compared to FP-free controls. When FP-reared males were mated with either female group in FP-free solution, these mating pairs displayed a 3-day delay in time to brood sac extrusion but ultimately did reproduce. As fipronil (1) has a high K(ow), (2) is persistent in sediments where meiobenthic copepods live, and (3) has been detected in estuarine waters >0.7 microg/L, it may pose high risk to copepod production in estuarine systems.

Protective Efficacy of Plasmodium vivax Radiation-Attenuated Sporozoites in Colombian Volunteers: A Randomized Controlled Trial.

PubMed

Arévalo-Herrera, Myriam; Vásquez-Jiménez, Juan M; Lopez-Perez, Mary; Vallejo, Andrés F; Amado-Garavito, Andrés B; Céspedes, Nora; Castellanos, Angélica; Molina, Karen; Trejos, Johanna; Oñate, José; Epstein, Judith E; Richie, Thomas L; Herrera, Sócrates

2016-10-01

Immunizing human volunteers by mosquito bite with radiation-attenuated Plasmodium falciparum sporozoites (RAS) results in high-level protection against infection. Only two volunteers have been similarly immunized with P. vivax (Pv) RAS, and both were protected. A phase 2 controlled clinical trial was conducted to assess the safety and protective efficacy of PvRAS immunization. A randomized, single-blinded trial was conducted. Duffy positive (Fy+; Pv susceptible) individuals were enrolled: 14 received bites from irradiated (150 ± 10 cGy) Pv-infected Anopheles mosquitoes (RAS) and 7 from non-irradiated non-infected mosquitoes (Ctl). An additional group of seven Fy- (Pv refractory) volunteers was immunized with bites from non-irradiated Pv-infected mosquitoes. A total of seven immunizations were carried out at mean intervals of nine weeks. Eight weeks after last immunization, a controlled human malaria infection (CHMI) with non-irradiated Pv-infected mosquitoes was performed. Nineteen volunteers completed seven immunizations (12 RAS, 2 Ctl, and 5 Fy-) and received a CHMI. Five of 12 (42%) RAS volunteers were protected (receiving a median of 434 infective bites) compared with 0/2 Ctl. None of the Fy- volunteers developed infection by the seventh immunization or after CHMI. All non-protected volunteers developed symptoms 8-13 days after CHMI with a mean pre-patent period of 12.8 days. No serious adverse events related to the immunizations were observed. Specific IgG1 anti-PvCS response was associated with protection. Immunization with PvRAS was safe, immunogenic, and induced sterile immunity in 42% of the Fy+ volunteers. Moreover, Fy- volunteers were refractory to Pv malaria. Identifier: NCT01082341.

In Vivo Killing Capacity of Cytotoxic T Cells Is Limited and Involves Dynamic Interactions and T Cell Cooperativity

PubMed Central

Halle, Stephan; Keyser, Kirsten Anja; Stahl, Felix Rolf; Busche, Andreas; Marquardt, Anja; Zheng, Xiang; Galla, Melanie; Heissmeyer, Vigo; Heller, Katrin; Boelter, Jasmin; Wagner, Karen; Bischoff, Yvonne; Martens, Rieke; Braun, Asolina; Werth, Kathrin; Uvarovskii, Alexey; Kempf, Harald; Meyer-Hermann, Michael; Arens, Ramon; Kremer, Melanie; Sutter, Gerd; Messerle, Martin; Förster, Reinhold

2016-01-01

Summary According to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2–16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8+ T cell immunity. PMID:26872694

Correlates of protection, antigen delivery and molecular epidemiology: basics for designing an HIV vaccine.

PubMed

Wagner, R; Shao, Y; Wolf, H

1999-03-26

Major obstacles in the development of HIV vaccines are the high variability of the virus and its complex interaction with the immune system. Recent studies demonstrated, that CTLs recognizing highly conserved epitopes in the group-specific antigen are capable of controlling HIV-replication in long-term nonprogressors. Necessary consequences for novel vaccine concepts are the presentation of a large repertoire of antigenic sites as well as the stimulation of different effectors of the immune system. Accordingly, different types of recombinant HIV-1 virus-like particles (VLPs) have been constructed stimulating the induction of neutralizing antibodies and HIV-specific CD8-positive CTL responses in preclinical studies. With respect to future vaccine trials, HIV vaccine formulations may need to be tailored to the local strains circulating within a geographical region. The expert group of the joint United Nations Programme on AIDS recently identified Yunnan, a southwestern province of China, as a region, in which the HIV epidemic is starting to gain speed, resembling to the situation in Thailand 10 years ago. A molecular clone of a representative virus strain is now available for the development of innovative antigen delivery systems aiming to be evaluated in future clinical vaccine trials throughout this area.

The TESS Input Catalog and Selection of Targets for the TESS Transit Search

NASA Astrophysics Data System (ADS)

Pepper, Joshua; Stassun, Keivan G.; Paegert, Martin; Oelkers, Ryan; De Lee, Nathan Michael; Torres, Guillermo; TESS Target Selection Working Group

2018-01-01

The TESS mission will photometrically survey millions of the brightest stars over almost the entire the sky to detect transiting exoplanets. A key step to enable that search is the creation of the TESS Input Catalog (TIC), a compiled catalog of 700 million stars and galaxies with observed and calculated parameters. From the TIC we derive the Candidate Target List (CTL) to identify target stars for the 2-minute TESS postage stamps. The CTL is designed to identify the best stars for the detection of small planets, which includes all bright cool dwarf stars in the sky. I will describe the target selection strategy, the distribution of stars in the current CTL, and how both the TIC and CTL will expand and improve going forward.

Mother Centriole Distal Appendages Mediate Centrosome Docking at the Immunological Synapse and Reveal Mechanistic Parallels with Ciliogenesis.

PubMed

Stinchcombe, Jane C; Randzavola, Lyra O; Angus, Karen L; Mantell, Judith M; Verkade, Paul; Griffiths, Gillian M

2015-12-21

Cytotoxic T lymphocytes (CTLs) are highly effective serial killers capable of destroying virally infected and cancerous targets by polarized release from secretory lysosomes. Upon target contact, the CTL centrosome rapidly moves to the immunological synapse, focusing microtubule-directed release at this point [1-3]. Striking similarities have been noted between centrosome polarization at the synapse and basal body docking during ciliogenesis [1, 4-8], suggesting that CTL centrosomes might dock with the plasma membrane during killing, in a manner analogous to primary cilia formation [1, 4]. However, questions remain regarding the extent and function of centrosome polarization at the synapse, and recent reports have challenged its role [9, 10]. Here, we use high-resolution transmission electron microscopy (TEM) tomography analysis to show that, as in ciliogenesis, the distal appendages of the CTL mother centriole contact the plasma membrane directly during synapse formation. This is functionally important as small interfering RNA (siRNA) targeting of the distal appendage protein, Cep83, required for membrane contact during ciliogenesis [11], impairs CTL secretion. Furthermore, the regulatory proteins CP110 and Cep97, which must dissociate from the mother centriole to allow cilia formation [12], remain associated with the mother centriole in CTLs, and neither axoneme nor transition zone ciliary structures form. Moreover, complete centrosome docking can occur in proliferating CTLs with multiple centriole pairs. Thus, in CTLs, centrosomes dock transiently with the membrane, within the cell cycle and without progression into ciliogenesis. We propose that this transient centrosome docking without cilia formation is important for CTLs to deliver rapid, repeated polarized secretion directed by the centrosome. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Discovery of novel targets for multi-epitope vaccines: Screening of HIV-1 genomes using association rule mining

PubMed Central

Paul, Sinu; Piontkivska, Helen

2009-01-01

Background Studies have shown that in the genome of human immunodeficiency virus (HIV-1) regions responsible for interactions with the host's immune system, namely, cytotoxic T-lymphocyte (CTL) epitopes tend to cluster together in relatively conserved regions. On the other hand, "epitope-less" regions or regions with relatively low density of epitopes tend to be more variable. However, very little is known about relationships among epitopes from different genes, in other words, whether particular epitopes from different genes would occur together in the same viral genome. To identify CTL epitopes in different genes that co-occur in HIV genomes, association rule mining was used. Results Using a set of 189 best-defined HIV-1 CTL/CD8+ epitopes from 9 different protein-coding genes, as described by Frahm, Linde & Brander (2007), we examined the complete genomic sequences of 62 reference HIV sequences (including 13 subtypes and sub-subtypes with approximately 4 representative sequences for each subtype or sub-subtype, and 18 circulating recombinant forms). The results showed that despite inclusion of recombinant sequences that would be expected to break-up associations of epitopes in different genes when two different genomes are recombined, there exist particular combinations of epitopes (epitope associations) that occur repeatedly across the world-wide population of HIV-1. For example, Pol epitope LFLDGIDKA is found to be significantly associated with epitopes GHQAAMQML and FLKEKGGL from Gag and Nef, respectively, and this association rule is observed even among circulating recombinant forms. Conclusion We have identified CTL epitope combinations co-occurring in HIV-1 genomes including different subtypes and recombinant forms. Such co-occurrence has important implications for design of complex vaccines (multi-epitope vaccines) and/or drugs that would target multiple HIV-1 regions at once and, thus, may be expected to overcome challenges associated with viral escape. PMID:19580659

NADPH Oxidase 4 Induces Cardiac Fibrosis and Hypertrophy through Activating Akt/mTOR and NFκB Signaling Pathways

PubMed Central

Zhao, Qingwei David; Viswanadhapalli, Suryavathi; Williams, Paul; Shi, Qian; Tan, Chunyan; Yi, Xiaolan; Bhandari, Basant; Abboud, Hanna E.

2015-01-01

Background NADPH oxidase 4 (Nox4) has been implicated in cardiac remodeling, but its precise role in cardiac injury remains controversial. Furthermore, little is known about the downstream effector signaling pathways activated by Nox4-derived ROS in the myocardium. We investigated the role of Nox4 and Nox4 associated signaling pathways in the development of cardiac remodeling. Methods and Results Cardiac-specific human Nox4 transgenic mice (c-hNox4Tg) were generated. Four groups of mice were studied: 1) control mice (CTL): littermates that are negative for hNox4 transgene but Cre positive; 2) c-hNox4 Tg mice; 3) angiotensin II (AngII)-infused CTL mice and 4) c-hNox4Tg mice infused with AngII. The c-hNox4Tg mice exhibited approximately 10-fold increase in Nox4 protein expression and 8-fold increase in the production of reactive oxygen species, and manifested cardiac interstitial fibrosis. AngII-infusion to CTL mice increased cardiac Nox4 expression and induced fibrosis and hypertrophy. The Tg mice receiving AngII exhibited more advanced cardiac remodeling and robust elevation in Nox4 expression, indicating that AngII worsens cardiac injury, at least partially by enhancing Nox4 expression. Moreover, hNox4 transgene and/or AngII-infusion induced the expression of cardiac fetal genes and activated the Akt-mTOR and NFκB signaling pathways. Treatment of AngII-infused c-hNox4Tg mice with GKT137831, a Nox4/Nox1 inhibitor, abolished the increase in oxidative stress, suppressed Akt-mTOR and NFκB signaling pathway and attenuated cardiac remodeling. Conclusion Upregulation of Nox4 in the myocardium causes cardiac remodeling through activating Akt-mTOR and NFκB signaling pathways. Inhibition of Nox4 has therapeutic potential to treat cardiac remodeling. PMID:25589557

Role of community tolerance level (CTL) in predicting the prevalence of the annoyance of road and rail noise.

PubMed

Schomer, Paul; Mestre, Vincent; Fidell, Sanford; Berry, Bernard; Gjestland, Truls; Vallet, Michel; Reid, Timothy

2012-04-01

Fidell et al. [(2011), J. Acoust. Soc. Am. 130(2), 791-806] have shown (1) that the rate of growth of annoyance with noise exposure reported in attitudinal surveys of the annoyance of aircraft noise closely resembles the exponential rate of change of loudness with sound level, and (2) that the proportion of a community highly annoyed and the variability in annoyance prevalence rates in communities are well accounted for by a simple model with a single free parameter: a community tolerance level (abbreviated CTL, and represented symbolically in mathematical expressions as L(ct)), expressed in units of DNL. The current study applies the same modeling approach to predicting the prevalence of annoyance of road traffic and rail noise. The prevalence of noise-induced annoyance of all forms of transportation noise is well accounted for by a simple, loudness-like exponential function with community-specific offsets. The model fits all of the road traffic findings well, but the prevalence of annoyance due to rail noise is more accurately predicted separately for interviewing sites with and without high levels of vibration and/or rattle.

Peptides of a major histocompatibility complex class I (Kb) molecule cause prolongation of skin graft survival and induce specific down-regulatory T cells demonstrable in the mixed lymphocyte reaction.

PubMed Central

Brondz, B D; Kazansky, D B; Chernyshova, A D; Ivanov, V S

1995-01-01

Six individual peptides of the major histocompatibility complex (MHC) class I molecule H-2Kb were synthesized. Intravenous injection of peptide 6 into mice prolonged the survival of Kb (BL/6 or B10.MBR) skin grafts on allogeneic R101 and B10.AKM mice, respectively. This was specific, as control skin grafts from Kk (B10.BR) or Kd (DBA/2) donors, respectively, were rejected at the same time in both control and peptide-treated mice. The optimal doses for peptide 6, which is from the alpha 2 domain, were defined. The test system was the inhibition of proliferation in vitro of naive lymph node cells by syngeneic mitomycin c-treated spleen cells from R101 mice preimmunized with irradiated stimulator splenocytes of Kb (BL/6) origin. Down-regulation was specific, as proliferation in response to third-party allogeneic stimulator Kk (B10.BR) splenocytes was not inhibited. Of the six peptides of H-2Kb tested, potent down-regulatory cells were induced by peptides 2 (alpha 1 domain) and 5 and 6 (alpha 2 domain). The greatest down-regulatory activity was obtained by giving peptide 2 to mice that had already been immunized against H-2Kb by injecting EL4 cells. Under the same conditions, injecting peptide 2 did not induce any cytotoxic T cells. In contrast, specific cytotoxic lymphocytes (CTL) were induced when cells from primed mice were incubated for 4 days with heated stimulator cells from BL/6 mice. The data suggest that peptides from MHC class I molecules activate precursors of down-regulatory T cells, but not of CTL, and this may explain their ability to prolong skin allograft survival. PMID:7490121

Effective Induction of Simian Immunodeficiency Virus-Specific Cytotoxic T Lymphocytes in Macaques by Using a Multiepitope Gene and DNA Prime-Modified Vaccinia Virus Ankara Boost Vaccination Regimen

PubMed Central

Hanke, Tomas; Samuel, Rachel V.; Blanchard, Tom J.; Neumann, Veronica C.; Allen, Todd M.; Boyson, Jon E.; Sharpe, Sally A.; Cook, Nicola; Smith, Geoffrey L.; Watkins, David I.; Cranage, Martin P.; McMichael, Andrew J.

1999-01-01

DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in humans. Here, by using a multicytotoxic T-lymphocyte (CTL) epitope gene and a DNA prime-MVA boost vaccination regimen, high levels of CTLs specific for a single simian immunodeficiency virus (SIV) gag-derived epitope were elicited in rhesus macaques. These vaccine-induced CTLs were capable of killing SIV-infected cells in vitro. Fluorescence-activated cell sorter analysis using soluble tetrameric major histocompatibility complex-peptide complexes showed that the vaccinated animals had 1 to 5% circulating CD8+ lymphocytes specific for the vaccine epitope, frequencies comparable to those in SIV-infected monkeys. Upon intrarectal challenge with pathogenic SIVmac251, no evidence for protection was observed in at least two of the three vaccinated animals. This study does not attempt to define correlates of protective immunity nor design a protective vaccine against immunodeficiency viruses, but it demonstrates clearly that the DNA prime-MVA boost regimen is an effective protocol for induction of CTLs in macaques. It also shows that powerful tools for studying the role of CTLs in the control of SIV and human immunodeficiency virus infections are now available: epitope-based vaccines, a protocol for an effective induction of CTLs in primates, and a simple and sensitive method for quantitation of epitope-specific T cells. The advantages of the DNA prime-MVA boost regimen as well as the correlations of tetramer staining of peripheral blood lymphocytes with CTL killing in vitro and postchallenge control of viremia are discussed. PMID:10438842

Human cytomegalovirus microRNA miR-US4-1 inhibits CD8+ T cell response by targeting the aminopeptidase ERAP1

PubMed Central

Kim, Sungchul; Lee, Sanghyun; Shin, Jinwook; Kim, Youngkyun; Evnouchidou, Irini; Kim, Donghyun; Kim, Young-Kook; Kim, Young-Eui; Ahn, Jin-Hyun; Riddell, Stanley R.; Stratikos, Efstratios; Kim, V. Narry; Ahn, Kwangseog

2012-01-01

The major histocompatibility complex (MHC) class I molecules present peptides on the cell surface by CD8+ T cells, which is critical for killing of virally infected or transformed cells. Precursors of MHC class I-presented peptides are trimmed to mature epitopes by endoplasmic reticulum aminopeptidase 1 (ERAP1). The US2-US11 genomic region of human cytomegalovirus (HCMV) is dispensable for viral replication and harbors 3 microRNAs (miRNAs). We show here the HCMV miR-US4-1 specifically down-regulates ERAP1 expression during viral infection. Accordingly, the trimming of HCMV-derived peptides is inhibited, leading to reduced susceptibility of infected cells to HCMV-specific cytotoxic T lymphocytes (CTLs). Our findings reveal a novel viral miRNA-based CTL evasion mechanism that targets a key step in the MHC class I antigen-processing pathway. PMID:21892175

Creatinine and HMH (5-hydroxy-1-methylhydantoin, NZ-419) as intrinsic hydroxyl radical scavengers.

PubMed

Ienaga, K; Yokozawa, T

2011-08-01

Creatinine (Crn) is one of the main intrinsic hydroxyl radical (•OH) scavengers and an ideal one for healthy or normal mammals, although this fact has not yet become widely accepted. Our results from urinary data estimated that ca. 0.4-0.6% of Crn is used daily to scavenge •OH in normal mammals [ca. 50 μmole and ca. 400 pmole of •OH in healthy subjects and normal rats, respectively]. In human subjects, Crn reacts non-enzymatically with •OH to form creatol (CTL: 5-hydroxycreatinine) and demethylcreatinine (DMC) in a one to one ratio, and CTL partially decomposes to methylguanidine (MG). And so, the scavenged mole of •OH by Crn is nearly equal to their molar total sum (CTL + MG + DMC) or 2 × (CTL + MG). The molar ratio of (scavenged •OH)/Crn in healthy subjects and normal rats are 4.4 and 6.0 mmole/mole, respectively, i.e. almost similar, but in patients with chronic kidney disease (CKD) the ratio increases up to ca. 60 mmole/mole in proportion to the severity of CKD. Since the level of Crn might not be enough to scavenge all •OH, and MG starts accumulating as a uremic toxin, Crn is not really the ideal scavenger. 5-Hydroxy-1-methylhydantoin (HMH, NZ-419), a Crn metabolite, is another antioxidant, having •OH scavenging ability, and has been shown to inhibit the progression of CKD in rats in stead of Crn, if sufficient amounts are given orally.

Impacts on soils and residual trees from cut-to-length thinning operations in California's redwood forests

Treesearch

Kyungrok Hwang; Han-sup Han; Susan E. Marshall; Deborah S. Page-Dumroese

2017-01-01

Cut-to-length (CTL) harvest systems have recently been introduced for thinning third-growth, young (<25 years old) redwood forests (Sequoia sempervirens (Lamb. ex D. Don) Endl.) in northern California. This type of harvesting can effective for thinning overstocked stands consisting of small-diameter trees. However, forestland managers and government agencies...

Modelling the Evolution and Spread of HIV Immune Escape Mutants

PubMed Central

Fryer, Helen R.; Frater, John; Duda, Anna; Roberts, Mick G.; Phillips, Rodney E.; McLean, Angela R.

2010-01-01

During infection with human immunodeficiency virus (HIV), immune pressure from cytotoxic T-lymphocytes (CTLs) selects for viral mutants that confer escape from CTL recognition. These escape variants can be transmitted between individuals where, depending upon their cost to viral fitness and the CTL responses made by the recipient, they may revert. The rates of within-host evolution and their concordant impact upon the rate of spread of escape mutants at the population level are uncertain. Here we present a mathematical model of within-host evolution of escape mutants, transmission of these variants between hosts and subsequent reversion in new hosts. The model is an extension of the well-known SI model of disease transmission and includes three further parameters that describe host immunogenetic heterogeneity and rates of within host viral evolution. We use the model to explain why some escape mutants appear to have stable prevalence whilst others are spreading through the population. Further, we use it to compare diverse datasets on CTL escape, highlighting where different sources agree or disagree on within-host evolutionary rates. The several dozen CTL epitopes we survey from HIV-1 gag, RT and nef reveal a relatively sedate rate of evolution with average rates of escape measured in years and reversion in decades. For many epitopes in HIV, occasional rapid within-host evolution is not reflected in fast evolution at the population level. PMID:21124991

Tracking in vivo migration and distribution of antigen-specific cytotoxic T lymphocytes by 5,6-carboxyfluorescein diacetate succinimidyl ester staining during cancer immunotherapy.

PubMed

Xu, Wei-li; Li, Suo-lin; Wen, Ming; Wen, Jun-ye; Han, Jie; Zhang, Hong-zhen; Gao, Fei; Cai, Jian-hui

2013-08-01

Killing of targeted tumors during adoptive cell transfer therapy is associated with cytotoxic T lymphocyte (CTL) numbers, immunophenotype, tumor-specificity, and in vivo residence time, migration, and distribution. Therefore, tracing in vivo persistence, migration, and distribution of CTLs is important for cancer immunotherapy. Optimal staining concentration for CTL proliferation was determined by cell counting kit-8 (CCK-8) assay and killing efficiencies of CTLs or carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled melanoma antigen-specific cytotoxic T lymphocytes (CFSE-CTLs) for malignant melanoma cells in vitro were compared. Additionally, CFSE-CTLs were intravenously transfused to mice receiving B16 melanoma, and their residence time, migration, and distribution in vivo were observed by measuring fluorescence intensities of CFSE-CTLs per gram of tissue (%FI/g) in various tissues and analyzing tumor/non-tumor (T/NT) values. Anti-tumor effects of transferred CTLs and correlation between %FI/g and D-value of tumor size were analyzed. Five-micromolar CFSE was optimal for labeling CTLs with minimal cytotoxicity. No significant difference occurred between CTLs and CFSE-CTLs for tumor cell killing (P = 0.849) or interleukin-2 (P = 0.318) and interferon-γ (P = 0.201) levels. Distribution of CTLs in vivo varied with time. A negative correlation between %FI/g in tumors and D-value of tumor sizes by Spearman correlation analysis was observed. CTLs were recruited to and killed tumors from 6 hours to 3 days after cell infusion. CTLs were observed up to three weeks later in the tumor, liver, kidneys, and spleen; this was related to the abundant blood supply or the nature of immune organs. CCK-8 assay is a novel method to select optimal CFSE staining concentrations. Fluorescence intensity of transferred CTLs reflects their killing efficiency of tumors. CFSE fluorescent markers can trace in vivo CTL persistence, migration, and distribution because of its stability, long half-life, and low toxicity.

Genetic characterization of the HIV-1 reservoir after Vacc-4x and romidepsin therapy in HIV-1 infected individuals.

PubMed

Winckelmann, Anni; Morcilla, Vincent; Shao, Wei; Schleimann, Mariane H; Højen, Jesper F; Schlub, Timothy E; Denton, Paul W; Østergaard, Lars; Søgaard, Ole S; Tolstrup, Martin; Palmer, Sarah

2018-05-11

Therapeutic HIV-1 immunization followed by latency reversal has been suggested as a strategy to eradicate HIV-1. Here we investigate the phylogenetic composition of the HIV-1 regions targeted by the therapeutic HIV-1 peptide vaccine Vacc-4x in participants in a clinical trial. Seventeen participants on suppressive antiretroviral therapy were vaccinated with six doses of Vacc-4x followed by three doses of romidepsin. Seven study participants were selected for sequencing analysis. All participants underwent an analytical treatment interruption. Single-genome/proviral sequencing of the p24-RT region was performed to genetically characterize proviral DNA, cell-associated (CA) RNA and outgrowth viruses during therapy as well as plasma HIV-1 RNA during an analytical treatment interruption. There were no changes in CA HIV-1 RNA (P = 0.83) and DNA (P = 0.09) diversity over the course of the study and no difference between CA HIV-1 RNA and DNA diversity (P = 0.32). Only one participant showed signs of potential vaccine-related selection in the rebounding plasma virus. In five of seven participants, we identified HLA-specific CTL epitopes containing non-silent mutations in 100% of the sequences. We detected no evidence of selective immune pressure reflected in proviral diversity or by occurrence of specific mutation in the vaccine-targeted epitopes. Pre-existing CTL epitope mutations may affect the potency of this therapeutic vaccine. This highlights the challenges of developing effective HIV-1 therapeutic vaccines.This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0.

Life cycle greenhouse gas emissions, consumptive water use and levelized costs of unconventional oil in North America

NASA Astrophysics Data System (ADS)

Mangmeechai, Aweewan

Conventional petroleum production in many countries that supply U.S. crude oil as well as domestic production has declined in recent years. Along with instability in the world oil market, this has stimulated the discussion of developing unconventional oil production, e.g., oil sands and oil shale. Expanding the U.S. energy mix to include oil sands and oil shale may be an important component in diversifying and securing the U.S. energy supply. At the same time, life cycle GHG emissions of these energy sources and consumptive water use are a concern. In this study, consumptive water use includes not only fresh water use but entire consumptive use including brackish water and seawater. The goal of this study is to determine the life cycle greenhouse gas (GHG) emissions and consumptive water use of synthetic crude oil (SCO) derived from Canadian oil sands and U.S. oil shale to be compared with U.S. domestic crude oil, U.S. imported crude oil, and coal-to-liquid (CTL). Levelized costs of SCO derived from Canadian oil sands and U.S. oil shale were also estimated. The results of this study suggest that CTL with no carbon capture and sequestration (CCS) and current electricity grid mix is the worst while crude oil imported from United Kingdom is the best in GHG emissions. The life cycle GHG emissions of oil shale surface mining, oil shale in-situ process, oil sands surface mining, and oil sands in-situ process are 43% to 62%, 13% to 32%, 5% to 22%, and 11% to 13% higher than those of U.S. domestic crude oil. Oil shale in-situ process has the largest consumptive water use among alternative fuels, evaluated due to consumptive water use in electricity generation. Life cycle consumptive water use of oil sands in-situ process is the lowest. Specifically, fresh water consumption in the production processes is the most concern given its scarcity. However, disaggregated data on fresh water consumption in the total water consumption of each fuel production process is not available. Given current information, it is inconclusive whether unconventional oil would require more or less consumptive fresh water use than U.S. domestic crude oil production. It depends on the water conservative strategy applied in each process. Increasing import of SCO derived from Canadian oil sands and U.S. oil shale would slightly increase life cycle GHG emissions of the U.S. petroleum status quo. The expected additional 2 million bpd of Canadian SCO from oil sands and U.S. oil shale would increase life cycle GHG emissions of the U.S. petroleum status quo on average only 10 and 40 kg CO2 equiv/bbl, or about 7.5 and 29 million tons CO2 equiv/year. However this increase represents less than 1 and 5% of U.S. transportation emissions in 2007. Because U.S. oil shale resources are located in areas experiencing water scarcity, methods to manage the issue were explored. The result also shows that trading water rights between Upper and Lower Colorado River basin and transporting synthetic crude shale oil to refinery elsewhere is the best scenario for life cycle GHG emissions and consumptive water use of U.S. oil shale production. GHG emissions and costs of water supply system contribute only 1-2% of life cycle GHG emissions and 1-6% of total levelized costs. The levelized costs of using SCO from oil shale as feedstock are greater than SCO from oil sands, and CTL. The levelized costs of producing liquid fuel (gasoline and diesel) using SCO derived from Canadian oil sands as feedstock are approximately 0.80-1.00/gal of liquid fuel. The levelized costs of SCO derived from oil shale are 1.6-4.5/gal of liquid fuel (oil shale surface mining process) and 1.6-5.2/gal of liquid fuel (oil shale in-situ process). From an energy security perspective, increasing the use of Canadian oil sands, U.S. oil shale, and CTL may be preferable to increasing Middle East imports. However, oil shale and CTL has the advantage security wise over Canadian oil sands because oil shale and coal are abundant U.S. resources. From a GHG emissions and consumptive water use perspective, CTL requires less consumptive water use than oil shale in-situ process but produces more GHG emissions than oil shale in-situ and surface mining process, unless CTL plant performs CCS and renewable electricity.

Low-pressure hydrocracking of coal-derived Fischer-Tropsch waxes to diesel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dieter Leckel

2007-06-15

Coal-derived low-temperature Fischer-Tropsch (LTFT) wax was hydrocracked at pressures of 3.5-7.0 MPa using silica-alumina-supported sulfided NiW/NiMo and an unsulfided noble metal catalyst, modified with MoO{sub 3}. A low-pressure operation at 3.5 MPa produced a highly isomerized diesel, having low cloud points (from -12 to -28{sup o}C) combined with high cetane numbers (69-73). These properties together with the extremely low sulfur ({lt}5 ppm) and aromatic ({lt}0.5%) contents place coal/liquid (CTL) derived distillates as highly valuable blending components to achieve Eurograde diesel specifications. The upgrading of coal-based LTFT waxes through hydrocracking to high-quality diesel fuel blend components in combination with commercial-feasible coal-integratedmore » gasification combined cycle (coal-IGCC) CO{sub 2} capture and storage schemes should make CTL technology more attractive. 28 refs., 7 figs., 8 tabs.« less

The primary immune response to Vaccinia virus vaccination includes cells with a distinct cytotoxic effector CD4 T-cell phenotype.

PubMed

Munier, C Mee Ling; van Bockel, David; Bailey, Michelle; Ip, Susanna; Xu, Yin; Alcantara, Sheilajen; Liu, Sue Min; Denyer, Gareth; Kaplan, Warren; Suzuki, Kazuo; Croft, Nathan; Purcell, Anthony; Tscharke, David; Cooper, David A; Kent, Stephen J; Zaunders, John J; Kelleher, Anthony D

2016-10-17

Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-stimulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Wider stall space affects behavior, lesion scores, and productivity of gestating sows.

PubMed

Salak-Johnson, J L; DeDecker, A E; Levitin, H A; McGarry, B M

2015-10-01

Limited space allowance within the standard gestation stall is an important welfare concern because it restricts the ability of the sow to make postural adjustments and hinders her ability to perform natural behaviors. Therefore, we evaluated the impacts of increasing stall space and/or providing sows the freedom to access a small pen area on sow well-being using multiple welfare metrics. A total of 96 primi- and multiparous crossbred sows were randomly assigned in groups of 4 sows/treatment across 8 replicates to 1 of 3 stall treatments (TRT): standard stall (CTL; dimensions: 61 by 216 cm), width-adjustable stall (flex stall [FLX]; dimensions: adjustable width of 56 to 79 cm by 216 cm), or an individual walk-in/lock-in stall with access to a small communal open-pen area at the rear of the stall (free-access stall [FAS]; dimensions: 69 by 226 cm). Lesion scores, behavior, and immune and productivity traits were measured at various gestational days throughout the study. Total lesion scores were greatest for sows in FAS and least for sows in FLX ( < 0.001). Higher-parity sows in FAS had the most severe lesion scores (TRT × parity, < 0.0001) and scores were greatest at all gestational days (TRT × day, < 0.05). Regardless of parity, sows in FLX had the least severe scores ( < 0.0001). As pregnancy progressed, lesion scores increased among sows in CTL ( < 0.05). Sow BW and backfat (BF) were greater for sows in FLX and FAS ( < 0.05), and BCS and BF were greater for parity 1 and 2 sows in FAS than the same parity sows in CTL (TRT × parity, < 0.05). Duration and frequency of some postural behaviors and sham chew behavior were affected by TRT ( < 0.05) and time of day (TRT × day, < 0.05). These data indicate that adequate stall space, especially late in gestation, may improve the well-being of higher-parity and heavier-bodied gestating sows as assessed by changes in postural behaviors, lesion severity scores, and other sow traits. Moreover, compromised welfare measures found among sows in various stall environments may be partly attributed to the specific constraints of each stall system such as restricted stall space in CTL, insufficient floor space in the open-pen area of the FAS system, and gate design of the FLX (e.g., direction of bars and feeder space). These results also indicate that parity and gestational day are additional factors that may exacerbate the effects of restricted stall space or insufficient pen space, further compromising sow well-being.

Memory CD8+ T Cells Protect Dendritic Cells from CTL Killing1

PubMed Central

Watchmaker, Payal B.; Urban, Julie A.; Berk, Erik; Nakamura, Yutaro; Mailliard, Robbie B.; Watkins, Simon C.; van Ham, S. Marieke; Kalinski, Pawel

2010-01-01

CD8+ T cells have been shown to be capable of either suppressing or promoting immune responses. To reconcile these contrasting regulatory functions, we compared the ability of human effector and memory CD8+ T cells to regulate survival and functions of dendritic cells (DC). We report that, in sharp contrast to the effector cells (CTLs) that kill DCs in a granzyme B- and perforin-dependent mechanism, memory CD8+ T cells enhance the ability of DCs to produce IL-12 and to induce functional Th1 and CTL responses in naive CD4+ and CD8+ T cell populations. Moreover, memory CD8+ T cells that release the DC-activating factor TNF-α before the release of cytotoxic granules induce DC expression of an endogenous granzyme B inhibitor PI-9 and protect DCs from CTL killing with similar efficacy as CD4+ Th cells. The currently identified DC-protective function of memory CD8+ T cells helps to explain the phenomenon of CD8+ T cell memory, reduced dependence of recall responses on CD4+ T cell help, and the importance of delayed administration of booster doses of vaccines for the optimal outcome of immunization. PMID:18322193

Gag-Positive Reservoir Cells Are Susceptible to HIV-Specific Cytotoxic T Lymphocyte Mediated Clearance In Vitro and Can Be Detected In Vivo

PubMed Central

Graf, Erin H.; Pace, Matthew J.; Peterson, Bennett A.; Lynch, Lindsay J.; Chukwulebe, Steve B.; Mexas, Angela M.; Shaheen, Farida; Martin, Jeffrey N.; Deeks, Steven G.; Connors, Mark; Migueles, Stephen A.; O’Doherty, Una

2013-01-01

Resting CD4+ T cells infected with HIV persist in the presence of suppressive anti-viral therapy (ART) and are barriers to a cure. One potential curative approach, therapeutic vaccination, is fueled by recognition of the ability of a subset of elite controllers (EC) to control virus without therapy due to robust anti-HIV immune responses. Controllers have low levels of integrated HIV DNA and low levels of replication competent virus, suggesting a small reservoir. As our recent data indicates some reservoir cells can produce HIV proteins (termed GPR cells for Gag-positive reservoir cells), we hypothesized that a fraction of HIV-expressing resting CD4+ T cells could be efficiently targeted and cleared in individuals who control HIV via anti-HIV cytotoxic T lymphocytes (CTL). To test this we examined if superinfected resting CD4+ T cells from EC express HIV Gag without producing infectious virus and the susceptibility of these cells to CTL. We found that resting CD4+ T cells expressed HIV Gag and were cleared by autologous CD8+ T cells from EC. Importantly, we found the extent of CTL clearance in our in vitro assay correlates with in vivo reservoir size and that a population of Gag expressing resting CD4+ T cells exists in vivo in patients well controlled on therapy. PMID:23951263

Immune Senescence Factors Associated with the Immunogenicity of a Live Attenuated Zoster Vaccine (ZV) in Older Adults

PubMed Central

Weinberg, Adriana; Schamder, Kenneth; Johnson, Michael; Popmihajlov, Zoran; Tovar-Salazar, Adriana; Caldas, Yupanqui; Pang, Lei; Cho, Alice; Levin, Myron

2017-01-01

Abstract Background ZV confers protection against herpes zoster by increasing the cell-mediated immunity (CMI) to varicella-zoster virus (VZV). ZV immunogenicity and protection decrease with increasing age. We investigated effects of age and immune senescence on ZV immunogenicity. Methods 399 adults ≥50 years had VZV T-cell helper 1 (Th1) CMI measured by ex vivo VZV-stimulated IL2/IFNg ELISPOT and blood T-cell nonspecific immune senescence by flow cytometric characterization of FOXP3, CD25, IL10, TGFb, PD1, CD28, CD57 and CD31 expression before and at 1, 6 and 52 weeks after ZV. In a subset of 95 vaccinees, VZV-stimulated T cell expression of CD107, Granzyme B, FOXP3, CD25, IL10, TGFb, CD39 and PD1 were also measured. Multivariate regression analysis was used to identify independent effects of age and immune senescence on VZV Th1 CMI (P < 0.025). Results IL2+ and IL2+IFNg+ Th1 memory VZV CMI peaked at 6 weeks after ZV and remained elevated at 1 year. Effectors, including VZV-specific IFNg+ Th1, and CD8+CD107+% and CD4+/CD8+Granzyme B+% cytotoxic T lymphocytes (CTL), peaked at 1 week, but only the IFNg+ Th1 effectors remained elevated at 1 year. There was also a transient increase in blood CD8+PD1+% exhausted T cells 1 week after ZV. Independent positive effects on peak memory Th1 VZV CMI included the baseline CMI and negative effects included blood CD4+FOXP3+% T regulatory (Treg) and CD8+PD1+% T exhausted cells. Independent positive effects on peak effector Th1 VZV CMI included baseline CMI and negative effects included blood CD8+CD25+FOXP3+% Treg. Age did not have an independent effect on peak CMI. Independent positive effects on persistent (1 year) memory Th1 included baseline CMI and negative effects included age, blood CD4+FOXP3+% Treg and CD8+PD1+% T exhausted cells. Persistent effector Th1 CMI was negatively affected by age only. Conclusion ZV generated VZV-specific Th1 and CTL responses. The early increase of CD8+ exhausted T cells in blood suggested that CTL responses to the vaccine virus may be compromised by immune senescence. The negative of age on VZV Th1 CMI was fully mediated by immune senescence at peak response, but age had a negative effect on CMI persistence that was independent from the markers of immune senescence included in this study. Disclosures A. Weinberg, merck: Grant Investigator, Research grant K. Schamder, merck: Grant Investigator, Research grant Z. Popmihajlov, Merck & Co., Inc.: Employee and Shareholder, Salary L. Pang, Merck: Employee and Shareholder, Salary M. Levin, merck: Grant Investigator and Scientific Advisor, Consulting fee and Research grant

The use of a magnesium-based bone adhesive for flexor tendon-to-bone healing

PubMed Central

Stavros, Thomopoulos; Emmanouil, Zampiakis; Rosalina, Das; Hyun-Min, Kim; J., Silva, Matthew; Necat, Havlioglu; H., Gelberman, Richard

2010-01-01

Purpose Our previous studies in a canine animal model demonstrated that the flexor tendon-to-bone insertion site has a poor capacity to heal. Magnesium based adhesives have the potential to improve tendon-to-bone healing. Therefore, we hypothesized that magnesium based bone adhesive (MBA) will improve the tendon-to-bone biomechanical properties initially and in the early period after repair. Methods Flexor digitorum profundus tendons were injured and repaired into bone tunnels in the distal phalanges of dogs. The bone tunnels were either filled with MBA prior to completing the repair or left empty (CTL). Histologic appearance, tensile properties, range of motion, and bone density were examined at time zero and 21 days after the repair. Results There was no histologic evidence of acute inflammation. There appeared to be more mast cells in the MBA group than in the CTL group. Chronic inflammatory infiltrate and fibrosis was slightly higher in the MBA group compared to the CTL group. Tensile properties at time zero were significantly higher in the MBA group compared to the CTL group. However, tensile properties were significantly lower in the MBA group compared to the CTL group at 21 days. Range of motion and bone density were significantly lower in the MBA and CTL groups compared to normal (i.e., uninjured) at 21 days; no differences were seen when comparing MBA to CTL. Conclusions We found that the initial biomechanical properties of flexor tendon-to-bone repairs can be improved with MBA. However, MBA use in vivo led to a decrease in the biomechanical properties of the repair. There was no effect of MBA on bone density or range of motion in the early period after repair. Our histologic analysis suggests that the poor healing in the MBA group may have been due to an allergic response or to increased chronic inflammation due to the foreign material. PMID:19643291

The effect of dietary supplementation of algae rich in docosahexaenoic acid on boar fertility.

PubMed

Murphy, E M; Stanton, C; Brien, C O '; Murphy, C; Holden, S; Murphy, R P; Varley, P; Boland, M P; Fair, S

2017-03-01

The objective of this study was to assess the effects of dietary supplementation of a commercial algal product rich in docosahexaenoic acid (DHA) on boar fertility as assessed in vitro and in vivo. Boars were fed one of three experimental diets for 19 weeks: (i) Control (Ctl) diet (n = 31), (ii) Ctl diet plus 75g All-G-Rich per day (n = 31) or (iii) Ctl diet plus 150g All-G-Rich per day (n = 30). Parameters assessed were (i) raw semen quality; volume, sperm concentration, total motility and morphology (ii) liquid semen quality; progressive motility, viability, hypotonic resistance and acrosomal integrity (iii) frozen-thawed semen quality; motility, thermal stress, viability, membrane fluidity and mitochondrial activity (iv) sperm and seminal plasma (SP) fatty acid composition (FAC) (v) total antioxidant capacity (TAC) of SP and (vi) farrowing rates and litter sizes of sows (n = 1158) inseminated with liquid semen. Boars consuming 75g All-G-Rich had a larger semen volume (P < 0.05) and a higher total sperm number (P < 0.01) than the Ctl treatment, however, there was no effect of treatment on any other semen quality parameter (P > 0.05). There was no effect of dietary treatment on the FAC and TAC of SP or on farrowing rate and litter size (P > 0.05). There was an effect of dietary treatment on the FAC of sperm, represented by an 1.72 and 1.60 fold increase in the DHA content for 75 and 150g treatments, respectively, compared to the Ctl treatment. In conclusion, a significant increase in semen volume and total sperm number in boars supplemented 75g All-G-Rich daily, resulted in an increase in production of 3 to 4 more doses per ejaculate, thus, indicating that the feeding regime described within this study has the potential for increasing the output of boar studs. Copyright © 2016 Elsevier Inc. All rights reserved.

Long-term adaptation of the influenza A virus by escaping cytotoxic T-cell recognition

NASA Astrophysics Data System (ADS)

Woolthuis, Rutger G.; van Dorp, Christiaan H.; Keşmir, Can; de Boer, Rob J.; van Boven, Michiel

2016-09-01

The evolutionary adaptation of the influenza A virus (IAV) to human antibodies is well characterised. Much less is known about the long-term evolution of cytotoxic T lymphocyte (CTL) epitopes, which are important antigens for clearance of infection. We construct an antigenic map of IAVs of all human subtypes using a compendium of 142 confirmed CTL epitopes, and show that IAV evolved gradually in the period 1932-2015, with infrequent antigenic jumps in the H3N2 subtype. Intriguingly, the number of CTL epitopes per virus decreases with more than one epitope per three years in the H3N2 subtype (from 84 epitopes per virus in 1968 to 64 in 2015), mostly attributed to the loss of HLA-B epitopes. We confirm these observations with epitope predictions. Our findings indicate that selection pressures imposed by CTL immunity shape the long-term evolution of IAV.

Antiatherogenic effect of quercetin is mediated by proteasome inhibition in the aorta and circulating leukocytes.

PubMed

Pashevin, Denis A; Tumanovska, Lesya V; Dosenko, Victor E; Nagibin, Vasyl S; Gurianova, Veronika L; Moibenko, Alexey A

2011-01-01

Quercetin, a plant-derived flavonoid, has attracted considerable attention as promising compound for heart disease prevention and therapy. It has been linked to decreased mortality from heart disease and decreased incidence of stroke. Here, we report new data showing the angioprotective properties of quercetin mediated by its effect on proteasomal proteolysis. This study was designed to investigate the ability of quercetin to modulate proteasomal activity in a rabbit model of cholesterol-induced atherosclerosis. First, we show proteasomal trypsin-like (TL) activity increased up to 2.4-fold, chymotrypsin-like (CTL) activity increased by up to 43% and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activity increased by up to 10% after 8 weeks of a cholesterol-rich diet. A single intravenous injection of the water-soluble form of quercetin (Corvitin) significantly decreased proteasomal TL activity 1.85-fold in monocytes, and decreased the CTL and PGPH activities more than 2-fold in polymorphonuclear leukocytes (PMNL) after 2 h. Prolonged administration (1 month) of Corvitin to animals following a cholesterol-rich diet significantly decreased all types of proteolytic proteasome activities both in tissues and in circulating leukocytes and was associated with the reduction of atherosclerotic lesion areas in the aorta. Additionally, the pharmacological form of quercetin (Quertin) was shown to have an antiatherogenic effect and an ability to inhibit proteasome activities.

THE EFFECTS OF A SIMPLE METHOD FOR CRYOPRESERVATION AND THAWING PROCEDURES ON CORD BLOOD DERIVED DC-BASED ESOPHAGEAL CARCINOMA VACCINE.

PubMed

Yu, J; Xie, L; Chen, S; Zhang, J; Guo, G; Chen, B

Producing sufficient numbers of DCs at one time point and subsequently cryopreserving the generated DCs in ready-for-use aliquots for clinical application is useful in cancer treatment. To study the effects of a simplified cryopreservation method and thawing procedures acting on the biological characteristics and specific cytotoxic activity of cord blood derived DC-based esophageal carcinoma vaccine. CD34+ hematopoietic stem cells were isolated from cord blood using CD34+ Progenitor Cell Isolation Kit by magnetic cell sorting system (MACS). The CD34+ cells were expanded with cytokines as DCs, and fused with EC109 cells by PEG-3600. The fused cells were transferred to a freezing tube without rate-controlled freezing and stored at -80 degree C for three weeks. During cryopreservation, 2.5% DMSO, 2.5% glucose and 10% FCS at final concentration was used as stock solution. After thawing, cells were assayed for Typan blue viability, morphology, immunophenotypes and T-cell stimulatory capacity, and specific CTL activity. Cryopreservation does not cause significant changes in the phenotypes expression or morphology of the fused cells, and the viability were well preserved (Typan blue viability was 77.2±1.8%). After being stimulated by DC-based esophageal carcinoma vaccine either before or after cryopreservation, the numbers of CD3+T/CD4+T and CD3+T/CD8+T lymphocytes increased obviously, especially for CD3+T/CD4+T, and the ratio of CD4/CD8 changed from 0.85 to 1.29 and 1.25 respectively. Specific CTL activity were well preserved (compare to the fresh fused vaccine, P>0.05). A simple -80 degree C freezing and storage method is practical for cord blood derived DC-based esophageal carcinoma vaccine. It will greatly facilitate the clinical use of DC-based vaccine for immunotherapy.

MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells

PubMed Central

Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

2015-01-01

Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach. PMID:25949869

MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells.

PubMed

Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

2015-01-01

Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach.

Multipotent adult germ-line stem cells, like other pluripotent stem cells, can be killed by cytotoxic T lymphocytes despite low expression of major histocompatibility complex class I molecules

PubMed Central

Dressel, Ralf; Guan, Kaomei; Nolte, Jessica; Elsner, Leslie; Monecke, Sebastian; Nayernia, Karim; Hasenfuss, Gerd; Engel, Wolfgang

2009-01-01

Background Multipotent adult germ-line stem cells (maGSCs) represent a new pluripotent cell type that can be derived without genetic manipulation from spermatogonial stem cells (SSCs) present in adult testis. Similarly to induced pluripotent stem cells (iPSCs), they could provide a source of cellular grafts for new transplantation therapies of a broad variety of diseases. To test whether these stem cells can be rejected by the recipients, we have analyzed whether maGSCs and iPSCs can become targets for cytotoxic T lymphocytes (CTL) or whether they are protected, as previously proposed for embryonic stem cells (ESCs). Results We have observed that maGSCs can be maintained in prolonged culture with or without leukemia inhibitory factor and/or feeder cells and still retain the capacity to form teratomas in immunodeficient recipients. They were, however, rejected in immunocompetent allogeneic recipients, and the immune response controlled teratoma growth. We analyzed the susceptibility of three maGSC lines to CTL in comparison to ESCs, iPSCs, and F9 teratocarcinoma cells. Major histocompatibility complex (MHC) class I molecules were not detectable by flow cytometry on these stem cell lines, apart from low levels on one maGSC line (maGSC Stra8 SSC5). However, using a quantitative real time PCR analysis H2K and B2m transcripts were detected in all pluripotent stem cell lines. All pluripotent stem cell lines were killed in a peptide-dependent manner by activated CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the SIINFEKL peptide. Conclusion Pluripotent stem cells, including maGSCs, ESCs, and iPSCs can become targets for CTLs, even if the expression level of MHC class I molecules is below the detection limit of flow cytometry. Thus they are not protected against CTL-mediated cytotoxicity. Therefore, pluripotent cells might be rejected after transplantation by this mechanism if specific antigens are presented and if specific activated CTLs are present. Our results show that the adaptive immune system has in principle the capacity to kill pluripotent and teratoma forming stem cells. This finding might help to develop new strategies to increase the safety of future transplantations of in vitro differentiated cells by exploiting a selective immune response against contaminating undifferentiated cells. Reviewers This article was reviewed by Bhagirath Singh, Etienne Joly and Lutz Walter. PMID:19715575

Fitness-Balanced Escape Determines Resolution of Dynamic Founder Virus Escape Processes in HIV-1 Infection

PubMed Central

Sunshine, Justine E.; Larsen, Brendan B.; Maust, Brandon; Casey, Ellie; Deng, Wenje; Chen, Lennie; Westfall, Dylan H.; Kim, Moon; Zhao, Hong; Ghorai, Suvankar; Lanxon-Cookson, Erinn; Rolland, Morgane; Collier, Ann C.; Maenza, Janine; Mullins, James I.

2015-01-01

ABSTRACT To understand the interplay between host cytotoxic T-lymphocyte (CTL) responses and the mechanisms by which HIV-1 evades them, we studied viral evolutionary patterns associated with host CTL responses in six linked transmission pairs. HIV-1 sequences corresponding to full-length p17 and p24 gag were generated by 454 pyrosequencing for all pairs near the time of transmission, and seroconverting partners were followed for a median of 847 days postinfection. T-cell responses were screened by gamma interferon/interleukin-2 (IFN-γ/IL-2) FluoroSpot using autologous peptide sets reflecting any Gag variant present in at least 5% of sequence reads in the individual's viral population. While we found little evidence for the occurrence of CTL reversions, CTL escape processes were found to be highly dynamic, with multiple epitope variants emerging simultaneously. We found a correlation between epitope entropy and the number of epitope variants per response (r = 0.43; P = 0.05). In cases in which multiple escape mutations developed within a targeted epitope, a variant with no fitness cost became fixed in the viral population. When multiple mutations within an epitope achieved fitness-balanced escape, these escape mutants were each maintained in the viral population. Additional mutations found to confer escape but undetected in viral populations incurred high fitness costs, suggesting that functional constraints limit the available sites tolerable to escape mutations. These results further our understanding of the impact of CTL escape and reversion from the founder virus in HIV infection and contribute to the identification of immunogenic Gag regions most vulnerable to a targeted T-cell attack. IMPORTANCE Rapid diversification of the viral population is a hallmark of HIV-1 infection, and understanding the selective forces driving the emergence of viral variants can provide critical insight into the interplay between host immune responses and viral evolution. We used deep sequencing to comprehensively follow viral evolution over time in six linked HIV transmission pairs. We then mapped T-cell responses to explore if mutations arose due to adaption to the host and found that escape processes were often highly dynamic, with multiple mutations arising within targeted epitopes. When we explored the impact of these mutations on replicative capacity, we found that dynamic escape processes only resolve with the selection of mutations that conferred escape with no fitness cost to the virus. These results provide further understanding of the complicated viral-host interactions that occur during early HIV-1 infection and may help inform the design of future vaccine immunogens. PMID:26223634

Vaccination with liposome-coupled glypican-3-derived epitope peptide stimulates cytotoxic T lymphocytes and inhibits GPC3-expressing tumor growth in mice.

PubMed

Iwama, Tatsuaki; Uchida, Tetsuya; Sawada, Yu; Tsuchiya, Nobuhiro; Sugai, Shiori; Fujinami, Norihiro; Shimomura, Manami; Yoshikawa, Toshiaki; Zhang, Rong; Uemura, Yasushi; Nakatsura, Tetsuya

2016-01-01

Because therapeutic manipulation of immunity can induce tumor regression, anti-cancer immunotherapy is considered a promising treatment modality. We previously reported that glypican-3 (GPC3), an oncofetal antigen overexpressed in hepatocellular carcinoma (HCC), is a useful target for cytotoxic T lymphocyte (CTL)-mediated cancer immunotherapy, and we have performed clinical trials using the GPC3-derived peptide vaccine. Although vaccine-induced GPC3-peptide-specific CTLs were often tumor reactive in vitro and were correlated with overall survival, no complete response was observed. In the current study, we synthesized liposome-coupled GPC3-derived CTL epitope peptide (pGPC3-lipsome) and investigated its antitumor potential. Vaccination with pGPC3-liposome induced peptide-specific CTLs at a lower dose than conventional vaccine emulsified in incomplete Freund's adjuvant. Coupling of pGPC3 to liposomes was essential for effective priming of GPC3-specific CTLs. In addition, immunization with pGPC3-liposome inhibited GPC3-expressing tumor growth. Thus, vaccination with tumor-associated antigen-derived epitope peptides coupled to the surfaces of liposomes may be a novel therapeutic strategy for cancer. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

A role for extracellular amastigotes in the immunopathology of Chagas disease.

PubMed

Scharfstein, J; Morrot, A

1999-01-01

In spite of the growing knowledge obtained about immune control of Trypanosoma cruzi infection, the mechanisms responsible for the variable clinico-pathological expression of Chagas disease remain unknown. In a twist from previous concepts, recent studies indicated that tissue parasitism is a pre-requisite for the development of chronic myocarditis. This fundamental concept, together with the realization that T. cruzi organisms consist of genetically heterogeneous clones, offers a new framework for studies of molecular pathogenesis. In the present article, we will discuss in general terms the possible implications of genetic variability of T. cruzi antigens and proteases to immunopathology. Peptide epitopes from a highly polymorphic subfamily of trans-sialidase (TS) antigens were recently identified as targets of killer T cell (CTL) responses, both in mice and humans. While some class I MHC restricted CTL recognize epitopes derived from amastigote-specific TS-related antigens (TSRA), others are targeted to peptide epitopes originating from trypomastigote-specific TSRA. A mechanistic hypothesis is proposed to explain how the functional activity and specificity of class I MHC restricted killer T cells may control the extent to which tissue are exposed to prematurely released amastigotes. Chronic immunopathology may be exacerbated due the progressive accumulation of amastigote-derived antigens and pro-inflammatory molecules (eg. GPI-mucins and kinin-releasing proteases) in dead macrophage bodies.

Protective antitumor activity through dendritic cell immunization is mediated by NK cell as well as CTL activation.

PubMed

Kim, K D; Kim, J K; Kim, S J; Choe, I S; Chung, T H; Choe, Y K; Lim, J S

1999-08-01

Dendritic cells (DCs) are potent professional antigen-presenting cells (APC) capable of inducing the primary T cell response to antigen. Although tumor cells express target antigens, they are incapable of stimulating a tumor-specific immune response due to a defect in the costimulatory signal that is required for optimal activation of T cells. In this work, we describe a new approach using tumor-DC coculture to improve the antigen presenting capacity of tumor cells, which does not require a source of tumor-associated antigen. Immunization of a weakly immunogenic and progressive tumor cocultured with bone marrow-derived DCs generated an effective tumor vaccine. Immunization with the cocultured DCs was able to induce complete protective immunity against tumor challenges and was effective for the induction of tumor-specific CTL (cytotoxic T lymphocyte) activity. Furthermore, high NK cell activity was observed in mice in which tumors were rejected. In addition, immunization with tumor-pulsed DCs induced delayed tumor growth, but not tumor eradication in tumor-bearing mice. Our results demonstrate that coculture of DCs with tumors generated antitumor immunity due to the NK cell activation as well as tumor-specific T cell. This approach would be useful for designing tumor vaccines using DCs when the information about tumor antigens is limited.

PLGA nanoparticle-mediated delivery of tumor antigenic peptides elicits effective immune responses

PubMed Central

Ma, Wenxue; Chen, Mingshui; Kaushal, Sharmeela; McElroy, Michele; Zhang, Yu; Ozkan, Cengiz; Bouvet, Michael; Kruse, Carol; Grotjahn, Douglas; Ichim, Thomas; Minev, Boris

2012-01-01

The peptide vaccine clinical trials encountered limited success because of difficulties associated with stability and delivery, resulting in inefficient antigen presentation and low response rates in patients with cancer. The purpose of this study was to develop a novel delivery approach for tumor antigenic peptides in order to elicit enhanced immune responses using poly(DL-lactide-co-glycolide) nanoparticles (PLGA-NPs) encapsulating tumor antigenic peptides. PLGA-NPs were made using the double emulsion-solvent evaporation method. Artificial antigen-presenting cells were generated by human dendritic cells (DCs) loaded with PLGA-NPs encapsulating tumor antigenic peptide(s). The efficiency of the antigen presentation was measured by interferon-γ ELISpot assay (Vector Laboratories, Burlingame, CA). Antigen-specific cytotoxic T lymphocytes (CTLs) were generated and evaluated by CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Fitchburg, WI). The efficiency of the peptide delivery was compared between the methods of emulsification in incomplete Freund’s adjuvant and encapsulation in PLGA-NPs. Our results showed that most of the PLGA-NPs were from 150 nm to 500 nm in diameter, and were negatively charged at pH 7.4 with a mean zeta potential of −15.53 ± 0.71 mV; the PLGA-NPs could be colocalized in human DCs in 30 minutes of incubation. Human DCs loaded with PLGA-NPs encapsulating peptide induced significantly stronger CTL cytotoxicity than those pulsed with free peptide, while human DCs loaded with PLGA-NPs encapsulating a three-peptide cocktail induced a significantly greater CTL response than those encapsulating a two-peptide cocktail. Most importantly, the peptide dose encapsulated in PLGA-NPs was 63 times less than that emulsified in incomplete Freund’s adjuvant, but it induced a more powerful CTL response in vivo. These results demonstrate that the delivery of peptides encapsulated in PLGA-NPs is a promising approach to induce effective antitumor CTL responses in vivo. PMID:22619507

PLGA nanoparticle-mediated delivery of tumor antigenic peptides elicits effective immune responses.

PubMed

Ma, Wenxue; Chen, Mingshui; Kaushal, Sharmeela; McElroy, Michele; Zhang, Yu; Ozkan, Cengiz; Bouvet, Michael; Kruse, Carol; Grotjahn, Douglas; Ichim, Thomas; Minev, Boris

2012-01-01

The peptide vaccine clinical trials encountered limited success because of difficulties associated with stability and delivery, resulting in inefficient antigen presentation and low response rates in patients with cancer. The purpose of this study was to develop a novel delivery approach for tumor antigenic peptides in order to elicit enhanced immune responses using poly(DL-lactide-co-glycolide) nanoparticles (PLGA-NPs) encapsulating tumor antigenic peptides. PLGA-NPs were made using the double emulsion-solvent evaporation method. Artificial antigen-presenting cells were generated by human dendritic cells (DCs) loaded with PLGA-NPs encapsulating tumor antigenic peptide(s). The efficiency of the antigen presentation was measured by interferon-γ ELISpot assay (Vector Laboratories, Burlingame, CA). Antigen-specific cytotoxic T lymphocytes (CTLs) were generated and evaluated by CytoTox 96(®) Non-Radioactive Cytotoxicity Assay (Promega, Fitchburg, WI). The efficiency of the peptide delivery was compared between the methods of emulsification in incomplete Freund's adjuvant and encapsulation in PLGA-NPs. Our results showed that most of the PLGA-NPs were from 150 nm to 500 nm in diameter, and were negatively charged at pH 7.4 with a mean zeta potential of -15.53 ± 0.71 mV; the PLGA-NPs could be colocalized in human DCs in 30 minutes of incubation. Human DCs loaded with PLGA-NPs encapsulating peptide induced significantly stronger CTL cytotoxicity than those pulsed with free peptide, while human DCs loaded with PLGA-NPs encapsulating a three-peptide cocktail induced a significantly greater CTL response than those encapsulating a two-peptide cocktail. Most importantly, the peptide dose encapsulated in PLGA-NPs was 63 times less than that emulsified in incomplete Freund's adjuvant, but it induced a more powerful CTL response in vivo. These results demonstrate that the delivery of peptides encapsulated in PLGA-NPs is a promising approach to induce effective antitumor CTL responses in vivo.

Establishment and operation of a Good Manufacturing Practice-compliant allogeneic Epstein-Barr virus (EBV)-specific cytotoxic cell bank for the treatment of EBV-associated lymphoproliferative disease

PubMed Central

Vickers, Mark A; Wilkie, Gwen M; Robinson, Nicolas; Rivera, Nadja; Haque, Tanzina; Crawford, Dorothy H; Barry, Jacqueline; Fraser, Neil; Turner, David M; Robertson, Victoria; Dyer, Phil; Flanagan, Peter; Newlands, Helen R; Campbell, John; Turner, Marc L

2014-01-01

Epstein-Barr virus (EBV) is associated with several malignancies, including post-transplant lymphoproliferative disorder (PTLD). Conventional treatments for PTLD are often successful, but risk organ rejection and cause significant side effects. EBV-specific cytotoxic T lymphocytes (CTLs) generated in vitro from peripheral blood lymphocytes provide an alternative treatment modality with few side effects, but autologous CTLs are difficult to use in clinical practice. Here we report the establishment and operation of a bank of EBV-specific CTLs derived from 25 blood donors with human leucocyte antigen (HLA) types found at high frequency in European populations. Since licensure, there have been enquiries about 37 patients, who shared a median of three class I and two class II HLA types with these donors. Cells have been infused into ten patients with lymphoproliferative disease, eight of whom achieved complete remission. Neither patient with refractory disease was matched for HLA class II. Both cases of EBV-associated non-haematopoietic sarcoma receiving cells failed to achieve complete remission. Thirteen patients died before any cells could be issued, emphasizing that the bank should be contacted before patients become pre-terminal. Thus, this third party donor-derived EBV-specific CTL cell bank can supply most patients with appropriately matched cells and most recipients have good outcomes. PMID:25066775

Effects of Developmental Bisphenol A Exposure on Reproductive-Related Behaviors in California Mice (Peromyscus californicus): A Monogamous Animal Model

PubMed Central

Williams, Scott A.; Jasarevic, Eldin; Vandas, Gregory M.; Warzak, Denise A.; Geary, David C.; Ellersieck, Mark R.; Roberts, R. Michael; Rosenfeld, Cheryl S.

2013-01-01

Bisphenol A (BPA), a pervasive, endocrine disrupting compound (EDC), acts as a mixed agonist- antagonist with respect to estrogens and other steroid hormones. We hypothesized that sexually selected traits would be particularly sensitive to EDC. Consistent with this concept, developmental exposure of males from the polygynous deer mouse, Peromyscus maniculatus, to BPA resulted in compromised spatial navigational ability and exploratory behaviors, while there was little effect on females. Here, we have examined a related, monogamous species, the California mouse (Peromyscus californicus), where we predicted that males would be less sensitive to BPA in terms of navigational and exploratory behaviors, while displaying other traits related to interactions with females and territorial marking that might be vulnerable to disruption. As in the deer mouse experiments, females were fed either a phytoestrogen-free CTL diet through pregnancy and lactation or the same diet supplemented with BPA (50 mg/kg feed weight) or ethinyl estradiol (EE) (0.1 part per billion) to provide a “pure” estrogen control. After weaning, pups were maintained on CTL diet until they had reached sexual maturity, at which time behaviors were evaluated. In addition, territorial marking was assessed in BPA-exposed males housed alone and when a control male was visible in the testing arena. In contrast to deer mice, BPA and EE exposure had no effect on spatial navigational skills in either male or female California mice. While CTL females exhibited greater exploratory behavior than CTL males, BPA exposure abolished this sex difference. BPA-exposed males, however, engaged in less territorial marking when CTL males were present. These studies demonstrate that developmental BPA exposure can disrupt adult behaviors in a sex- and species-dependent manner and are consistent with the hypothesis that sexually selected traits are particularly vulnerable to endocrine disruption and should be a consideration in risk assessment studies. PMID:23405200

Elicitation of anti-viral cytotoxic T lymphocytes with purified viral and H-2 antigens.

PubMed

Hale, A H; Ruebush, M J; Harris, D T

1980-07-01

The minimal molecular requirements for elicitation of secondary anti-Sendai virus CTL were investigated. The hemagglutinin-neuraminidase (HN) glycoprotein of Sendai virus and the H-2Kk glycoprotein of YAC tumor cells were purified and incorporated into phospholipid vesicles. These unilamellar liposomes were then tested for the ability to elicit H-2 restricted secondary anti-Sendai virus CTL. The results indicate that these well-defined vesicles were capable of eliciting secondary anti-Sendai virus CTL which lysed only target cells possessing the H-2Kk haplotype and modified with inactivated Sendai virus.

DR3 regulation of apoptosis of naive T-lymphocytes in children with acute infectious mononucleosis.

PubMed

Filatova, Elena Nikolaevna; Anisenkova, Elena Viktorovna; Presnyakova, Nataliya Borisovna; Utkin, Oleg Vladimirovich

2016-09-01

Acute infectious mononucleosis (AIM) is a widespread viral disease that mostly affects children. Development of AIM is accompanied by a change in the ratio of immune cells. This is provided by means of different biological processes including the regulation of apoptosis of naive T-cells. One of the potential regulators of apoptosis of T-lymphocytes is a death receptor 3 (DR3). We have studied the role of DR3 in the regulation of apoptosis of naive CD4 + (nTh) and CD8 + (nCTL) T-cells in healthy children and children with AIM. In healthy children as well as in children with AIM, the activation of DR3 is accompanied by inhibition of apoptosis of nTh. In healthy children, the stimulation of DR3 resulted in the increase in apoptosis of nCTL. On the contrary, in children with AIM, the level of apoptosis of nCTL decreased after DR3 activation, which is a positive contribution to the antiviral immune response. In children with AIM, nCTL are characterized by reduced level of apoptosis as compared with healthy children. These results indicate that DR3 can be involved in the reduction of sensitivity of nCTL to apoptosis in children with AIM.

Implementing the Simple Biosphere Model (SiB) in a general circulation model: Methodologies and results

NASA Technical Reports Server (NTRS)

Sato, N.; Sellers, P. J.; Randall, D. A.; Schneider, E. K.; Shukla, J.; Kinter, J. L., III; Hou, Y.-T.; Albertazzi, E.

1989-01-01

The Simple Biosphere MOdel (SiB) of Sellers et al., (1986) was designed to simulate the interactions between the Earth's land surface and the atmosphere by treating the vegetation explicitly and relistically, thereby incorporating biophysical controls on the exchanges of radiation, momentum, sensible and latent heat between the two systems. The steps taken to implement SiB in a modified version of the National Meteorological Center's spectral GCM are described. The coupled model (SiB-GCM) was used with a conventional hydrological model (Ctl-GCM) to produce summer and winter simulations. The same GCM was used with a conventional hydrological model (Ctl-GCM) to produce comparable 'control' summer and winter variations. It was found that SiB-GCM produced a more realistic partitioning of energy at the land surface than Ctl-GCM. Generally, SiB-GCM produced more sensible heat flux and less latent heat flux over vegetated land than did Ctl-GCM and this resulted in the development of a much deeper daytime planetary boundary and reduced precipitation rates over the continents in SiB-GCM. In the summer simulation, the 200 mb jet stream and the wind speed at 850 mb were slightly weakened in the SiB-GCM relative to the Ctl-GCM results and equivalent analyses from observations.

Vitamin D prevents articular cartilage erosion by regulating collagen II turnover through TGF-β1 in ovariectomized rats.

PubMed

Li, S; Niu, G; Wu, Y; Du, G; Huang, C; Yin, X; Liu, Z; Song, C; Leng, H

2016-02-01

To explore the effect of vitamin D on turnover of articular cartilage with ovariectomy (OVX) induced OA, and to investigate transforming growth factor-β1 (TGF-β1) as a possible underlying mechanism mediated by 1α,25(OH)2D3. Sixty-six rats were randomly allocated into seven groups: sham plus control diet (SHAM+CTL), OVX+CTL diet, sham plus vitamin D-deficient (VDD) diet, OVX+VDD diet, and three groups of ovariectomized rats treated with different doses of 1α,25(OH)2D3. The cartilage erosion and the levels of serum 17β-estradiol, 1α,25(OH)2D3 and C-telopeptide of type II collagen (CTX-II) were measured. TGF-β1, type II Collagen (CII), matrix metalloproteinases (MMP)-9,-13 in articular cartilage were assessed by immunohistochemistry. TGF-β1 and CTX-II expression were measured in articular cartilage chondrocytes treated with/without tumor necrosis factor (TNF-α), 1α,25(OH)2D3, and TGF-β receptor inhibitor (SB505124) in vitro. Cartilage erosion due to OVX was significantly reduced in a dose-dependent manner by 1α,25(OH)2D3 supplementation, and exacerbated by VDD. The expressions of TGF-β1 and CII in articular cartilage were suppressed by OVX and VDD, and rescued by 1α,25(OH)2D3 supplementation. The expression of MMP-9,-13 in articular cartilage increased with OVX and VDD, and decreased with 1α,25(OH)2D3 supplementation. In vitro experiments showed that 1α,25(OH)2D3 increased the TGF-β1 expression of TNF-α stimulated chondrocytes in a dose-dependent manner. 1α,25(OH)2D3 significantly counteracted the increased CTX-II release due to TNF-α stimulation, and this effect was significantly suppressed by SB505124. VDD aggravated cartilage erosion, and 1α,25(OH)2D3 supplementation showed protective effects in OVX-induced OA partly through the TGF-β1 pathway. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Assessment of DNA damage as an index of genetic toxicity in welding microenvironments among iron-based industries.

PubMed

Singh, Zorawar; Chadha, Pooja

2016-10-01

Welding is used extensively in different industries. Welders are always at a risk of exposure to a number of gases and metal-containing fumes in their respective microenvironments in which they work. Welding fumes consist of a wide range of complex metal oxide particles which can deposit in different parts of their bodies causing serious health problems. In the present study, 35 welders (age: 33.80 ± 1.04 years) from two iron-based industries have been assessed for DNA damage in peripheral blood lymphocytes using single-cell gel electrophoresis. An equal number of subjects (N = 35; age: 30.40 ± 1.51 years) matched to exposed subjects with respect to sex, age, socioeconomic status, smoking, and alcoholic habits were taken as controls. The results revealed that the damaged cell frequency (DCF) and mean comet tail length (CTL) in welders were significantly higher as compared to the controls (DCF: 69.74 ± 1.68 vs. 31.14 ± 1.67 and CTL: 29.21 ± 1.48 vs. 1.47 ± 0.08; p < 0.05). The effect of confounding factors such as age, duration of exposure, smoking, and drinking habits was also studied. Blood lead levels also showed a positive correlation with duration of exposure and CTL, and the overall results indicated an increased genetic damage as an index of genotoxicity in workers occupationally engaged in welding microenvironments. © The Author(s) 2015.

M. tuberculosis-Initiated Human Mannose Receptor Signaling Regulates Macrophage Recognition and Vesicle Trafficking by FcRγ-Chain, Grb2, and SHP-1.

PubMed

Rajaram, Murugesan V S; Arnett, Eusondia; Azad, Abul K; Guirado, Evelyn; Ni, Bin; Gerberick, Abigail D; He, Li-Zhen; Keler, Tibor; Thomas, Lawrence J; Lafuse, William P; Schlesinger, Larry S

2017-10-03

Despite its prominent role as a C-type lectin (CTL) pattern recognition receptor, mannose receptor (MR, CD206)-specific signaling molecules and pathways are unknown. The MR is highly expressed on human macrophages, regulating endocytosis, phagocytosis, and immune responses and mediating Mycobacterium tuberculosis (M.tb) phagocytosis by human macrophages, thereby limiting phagosome-lysosome (P-L) fusion. We identified human MR-associated proteins using phosphorylated and non-phosphorylated MR cytoplasmic tail peptides. We found that MR binds FcRγ-chain, which is required for MR plasma membrane localization and M.tb cell association. Additionally, we discovered that MR-mediated M.tb association triggers immediate MR tyrosine residue phosphorylation and Grb2 recruitment, activating the Rac/Pak/Cdc-42 signaling cascade important for M.tb uptake. MR activation subsequently recruits SHP-1 to the M.tb-containing phagosome, where its activity limits PI(3)P generation at the phagosome and M.tb P-L fusion and promotes M.tb growth. In sum, we identify human MR signaling pathways that temporally regulate phagocytosis and P-L fusion during M.tb infection. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Tumor regression induced by intratumor therapy with a disabled infectious single cycle (DISC) herpes simplex virus (HSV) vector, DISC/HSV/murine granulocyte-macrophage colony-stimulating factor, correlates with antigen-specific adaptive immunity.

PubMed

Ali, Selman A; Lynam, June; McLean, Cornelia S; Entwisle, Claire; Loudon, Peter; Rojas, José M; McArdle, Stephanie E B; Li, Geng; Mian, Shahid; Rees, Robert C

2002-04-01

Direct intratumor injection of a disabled infectious single cycle HSV-2 virus encoding the murine GM-CSF gene (DISC/mGM-CSF) into established murine colon carcinoma CT26 tumors induced a significant delay in tumor growth and complete tumor regression in up to 70% of animals. Pre-existing immunity to HSV did not reduce the therapeutic efficacy of DISC/mGM-CSF, and, when administered in combination with syngeneic dendritic cells, further decreased tumor growth and increased the incidence of complete tumor regression. Direct intratumor injection of DISC/mGM-CSF also inhibited the growth of CT26 tumor cells implanted on the contralateral flank or seeded into the lungs following i.v. injection of tumor cells (experimental lung metastasis). Proliferation of splenocytes in response to Con A was impaired in progressor and tumor-bearer, but not regressor, mice. A potent tumor-specific CTL response was generated from splenocytes of all mice with regressing, but not progressing tumors following in vitro peptide stimulation; this response was specific for the gp70 AH-1 peptide SPSYVYHQF and correlated with IFN-gamma, but not IL-4 cytokine production. Depletion of CD8(+) T cells from regressor splenocytes before in vitro stimulation with the relevant peptide abolished their cytolytic activity, while depletion of CD4(+) T cells only partially inhibited CTL generation. Tumor regression induced by DISC/mGM-CSF virus immunotherapy provides a unique model for evaluating the immune mechanism(s) involved in tumor rejection, upon which tumor immunotherapy regimes may be based.

Morphology of brood pouch formation in the pot-bellied seahorse Hippocampus abdominalis.

PubMed

Kawaguchi, Mari; Okubo, Ryohei; Harada, Akari; Miyasaka, Kazuki; Takada, Kensuke; Hiroi, Junya; Yasumasu, Shigeki

2017-01-01

The reproductive strategies of vertebrates are diverse. Seahorses (Pisces: Syngnathidae) possess the unique characteristic of male pregnancy; i.e., males, not females, incubate embryos in a specialized structure called a 'brood pouch'. The brood pouch is formed along the ventral midline of the tail. The lumen of the brood pouch is surrounded by loose connective tissue, called pseudoplacenta, and dermis. We visualized and evaluated the morphology of brood pouch formation in Hippocampus abdominalis to gain generalizable insights into this process in seahorses. First, we employed several staining methods to characterize the pseudoplacenta and dermis of the brood pouch of mature male seahorses. The pseudoplacenta is composed mainly of reticular fibers, while the dermis is composed mainly of collagenous fibers. Further observations showed that pouch formation is initiated by linear projections of epithelia on both ventrolateral sides of the body. These projections elongated toward the ventral midline, eventually fused together, and then formed a baggy structure composed of a single dermis layer with neither smooth muscle nor pseudoplacenta. Finally, the pseudoplacenta was formed, together with two layers of dermis and smooth muscle. Thus, a fully developed brood pouch was established. The morphology of the luminal epithelium also changed during pouch formation. We analyzed the localization of C-type lectins as markers; haCTL II was localized in both the outer and luminal epithelia of the brood pouch throughout development in the male seahorse, whereas haCTL IV, which was not detected in the early stage of seahorse development, became localized only in the luminal epithelium as development proceeded. We categorized the processes of brood pouch formation during male seahorse development into three stages: (1) the early stage, characterized by formation of a baggy structure from the primordium; (2) the middle stage, characterized by the differentiation and establishment of brood pouch-specific tissues; and (3) the late stage, characterized by a fully formed pouch with developing blood vessels and a pouch fold ultimately capable of carrying and incubating embryos.

The Theory and Application of Contextualized Teaching and Learning in Relation to Programs of Study and Career Pathways. Transition Highlights. Issue 2

ERIC Educational Resources Information Center

Kalchik, Stephanie; Oertle, Kathleen Marie

2010-01-01

Contextualized Teaching and Learning (CTL), also known as Contextualized Instruction, is defined as a "diverse family of instructional strategies designed to more seamlessly link the learning of foundational skills and academic or occupational content by focusing teaching and learning squarely on concrete applications in a specific context that is…

Immunogenicity of Recombinant Classic Swine Fever Virus CD8+ T Lymphocyte Epitope and Porcine Parvovirus VP2 Antigen Coexpressed by Lactobacillus casei in Swine via Oral Vaccination ▿

PubMed Central

Xu, Yigang; Cui, Lichun; Tian, Changyong; Zhang, Guocai; Huo, Guicheng; Tang, Lijie; Li, Yijing

2011-01-01

Classical swine fever virus (CSFV) and porcine parvovirus (PPV) are highly contagious pathogens, resulting in enormous economic losses in pig industries worldwide. Because vaccines play an important role in disease control, researchers are seeking improved vaccines that could induce antiviral immune responses against CSFV and PPV at the mucosal and systemic levels simultaneously. In this study, a genetically engineered Lactobacillus strain coexpressing the CSFV-specific cytotoxic T lymphocyte (CTL) epitope 290 and the VP2 antigen of PPV was developed, and its immunopotentiating capacity as an oral vaccine in pigs was analyzed. The data demonstrated that in the absence of any adjuvant, the recombinant Lactobacillus strain can efficiently stimulate mucosal and systemic CSFV-specific CD8+ CTL responses to protect pigs against CSFV challenge. Moreover, anti-PPV-VP2 serum IgG and mucosal IgA were induced in pigs immunized orally with the recombinant Lactobacillus strain, showing a neutralizing effect on PPV infection. The results suggest that the recombinant Lactobacillus microecological agent may be a valuable component of a strategy for development of a vaccine against CSFV and PPV. PMID:21940406

Complete thoracoscopic lobectomy for cancer: comparative study of three-dimensional high-definition with two-dimensional high-definition video systems †.

PubMed

Bagan, Patrick; De Dominicis, Florence; Hernigou, Jacques; Dakhil, Bassel; Zaimi, Rym; Pricopi, Ciprian; Le Pimpec Barthes, Françoise; Berna, Pascal

2015-06-01

Common video systems for video-assisted thoracic surgery (VATS) provide the surgeon a two-dimensional (2D) image. This study aimed to evaluate performances of a new three-dimensional high definition (3D-HD) system in comparison with a two-dimensional high definition (2D-HD) system when conducting a complete thoracoscopic lobectomy (CTL). This multi-institutional comparative study trialled two video systems: 2D-HD and 3D-HD video systems used to conduct the same type of CTL. The inclusion criteria were T1N0M0 non-small-cell lung carcinoma (NSCLC) in the left lower lobe and suitable for thoracoscopic resection. The CTL was performed by the same surgeon using either a 3D-HD or 2D-HD system. Eighteen patients with NSCLC were included in the study between January and December 2013: 14 males, 4 females, with a median age of 65.6 years (range: 49-81). The patients were randomized before inclusion into two groups: to undergo surgery with the use of a 2D-HD or 3D-HD system. We compared operating time, the drainage duration, hospital stay and the N upstaging rate from the definitive histology. The use of the 3D-HD system significantly reduced the surgical time (by 17%). However, chest-tube drainage, hospital stay, the number of lymph-node stations and upstaging were similar in both groups. The main finding was that 3D-HD system significantly reduced the surgical time needed to complete the lobectomy. Thus, future integration of 3D-HD systems should improve thoracoscopic surgery, and enable more complex resections to be performed. It will also help advance the field of endoscopically assisted surgery. © The Author 2015. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

Development of a novel immunoPET tracer to image human PD-1 checkpoint expression on tumor infiltrating lymphocytes in a humanized mouse model

PubMed Central

Natarajan, Arutselvan; Mayer, Aaron T; Reeves, Robert E; Nagamine, Claude M; Gambhir, Sanjiv S.

2017-01-01

Purpose It is well known that cancers exploit immune checkpoints (programmed death 1 receptor (PD-1) and its ligand (PD-L1)) to evade anti-tumor immune responses. Although immune checkpoint (IC) blockade is a promising approach, not all patients respond. Hence, the purpose of this study is imaging of tumor infiltrating lymphocytes (TILs), as they are known to express PD-1 during activation and subsequent exhaustion in the tumor microenvironment and are thought to be potentially predictive of therapeutic responses to IC blockade. Procedures We developed immunoPET tracers to image hPD-1 status of human peripheral blood mononuclear cells (hPBMC) adoptively transferred to NOD-scid IL-2Rγnull (NSG) mice (hNSG) bearing A375 human skin melanoma tumors. The anti-PD-1 human antibody (IgG; keytruda) labeled with either [89Zr]- or [64Cu]- radiometals to image PD-1 expressing human TILs in vivo. Results [89Zr]keytruda (groups = 2; NSG-ctl [control] and hNSG-nblk [non-blocking], n=3-5, 3.2 ± 0.4 MBq/15-16 μg/200 μL, and [64Cu]keytruda (groups = 3; NSG-ctl, NSG-blk [blocking], and hNSG-nblk) n=4, 7.4 ± 0.4 MBq /20-25μg/200 μL) were administered in mice. PET-CT scans were performed over 1-144 h ([89Zr]keytruda) and 1-48 h ([64Cu]keytruda) on mice. hNSG mice exhibited a high tracer uptake in the spleen lymphoid organs and tumors. At 24h, human TILs homing into melanoma of hNSG-nblk mice exhibited high signal (mean %ID/g ± SD) of 3.8 ± 0.4 ([89Zr]keytruda), and 6.4 ± 0.7 ([64Cu]keytruda), which was 1.5- and 3-fold higher uptake compared to NSG-ctl mice (p = 0.01), respectively. Biodistribution measurements of hNSG-nblk mice performed at 144 h ([89Zr]keytruda), and 48 h ([64Cu]keytruda) p.i. revealed tumor to muscle ratios as high as 45 and 12-fold, respectively. Conclusion This study clearly demonstrates specific imaging of human PD-1 expressing TILs within the tumor and lymphoid tissues. This suggests anti-human-PD-1 tracer could be clinically translatable to monitor cancer treatment response to IC blockade therapy. PMID:28247187

Immunogenicity in Swine of Orally Administered Recombinant Lactobacillus plantarum Expressing Classical Swine Fever Virus E2 Protein in Conjunction with Thymosin α-1 as an Adjuvant

PubMed Central

Xu, Yi-Gang; Guan, Xue-Ting; Liu, Zhong-Mei; Tian, Chang-Yong

2015-01-01

Classical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious disease that results in enormous economic losses in pig industries. The E2 protein is one of the main structural proteins of CSFV and is capable of inducing CSFV-neutralizing antibodies and cytotoxic T lymphocyte (CTL) activities in vivo. Thymosin α-1 (Tα1), an immune-modifier peptide, plays a very important role in the cellular immune response. In this study, genetically engineered Lactobacillus plantarum bacteria expressing CSFV E2 protein alone (L. plantarum/pYG-E2) and in combination with Tα1 (L. plantarum/pYG-E2-Tα1) were developed, and the immunogenicity of each as an oral vaccine to induce protective immunity against CSFV in pigs was evaluated. The results showed that recombinant L. plantarum/pYG-E2 and L. plantarum/pYG-E2-Tα1 were both able to effectively induce protective immune responses in pigs against CSFV infection by eliciting immunoglobulin A (IgA)-based mucosal, immunoglobulin G (IgG)-based humoral, and CTL-based cellular immune responses via oral vaccination. Significant differences (P < 0.05) in the levels of immune responses were observed between L. plantarum/pYG-E2-Tα1 and L. plantarum/pYG-E2, suggesting a better immunogenicity of L. plantarum/pYG-E2-Tα1 as a result of the Tα1 molecular adjuvant that can enhance immune responsiveness and augment specific lymphocyte functions. Our data suggest that the recombinant Lactobacillus microecological agent expressing CSFV E2 protein combined with Tα1 as an adjuvant provides a promising strategy for vaccine development against CSFV. PMID:25819954

Selective Downregulation of Rhesus Macaque and Sooty Mangabey Major Histocompatibility Complex Class I Molecules by Nef Alleles of Simian Immunodeficiency Virus and Human Immunodeficiency Virus Type 2▿

PubMed Central

DeGottardi, M. Quinn; Specht, Anke; Metcalf, Benjamin; Kaur, Amitinder; Kirchhoff, Frank; Evans, David T.

2008-01-01

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates HLA-A and -B molecules, but not HLA-C or -E molecules, based on amino acid differences in their cytoplasmic domains to simultaneously evade cytotoxic T lymphocyte (CTL) and natural killer cell surveillance. Rhesus macaques and sooty mangabeys express orthologues of HLA-A, -B, and -E, but not HLA-C, and many of these molecules have unique amino acid differences in their cytoplasmic tails. We found that these differences also resulted in differential downregulation by primary simian immunodeficiency virus (SIV) SIVsmm/mac and HIV-2 Nef alleles. Thus, selective major histocompatibility complex class I downregulation is a conserved mechanism of immune evasion for pathogenic SIV infection of rhesus macaques and nonpathogenic SIV infection of sooty mangabeys. PMID:18199657

Contribution of advances in immunology to vaccine development.

PubMed

Morrison, W I; Taylor, G; Gaddum, R M; Ellis, S A

1999-01-01

During the last 10 years, investigation of the bovine immune system has generated knowledge and reagents that can now be applied to study the mechanisms of immunity to disease and the identity of antigens recognized by protective immune responses. Such studies can indicate which antigens are likely to be effective in subunit vaccines and also highlight the type of antigen delivery system that will be required for a vaccine to induce a protective immune response. In the case of bovine RSV, studies of immune responses in the target host have demonstrated that both antibody and CTL responses play an important role in immunity. Both the F and G glycoproteins have been identified as targets of protective antibodies, and systems have been established that will allow the identification of the viral antigens recognized by CTL. Further studies of CD4+ T-cell responses to the virus are required to determine whether or not components of the response have the potential to enhance disease and, therefore, need to be avoided in vaccination strategies.

Blood flow dynamics in heart failure

NASA Technical Reports Server (NTRS)

Shoemaker, J. K.; Naylor, H. L.; Hogeman, C. S.; Sinoway, L. I.

1999-01-01

BACKGROUND: Exercise intolerance in heart failure (HF) may be due to inadequate vasodilation, augmented vasoconstriction, and/or altered muscle metabolic responses that lead to fatigue. METHODS AND RESULTS: Vascular and metabolic responses to rhythmic forearm exercise were tested in 9 HF patients and 9 control subjects (CTL) during 2 protocols designed to examine the effect of HF on the time course of oxygen delivery versus uptake (protocol 1) and on vasoconstriction during exercise with 50 mm Hg pressure about the forearm to evoke a metaboreflex (protocol 2). In protocol 1, venous lactate and H+ were greater at 4 minutes of exercise in HF versus CTL (P<0.05) despite similar blood flow and oxygen uptake responses. In protocol 2, mean arterial pressure increased similarly in each group during ischemic exercise. In CTL, forearm blood flow and vascular conductance were similar at the end of ischemic and ambient exercise. In HF, forearm blood flow and vascular conductance were reduced during ischemic exercise compared with the ambient trial. CONCLUSIONS: Intrinsic differences in skeletal muscle metabolism, not vasodilatory dynamics, must account for the augmented glycolytic metabolic responses to moderate-intensity exercise in class II and III HF. The inability to increase forearm vascular conductance during ischemic handgrip exercise, despite a normal pressor response, suggests that enhanced vasoconstriction of strenuously exercising skeletal muscle contributes to exertional fatigue in HF.

Minimizing performance degradation induced by interfacial recombination in perovskite solar cells through tailoring of the transport layer electronic properties

NASA Astrophysics Data System (ADS)

Xu, Liang; Molaei Imenabadi, Rouzbeh; Vandenberghe, William G.; Hsu, Julia W. P.

2018-03-01

The performance of hybrid organic-inorganic metal halide perovskite solar cells is investigated using one-dimensional drift-diffusion device simulations. We study the effects of interfacial defect density, doping concentration, and electronic level positions of the charge transport layer (CTL). Choosing CTLs with a favorable band alignment, rather than passivating CTL-perovskite interfacial defects, is shown to be beneficial for maintaining high power-conversion efficiency, due to reduced minority carrier density arising from a favorable local electric field profile. Insights from this study provide theoretical guidance on practical selection of CTL materials for achieving high-performance perovskite solar cells.

Functional PET Evaluation of the Photosensitive Baboon

PubMed Central

Szabó, C. Ákos; Salinas, Felipe S; Narayana, Shalini

2011-01-01

The baboon provides a unique, natural model of epilepsy in nonhuman primates. Additionally, photosensitivity of the epileptic baboon provides an important window into the mechanism of human idiopathic generalized epilepsies. In order to better understand the networks underlying this model, our group utilized functional positron emission tomography (PET) to compare cerebral blood flow (CBF) changes occurring during intermittent light stimulation (ILS) and rest between baboons photosensitive, epileptic (PS) and asymptomatic, control (CTL) animals. Our studies utilized subtraction and covariance analyses to evaluate CBF changes occurring during ILS across activation and resting states, but also evaluated CBF correlations with ketamine doses and interictal epileptic discharge (IED) rate during the resting state. Furthermore, our group also assessed the CBF responses related to variation of ILS in PS and CTL animals. CBF changes in the subtraction and covariance analyses reveal the physiological response and visual connectivity in CTL animals and pathophysiological networks underlying responses associated with the activation of ictal and interictal epileptic discharges in PS animals. The correlation with ketamine dose is essential to understanding differences in CBF responses between both groups, and correlations with IED rate provides an insight into an epileptic network independent of visual activation. Finally, the ILS frequency dependent changes can help develop a framework to study not only spatial connectivity but also the temporal sequence of regional activations and deactivations related to ILS. The maps generated by the CBF analyses will be used to target specific nodes in the epileptic network for electrophysiological evaluation using intracranial electrodes. PMID:22276085

Assessing the feasibility and profitability of cut-to-length harvests in eastern hardwoods

Treesearch

Chris B. LoDoux

2002-01-01

Cut-to-length (CTL) logging applications are becoming more popular in hardwood forests. CTL harvesting causes much less damage to the residual stand than conventional harvesting because logs and trees are not pulled through the stand and trees can be felled directionally.

Characterization of Plasmodium falciparum Choline Transporters

DTIC Science & Technology

2005-04-01

ElO PfCTL v.1 transcript El IE2E31E41 E5 E6EE EJ PfCTL v.2 transcript B 1/1 31/11 61/21 91/31 ATG AAT TAC ATC GAG ATG GAA GA CGT GAA TAT AAA CCA CTT...ATA GA GAA GTGBGAT AAT GGA AC AT ATT ATA ATA MT MC MG GM TAT TAT AAC ATG TAT GA AAC AT MT ATA M N Y I E M4 E E R E Y K P L I EK E V D N G N N I B I N N...GGT ATA AAT TAC MT GGG AM ATA TGT GGA AAG GAT CTA CAT AA TAT CCA TAT TTA TAC TTC CCT CTT ACT CCT MA MT CCT MA CCT GA ATA TTA AGT ACC TAT GCT MA TGC YO G