Sample records for antigen specific ctl
-
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
-
Antigen delivery by α2-macroglobulin enhances the cytotoxic T lymphocyte response
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
-
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
-
Eberle, R; Russell, R G; Rouse, B T
1981-01-01
In this communication, we examine the specificity of anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL). Serological studies of the two related HSV serotypes (HSV-1 and HSV-2) have revealed both type-specific and cross-reactive antigenic determinants in the viral envelope and on the surface of infected cells. By analysis of cytotoxicity of CTL, generated in vitro by restimulation of splenocytes from mice primed with one or the other HSV serotype, the recognition of both type-specific and cross-reactive determinants on infected target cells by anti-HSV CTL was detectable. Thus, effector cells generated by priming and restimulating with the same virus recognized both type-specific and cross-reactive determinants on target cells infected with the homologous virus, but only cross-reactive determinants on target cells infected with the heterologous HSV serotype. CTL generated by restimulation with the heterologous virus were capable of recognizing only the cross-reactive determinants on either HSV-1- or HSV-2-infected target cells. These results indicate that two subpopulations of CTL exist in a population of anti-HSV immune spleen cells--those which recognize type-specific determinants and those specific for cross-reactive antigenic determinants present on the surface of HSV infected cells. The type-specific subset of anti-HSV CTL was shown to recognize the gC glycoprotein of HSV-1 infected target cells. In addition to the gC glycoprotein, at least one other type-specific surface antigen was also recognized by anti-HSV CTL in addition to the cross-reactive determinants recognized by anti-HSV CTL. PMID:6277790
-
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.
-
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
-
Identification of immunodominant antigens for the laboratory diagnosis of toxocariasis.
Zhan, Bin; Ajmera, Ravi; Geiger, Stefan Michael; Gonçalves, Marco Túlio Porto; Liu, Zhuyun; Wei, Junfei; Wilkins, Patricia P; Fujiwara, Ricardo; Gazzinelli-Guimaraes, Pedro Henrique; Bottazzi, Maria Elena; Hotez, Peter
2015-12-01
To identify immunodominant antigens of Toxocara canis recognised by Toxocara-infected sera as recombinant reagents for immunodiagnosis of toxocariasis. Pooled sera from human cases of toxocariasis were used to identify immunodominant antigens by immunoscreening a T. canis larval expression cDNA library. The positive clones were sequenced to reveal the identity of the antigens. The recombinant proteins were expressed in E. coli and then used to confirm their immunoreaction with sera of humans with toxocariasis. Two chosen antigens were also used to differentiate Toxocara infection from other helminth infections in mice. Eleven antigens with immunodiagnostic potential were identified, including two C-type lectins (CTLs) that reacted strongly with the Toxocara-positive serum pool. The first CTL (Tc-CTL-1) is the same as TES-32, previously identified as a major immunodominant component of TES; the second CTL (Tc-CTL-2) is a novel C-type lectin sharing 83% amino acid sequence identity within the functional domain of Tc-CTL-1. The E. coli-expressed recombinant Tc-CTL-1 was strongly recognised by the Toxocara-positive serum pool or sera from animals experimentally infected with T. canis. Reactivity with recombinant Tc-CTL-1 was higher when the unreduced protein was used in an enzyme-linked immunosorbent assay (ELISA), dot-blot assay or Western blot test compared to the protein under reduced condition. Both recombinant Tc-CTL-1- and Tc-CTL-2-based ELISAs were able to differentiate T. canis infection from other helminth infections in experimentally infected mice. Both Tc-CTL-1 and Tc-CTL-2 were able to differentiate Toxocara infection from other helminth infections and could potentially be used as sensitive and specific immunodiagnostic antigens. © 2015 John Wiley & Sons Ltd.
-
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.
-
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.
-
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
-
TNF-induced target cell killing by CTL activated through cross-presentation.
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.
-
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.
-
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
-
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
-
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.
-
Antigen-specific CTLs: to produce autologous cells product for adoptive cellular therapy.
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.
-
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.
-
Ioannides, C G; Freedman, R S; Platsoucas, C D; Rashed, S; Kim, Y P
1991-03-01
CTL clones were developed from tumor infiltrating lymphocytes (TIL) from the ascites of a patient with ovarian carcinoma by coculture of TIL with autologous tumor cells and subsequent cloning in the presence of autologous tumor cells. These CTL clones expressed preferential cytolytic activity against autologous tumor cells but not against allogeneic ovarian tumor cells and the NK-sensitive cell line K562. The cytolytic activity of these CTL against autologous tumors was inhibited by anti-TCR (WT31 mAb), anti-HLA class I, and anti-CD3 mAb but not by the NK function antibody Leu 11b. Cloning of the autologous tumor cells in vitro revealed that the CTL clones of the ovarian TIL expressed differential abilities to lyse autologous tumor cell clones. The specificity analysis of these autologous tumor specific CTL suggested that they recognize several antigenic determinants present on the ovarian tumor cells. Our results indicate the presence of at least three antigenic epitopes on the tumor cells (designated OVA-1A, OVA-1B, and OVA-1C), one of which (OVA-1C) is unstable. These determinants are present either simultaneously or separately, and six types of ovarian clones can be distinguished on the basis of their expression. These results indicate that CTL of the TIL detect intratumor antigenic heterogeneity. The novel heterogeneity identified within the ovarian tumor cells in this report may be of significance for understanding cellular immunity in ovarian cancer and developing adoptive specific immunotherapeutic approaches in ovarian cancer.
-
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
-
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.
-
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
-
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.
-
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.
-
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
-
Autologous cellular vaccine overcomes cancer immunoediting in a mouse model of myeloma.
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
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
-
Therapeutic limitations in tumor-specific CD8+ memory T cell engraftment
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
-
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).
-
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.
-
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
-
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.
-
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.
-
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.
-
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
-
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.
-
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.
-
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
-
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.
-
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
-
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
-
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
-
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.
-
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
-
Recognition and killing of brain tumor stem-like initiating cells by CD8+ cytolytic T cells.
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.
-
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
-
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.
-
Establishment of anti-tumor memory in humans using in vitro-educated CD8+ T cells
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
-
Cotransfection of DC with TLR4 and MART-1 RNA induces MART-1-specific responses.
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.
-
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.
-
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.
-
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.
-
Quantitative Expression and Immunogenicity of MAGE-3 and -6 in Upper Aerodigestive Tract Cancer
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.
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.
-
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.
-
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.
-
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.
-
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
-
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
-
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.
-
Antigen Cross-Presentation of Immune Complexes
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
-
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
-
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.
-
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.
-
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
-
Positive Selection of γδ CTL by TL Antigen Expressed in the Thymus
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
-
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.
-
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
-
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
-
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.
-
Identification of HLA-A2–restricted CD8+ Cytotoxic T Cell Responses in Primary Biliary Cirrhosis
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
-
FLT3-regulated antigens as targets for leukemia-reactive cytotoxic T lymphocytes
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
-
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.
-
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.
-
PLGA nanoparticle-mediated delivery of tumor antigenic peptides elicits effective immune responses
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.
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.
-
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.
-
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
-
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
-
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.
-
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.
-
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
-
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
-
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
-
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.
-
Abuknesha, Ram; Uematsu, Satoshi; Akira, Shizuo; Nestle, Frank O.; Diebold, Sandra S.
2012-01-01
Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC) that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA) and CpG oligodeoxynucleotides (ODN). We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL) responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses. PMID:22808118
-
A role for extracellular amastigotes in the immunopathology of Chagas disease.
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.
-
Adeno-associated virus capsid antigen presentation is dependent on endosomal escape
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
-
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.
-
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
-
Chimeric Antigen Receptor–Modified T Cells for Acute Lymphoid Leukemia
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.
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.
-
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.
-
Serine Proteases Enhance Immunogenic Antigen Presentation on Lung Cancer Cells
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
-
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
-
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
-
Natural killer cells facilitate PRAME-specific T-cell reactivity against neuroblastoma
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
-
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.
-
Brander, Christian; Yang, Otto O.; Jones, Norman G.; Lee, Yun; Goulder, Philip; Johnson, R. Paul; Trocha, Alicja; Colbert, David; Hay, Christine; Buchbinder, Susan; Bergmann, Cornelia C.; Zweerink, Hans J.; Wolinsky, Steven; Blattner, William A.; Kalams, Spyros A.; Walker, Bruce D.
1999-01-01
Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences. PMID:10559335
-
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.
-
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
-
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
-
Tarokhian, Batoul; Sherkat, Roya; Nasr Esfahani, Mohamma Hossein; Adib, Minoo; Kiani Esfahani, Abbas; Ataei, Behrooz
2014-01-01
Background: Some evidence has shown a relationship between primary human cytomegalovirus (CMV) infection and pregnancy loss. The impact of CMV infection reactivation during pregnancy on adverse pregnancy outcomes is not completely understood. It is proposed that altered immune response, and therefore, recurrence or reactivation of latent CMV infection may relate to recurrent spontaneous abortion (RSA); however, few data are available in this regard. To find out about any cell mediated defect and reactivation of latent CMV infection in women with RPL, cellular immunity to the virus has been evaluated by specific cytotoxic T lymphocyte (CTL) response to CMV. Materials and Methods: In a case control study, CTL CD107a expression and in- tercellular IFN-γ production in response to CMV pp65 antigen and staphylococcus enterotoxin B (SEB) in women with RSA were assessed by flow cytometric analysis. Forty-four cases with history of recurrent pregnancy and forty-four controls with history of successful pregnancies were included. The FACSCaliber flow cytometer were used for analysis. Results: No significant difference was observed between CD107a expression and IFN-γ production in response to CMV PP65 antigen in RPL patients and control group. How- ever, the cytotoxic response to SEB antigen in patients with RPL was significantly lower than control group (p=0.042). Conclusion: The results of this study show that impaired CD107a expression and IFN-γ production as CTL response to CMV does not appear to be a major contrib- uting and immune incompetence factor in patients with RPL, but cytotoxic T cell response defect to other antigens requires to be assessed further in these patients. PMID:24520502
-
Tarokhian, Batoul; Sherkat, Roya; Nasr Esfahani, Mohamma Hossein; Adib, Minoo; Kiani Esfahani, Abbas; Ataei, Behrooz
2014-01-01
Some evidence has shown a relationship between primary human cytomegalovirus (CMV) infection and pregnancy loss. The impact of CMV infection reactivation during pregnancy on adverse pregnancy outcomes is not completely understood. It is proposed that altered immune response, and therefore, recurrence or reactivation of latent CMV infection may relate to recurrent spontaneous abortion (RSA); however, few data are available in this regard. To find out about any cell mediated defect and reactivation of latent CMV infection in women with RPL, cellular immunity to the virus has been evaluated by specific cytotoxic T lymphocyte (CTL) response to CMV. In a case control study, CTL CD107a expression and in- tercellular IFN-γ production in response to CMV pp65 antigen and staphylococcus enterotoxin B (SEB) in women with RSA were assessed by flow cytometric analysis. Forty-four cases with history of recurrent pregnancy and forty-four controls with history of successful pregnancies were included. The FACSCaliber flow cytometer were used for analysis. No significant difference was observed between CD107a expression and IFN-γ production in response to CMV PP65 antigen in RPL patients and control group. How- ever, the cytotoxic response to SEB antigen in patients with RPL was significantly lower than control group (p=0.042). The results of this study show that impaired CD107a expression and IFN-γ production as CTL response to CMV does not appear to be a major contrib- uting and immune incompetence factor in patients with RPL, but cytotoxic T cell response defect to other antigens requires to be assessed further in these patients.
-
Clinical-Grade Human Multipotent Adult Progenitor Cells Block CD8+ Cytotoxic T Lymphocytes
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.
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.
-
In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor.
Boissonnas, Alexandre; Fetler, Luc; Zeelenberg, Ingrid S; Hugues, Stéphanie; Amigorena, Sebastian
2007-02-19
Although the immune system evolved to fight infections, it may also attack and destroy solid tumors. In most cases, tumor rejection is initiated by CD8(+) cytotoxic T lymphocytes (CTLs), which infiltrate solid tumors, recognize tumor antigens, and kill tumor cells. We use a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs. We show that in the periphery of a thymoma growing subcutaneously, activated CTLs migrate with high instantaneous velocities. The CTLs arrest in close contact to tumor cells expressing their cognate antigen. In regions where most tumor cells are dead, CTLs resume migration, sometimes following collagen fibers or blood vessels. CTLs migrating along blood vessels preferentially adopt an elongated morphology. CTLs also infiltrate tumors in depth, but only when the tumor cells express the cognate CTL antigen. In tumors that do not express the cognate antigen, CTL infiltration is restricted to peripheral regions, and lymphocytes neither stop moving nor kill tumor cells. Antigen expression by tumor cells therefore determines both CTL motility within the tumor and profound tumor infiltration.
-
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.
-
MUC1-specific CTLs are non-functional within a pancreatic tumor microenvironment.
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.
-
An Exosome-Based Vaccine Platform Imparts Cytotoxic T Lymphocyte Immunity Against Viral Antigens.
Anticoli, Simona; Manfredi, Francesco; Chiozzini, Chiara; Arenaccio, Claudia; Olivetta, Eleonora; Ferrantelli, Flavia; Capocefalo, Antonio; Falcone, Emiliana; Ruggieri, Anna; Federico, Maurizio
2018-04-01
Exosomes are 50-150 nm sized nanovesicles released by all eukaryotic cells. The authors very recently described a method to engineer exosomes in vivo with the E7 protein of Human Papilloma Virus (HPV). This technique consists in the intramuscular injection of a DNA vector expressing HPV-E7 fused at the C-terminus of an exosome-anchoring protein, that is, Nef mut , the authors previously characterized for its high levels of incorporation in exosomes. In this configuration, the ≈11 kDa E7 protein elicited a both strong and effective antigen-specific cytotoxic T lymphocyte (CTL) immunity. Attempting to establish whether this method could have general applicability, the authors expanded the immunogenicity studies toward an array of viral products of various origin and size including Ebola Virus VP24, VP40 and NP, Influenza Virus NP, Crimean-Congo Hemorrhagic Fever NP, West Nile Virus NS3, and Hepatitis C Virus NS3. All antigens appeared stable upon fusion with Nef mut , and are uploaded in exosomes at levels comparable to Nef mut . When injected in mice, DNA vectors expressing the diverse fusion products elicited a well detectable antigen-specific CD8 + T cell response associating with a cytotoxic activity potent enough to kill peptide-loaded and/or antigen-expressing syngeneic cells. These data definitely proven both effectiveness and flexibility of this innovative CTL vaccine platform. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
-
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
-
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.
-
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
-
Smyth, Mark J.; Krasovskis, Erika; Johnstone, Ricky W.
1998-01-01
Mouse cytotoxic T lymphocytes (CTL) reactive with a H-2Db-presented 9-mer peptide of the human papillomavirus type 16 protein E749-57 (RAHYNIVTF) were generated from the spleen cells of wild-type C57BL/6 (B6) or B6 perforin-deficient (B6.P0) mice. CD8+ B6 CTL displayed peptide-specific perforin- and Fas-mediated lysis of E7-transfected mouse RMA lymphoma cells (RMA-E7), while CD8+ CTL from B6.P0 mice lysed RMA-E7 cells via Fas ligand (FasL) exclusively. Rapid and efficient lysis of syngeneic bystander B6 blasts or RMA cells by either B6 or B6.P0 Ag-activated CTL was mediated by a FasL-Fas mechanism. Fas-resistant bystanders were not lysed, nor were allogeneic Fas-sensitive C3H/HeJ (H-2k) or BALB/c (H-2d) bystander blasts. Interestingly, however, phorbol myristate acetate-ionomycin preactivation of B6.P0 effectors enabled lysis of allogeneic H-2k and H-2d bystanders even in the absence of antigenic stimulation. Lysis of syngeneic bystander cells was always FasL-Fas dependent and required effector-bystander contact and, in particular, an interaction between CTL LFA-1 and bystander ICAM-1. Thus, in the context of major histocompatibility complex class I molecule-peptide ligation of the T-cell receptors of CD8+ CTL, neighboring bystander cells that are syngeneic and Fas sensitive and express the adhesion molecule ICAM-1 are potential targets of CTL attack. PMID:9621057
-
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.
-
Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.
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.
-
In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor
Boissonnas, Alexandre; Fetler, Luc; Zeelenberg, Ingrid S.; Hugues, Stéphanie; Amigorena, Sebastian
2007-01-01
Although the immune system evolved to fight infections, it may also attack and destroy solid tumors. In most cases, tumor rejection is initiated by CD8+ cytotoxic T lymphocytes (CTLs), which infiltrate solid tumors, recognize tumor antigens, and kill tumor cells. We use a combination of two-photon intravital microscopy and immunofluorescence on ordered sequential sections to analyze the infiltration and destruction of solid tumors by CTLs. We show that in the periphery of a thymoma growing subcutaneously, activated CTLs migrate with high instantaneous velocities. The CTLs arrest in close contact to tumor cells expressing their cognate antigen. In regions where most tumor cells are dead, CTLs resume migration, sometimes following collagen fibers or blood vessels. CTLs migrating along blood vessels preferentially adopt an elongated morphology. CTLs also infiltrate tumors in depth, but only when the tumor cells express the cognate CTL antigen. In tumors that do not express the cognate antigen, CTL infiltration is restricted to peripheral regions, and lymphocytes neither stop moving nor kill tumor cells. Antigen expression by tumor cells therefore determines both CTL motility within the tumor and profound tumor infiltration. PMID:17261634
-
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.
-
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.
-
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
-
NetCTLpan: pan-specific MHC class I pathway epitope predictions
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
-
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
-
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.
-
Immunostimulatory activities of dendritic cells loaded with adenovirus vector carrying HBcAg/HBsAg
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
-
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.
-
Raafat, Nermin; Sadowski-Cron, Charlotte; Mengus, Chantal; Heberer, Michael; Spagnoli, Giulio C; Zajac, Paul
2012-09-01
Herpes simplex virus protein ICP47, encoded by US12 gene, strongly downregulates major histocompatibility complex (MHC) class-I antigen restricted presentation by blocking transporter associated with antigen processing (TAP) protein. To decrease viral vector antigenic immunodominance and MHC class-I driven clearance, we engineered recombinant vaccinia viruses (rVV) expressing ICP47 alone (rVV-US12) or together with endoplasmic reticulum (ER)-targeted Melan-A/MART-1(27-35) model tumor epitope (rVV-MUS12). In this study, we show that antigen presenting cells (APC), infected with rVV-US12, display a decreased ability to present TAP dependent MHC class-I restricted viral antigens to CD8+ T-cells. While HLA class-I cell surface expression is strongly downregulated, other important immune related molecules such as CD80, CD44 and, most importantly, MHC class-II are unaffected. Characterization of rVV-MUS12 infected cells demonstrates that over-expression of a TAP-independent peptide, partially compensates for ICP47 induced surface MHC class-I downregulation (30% vs. 70% respectively). Most importantly, in conditions where clearance of infected APC by virus-specific CTL represents a limiting factor, a significant enhancement of CTL responses to the tumor epitope can be detected in cultures stimulated with rVV-MUS12, as compared to those stimulated by rVV-MART alone. Such reagents could become of high relevance in multiple boost protocols required for cancer immunotherapy, to limit vector-specific responsiveness. Copyright © 2011 UICC.
-
Lin, Xiaoyun; Chen, Shao; Xue, Xiangyang; Lu, Lijun; Zhu, Shanli; Li, Wenshu; Chen, Xiangmin; Zhong, Xiaozhi; Jiang, Pengfei; Sename, Torsoo Sophia; Zheng, Yi; Zhang, Lifang
2016-01-01
Epstein–Barr virus (EBV) is prevalent throughout the world and is associated with several malignant diseases in humans. Latent membrane protein 2 (LMP2) of EBV plays a crucial role in the pathogenesis of EBV-associated tumors; therefore, LMP2 has been considered to be a potential immunodiagnostic and immunotherapeutic target. A multi-epitope-based antigen is a promising option for therapeutic vaccines and diagnoses of such malignancies. In this study, we systematically screened cytotoxic T lymphocyte (CTL), helper T cell (Th) and B-cell epitopes within EBV-LMP2 using bioinformatics. Based on the screen, two peptides rich in overlapping epitopes of both T cells and B cells were selected to construct a plasmid containing the sequence for a chimeric multi-epitope protein referred to as EBV-LMP2m, which is composed of LMP2aa195∼232 and LMP2aa419∼436. The EBV-LMP2m protein was expressed in E. coli BL21 (DE3) after prokaryotic codon optimization. Inoculation of the purified chimeric antigen in BALB/c mice induced not only high levels of specific IgG in the serum and secretory IgA in the vaginal mucus but also a specific CTL response. By using purified EBV-LMP2m as an antigen, the presence of specific IgG in the serum specimens of 202 nasopharyngeal carcinoma (NPC) patients was effectively detected with 52.84% sensitivity and 95.40% specificity, which represents an improvement over the traditional detection method based on VCA-IgA (60.53% sensitivity and 76.86% specificity). The above results indicate that EBV-LMP2m may be used not only as a potential target antigen for EBV-associated tumors but also a diagnostic agent for NPC patients. PMID:25864917
-
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.
-
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.
-
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
-
Design of therapeutic vaccines as a novel antibody therapy for cardiovascular diseases.
Nakagami, Hironori
2017-09-01
Vaccines are primarily used worldwide as a preventive medicine for infectious diseases and have recently been applied to cancer. We and others have developed therapeutic vaccines designed for cardiovascular diseases that are notably different from previous vaccines. In the case of cancer vaccines, a specific protein in cancer cells is a target antigen, and the activation of cytotoxic T cells (CTL) is required to kill and remove the antigen-presenting cancer cells. Our therapeutic vaccines work against hypertension by targeting angiotensin II (Ang II) as the antigen, which is an endogenous hormone. Therapeutic vaccines must avoid CTL activation and induce the blocking antibodies for Ang II. The goal of our therapeutic vaccine for cardiovascular diseases is to induce the specific antibody response toward the target protein without inducing T-cell or antibody-mediated inflammation through the careful selection of the target antigen, carrier protein and adjuvants. The goal of our therapeutic vaccine is similar to that of antibody therapy. Recently, multiple antibody-based drugs have been developed for cancer, immune-related diseases, and dyslipidemia, which are efficient but expensive. If the effect of a therapeutic vaccine is nearly equivalent to antibody therapy as an alternative approach, the lower medical cost and improvement in drug adherence can be advantages of therapeutic vaccines. In this review, we will describe our concept of therapeutic vaccines for cardiovascular diseases and the future directions of therapeutic vaccines as novel antibody therapies. Copyright © 2017. Published by Elsevier Ltd.
-
Controlling Influenza by Cytotoxic T-Cells: Calling for Help from Destroyers
Schotsaert, Michael; Ibañez, Lorena Itatí; Fiers, Walter; Saelens, Xavier
2010-01-01
Influenza is a vaccine preventable disease that causes severe illness and excess mortality in humans. Licensed influenza vaccines induce humoral immunity and protect against strains that antigenically match the major antigenic components of the vaccine, but much less against antigenically diverse influenza strains. A vaccine that protects against different influenza viruses belonging to the same subtype or even against viruses belonging to more than one subtype would be a major advance in our battle against influenza. Heterosubtypic immunity could be obtained by cytotoxic T-cell (CTL) responses against conserved influenza virus epitopes. The molecular mechanisms involved in inducing protective CTL responses are discussed here. We also focus on CTL vaccine design and point to the importance of immune-related databases and immunoinformatics tools in the quest for new vaccine candidates. Some techniques for analysis of T-cell responses are also highlighted, as they allow estimation of cellular immune responses induced by vaccine preparations and can provide correlates of protection. PMID:20508820
-
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.
-
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
-
HIV: current opinion in escapology.
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.
-
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
-
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.
-
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
-
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.
-
HIV-I Nef inhibitors: a novel class of HIV-specific immune adjuvants in support of a cure.
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.
-
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.
-
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
-
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.
-
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
-
Kessler, Jan H.; Beekman, Nico J.; Bres-Vloemans, Sandra A.; Verdijk, Pauline; van Veelen, Peter A.; Kloosterman-Joosten, Antoinette M.; Vissers, Debby C.J.; ten Bosch, George J.A.; Kester, Michel G.D.; Sijts, Alice; Drijfhout, Jan Wouter; Ossendorp, Ferry; Offringa, Rienk; Melief, Cornelis J.M.
2001-01-01
We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A*0201–presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved “reverse immunology” strategy. Next to motif-based HLA-A*0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A*0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA100–108; SLYSFPEPEA, PRA142–151; ALYVDSLFFL, PRA300–309; and SLLQHLIGL, PRA425–433) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A*0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer. PMID:11136822
-
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.
-
Dose-dependent modulation of CD8 and functional avidity as a result of peptide encounter
Kroger, Charles J; Alexander-Miller, Martha A
2007-01-01
The generation of an optimal CD8+ cytotoxic T lymphocyte (CTL) response is critical for the clearance of many intracellular pathogens. Previous studies suggest that one contributor to an optimal immune response is the presence of CD8+ cells exhibiting high functional avidity. In this regard, CD8 expression has been shown to contribute to peptide sensitivity. Here, we investigated the ability of naive splenocytes to modulate CD8 expression according to the concentration of stimulatory peptide antigen. Our results showed that the level of CD8 expressed was inversely correlated with the amount of peptide used for the primary stimulation, with higher concentrations of antigen resulting in lower expression of both CD8α and CD8β. Importantly the ensuing CD8low and CD8high CTL populations were not the result of the selective outgrowth of naive CD8+ T-cell subpopulations expressing distinct levels of CD8. Subsequent encounter with peptide antigen resulted in continued modulation of both the absolute level and the isoform of CD8 expressed and in the functional avidity of the responding cells. We propose that CD8 cell surface expression is not a static property, but can be modulated to ‘fine tune’ the sensitivity of responding CTL to a defined concentration of antigen. PMID:17484768
-
Bougie, Daniel W; Peterson, Julie A; Kanack, Adam J; Curtis, Brian R; Aster, Richard H
2014-01-01
Background HNA-3a specific antibodies can cause severe, sometimes fatal, transfusion related acute lung injury (TRALI) when present in transfused blood. The HNA3-a/b antigens are determined by an R154Q polymorphism in the first of five extracellular loops of the 10-membrane spanning choline transporter-like protein 2 (CTL2) expressed on neutrophils, lymphocytes and other tissues. About 50% of HNA-3a antibodies (Type 1) can be detected using CTL2 Loop 1 peptides containing R154; the remaining 50% (Type 2) fail to recognize this target. Understanding the basis for this difference could guide efforts to develop practical assays to screen blood donors for HNA-3 antibodies. Study design and methods Reactions of HNA-3a antibodies against recombinant versions of human, mouse, and human/mouse (chimeric) CTL2 were characterized using flow cytometry and various solid phase assays. Results Findings made show that, for binding to CTL2, Type 2 HNA-3a antibodies require non-polymorphic amino acid residues in the third, and possibly the second, extracellular loops of CTL2 to be in a configuration comparable to that found naturally in the cell membrane. In contrast, Type 1 antibodies require only peptides from the first extracellular loop that contain R154 for recognition. Conclusion Although Type 1 HNA-3a antibodies can readily be detected in solid phase assays that use a CTL2 peptide containing R154 as a target, development of a practical test to screen blood donors for Type 2 antibodies will pose a serious technical challenge because of the complex nature of the epitope(s) recognized by this antibody sub-group. PMID:24846273
-
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.
-
Shurin, Michael R.; Potapovich, Alla I.; Tyurina, Yulia Y.; Tourkova, Irina L.; Shurin, Galina V.; Kagan, Valerian E.
2014-01-01
Dendritic cells (DC) loaded with tumor antigens from apoptotic/necrotic tumor cells are commonly used as vaccines for cancer therapy. However, the use of dead tumor cells may cause both tolerance and immunity, making the effect of vaccination unpredictable. To deliver live tumor “cargoes” into DC, we developed a new approach based on the “labeling” of tumors with a phospholipid “eat-me” signal, phosphatidylserine. Expression of phosphatidylserine on live tumor cells mediated their recognition and endocytosis by DC resulting in the presentation of tumor antigens to antigen-specific T cells. In mice, topical application of phosphatidylserine-containing ointment over melanoma induced tumor-specific CTL, local and systemic antitumor immunity, and inhibited tumor growth. Thus, labeling of tumors with phosphatidylserine is a promising strategy for cancer immunotherapy. PMID:19276376
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Chandra, Subhash; Kaur, Manpreet; Midha, Shuchi
2007-05-25
We have investigated the efficiency of N-terminal 1-260 residues of Edema factor (EFn) as a delivery system for ESAT-6, an antigenic protein of Mycobacterium tuberculosis H{sub 37}R{sub v}, into the cytosol of mammalian cells. The EFn.ESAT-6 recombinant protein was obtained by genetic fusion of EFn and ESAT-6 DNA. Our data shows that in the presence of PA, EFn.ESAT-6 fusion protein is internalized into the cytosol of antigen presenting cells, and the splenocytes produced both Th1 and Th2 cytokines in vitro. Further, EFn.ESAT-6 elicited effective cytotoxicT lymphocyte (CTL) response in an in vitro CTL assay. This study for the first timemore » demonstrates that EFn can be used as a vehicle to deliver heterologous proteins of therapeutic importance.« less
-
Kim, Jin Hee; Kang, Tae Heung; Noh, Kyung Hee; Bae, Hyun Cheol; Kim, Seok-Ho; Yoo, Young Do; Seong, Seung-Yong; Kim, Tae Woo
2009-01-29
Dendritic cells (DCs) have become an important measure for the treatment of malignancies. Current DC preparations, however, generate short-lived DCs because they are subject to cell death from various apoptotic pressures. Antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) is one of the main obstacles to limit the DC-mediated immune priming since CTLs can recognize the target antigen expressing DCs as target cells and kill the DCs. CTLs secret perforin and serine protease granzymes during CTL killing. Perforin and serine protease granzymes induce the release of a number of mitochondrial pro-apoptotic factors, which are controlled by members of the BCL-2 family, such as BAK, BAX and BIM. FasL linking to Fas on DCs triggers the activation of caspase-8, which eventually leads to mitochondria-mediated apoptosis via truncation of BID. In this study, we tried to enhance the DC priming capacity by prolonging DC survival using anti-apoptotic siRNA targeting these key pro-apoptotic molecules in CTL killing. Human papillomavirus (HPV)-16 E7 antigen presenting DCs that were transfected with these anti-apoptotic siRNAs showed increased resistance to T cell-mediated death, leading to enhanced E7-specific CD8(+) T cell activation in vitro and in vivo. Among them, siRNA targeting BIM (siBIM) generated strongest E7-specific E7-specific CD8(+) T cell immunity. More importantly, vaccination with E7 presenting DCs transfected with siBIM was capable of generating a marked therapeutic effect in vaccinated mice. Our data indicate that ex vivo manipulation of DCs with siBIM may represent a plausible strategy for enhancing dendritic cell-based vaccine potency.
-
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.
-
Equine infectious anemia virus-infected dendritic cells retain antigen presentation capability
DOE Office of Scientific and Technical Information (OSTI.GOV)
Rivera, Julie A.; McGuire, Travis C.
2005-05-10
To determine if equine monocyte-derived dendritic cells (DC) were susceptible to equine infectious anemia virus (EIAV) infection, ex vivo-generated DC were infected with virus in vitro. EIAV antigen was detected by immunofluorescence 3 days post-infection with maximum antigen being detected on day 4, whereas there was no antigen detected in DC incubated with the same amount of heat-inactivated EIAV. No cytolytic activity was observed after EIAV{sub WSU5} infection of DC. These monocyte-derived DC were more effective than macrophages and B cells in stimulating allogenic T lymphocytes. Both infected macrophages and DC stimulated similar levels of memory CTL responses in mixturesmore » of CD8+ and CD4+ cells as detected with {sup 51}Cr-release assays indicating that EIAV infection of DC did not alter antigen presentation. However, EIAV-infected DC were more effective than infected macrophages when used to stimulate memory CTL in isolated CD8+ cells. The maintenance of antigen processing and presenting function by EIAV-infected DC in vitro suggests that this function is maintained during in vivo infection.« less
-
Duong, Ellen; Bracho-Sanchez, Edith; Rucevic, Marijana; Liebesny, Paul H.; Xu, Yang; Shimada, Mariko; Ghebremichael, Musie; Kavanagh, Daniel G.; Le Gall, Sylvie
2014-01-01
Dendritic cells (DCs), macrophages (MPs) and monocytes are permissive to HIV. Whether they similarly process and present HIV epitopes to HIV-specific CD8 T cells is unknown despite the critical role of peptide processing and presentation for recognition and clearance of infected cells. Cytosolic peptidases degrade endogenous proteins originating from self or pathogens, exogenous antigens preprocessed in endolysosomes, thus shaping the peptidome available for endoplasmic reticulum (ER) translocation, trimming and MHC-I presentation. Here we compared the capacity of DCs, MPs and monocyte cytosolic extracts to produce epitope precursors and epitopes. We showed differences in the proteolytic activities and expression levels of cytosolic proteases between monocyte-derived DCs and MPs and upon maturation with LPS, R848 and CL097, with mature MPs having the highest activities. Using cytosol as a source of proteases to degrade epitope-containing HIV peptides, we showed by mass spectrometry that the degradation patterns of long peptides and the kinetics and amount of antigenic peptides produced differed among DCs, MPs and monocytes. Additionally, variable intracellular stability of HIV peptides prior to loading onto MHC may accentuate the differences in epitope availability for presentation by MHC-I between these subsets. Differences in peptide degradation led to 2- to 25-fold differences in the CTL responses elicited by the degradation peptides generated in DCs, MPs and monocytes. Differences in antigen processing activities between these subsets might lead to variations in the timing and efficiency of recognition of HIV-infected cells by CTLs and contribute to the unequal capacity of HIV-specific CTLs to control viral load. PMID:25230751
-
Nicotine Inhibits Memory CTL Programming
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
-
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
-
Elicitation of anti-viral cytotoxic T lymphocytes with purified viral and H-2 antigens.
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.
-
Molecular basis for universal HLA-A*0201-restricted CD8+ T-cell immunity against influenza viruses.
Valkenburg, Sophie A; Josephs, Tracy M; Clemens, E Bridie; Grant, Emma J; Nguyen, Thi H O; Wang, George C; Price, David A; Miller, Adrian; Tong, Steven Y C; Thomas, Paul G; Doherty, Peter C; Rossjohn, Jamie; Gras, Stephanie; Kedzierska, Katherine
2016-04-19
Memory CD8(+)T lymphocytes (CTLs) specific for antigenic peptides derived from internal viral proteins confer broad protection against distinct strains of influenza A virus (IAV). However, immune efficacy can be undermined by the emergence of escape mutants. To determine how T-cell receptor (TCR) composition relates to IAV epitope variability, we used ex vivo peptide-HLA tetramer enrichment and single-cell multiplex analysis to compare TCRs targeted to the largely conserved HLA-A*0201-M158and the hypervariable HLA-B*3501-NP418antigens. The TCRαβs for HLA-B*3501-NP418 (+)CTLs varied among individuals and across IAV strains, indicating that a range of mutated peptides will prime different NP418-specific CTL sets. Conversely, a dominant public TRAV27/TRBV19(+)TCRαβ was selected in HLA-A*0201(+)donors responding to M158 This public TCR cross-recognized naturally occurring M158variants complexed with HLA-A*0201. Ternary structures showed that induced-fit molecular mimicry underpins TRAV27/TRBV19(+)TCR specificity for the WT and mutant M158peptides, suggesting the possibility of universal CTL immunity in HLA-A*0201(+)individuals. Combined with the high population frequency of HLA-A*0201, these data potentially explain the relative conservation of M158 Moreover, our results suggest that vaccination strategies aimed at generating broad protection should incorporate variant peptides to elicit cross-reactive responses against other specificities, especially those that may be relatively infrequent among IAV-primed memory CTLs.
-
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
-
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.
-
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
-
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.
-
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.
-
Contribution of advances in immunology to vaccine development.
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.
-
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
-
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.
-
Singh, Satwinder Kaur; Meyering, Maaike; Ramwadhdoebe, Tamara H; Stynenbosch, Linda F M; Redeker, Anke; Kuppen, Peter J K; Melief, Cornelis J M; Welters, Marij J P; van der Burg, Sjoerd H
2012-11-01
The ability to measure antigen-specific T cells at the single-cell level by intracellular cytokine staining (ICS) is a promising immunomonitoring tool and is extensively applied in the evaluation of immunotherapy of cancer. The protocols used to detect antigen-specific CD8+ T-cell responses generally work for the detection of antigen-specific T cells in samples that have undergone at least one round of in vitro pre-stimulation. Application of a common protocol but now using long peptides as antigens was not suitable to simultaneously detect antigen-specific CD8+ and CD4+ T cells directly ex vivo in cryopreserved samples. CD8 T-cell reactivity to monocytes pulsed with long peptides as antigens ranged between 5 and 25 % of that observed against monocytes pulsed with a direct HLA class I fitting minimal CTL peptide epitope. Therefore, we adapted our ICS protocol and show that the use of tenfold higher concentration of long peptides to load APC, the use of IFN-α and poly(I:C) to promote antigen processing and improve T-cell stimulation, does allow for the ex vivo detection of low-frequency antigen-specific CD8+ and CD4+ T cells in an HLA-independent setting. While most of the improvements were related to increasing the ability to measure CD8+ T-cell reactivity following stimulation with long peptides to at least 50 % of the response detected when using a minimal peptide epitope, the final analysis of blood samples from vaccinated patients successfully showed that the adapted ICS protocol also increases the ability to ex vivo detect low-frequency p53-specific CD4+ T-cell responses in cryopreserved PBMC samples.
-
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
-
Tumor-targeting CTL expressing a single-chain Fv specific for VEGFR2.
Kanagawa, Naoko; Yanagawa, Tatsuya; Mukai, Yohei; Yoshioka, Yasuo; Okada, Naoki; Nakagawa, Shinsaku
2010-03-26
Cytotoxic T lymphocytes (CTL) are critical effector cells in tumor immunity. Adoptive transfer therapy with in vitro-expanded tumor-specific CTL is a promising approach for preventing cancer metastasis and recurrence. Transferred CTL are not effective in clinical trials, however, due to inadequate tumor-infiltration. Therefore, the development of functionally modified CTL, such as tumor-targeting CTL, is widely desired. Here, we designed the tumor-targeting CTL expressing a single-chain antibody fragment (scFv-CTL) specific for vascular endothelial growth factor receptor 2 (VEGFR2/flk1) by transducing the CTL with a retroviral vector. The scFv-CTL bound to VEGFR2/flk1-expressing cells and retained their cytotoxic activity against tumor cells. In addition, adoptive transfer of scFv-CTL into tumor-bearing mice effectively suppressed tumor growth due to the augmented accumulation of the transferred CTL in the tumor tissue. These findings indicate that the creation of CTL capable of targeting tumor vascular endothelial cells by scFv-expression technique is considerably promising for improvement of efficacy in adoptive immunotherapy. Copyright (c) 2010 Elsevier Inc. All rights reserved.
-
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.
-
In vitro reactivity of allospecific cytotoxic T lymphocytes does not explain the taboo phenomenon.
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.
-
Zhao, Jun; Lu, Jing; Liu, Ya-qin; Yang, Hong-yan; Huang, You-tian; Zhao, Ji-min; Li, Shan; Zhai, Jing-ming; Zhao, Ming-yao; Zhang, Xi; Dong, Zi-ming
2011-01-01
To explore the specific cellular and humoral immunity induced by dendritic cells (DC) vaccine loading allogenic microvascular endothelial cell bEnd.3 antigen against U14 cervical cancer cell of mice. Mouse brain microvascular endothelial cell bEnd.3 was cultured and identified for preparation endothelial cell bEnd.3 antigen. The level of mRNA expression of vascular endothelial growth factor receptor 2 (VEGF-R₂) and integrin αV was detected by reverse transcription (RT)-PCR. The BALB/c mice were immuned with DC loading bEnd.3 antigen 4 times in 4 weeks (bEnd.3-DC group), while the mice only were immuned with DC or injected with phosphate buffer saline (PBS group) as control group. One week after last vaccination, U14 cervical cancer cells were injected subcutaneously into the mice. The tumor size, cytotoxic T lymphocyte (CTL) response of spleen lymphocytes in vitro, the percentage of CD₃+CD₈+ surface markers of spleen lymphocytes, and the titer of serum antibody were detected. The specific immunity was examined by immunocytochemistry and western blot. The expression of VEGF-R₂ and integrin αV gene in bEnd.3 cells were expressed highly. After the vaccine was injected, the tumors of mice in PBS group grew faster than those in other groups, while the tumors in bEnd.3-DC group grew slowly and disappeared after 2 weeks. The volume of tumors in DC group grew slower than those in PBS group [(0.11 ± 0.13) cm³ versus (3.38 ± 0.34) cm³]. The CTL response of spleen lymphocytes in vitro showed that bEnd.3-DC cells could kill bEnd.3 cells, the special lysis rate was more than 60%. The percentage of CD₃+CD₈+ spleen lymphocytes in bEnd.3-DC group [(38.6 ± 0.7)%] was higher than those in other groups (P < 0.05). The titer of serum antibody of bEnd.3-DC group was 1:3200, while it was 1:800 in DC group and there were not any in PBS group. Immunocytochemistry analysis indicated there were specific antigen-antibody reaction to bEnd.3 cell in bEnd.3-DC group. Western blot analysis revealed that there were specific bands at 220,000 (VEGF-R₂). bEnd.3-DC vaccine can inhibit the tumor growth of U14 cervical cancer cell of mice, which indicates that the special cellular and humoral immunity are induced by bEnd.3-DC antigen which maybe have some antigens in bEnd.3 cells that reacts with endothelial cell proliferation-related antigens.
-
Hu, Shijie; Li, Bing; Shen, Xuefeng; Zhang, Rui; Gao, Dakuan; Guo, Qingdong; Jin, Yan; Fei, Zhou
2016-04-01
The present study aimed to investigate the feasibility of using ecto-mesenchymal stem cell (EMSC)-derived dendritic cells (DCs) for glioma immunotherapy following infection by a recombinant adenovirus containing the melanoma-associated antigen D4a (MAGE-D4a) gene. The ex vivo cultured EMSCs were infected by the adenoviral plasmid containing MAGE-D4a (pAd/MAGE-D4a). Efficiency of transfection was evaluated through the detection of green fluorescent protein-marked MAGE-D4a. The MAGE-EMSCs were induced to differentiate into DCs, termed as MAGE-EMSCs-DCs. The morphology was subsequently analyzed under a microscope, and methyl thiazolyl tetrazolium (MTT) and interferon-γ (IFN-γ) assays were performed to analyze the cytotoxicity of the MAGE-EMSC-DCs on the human glioma U251 cell line. Following purification by magnetic-activated cell sorting, the EMSCs grew into swirls, with a long spindle shape and were fibroblast-like. The gene transfected with recombinant adenovirus vectors maintained high and stable expression levels of MAGE-D4a, and its efficiency was increased in a multiplicity of infection-dependent manner. The results of the MTT assay indicated that the T cells, primed by the recombinant MAGE-D4a-infected EMSC-DCs in vitro , recognized MAGE-D4a-expressing tumor cell lines in a human leukocyte antigen class I-restricted manner, and evoked a higher cytotoxic T cell (CTL) response. The CTL response induced by the MAGE-EMSC-DCs, co-cultured with the U251 cells for 24 h, produced 765.0 pg/ml IFN-γ, which was significantly greater when compared to the control wells. T lymphocytes stimulated by MAGE-EMSC-DCs evoke a higher CTL response to human glioma cell lines, and may serve as a promising therapeutic modality for the treatment of MAGE-D4a-expressing glioma.
-
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
-
Ulivieri, Cristina; Citro, Alessandra; Ivaldi, Federico; Mascolo, Dina; Ghittoni, Raffaella; Fanigliulo, Daniela; Manca, Fabrizio; Baldari, Cosima Tatiana; Li Pira, Giuseppina; Del Pozzo, Giovanna
2008-08-15
Several efforts have been invested in the identification of CTL and Th epitopes, as well as in the characterization of their immunodominance and MHC restriction, for the generation of a peptide-based HCMV vaccine. Small synthetic peptides are, however, poor antigens and carrier proteins are important for improving the efficacy of synthetic peptide vaccines. Recombinant bacteriophages appear as promising tools in the design of subunit vaccines. To investigate the antigenicity of peptides carried by recombinant bacteriophages we displayed different HCMV MHCII restricted peptides on the capsid of filamentous bacteriophage (fd) and found that hybrid bacteriophages are processed by human APC and activate HCMV-specific CD4 T-cells. Furthermore we constructed a reporter T-cell hybridoma expressing a chimeric TCR comprising murine alphabeta constant regions and human variable regions specific for the HLA-A2 restricted immunodominant NLV peptide of HCMV. Using the filamentous bacteriophage as an epitope carrier, we detected a more robust and long lasting response of the reporter T-cell hybridoma compared to peptide stimulation. Our results show a general enhancement of T-cell responses when antigenic peptides are carried by phages.
-
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
-
Protective Role of Cytotoxic T Lymphocytes in Filovirus Hemorrhagic Fever
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
-
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.
-
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.
-
Huang, Mengmeng; Mu, Changkao; Wu, Yuehong; Ye, Fei; Wang, Dan; Sun, Cong; Lv, Zhengbing; Han, Bingnan; Wang, Chunlin; Xu, Xue-Wei
2017-11-01
C-type lectins are a superfamily of Ca 2+ -dependent carbohydrate-recognition proteins, which play crucial roles in innate immunity including nonself-recognition and pathogen elimination. In the present study, two single-CRD containing C-type lectins were identified from swimming crab Portunus trituberculatus (designated as PtCTL-2 and PtCTL-3). The open reading frame (ORF) of PtCTL-2 encoded polypeptides of 485 amino acids with a signal peptide and a single carbohydrate-recognition domain (CRD), while PtCTL-3's ORF encoded polypeptides of 241 amino acids with a coiled-coil region and a single-CRD. The key motifs determining carbohydrate binding specificity in PtCTL-2 and PtCTL-3 were EPR (Glu-Pro-Arg) and QPD (Gln-Pro-Asp). EPR is a motif being identified for the first time, whereas QPD is a typical motif in C-type lectins. Different PAMPs binding features of the two recombinant proteins - PtCTL-2 (rPtCTL-2) and PtCTL-3 (rPtCTL-3) have been observed in our experiments. rPtCTL-2 could bind three pathogen-associated molecular patterns (PAMPs) with relatively high affinity, including glucan, lipopolysaccharide (LPS) and peptidoglycan (PGN), while rPtCTL-3 could barely bind any of them. However, rPtCTL-2 could bind seven kinds of microbes and rPtCTL-3 could bind six kinds in microbe binding assay. Moreover, rPtCTL-2 and rPtCTL-3 exhibited similar agglutination activity against Gram-positive bacteria, Gram-negative bacteria and fungi in agglutination assay. All these results illustrated that PtCTL-2 and PtCTL-3 could function as important pattern-recognition receptors (PRR) with broad nonself-recognition spectrum involved in immune defense against invaders. In addition, the results of carbohydrate binding specificity showed that PtCTL-2 with novel key motif had broad carbohydrate binding specificity, while PtCTL-3 with typical key motif possessed different carbohydrate binding specificity from the classical binding rule. Furthermore, PtCTL-2 and PtCTL-3 could also function as opsonin to enhance encapsulation of hemocytes against Ni-NTA beads. Copyright © 2017 Elsevier Ltd. All rights reserved.
-
1984-01-01
We studied the mechanism of lymphocytic choriomeningitis virus (LCMV) persistence and the suppression of cytotoxic T lymphocyte (CTL) responses in BALB/c WEHI mice infected at birth with LCMV Armstrong strain. Using adoptive transfer experiments we found that spleen cells from persistently infected (carrier) mice actively suppressed the expected LCMV-specific CTL response of spleen cells from normal adult mice. The suppression was specific for the CTL response and LCMV - specific antibody responses were not affected. Associated with the specific CTL suppression was the establishment of persistent LCMV infection. The transfer of spleen or lymph node cells containing LCMV - specific CTL resulted in virus clearance and prevented establishment of the carrier state. The suppression of LCMV -specific CTL responses by carrier spleen cells is not mediated by a suppressor cell, but is due to the presence of genetic variants of LCMV in spleens of carrier mice. Such virus variants selectively suppress LCMV-specific CTL responses and cause persistent infections in immunocompetent mice. In striking contrast, wild-type LCMV Armstrong, from which these variants were generated, induces a potent CTL response in immunocompetent mice and the LCMV infection is rapidly cleared. Our results show that LCMV variants that emerge during infection in vivo play a crucial role in the suppression of virus-specific CTL responses and in the maintenance of virus persistence. PMID:6332167
-
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.
-
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.
-
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.
-
Mudd, Philip A; Piaskowski, Shari M; Neves, Patricia C Costa; Rudersdorf, Richard; Kolar, Holly L; Eernisse, Christopher M; Weisgrau, Kim L; de Santana, Marlon G Veloso; Wilson, Nancy A; Bonaldo, Myrna C; Galler, Ricardo; Rakasz, Eva G; Watkins, David I
2010-09-01
The yellow fever vaccine 17D (YF17D) is one of the most effective vaccines. Its wide use and favorable safety profile make it a prime candidate for recombinant vaccines. It is believed that neutralizing antibodies account for a large measure of the protection afforded to YF17D-vaccinated individuals, however cytotoxic T lymphocyte (CTL) responses have been described in the setting of YF17D vaccination. YF17D is an ssRNA flavivirus that is translated as a full-length polyprotein, several domains of which pass into the lumen of the endoplasmic reticulum (ER). The processing and presentation machinery for MHC class I-restricted CTL responses favor cytoplasmic peptides that are transported into the ER by the transporter associated with antigen presentation proteins. In order to inform recombinant vaccine design, we sought to determine if YF17D-induced CTL responses preferentially targeted viral domains that remain within the cytoplasm. We performed whole YF17D proteome mapping of CTL responses in six Indian rhesus macaques vaccinated with YF17D using overlapping YF17D peptides. We found that the ER luminal E protein was the most immunogenic viral protein followed closely by the cytoplasmic NS3 and NS5 proteins. These results suggest that antigen processing and presentation in this model system is not preferentially affected by the subcellular location of the viral proteins that are the source of CTL epitopes. The data also suggest potential immunogenic regions of YF17D that could serve as the focus of recombinant T cell vaccine development.
-
Jessen, Birthe; Faller, Simone; Krempl, Christine D; Ehl, Stephan
2011-10-01
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-2(d) 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-2(d) 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-2(b) mice only 20% of antiviral CTL were Vβ9(+) CTL specific for the immunodominant epitope M187. The immunodominant H-2(d)-restricted CTL lysed target cells less efficiently than the immunodominant H-2(b) 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-2(d) mating (C57BL/6-H-2(dxb) mice) attenuated disease. Moreover, disease in H-2(d) 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.
-
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
-
Chen, Jiang; Li, Hong-Yu; Wang, Di; Shao, Xiao-Dong
2015-01-01
Pancreatic cancer (PC) is a deadly human malignancy. Dendritic cell (DC)-based immunotherapy with whole tumor antigens demonstrates potential efficiency in cancer treatment. Tumor RNA and tumor fusion hybrid cells are sources of whole tumor antigens for preparing DC tumor vaccines. However, the efficacy of these sources in eliciting immune responses against PC has not yet to be directly compared. In the present study, patient-derived PC cells and DCs were fused (DC–tumor hybrids) and primary cultured PC cell-derived total RNA was electroporated into autologous DCs (DC–tumor RNA). The antitumor immune responses induced by DC–tumor hybrids and DC–tumor RNA were compared directly. The results showed that both RNA and hybrid methodologies could induce tumor-specific cytotoxic T lymphocyte (CTL) responses, but pulsing DCs with total tumor RNA could induce a higher frequency of activated CTLs and T-helper cells than fusing DCs with autologous tumor cells. In addition, DC–tumor RNA triggered stronger autologous tumor cell lysis than DC–tumor hybrids. It could be concluded that DCs pulsed with whole tumor RNA are superior to those fused with tumor cells in priming anti-PC CTL responses. Electroporation with total tumor RNA may be more suitable for DC-based PC vaccination. PMID:25736302
-
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.
-
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
-
Induced pluripotent stem cells: challenges and opportunities for cancer immunotherapy.
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.
-
Posavad, C M; Huang, M L; Barcy, S; Koelle, D M; Corey, L
2000-07-15
Herpes simplex virus (HSV) establishes a lifelong infection in humans. Reactivation of latent virus occurs intermittently so that the immune system is frequently exposed to viral Ag, providing an opportunity to evaluate memory T cells to a persistent human pathogen. We studied the persistence of genital herpes lesion-derived HSV-specific CD8+ CTL from three immunocompetent individuals with frequently recurring genital HSV-2 infection. All CTL clones were HSV-2 type specific and only one to three unique clonotypes were identified from any single biopsy specimen. The TCRBV genes utilized by these clonotypes were sequenced, and clonotype-specific probes were used to longitudinally track these clonotypes in PBMC and genital lesions. CTL clonotypes were consistently detected in PBMC and lesions for at least 2 and up to 7 years, and identical clonotypes infiltrated herpes lesions spaced as long as 7.5 years apart. Moreover, these clones were functionally lytic in vivo over these time periods. Additionally, CTL clones killed target cells infected with autologous viral isolates obtained 6.5 years after CTL clones were established, suggesting that selective pressure by these CTL did not result in the mutation of CTL epitopes. Thus, HSV recurs in the face of persistent CD8+ CTL with no evidence of clonal exhaustion or mutation of CTL epitopes as mechanisms of viral persistence.
-
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
-
Genome-Editing Technologies in Adoptive T Cell Immunotherapy for Cancer.
Singh, Nathan; Shi, Junwei; June, Carl H; Ruella, Marco
2017-12-01
In this review, we discuss the most recent developments in gene-editing technology and discuss their application to adoptive T cell immunotherapy. Engineered T cell therapies targeting cancer antigens have demonstrated significant efficacy in specific patient populations. Most impressively, CD19-directed chimeric antigen receptor T cells (CART19) have led to impressive responses in patients with B-cell leukemia and lymphoma. CTL019, or KYMRIAH™ (tisagenlecleucel), a CD19 CAR T cell product developed by Novartis and the University of Pennsylvania, was recently approved for clinical use by the Food and Drug Administration, representing a landmark in the application of adoptive T cell therapies. As CART19 enters routine clinical use, improving the efficacy of this exciting platform is the next step in broader application. Novel gene-editing technologies like CRISPR-Cas9 allow facile editing of specific genes within the genome, generating a powerful platform to further optimize the activity of engineered T cells.
-
Ito, Masaki; Hayashi, Kazumi; Minamisawa, Tamiko; Homma, Sadamu; Koido, Shigeo; Shiba, Kiyotaka
2017-01-01
Adjuvants are indispensable for achieving a sufficient immune response from vaccinations. From a functional viewpoint, adjuvants are classified into two categories: "physical adjuvants" increase the efficacy of antigen presentation by antigen-presenting cells (APC) and "signal adjuvants" induce the maturation of APC. Our previous study has demonstrated that a physical adjuvant can be encrypted into proteinous antigens by creating artificial proteins from combinatorial assemblages of epitope peptides and those peptide sequences having propensities to form certain protein structures (motif programming). However, the artificial antigens still require a signal adjuvant to maturate the APC; for example, co-administration of the Toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) was required to induce an in vivo immunoreaction. In this study, we further modified the previous artificial antigens by appending the peptide motifs, which have been reported to have agonistic activity for TLR4, to create "adjuvant-free" antigens. The created antigens with triple TLR4 agonistic motifs in their C-terminus have activated NF-κB signaling pathways through TLR4. These proteins also induced the production of the inflammatory cytokine TNF-α, and the expression of the co-stimulatory molecule CD40 in APC, supporting the maturation of APC in vitro. Unexpectedly, these signal adjuvant-encrypted proteins have lost their ability to be physical adjuvants because they did not induce cytotoxic T lymphocytes (CTL) in vivo, while the parental proteins induced CTL. These results confirmed that the manifestation of a motif's function is context-dependent and simple addition does not always work for motif-programing. Further optimization of the molecular context of the TLR4 agonistic motifs in antigens should be required to create adjuvant-free antigens.
-
Hayashi, Kazumi; Minamisawa, Tamiko; Homma, Sadamu; Koido, Shigeo; Shiba, Kiyotaka
2017-01-01
Adjuvants are indispensable for achieving a sufficient immune response from vaccinations. From a functional viewpoint, adjuvants are classified into two categories: “physical adjuvants” increase the efficacy of antigen presentation by antigen-presenting cells (APC) and “signal adjuvants” induce the maturation of APC. Our previous study has demonstrated that a physical adjuvant can be encrypted into proteinous antigens by creating artificial proteins from combinatorial assemblages of epitope peptides and those peptide sequences having propensities to form certain protein structures (motif programming). However, the artificial antigens still require a signal adjuvant to maturate the APC; for example, co-administration of the Toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) was required to induce an in vivo immunoreaction. In this study, we further modified the previous artificial antigens by appending the peptide motifs, which have been reported to have agonistic activity for TLR4, to create “adjuvant-free” antigens. The created antigens with triple TLR4 agonistic motifs in their C-terminus have activated NF-κB signaling pathways through TLR4. These proteins also induced the production of the inflammatory cytokine TNF-α, and the expression of the co-stimulatory molecule CD40 in APC, supporting the maturation of APC in vitro. Unexpectedly, these signal adjuvant-encrypted proteins have lost their ability to be physical adjuvants because they did not induce cytotoxic T lymphocytes (CTL) in vivo, while the parental proteins induced CTL. These results confirmed that the manifestation of a motif’s function is context-dependent and simple addition does not always work for motif-programing. Further optimization of the molecular context of the TLR4 agonistic motifs in antigens should be required to create adjuvant-free antigens. PMID:29190754
-
Usman Mirza, Muhammad; Rafique, Shazia; Ali, Amjad; Munir, Mobeen; Ikram, Nazia; Manan, Abdul; Salo-Ahen, Outi M H; Idrees, Muhammad
2016-12-09
The recent outbreak of Zika virus (ZIKV) infection in Brazil has developed to a global health concern due to its likely association with birth defects (primary microcephaly) and neurological complications. Consequently, there is an urgent need to develop a vaccine to prevent or a medicine to treat the infection. In this study, immunoinformatics approach was employed to predict antigenic epitopes of Zika viral proteins to aid in development of a peptide vaccine against ZIKV. Both linear and conformational B-cell epitopes as well as cytotoxic T-lymphocyte (CTL) epitopes were predicted for ZIKV Envelope (E), NS3 and NS5 proteins. We further investigated the binding interactions of altogether 15 antigenic CTL epitopes with three class I major histocompatibility complex (MHC I) proteins after docking the peptides to the binding groove of the MHC I proteins. The stability of the resulting peptide-MHC I complexes was further studied by molecular dynamics simulations. The simulation results highlight the limits of rigid-body docking methods. Some of the antigenic epitopes predicted and analyzed in this work might present a preliminary set of peptides for future vaccine development against ZIKV.
-
Junaid, Muhammad; Kaushik, Aman Chandra; Ali, Arif; Ali, Syed Shujait; Mehmood, Aamir; Wei, Dong-Qing
2018-01-01
High-risk human papillomaviruses (hrHPVs) are the most prevalent viruses in human diseases including cervical cancers. Expression of E6 protein has already been reported in cervical cancer cases, excluding normal tissues. Continuous expression of E6 protein is making it ideal to develop therapeutic vaccines against hrHPVs infection and cervical cancer. Therefore, we carried out a meta-analysis of multiple hrHPVs to predict the most potential prophylactic peptide vaccines. In this study, immunoinformatics approach was employed to predict antigenic epitopes of hrHPVs E6 proteins restricted to 12 Human HLAs to aid the development of peptide vaccines against hrHPVs. Conformational B-cell and CTL epitopes were predicted for hrHPVs E6 proteins using ElliPro and NetCTL. The potential of the predicted peptides were tested and validated by using systems biology approach considering experimental concentration. We also investigated the binding interactions of the antigenic CTL epitopes by using docking. The stability of the resulting peptide-MHC I complexes was further studied by molecular dynamics simulations. The simulation results highlighted the regions from 46–62 and 65–76 that could be the first choice for the development of prophylactic peptide vaccines against hrHPVs. To overcome the worldwide distribution, the predicted epitopes restricted to different HLAs could cover most of the vaccination and would help to explore the possibility of these epitopes for adaptive immunotherapy against HPVs infections. PMID:29715318
-
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.
-
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
-
Li, Dan; Li, Yufeng; Hernandez, Jessica A.; Patenia, Rebecca; Kim, Tae Kon; Khalili, Jahan; Dougherty, Mark C.; Hanley, Patrick J.; Bollard, Catherine M.; Komanduri, Krishna V.; Hwu, Patrick; Champlin, Richard E.; Radvanyi, Laszlo G.; Molldrem, Jeffrey J.; Ma, Qing
2016-01-01
Statin treatment has been shown to reduce graft-versus-host disease (GVHD) while preserving graft-versus-tumor (GVT) effect in allogeneic stem cell transplantation (allo-HCT). Herein, we investigated whether lovastatin treatment affects the function of human cytolytic T lymphocytes (CTLs). Upon TCR stimulation, lovastatin significantly inhibited the proliferation of both CD4+ and CD8+ T cells from healthy donors while their intracellular cytokine production including IFN-γ and TNF-α remained the same with a slight decrease of IL-2. Moreover, the specific lysis of target cells by CTL lines derived from patients and normal donors specific for EBV-encoded antigen LMP2 or CMV-encoded antigen pp65 was uncompromised in the presence of lovastatin. In addition, we evaluated the effect of lovastatin on the proliferation and effector function of the CD8+ tumor–infiltrating lymphocytes (TILs) derived from melanoma patients specific for MART-1 antigen. Lovastatin significantly reduced the expansion of antigen-specific TILs upon MART-1 stimulation. However, the effector function of TILs, including the specific lysis of target cells and secretion of cytokine IFN-γ, remained intact with lovastatin treatment. Taken together, these data demonstrated that lovastatin inhibits the proliferation of EBV-, CMV- and MART-1-specific CTLs without affecting cytolytic capacity. The differential effect of lovastatin on the proliferation versus cytoxicity of CTLs might shed some light on elucidating the possible mechanisms of GVHD and GVT effect elicited by alloimmune responses. PMID:20948439
-
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
-
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.
-
Ichikawa, Kosuke; Kagamu, Hiroshi; Koyama, Kenichi; Miyabayashi, Takao; Koshio, Jun; Miura, Satoru; Watanabe, Satoshi; Yoshizawa, Hirohisa; Narita, Ichiei
2012-09-21
MHC class I-restricted peptide-based vaccination therapies have been conducted to treat cancer patients, because CD8⁺ CTL can efficiently induce apoptosis of tumor cells in an MHC class I-restricted epitope-specific manner. Interestingly, clinical responders are known to demonstrate reactivity to epitopes other than those used for vaccination; however, the mechanism underlying how antitumor T cells with diverse specificity are induced is unclear. In this study, we demonstrated that dendritic cells (DCs) that engulfed apoptotic tumor cells in the presence of non-tumor MHC class II-restricted epitope peptides, OVA(323-339), efficiently presented tumor-associated antigens upon effector-dominant CD4⁺ T cell balance against regulatory T cells (Treg) for the OVA(323-339) epitope. Th1 and Th17 induced tumor-associated antigens presentation of DC, while Th2 ameliorated tumor-antigen presentation for CD8⁺ T cells. Blocking experiments with anti-IL-23p19 antibody and anti-IL-23 receptor indicated that an autocrine mechanism of IL-23 likely mediated the diverted tumor-associated antigens presentation of DC. Tumor-associated antigens presentation of DC induced by OVA(323-339) epitope-specific CD4⁺ T cells resulted in facilitated antitumor immunity in both priming and effector phase in vivo. Notably, this immunotherapy did not require pretreatment to reduce Treg induced by tumor. This strategy may have clinical implications for designing effective antitumor immunotherapies. Copyright © 2012 Elsevier Ltd. All rights reserved.
-
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.
-
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.
-
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.
-
HIV-specific CD8+ T cells: serial killers condemned to die?
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.
-
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.
-
The B-Cell Follicle in HIV Infection: Barrier to a Cure.
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.
-
Sarkar, Koustav; Goswami, Shyamal; Roy, Soumyabrata; Mallick, Atanu; Chakraborty, Krishnendu; Bose, Anamika; Baral, Rathindranath
2010-08-01
Vaccination with neem leaf glycoprotein matured carcinoembryonic antigen (CEA) pulsed dendritic cells (DCs) enhances antigen-specific humoral and cellular immunity against CEA and restricts the growth of CEA(+) murine tumors. NLGP helps better CEA uptake, processing and presentation to T/B cells. This vaccination (DCNLGPCEA) elicits mitogen induced and CEA specific T cell proliferation, IFN gamma secretion and induces specific cytotoxic reactions to CEA(+) colon tumor cells. In addition to T cell response, DCNLGPCEA vaccine generates anti-CEA antibody response, which is principally IgG2a in nature. This antibody participates in cytotoxicity of CEA(+) cells in antibody-dependent manner. This strong anti-CEA cellular and humoral immunity protects mice from tumor development and these mice remained tumor free following second tumor inoculation, indicating generation of effector memory response. Evaluation of underlying mechanism suggests vaccination generates strong CEA specific CTL and antibody response that can completely prevent the tumor growth following adoptive transfer. In support, significant upregulation of CD44 on the surface of lymphocytes from DCNLGPCEA immunized mice was noticed with a substantial reduction in L-selectin (CD62L). (c) 2010 Elsevier B.V. All rights reserved.
-
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
-
Heiser, Axel; Coleman, Doris; Dannull, Jens; Yancey, Donna; Maurice, Margaret A.; Lallas, Costas D.; Dahm, Philipp; Niedzwiecki, Donna; Gilboa, Eli; Vieweg, Johannes
2002-01-01
Autologous dendritic cells (DCs) transfected with mRNA encoding prostate-specific antigen (PSA) are able to stimulate potent, T cell–mediated antitumor immune responses in vitro. A phase I trial was performed to evaluate this strategy for safety, feasibility, and efficacy to induce T cell responses against the self-protein PSA in patients with metastatic prostate cancer. In 13 study subjects, escalating doses of PSA mRNA–transfected DCs were administered with no evidence of dose-limiting toxicity or adverse effects, including autoimmunity. Induction of PSA-specific T cell responses was consistently detected in all patients, suggesting in vivo bioactivity of the vaccine. Vaccination was further associated with a significant decrease in the log slope PSA in six of seven subjects; three patients that could be analyzed exhibited a transient molecular clearance of circulating tumor cells. The demonstration of vaccine safety, successful in vivo induction of PSA-specific immunity, and impact on surrogate clinical endpoints provides a scientific rationale for further clinical investigation of RNA-transfected DCs in the treatment of human cancer. PMID:11828001
-
Granzyme B mediated function of Parvovirus B19-specific CD4+ T cells
Kumar, Arun; Perdomo, Maria F; Kantele, Anu; Hedman, Lea; Hedman, Klaus; Franssila, Rauli
2015-01-01
A novel conception of CD4+ T cells with cytolytic potential (CD4+ CTL) is emerging. These cells appear to have a part in controlling malignancies and chronic infections. Human parvovirus B19 can cause a persistent infection, yet no data exist on the presence of B19-specific CD4+ CTLs. Such cells could have a role in the pathogenesis of some autoimmune disorders reported to be associated with B19. We explored the cytolytic potential of human parvovirus B19-specific T cells by stimulating peripheral blood mononuclear cell (PBMC) with recombinant B19-VP2 virus-like particles. The cytolytic potential was determined by enzyme immunoassay-based quantitation of granzyme B (GrB) and perforin from the tissue culture supernatants, by intracellular cytokine staining (ICS) and by detecting direct cytotoxicity. GrB and perforin responses with the B19 antigen were readily detectable in B19-seropositive individuals. T-cell depletion, HLA blocking and ICS experiments showed GrB and perforin to be secreted by CD4+ T cells. CD4+ T cells with strong GrB responses were found to exhibit direct cytotoxicity. As anticipated, ICS of B19-specific CD4+ T cells showed expected co-expression of GrB, perforin and interferon gamma (IFN-γ). Unexpectedly, also a strong co-expression of GrB and interleukin 17 (IL-17) was detected. These cells expressed natural killer (NK) cell surface marker CD56, together with the CD4 surface marker. To our knowledge, this is the first report on virus-specific CD4+ CTLs co-expressing CD56 antigen. Our results suggest a role for CD4+ CTL in B19 immunity. Such cells could function within both immune regulation and triggering of autoimmune phenomena such as systemic lupus erythematosus (SLE) or rheumatoid arthritis. PMID:26246896
-
Immunomodulatory Effects of dsRNA and Its Potential as Vaccine Adjuvant
Jin, Bo; Sun, Tao; Yu, Xiao-Hong; Liu, Chao-Qun; Yang, Ying-Xiang; Lu, Ping; Fu, Shan-Feng; Qiu, Hui-Bin; Yeo, Anthony E. T.
2010-01-01
dsRNA can be detected by pattern recognition receptors, for example, TLR3, MDA-5, NLRP3 to induce proinflammatory cytokines responsible for innate/adaptive immunity. Recognized by endosomal TLR3 in myeloid DCs (mDCs), dsRNA can activate mDCs into mature antigen presenting cells (mAPCs) which in turn present antigen epitopes with MHC-I molecules to naïve T cells. Coadministration of protein and synthetic dsRNA analogues can elicit an antigen-specific Th1-polarized immune response which stimulates the CD8+ CTL response and possibly dampen Th17 response. Synthetic dsRNA analogues have been tested as vaccine adjuvant against viral infections in animal models. However, a dsRNA receptor, TLR3 can be expressed in tumor cells while other members of TLR family, for example, TLR4 and TLR2 have been shown to promote tumor progression, metastasis, and chemoresistance. Thus, the promising potential of dsRNA analogues as a tumor therapeutic vaccine adjuvant should be evaluated cautiously. PMID:20671921
-
Holubova, Jana; Jelinek, Jiri; Tomala, Jakub; Masin, Jiri; Kosova, Martina; Stanek, Ondrej; Bumba, Ladislav; Michalek, Jaroslav; Kovar, Marek; Sebo, Peter
2012-01-01
The Bordetella adenylate cyclase toxin-hemolysin (CyaA; also called ACT or AC-Hly) targets CD11b-expressing phagocytes and translocates into their cytosol an adenylyl cyclase (AC) that hijacks cellular signaling by conversion of ATP to cyclic AMP (cAMP). Intriguingly, insertion of large passenger peptides removes the enzymatic activity but not the cell-invasive capacity of the AC domain. This has repeatedly been exploited for delivery of heterologous antigens into the cytosolic pathway of CD11b-expressing dendritic cells by CyaA/AC− toxoids, thus enabling their processing and presentation on major histocompatibility complex (MHC) class I molecules to cytotoxic CD8+ T lymphocytes (CTLs). We produced a set of toxoids with overlapping deletions within the first 371 residues of CyaA and showed that the structure of the AC enzyme does not contain any sequences indispensable for its translocation across target cell membrane. Moreover, replacement of the AC domain (residues 1 to 371) with heterologous polypeptides of 40, 146, or 203 residues yielded CyaAΔAC constructs that delivered passenger CTL epitopes into antigen-presenting cells (APCs) and induced strong antigen-specific CD8+ CTL responses in vivo in mice and ex vivo in human peripheral blood mononuclear cell cultures. This shows that the RTX (repeats in toxin) hemolysin moiety, consisting of residues 374 to 1706 of CyaA, harbors all structural information involved in translocation of the N-terminal AC domain across target cell membranes. These results decipher the extraordinary capacity of the AC domain of CyaA to transport large heterologous cargo polypeptides into the cytosol of CD11b+ target cells and pave the way for the construction of CyaAΔAC-based polyvalent immunotherapeutic T cell vaccines. PMID:22215742
-
Altfeld, M A; Trocha, A; Eldridge, R L; Rosenberg, E S; Phillips, M N; Addo, M M; Sekaly, R P; Kalams, S A; Burchett, S A; McIntosh, K; Walker, B D; Goulder, P J
2000-09-01
Human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte (CTL) responses play a major role in the antiviral immune response, but the relative contribution of CTL responses restricted by different HLA class I molecules is less well defined. HLA-B60 or the related allele B61 is expressed in 10 to 20% of Caucasoid populations and is even more highly prevalent in Asian populations, but yet no CTL epitopes restricted by these alleles have been defined. Here we report the definition of five novel HLA-B60-restricted HIV-1-specific CTL epitopes, using peripheral blood mononuclear cells in enzyme-linked immunospot (Elispot) assays and using CTL clones and lines in cytolytic assays. The dominant HLA-B60-restricted epitope, Nef peptide KEKGGLEGL, was targeted by all eight subjects with B60 and also by both subjects with B61 studied. This study additionally establishes the utility of the Elispot assay as a more rapid and efficient method of defining novel CTL epitopes. This approach will help to define new CTL epitopes that may play an important role in the immune control of HIV-1.
-
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.
-
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.
-
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
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Somamoto, Tomonori, E-mail: somamoto@agr.kyushu-u.ac.j; Okamoto, Nobuaki; Nakanishi, Teruyuki
2009-06-20
Little is known about antigen-specific T-cell responses to viruses in teleosts due to a lack of a suitable experimental system using inbred or clonal animals. In the present study we have successfully induced an in vitro generation of virus-specific cytotoxic T-cells (CTLs) from isogeneic ginbuna crucian carp. Responder cells (primarily lymphocytes) from crucian carp haematopoietic necrosis virus (CHNV)-infected fish were capable of proliferating after stimulation in vitro with CHNV-infected syngeneic stimulator cells (primarily lymphocytes and macrophages). The effector cells collected 8 and 12 days after the in vitro stimulation efficiently lysed CHNV-infected syngeneic cells, but not CHNV-infected allogeneic cells ormore » different virus (EVA)-infected syngeneic cells. Furthermore, in situ hybridization analysis showed that some effector cells binding to a CHNV-infected target were TCRbeta or CD8alpha positive. These results provide evidence that the teleost effector cells generated in vitro correspond to virus-specific CTL and they recognize virus-infected target cells in a similar manner of mammalian counterparts.« less
-
Wells, James W; Cowled, Chris J; Darling, David; Guinn, Barbara-Ann; Farzaneh, Farzin; Noble, Alistair; Galea-Lauri, Joanna
2007-12-01
Alloreactive T-cell responses are known to result in the production of large amounts of proinflammatory cytokines capable of activating and maturing dendritic cells (DC). However, it is unclear whether these allogeneic responses could also act as an adjuvant for concurrent antigen-specific responses. To examine effects of simultaneous alloreactive and antigen-specific T-cell responses induced by semi-allogeneic DC. Semi-allogeneic DC were generated from the F(1) progeny of inbred strains of mice (C57BL/6 and C3H, or C57BL/6 and DBA). We directly primed antigen-specific CD8(+) and CD4(+) T-cells from OT-I and OT-II mice, respectively, in the absence of allogeneic responses, in vitro, and in the presence or absence of alloreactivity in vivo. In vitro, semi-allogeneic DC cross-presented ovalbumin (OVA) to naïve CD8(+) OT-I transgenic T-cells, primed naïve CD4(+) OT-II transgenic T-cells and could stimulate strong alloreactive T-cell proliferation in a primary mixed lymphocyte reaction (MLR). In vivo, semi-allogeneic DC migrated efficiently to regional lymph nodes but did not survive there as long as autologous DC. In addition, they were not able to induce cytotoxic T-lymphocyte (CTL) activity to a target peptide, and only weakly stimulated adoptively transferred OT-II cells. The CD4(+) response was unchanged in allo-tolerized mice, indicating that alloreactive T-cell responses could not provide help for concurrently activated antigen-specific responses. In an EL4 tumour-treatment model, vaccination with semi-allogeneic DC/EL4 fusion hybrids, but not allogeneic DC/EL4 hybrids, significantly increased mouse survival. Expression of self-Major histocompatibility complex (MHC) by semi-allogeneic DC can cause the induction of antigen-specific immunity, however, concurrently activated allogeneic bystander responses do not provide helper or adjuvant effects.
-
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
-
Kim, Peter S.; Lee, Peter P.
2012-01-01
A next generation approach to cancer envisions developing preventative vaccinations to stimulate a person's immune cells, particularly cytotoxic T lymphocytes (CTLs), to eliminate incipient tumors before clinical detection. The purpose of our study is to quantitatively assess whether such an approach would be feasible, and if so, how many anti-cancer CTLs would have to be primed against tumor antigen to provide significant protection. To understand the relevant dynamics, we develop a two-compartment model of tumor-immune interactions at the tumor site and the draining lymph node. We model interactions at the tumor site using an agent-based model (ABM) and dynamics in the lymph node using a system of delay differential equations (DDEs). We combine the models into a hybrid ABM-DDE system and investigate dynamics over a wide range of parameters, including cell proliferation rates, tumor antigenicity, CTL recruitment times, and initial memory CTL populations. Our results indicate that an anti-cancer memory CTL pool of 3% or less can successfully eradicate a tumor population over a wide range of model parameters, implying that a vaccination approach is feasible. In addition, sensitivity analysis of our model reveals conditions that will result in rapid tumor destruction, oscillation, and polynomial rather than exponential decline in the tumor population due to tumor geometry. PMID:23133347
-
Intravital imaging of CTLs killing islet cells in diabetic mice
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
-
Argilaguet, Jordi M.; Pérez-Martín, Eva; Nofrarías, Miquel; Gallardo, Carmina; Accensi, Francesc; Lacasta, Anna; Mora, Mercedes; Ballester, Maria; Galindo-Cardiel, Ivan; López-Soria, Sergio; Escribano, José M.; Reche, Pedro A.; Rodríguez, Fernando
2012-01-01
The lack of available vaccines against African swine fever virus (ASFV) means that the evaluation of new immunization strategies is required. Here we show that fusion of the extracellular domain of the ASFV Hemagglutinin (sHA) to p54 and p30, two immunodominant structural viral antigens, exponentially improved both the humoral and the cellular responses induced in pigs after DNA immunization. However, immunization with the resulting plasmid (pCMV-sHAPQ) did not confer protection against lethal challenge with the virulent E75 ASFV-strain. Due to the fact that CD8+ T-cell responses are emerging as key components for ASFV protection, we designed a new plasmid construct, pCMV-UbsHAPQ, encoding the three viral determinants above mentioned (sHA, p54 and p30) fused to ubiquitin, aiming to improve Class I antigen presentation and to enhance the CTL responses induced. As expected, immunization with pCMV-UbsHAPQ induced specific T-cell responses in the absence of antibodies and, more important, protected a proportion of immunized-pigs from lethal challenge with ASFV. In contrast with control pigs, survivor animals showed a peak of CD8+ T-cells at day 3 post-infection, coinciding with the absence of viremia at this time point. Finally, an in silico prediction of CTL peptides has allowed the identification of two SLA I-restricted 9-mer peptides within the hemagglutinin of the virus, capable of in vitro stimulating the specific secretion of IFNγ when using PBMCs from survivor pigs. Our results confirm the relevance of T-cell responses in protection against ASF and open new expectations for the future development of more efficient recombinant vaccines against this disease. PMID:23049728
-
Altfeld, Marcus A.; Trocha, Alicja; Eldridge, Robert L.; Rosenberg, Eric S.; Phillips, Mary N.; Addo, Marylyn M.; Sekaly, Rafick P.; Kalams, Spyros A.; Burchett, Sandra A.; McIntosh, Kenneth; Walker, Bruce D.; Goulder, Philip J. R.
2000-01-01
Human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte (CTL) responses play a major role in the antiviral immune response, but the relative contribution of CTL responses restricted by different HLA class I molecules is less well defined. HLA-B60 or the related allele B61 is expressed in 10 to 20% of Caucasoid populations and is even more highly prevalent in Asian populations, but yet no CTL epitopes restricted by these alleles have been defined. Here we report the definition of five novel HLA-B60-restricted HIV-1-specific CTL epitopes, using peripheral blood mononuclear cells in enzyme-linked immunospot (Elispot) assays and using CTL clones and lines in cytolytic assays. The dominant HLA-B60-restricted epitope, Nef peptide KEKGGLEGL, was targeted by all eight subjects with B60 and also by both subjects with B61 studied. This study additionally establishes the utility of the Elispot assay as a more rapid and efficient method of defining novel CTL epitopes. This approach will help to define new CTL epitopes that may play an important role in the immune control of HIV-1. PMID:10954555
-
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
-
Wang, Yue-Dan; Chen, Wei Feng
2004-11-01
To assess specific cytotoxic T lymphocytes (CTLs) against Severe acute respiratory syndrome (SARS)-coronavirus, a modified DimerX flow cytometry assay was performed with peripheral blood mononuclear cell (PBMC) from HLA-A2+ SARS-recovered donors at different time points post disease. CD8+DimerX-S1203+ CTLs were detected in the PBMC from these donors up to 3 months after recovery. The percentages of CD8+DimerX-S1203+ cells paralleled the numbers of interferon-gamma-positive spots in an ELISPOT assay using the same antigenic peptide. In conclusion, DimerX-based flow cytometry staining may prove to be a real-time method to screen for CTL directed at epitopes from a newly identified virus.
-
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.
-
Chemotherapy Enhances Cross-Presentation of Nuclear Tumor Antigens
Anyaegbu, Chidozie C.; Lake, Richard A.; Heel, Kathy; Robinson, Bruce W.; Fisher, Scott A.
2014-01-01
Cross-presentation of tumor antigen is essential for efficient priming of naïve CD8+ T lymphocytes and induction of effective anti-tumor immunity. We hypothesized that the subcellular location of a tumor antigen could affect the efficiency of cross-presentation, and hence the outcome of anti-tumor responses to that antigen. We compared cross-presentation of a nominal antigen expressed in the nuclear, secretory, or cytoplasmic compartments of B16 melanoma tumors. All tumors expressed similar levels of the antigen. The antigen was cross-presented from all compartments but when the concentration was low, nuclear antigen was less efficiently cross-presented than antigen from other cellular locations. The efficiency of cross-presentation of the nuclear antigen was improved following chemotherapy-induced tumor cell apoptosis and this correlated with an increase in the proportion of effector CTL. These data demonstrate that chemotherapy improves nuclear tumor antigen cross-presentation and could be important for anti-cancer immunotherapies that target nuclear antigens. PMID:25243472
-
BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes.
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.
-
Buschow, Christian; Charo, Jehad; Anders, Kathleen; Loddenkemper, Christoph; Jukica, Ana; Alsamah, Wisam; Perez, Cynthia; Willimsky, Gerald; Blankenstein, Thomas
2010-03-15
Visualizing oncogene/tumor Ag expression by noninvasive imaging is of great interest for understanding processes of tumor development and therapy. We established transgenic (Tg) mice conditionally expressing a fusion protein of the SV40 large T Ag and luciferase (TagLuc) that allows monitoring of oncogene/tumor Ag expression by bioluminescent imaging upon Cre recombinase-mediated activation. Independent of Cre-mediated recombination, the TagLuc gene was expressed at low levels in different tissues, probably due to the leakiness of the stop cassette. The level of spontaneous TagLuc expression, detected by bioluminescent imaging, varied between the different Tg lines, depended on the nature of the Tg expression cassette, and correlated with Tag-specific CTL tolerance. Following liver-specific Cre-loxP site-mediated excision of the stop cassette that separated the promoter from the TagLuc fusion gene, hepatocellular carcinoma development was visualized. The ubiquitous low level TagLuc expression caused the failure of transferred effector T cells to reject Tag-expressing tumors rather than causing graft-versus-host disease. This model may be useful to study different levels of tolerance, monitor tumor development at an early stage, and rapidly visualize the efficacy of therapeutic intervention versus potential side effects of low-level Ag expression in normal tissues.
-
Loss of DNAM-1 contributes to CD8+ T cell exhaustion in chronic HIV-1 infection
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
-
1986-01-01
The UV-induced, C3H fibrosarcoma, 1591, expresses at least three unique MHC class I antigens not found on normal C3H tissue. Here we report the complete DNA sequence of the three novel class I genes encoding these molecules, and describe in detail the recognition of the individual products by tumor-reactive and allospecific CTL. Remarkably, although C3H does not appear to express H-2L locus information, this C3H tumor expresses two distinct antigens, termed A149 and A166, which are extremely homologous to each other and to the H-2Ld antigen from BALB/c. The gene encoding the third novel class I antigen from 1591, A216, is quite homologous to H-2Kk) throughout its 3' end. Since all three of these genes account for polymorphic restriction fragments not found in C3H, it is likely that they were derived by recombination from the endogenous class I genes of C3H. The DNA sequence homology of A149, A166, and H-2Ld is especially significant given the functional conservation observed between the products of these genes. Limited sequence substitutions appear to correlate with some of the discrete serological differences observed between these molecules. In addition, both A149 and A166 crossreact, but to differing extents, with H-2Ld at the level of T cell recognition. Our results are consistent with the view that CTL recognize complex conformational determinants on class I molecules, but extend previous observations by comparing a set of antigens with discrete and overlapping structural and functional differences. PMID:3489061
-
Balancing Immune Protection and Immune Pathology by CD8+ T-Cell Responses to Influenza Infection
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
-
2012-01-01
Background Merkel cell carcinoma (MCC) is a relatively new addition to the expanding category of oncovirus-induced cancers. Although still comparably rare, the number of cases has risen dramatically in recent years. Further complicating this trend is that MCC is an extremely aggressive neoplasm with poor patient prognosis and limited treatment options for advanced disease. The causative agent of MCC has been identified as the merkel cell polyomavirus (MCPyV). The MCPyV-encoded large T (LT) antigen is an oncoprotein that is theorized to be essential for virus-mediated tumorigenesis and is therefore, an excellent MCC antigen for the generation of antitumor immune responses. As a foreign antigen, the LT oncoprotein avoids the obstacle of immune tolerance, which normally impedes the development of antitumor immunity. Ergo, it is an excellent target for anti-MCC immunotherapy. Since tumor-specific CD8+ T cells lead to better prognosis for MCC and numerous other cancers, we have generated a DNA vaccine that is capable of eliciting LT-specific CD8+ T cells. The DNA vaccine (pcDNA3-CRT/LT) encodes the LT antigen linked to a damage-associated molecular pattern, calreticulin (CRT), as it has been demonstrated that the linkage of CRT to antigens promotes the induction of antigen-specific CD8+ T cells. Results The present study shows that DNA vaccine-induced generation of LT-specific CD8+ T cells is augmented by linking CRT to the LT antigen. This is relevant since the therapeutic effects of the pcDNA3-CRT/LT DNA vaccine is mediated by LT-specific CD8+ T cells. Mice vaccinated with the DNA vaccine produced demonstrably more LT-specific CD8+ T cells. The DNA vaccine was also able to confer LT-specific CD8+ T cell-mediated protective and therapeutic effects to prolong the survival of mice with LT-expressing tumors. In the interest of determining the LT epitope which most MCC-specific CD8+ T cells recognize, we identified the amino acid sequence of the immunodominant LT epitope as aa19-27 (IAPNCYGNI) and found that it is H-2kb-restricted. Conclusion The results of this study can facilitate the development of other modes of MCC treatment such as peptide-based vaccines and adoptive transfer of LT-specific CD8+ T cells. Likewise, the MCC DNA vaccine has great potential for clinical translation as the immunologic specificity is high and the treatment strategy can be exported to address other virus-induced tumors. PMID:23095249
-
De Sanctis, Francesco; Sandri, Sara; Martini, Matteo; Mazzocco, Marta; Fiore, Alessandra; Trovato, Rosalinda; Garetto, Stefano; Brusa, Davide; Ugel, Stefano; Sartoris, Silvia
2018-06-14
Most active cancer immunotherapies able to induce a long-lasting protection against tumours are based on the activation of tumour-specific cytotoxic T lymphocytes (CTLs). Cell death by hyperthermia induces apoptosis followed by secondary necrosis, with the production of factors named "danger associated molecular pattern" (DAMP) molecules (DAMPs), that activate dendritic cells (DCs) to perform antigen uptake, processing and presentation, followed by CTLs cross priming. In many published studies, hyperthermia treatment of tumour cells is performed at 42-45 °C; these temperatures mainly promote cell surface expression of DAMPs. Treatment at 56 °C of tumour cells was shown to induce DAMPs secretion rather than their cell surface expression, improving DC activation and CTL cross priming in vitro. Thus we tested the relevance of this finding in vivo on the generation of a tumour-specific memory immune response, in the TRAMP-C2 mouse prostate carcinoma transplantable model. TRAMP-C2 tumour cells treated at 56 °C were able not only to activate DCs in vitro but also to trigger a tumour-specific CTL-dependent immune response in vivo. Prophylactic vaccination with 56 °C-treated TRAMP-C2 tumour cells alone provided protection against TRAMP-C2 tumour growth in vivo, whilst in the therapeutic regimen, control of tumour growth was achieved combining immunization with adjuvant chemotherapy. Copyright © 2018 Elsevier Ltd. All rights reserved.
-
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)
-
On the dynamics of acute EBV infection and the pathogenesis of infectious mononucleosis
Hadinoto, Vey; Shapiro, Michael; Greenough, Thomas C.; Sullivan, John L.; Luzuriaga, Katherine
2008-01-01
Memory B cells latently infected with Epstein-Barr virus (mBLats) in the blood disappear rapidly on presentation with acute symptomatic primary infection (acute infectious mononucleosis [AIM]). They undergo a simple exponential decay (average half-life: 7.5 ± 3.7 days) similar to that of normal memory B cells. The cytotoxic T lymphocyte (CTL) response to immediate early (IE) lytic antigens (CTLIEs) also decays over this time period, but no such correlation was observed for the CTL response to lytic or latent antigens or to the levels of virions shed into saliva. We have estimated the average half-life of CTLIEs to be 73 (± 23) days. We propose that cycles of infection and reactivation occur in the initial stages of infection that produce high levels of mBLats in the circulation. Eventually the immune response arises and minimizes these cycles leaving the high levels of mBLats in the blood to decay through simple memory B-cell homeostasis mechanisms. This triggers the cells to reactivate the virus whereupon most are killed by CTLIEs before they can release virus and infect new cells. The release of antigens caused by this large-scale destruction of infected cells may trigger the symptoms of AIM and be a cofactor in other AIM-associated diseases. PMID:17991806
-
Mokshina, Natalia; Gorshkova, Tatyana; Deyholos, Michael K
2014-01-01
Plant chitinases (EC 3.2.1.14) and chitinase-like (CTL) proteins have diverse functions including cell wall biosynthesis and disease resistance. We analyzed the expression of 34 chitinase and chitinase-like genes of flax (collectively referred to as LusCTLs), belonging to glycoside hydrolase family 19 (GH19). Analysis of the transcript expression patterns of LusCTLs in the stem and other tissues identified three transcripts (LusCTL19, LusCTL20, LusCTL21) that were highly enriched in developing bast fibers, which form cellulose-rich gelatinous-type cell walls. The same three genes had low relative expression in tissues with primary cell walls and in xylem, which forms a xylan type of secondary cell wall. Phylogenetic analysis of the LusCTLs identified a flax-specific sub-group that was not represented in any of other genomes queried. To provide further context for the gene expression analysis, we also conducted phylogenetic and expression analysis of the cellulose synthase (CESA) family genes of flax, and found that expression of secondary wall-type LusCESAs (LusCESA4, LusCESA7 and LusCESA8) was correlated with the expression of two LusCTLs (LusCTL1, LusCTL2) that were the most highly enriched in xylem. The expression of LusCTL19, LusCTL20, and LusCTL21 was not correlated with that of any CESA subgroup. These results defined a distinct type of CTLs that may have novel functions specific to the development of the gelatinous (G-type) cellulosic walls.
-
Ren, Wei-na; Chang, Chun-kang; Fan, Hua-hua; Guo, Fang; Ren, Ya-na; Yang, Jie; Guo, Juan; Li, Xiao
2011-01-01
To improve its antitumor effect, we used human leukocyte antigen -A2 (HLA-A2)-positive human dendritic cell (DC)-derived DEXs (DC-derived exosomes) to support NY-ESO-1 antigen and polyI:C, with the aim of increasing the proliferation of specific cytotoxic T lymphocytes (CTL) in transgenic mice. Mature dendritic cells derived from peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy adults with positive HLA-2A. Using centrifuge and membrane ultrafiltration, EXO (exosomes) were extracted from the supernatant of DCs secretions. Transgenic C57 mice were immunized with human-derived tumor testis antigen NY-ESO-1/EXO, with or without polyI:C. Mice were sacrificed four weeks after immunization, and spleen cells were isolated and tested for function. The experiments included antigen-specific CTL proliferation, as tested by dimerization and antitumor effects for K562 cells as well as melanoma, tested at different ratios of effected cells:target cells (0:1, 10:1, 50:1, and 100:1). Dimerization experiments indicated that the effect of DEX/TSA (tumor specific antigens) + PolyI:C was 2.36 ± 1.10% and the control was 0.38 ± 0.31%, while the effect of DEX/TSA was 1.97 ± 0.63% and the control was 0.36 ± 0.07%. Antitumor effects by DEX/TSA: PolyI:C for the cell ratios of 0:1, 10:1, 50:1, and 100:1 were 11.14 ± 1.36%, 14.17 ± 0.62%, 15.71 ± 2.48%, and 24.31 ± 2.91%, respectively, for K562 cells. The antitumor effects for DEX/TSA for the cell ratios of 0:1, 10:1, 50:1, and 100:1 were 12.23 ± 2.25%, 13.10 ± 1.57%, 15.27 ± 2.93%, and 19.87 ± 2.72%, respectively, for K562 cells. With ratios of 10:1 and 100:1, the antitumor effects of DEX/TSA + PolyI:C were better than for the DEX/TSA group (P < 0.05). However, higher ratios of effecter cells to target cells increased, and there were no significant improvements in antitumor effect for control cells. Combining PolyI:C with DEX/TSA derived from healthy human blood positive for HLA-A2 is a promising strategy for developing new subcellular antitumor vaccination.
-
Porter, David L.; Hwang, Wei-Ting; Frey, Noelle V.; Lacey, Simon F.; Shaw, Pamela A.; Loren, Alison W.; Bagg, Adam; Marcucci, Katherine T.; Shen, Angela; Gonzalez, Vanessa; Ambrose, David; Grupp, Stephan A.; Chew, Anne; Zheng, Zhaohui; Milone, Michael C.; Levine, Bruce L.; Melenhorst, Jan J.; June, Carl H.
2018-01-01
Patients with multiply relapsed or refractory chronic lymphocytic leukemia (CLL) have a poor prognosis. Chimeric antigen receptor (CAR)–modified T cells targeting CD19 have the potential to improve on the low complete response rates with conventional therapies by inducing sustained remissions in patients with refractory B cell malignancies. We previously reported preliminary results on three patients with refractory CLL. We report the mature results from our initial trial using CAR-modified T cells to treat 14 patients with relapsed and refractory CLL. Autologous T cells transduced with a CD19-directed CAR (CTL019) lentiviral vector were infused into patients with relapsed/refractory CLL at doses of 0.14 × 108 to 11 × 108 CTL019 cells (median, 1.6 × 108 cells). Patients were monitored for toxicity, response, expansion, and persistence of circulating CTL019 T cells. The overall response rate in these heavily pretreated CLL patients was 8 of 14 (57%), with 4 complete remissions (CR) and 4 partial remissions (PR). The in vivo expansion of the CAR T cells correlated with clinical responses, and the CAR T cells persisted and remained functional beyond 4 years in the first two patients achieving CR. No patient in CR has relapsed. All responding patients developed B cell aplasia and experienced cytokine release syndrome, coincident with T cell proliferation. Minimal residual disease was not detectable in patients who achieved CR, suggesting that disease eradication may be possible in some patients with advanced CLL. PMID:26333935
-
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.
-
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
-
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
-
Peptide-independent Recognition by Alloreactive Cytotoxic T Lymphocytes (CTL)
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
-
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
-
Vergleich von rekombinanten Vaccinia- und DNA-Vektoren zur Tumorimmuntherapie im C57BL/6-Mausmodell
NASA Astrophysics Data System (ADS)
Johnen, Heiko
2002-10-01
In der vorliegenden Arbeit wurden Tumorimpfstoffe auf der Basis des Plasmid-Vektors pCI, modified vaccinia virus Ankara (MVA) und MVA-infizierten dendritischen Zellen entwickelt und durch Sequenzierung, Western blotting und durchflußzytometrische Analyse überprüft. Die in vivo Wirksamkeit der Vakzinen wurde in verschiedenen Tumormodellen in C57BL/6 Mäusen verglichen. Die auf dem eukaryotischen Expressionsvektor pCI basierende DNA-Vakzinierung induzierte einen sehr wirksamen, antigenspezifischen und langfristigen Schutz vor Muzin, CEA oder beta-Galactosidase exprimierenden Tumoren. Eine MVA-Vakzinierung bietet in den in dieser Arbeit durchgeführten Tumormodellen keinen signifikanten Schutz vor Muzin oder beta-Galactosidase exprimierenden Tumoren. Sowohl humane, als auch murine in vitro generierte dendritische Zellen lassen sich mit MVA – im Vergleich zu anderen viralen Vektoren – sehr gut infizieren. Die Expressionsrate der eingefügten Gene ist aber gering im Vergleich zur Expression in permissiven Wirtszellen des Virus (embryonale Hühnerfibroblasten). Es konnte gezeigt werden, daß eine MVA-Infektion dendritischer Zellen ähnliche Auswirkungen auf den Reifezustand humaner und muriner dendritischer Zellen hat, wie eine Infektion mit replikationskompetenten Vakzinia-Stämmen, und außerdem die Hochregulation von CD40 während der terminalen Reifung von murinen dendritischen Zellen inhibiert wird. Die während der langfristigen in vitro Kultur auf CEF-Zellen entstandenen Deletionen im MVA Genom führten zu einer starken Attenuierung und dem Verlust einiger Gene, die immunmodulatorische Proteine kodieren, jedoch nicht zu einer Verminderung des zytopathischen Effekts in dendritischen Zellen. Die geringe Expressionsrate und die beobachtete Inhibition der Expression kostimulatorischer Moleküle auf dendritischen Zellen kann für eine wenig effektive Induktion einer Immunantwort in MVA vakzinierten Tieren durch cross priming oder die direkte Infektion antigenpräsentierender Zellen verantwortlich sein. Durch die Modifikation einer Methode zur intrazellulären IFN-gamma Färbung konnten in vakzinierten Mäusen tumorantigenspezifische CTL sensitiv und quantitativ detektiert werden. Die so bestimmte CTL-Frequenz, nicht jedoch die humorale Antwort, korrelierte mit der in vivo Wirksamkeit der verschiedenen Vakzinen: DNA vakzinierte Tiere entwickeln starke tumorantigenspezifische CTL-Antworten, wohingegen in MVA-vakzinierten Tieren überwiegend gegen virale Epitope gerichtete CD4 und CD8-T-Zellen detektiert wurden. Die Wirksamkeit der pCI-DNA-Vakzine spricht für die Weiterentwicklung in weiteren präklinischen Mausmodellen, beispielsweise unter Verwendung von MUC1 oder HLA-A2 transgenen Mäusen. Die Methoden zur Detektion Tumorantigen-spezifischer CTL in 96-Loch-Mikrotiterplatten können dabei zur systematischen Suche nach im Menschen immundominanten T-Zell-Epitopen im Muzin-Molekül genutzt werden. Der durchgeführte Vergleich der auf den Vektoren pCI und MVA basierenden Vakzinen und die Analyse neuerer Publikationen führen zu dem Ergebniss, daß vor allem DNA-Vakzinen in Zukunft eine wichtige Rolle bei der Entwicklung von aktiven Tumorimpfstoffen spielen werden. Rekombinante MVA-Viren, eventuell in Kombination mit DNA- oder anderen Vektoren, haben sich dagegen in zahlreichen Studien als wirksame Impfstoffe zur Kontrolle von durch Pathogene hervorgerufenen Infektionserkrankungen erwiesen. In this study, tumor vaccines based on the plasmid pCI, the attenuated vaccinia virus strain modified vaccinia virus Ankara (MVA) and MVA-infected dendritic cells were constructed and characterized by sequencing, Western blot and flow cytometric analysis. The efficiency to induce tumor immunity in vivo was compared in several C57BL/6 mouse tumor models. Naked DNA Vaccination based on the eukaryotic expression vector pCI did induce very effective, antigen-specific and long-term protection against tumor cell lines expressing mucin, CEA or beta-Gal whereas MVA vaccination did not elicit protective immunity against Mucin or beta-Gal expressing tumors. MVA does infect human or murine in vitro generated dendritic cells very efficiently compared to other viral vectors, however expression levels of the inserted antigens in dendritic cells are significantly lower than in permissive host cells (chicken embryo fibroblasts). It could be shown that the effect of MVA infection on the maturation status of dendritic cells is similar to the effects described for dendritic cells infected with replication competent vaccinia strains. In addition it was shown that the upregulation of the important costimulatory molecule CD40 through LPS stimulation is strongly inhibited in MVA infected cells. During passage in tissue culture, MVA has accumulated a number of large deletions, including a number of immunomodulatory molecules and resulting in a strong attenuation. However the strong cytopathic effect on dendritic cells is maintained. The low level of expression and the effect on dendritic cell maturation may be responsible for the failure of MVA to induce tumor immunity through either cross presentation or direct infection of antigen presenting cells. To detect and quantify tumor-antigen-specific CTL a method based on intracellular IFN-gamma staining was modified and it could be shown that the cellular – but not the humoral – response does correlate with in vivo protection: DNA but not MVA vaccines do induce high levels of tumorantigen-specific CTL whereas MVA-vaccines do induce strong and long lasting CD4 and CD8-T-cell responses against vaccinia antigens. The excellent protection induced by pCI-DNA-vaccination in different tumor models does encourage us to further investigate the elicitation of tumor immunity in MUC1 or HLA-A2 transgenic mice. In mice transgenic for human MHC-I, the IFN-gamma staining protocol could be used to systematically screen for mucin T-cell epitopes that are relevant in humans.
-
Computational elucidation of potential antigenic CTL epitopes in Ebola virus.
Dikhit, Manas R; Kumar, Santosh; Vijaymahantesh; Sahoo, Bikash R; Mansuri, Rani; Amit, Ajay; Yousuf Ansari, Md; Sahoo, Ganesh C; Bimal, Sanjiva; Das, Pradeep
2015-12-01
Cell-mediated immunity is important for the control of Ebola virus infection. We hypothesized that those HLA A0201 and HLA B40 restricted epitopes derived from Ebola virus proteins, would mount a good antigenic response. Here we employed an immunoinformatics approach to identify specific 9mer amino acid which may be capable of inducing a robust cell-mediated immune response in humans. We identified a set of 28 epitopes that had no homologs in humans. Specifically, the epitopes derived from NP, RdRp, GP and VP40 share population coverage of 93.40%, 84.15%, 74.94% and 77.12%, respectively. Based on the other HLA binding specificity and population coverage, seven novel promiscuous epitopes were identified. These 7 promiscuous epitopes from NP, RdRp and GP were found to have world-wide population coverage of more than 95% indicating their potential significance as useful candidates for vaccine design. Epitope conservancy analysis also suggested that most of the peptides are highly conserved (100%) in other virulent Ebola strain (Mayinga-76, Kikwit-95 and Makona-G3816- 2014) and can therefore be further investigated for their immunological relevance and usefulness as vaccine candidates. Copyright © 2015 Elsevier B.V. All rights reserved.
-
Engineering HIV-Specific Immunity with Chimeric Antigen Receptors.
Kitchen, Scott G; Zack, Jerome A
2016-12-01
HIV remains a highly important public health and clinical issue despite many recent advances in attempting to develop a cure, which has remained elusive for most people infected with HIV. HIV disease can be controlled with pharmacologic therapies; however, these treatments are expensive, may have severe side effects, and are not curative. Consequently, an improved means to control or eliminate HIV replication is needed. Cytotoxic T lymphocytes (CTLs) play a critical role in controlling viral replication and are an important part in the ability of the immune response to eradicate most viral infections. There are considerable efforts to enhance CTL responses in HIV-infected individuals in hopes of providing the immune response with armaments to more effectively control viral replication. In this review, we discuss some of these efforts and focus on the development of a gene therapy-based approach to engineer hematopoietic stem cells with an HIV-1-specific chimeric antigen receptor, which seeks to provide an inexhaustible source of HIV-1-specific immune cells that are MHC unrestricted and superior to natural antiviral T cell responses. These efforts provide the basis for further development of T cell functional enhancement to target and treat chronic HIV infection in hopes of eradicating the virus from the body.
-
MUC1-specific cytotoxic T lymphocytes eradicate tumors when adoptively transferred in vivo.
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.
-
Beloki, Lorea; Ciaurriz, Miriam; Mansilla, Cristina; Zabalza, Amaya; Perez-Valderrama, Estela; Samuel, Edward R; Lowdell, Mark W; Ramirez, Natalia; Olavarria, Eduardo
2015-05-20
Adoptive transfer of CMV-specific T cells has shown promising results in preventing pathological effects caused by opportunistic CMV infection in immunocompromised patients following allogeneic hematopoietic stem cell transplantation. The majority of studies have used steady-state leukapheresis for CMV-reactive product manufacture, a collection obtained prior to or months after G-CSF mobilization, but the procurement of this additional sample is often not available in the unrelated donor setting. If the cellular product for adoptive immunotherapy could be generated from the same G-CSF mobilized collection, the problems associated with the additional harvest could be overcome. Despite the tolerogenic effects associated with G-CSF mobilization, recent studies described that CMV-primed T cells generated from mobilized donors remain functional. MHC-multimers are potent tools that allow the rapid production of antigen-specific CTLs. Therefore, in the present study we have assessed the feasibility and efficacy of CMV-specific CTL manufacture from G-CSF mobilized apheresis using MHC-multimers. CMV-specific CTLs can be efficiently isolated from G-CSF mobilized samples with Streptamers and are able to express activation markers and produce cytokines in response to antigenic stimulation. However, this anti-viral functionality is moderately reduced when compared to non-mobilized products. The translation of Streptamer technology for the isolation of anti-viral CTLs from G-CSF mobilized PBMCs into clinical practice would widen the number of patients that could benefit from this therapeutic strategy, although our results need to be taken into consideration before the infusion of antigen-specific T cells obtained from G-CSF mobilized samples.
-
Cheng, Wing Ki; Wee, Kathleen; Kollmann, Tobias R.
2014-01-01
Robust CD8+ T cell responses are essential for immune protection against intracellular pathogens. Using parenteral administration of ovalbumin (OVA) protein as a model antigen, the effect of the Toll-like receptor 9 (TLR9) agonist, CpG oligodeoxynucleotide (ODN) 1826, as an adjuvant delivered either topically, subcutaneously, or intramuscularly on antigen-specific CD8+ T cell responses in a mouse model was evaluated. Topical CpG adjuvant increased the frequency of OVA-specific CD8+ T cells in the peripheral blood and in the spleen. The more effective strategy to administer topical CpG adjuvant to enhance CD8+ T cell responses was single-dose administration at the time of antigen injection with a prime-boost regimen. Topical CpG adjuvant conferred both rapid and long-lasting protection against systemic challenge with recombinant Listeria monocytogenes expressing the cytotoxic T lymphocyte (CTL) epitope of OVA257–264 (strain Lm-OVA) in a TLR9-dependent manner. Topical CpG adjuvant induced a higher proportion of CD8+ effector memory T cells than parenteral administration of the adjuvant. Although traditional vaccination strategies involve coformulation of antigen and adjuvant, split administration using topical adjuvant is effective and has advantages of safety and flexibility. Split administration of topical CpG ODN 1826 with parenteral protein antigen is superior to other administration strategies in enhancing both acute and memory protective CD8+ T cell immune responses to subcutaneous protein vaccines. This vaccination strategy induces rapid and persistent protective immune responses against the intracellular organism L. monocytogenes. PMID:24391136
-
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
-
Two roles for CD4 cells in the control of the generation of cytotoxic T lymphocytes.
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.
-
Chapuis, Aude G.; Roberts, Ilana M.; Thompson, John A.; Margolin, Kim A.; Bhatia, Shailender; Lee, Sylvia M.; Sloan, Heather L.; Lai, Ivy P.; Farrar, Erik A.; Wagener, Felecia; Shibuya, Kendall C.; Cao, Jianhong; Wolchok, Jedd D.; Greenberg, Philip D.
2016-01-01
Purpose Peripheral blood–derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti–CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8+ T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses, highlighting the promise of this strategy. PMID:27269940
-
Chapuis, Aude G; Roberts, Ilana M; Thompson, John A; Margolin, Kim A; Bhatia, Shailender; Lee, Sylvia M; Sloan, Heather L; Lai, Ivy P; Farrar, Erik A; Wagener, Felecia; Shibuya, Kendall C; Cao, Jianhong; Wolchok, Jedd D; Greenberg, Philip D; Yee, Cassian
2016-11-01
Purpose Peripheral blood-derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti-CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8 + T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses, highlighting the promise of this strategy.
-
Yuba, Eiji; Harada, Atsushi; Sakanishi, Yuichi; Watarai, Shinobu; Kono, Kenji
2013-04-01
Highly pH-sensitive liposomes that deliver antigenic molecules into cytosol through fusion with or destabilization of endosome were prepared by surface modification of egg yolk phosphatidylcholine/dioleoylphosphatidylethanolamine (1/1, mol/mol) liposomes with 3-methylglutarylated poly(glycidol) of linear (MGlu-LPG) or hyperbranched structure (MGlu-HPG). These polymer-modified liposomes were stable at neutral pH, but they became strongly destabilized below pH 6, which corresponds to the pH of endosome. These polymer-modified liposomes were taken up by murine dendritic cells (DCs) more efficiently than the unmodified liposomes were through an endocytic pathway. They introduced entrapped ovalbumin (OVA) molecules into cytosol. Subcutaneous or nasal administration of the polymer-modified liposomes loaded with OVA induced generation of OVA-specific cytotoxic T cells (CTL) much more effectively than the unmodified liposomes loaded with OVA. Furthermore, administration of the polymer-modified OVA-loaded liposomes to mice bearing E.G7-OVA tumor significantly reduced the tumor burden, although the OVA-loaded unmodified liposomes only slightly affected tumor growth. Results suggest that the polymer-modified liposomes with highly pH-sensitive destabilizing property are promising as antigen carriers for efficient cancer immunotherapy. Copyright © 2012 Elsevier Ltd. All rights reserved.
-
Yoshizaki, Yuta; Yuba, Eiji; Komatsu, Toshihiro; Udaka, Keiko; Harada, Atsushi; Kono, Kenji
2016-09-26
To establish peptide vaccine-based cancer immunotherapy, we investigated the improvement of antigenic peptides by encapsulation with pH-sensitive fusogenic polymer-modified liposomes for induction of antigen-specific immunity. The liposomes were prepared by modification of egg yolk phosphatidylcholine and l-dioleoyl phosphatidylethanolamine with 3-methyl-glutarylated hyperbranched poly(glycidol) (MGlu-HPG) and were loaded with antigenic peptides derived from ovalbumin (OVA) OVA-I (SIINFEKL), and OVA-II (PSISQAVHAAHAEINEAP β A), which bind, respectively, to major histocompatibility complex (MHC) class I and class II molecules on dendritic cell (DCs). The peptide-loaded liposomes were taken up efficiently by DCs. The peptides were delivered into their cytosol. Administration of OVA-I-loaded MGlu-HPG-modified liposomes to mice bearing OVA-expressing E.G7-OVA tumors induced the activation of OVA-specific CTLs much more efficiently than the administration of free OVA-I peptide did. Mice strongly rejected E.G7-OVA cells after immunization with OVA-I peptide-loaded MGlu-HPG liposomes, although mice treated with free OVA-I peptide only slightly rejected the cells. Furthermore, efficient suppression of tumor volume was observed when tumor-bearing mice were immunized with OVA-I-peptide-loaded liposomes. Immunization with OVA-II-loaded MGlu-HPG-modified liposomes exhibited much lower tumor-suppressive effects. Results indicate that MGlu-HPG liposomes might be useful for improvement of CTL-inducing peptides for efficient cancer immunotherapy.
-
Casein expression in cytotoxic T lymphocytes.
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
-
Tang, Baishan; Zhou, Weijun; Du, Jingwen; He, Yanjie; Li, Yuhua
2015-08-01
T-cell-mediated immunotherapy of hematological malignancies requires selection of targeted tumor-associated antigens and T-cell epitopes contained in these tumor proteins. Epidermal growth factor receptor pathway substrate 8 (EPS8), whose function is pivotal for tumor proliferation, progression and metastasis, has been found to be overexpressed in most human tumor types, while its expression in normal tissue is low. The aim of the present study was to identify human leukemia antigen (HLA)-A*0201-restricted epitopes of EPS8 by using a reverse immunology approach. To achieve this, computer algorithms were used to predict HLA-A*0201 molecular binding, proteasome cleavage patterns as well as translocation of transporters associated with antigen processing. Candidate peptides were experimentally validated by T2 binding affinity assay and brefeldin-A decay assay. The functional avidity of peptide-specific cytotoxic T lymphocytes (CTLs) induced from peripheral blood mononuclear cells of healthy volunteers were evaluated by using an enzyme-linked immunosorbent spot assay and a cytotoxicity assay. Four peptides, designated as P455, P92, P276 and P360, had high affinity and stability of binding towards the HLA-A*0201 molecule, and specific CTLs induced by them significantly responded to the corresponding peptides and secreted IFN-γ. At the same time, the CTLs were able to specifically lyse EPS8-expressing cell lines in an HLA-A*0201-restricted manner. The present study demonstrated that P455, P92, P276 and P360 were CTL epitopes of EPS8, and were able to be used for epitope-defined adoptive T-cell transfer and multi-epitope-based vaccine design.
-
Lin, Su-I; Huang, Ming-Hsi; Chang, Yu-Wen; Chen, I-Hua; Roffler, Steve; Chen, Bing-Mae; Sher, Yuh-Pyng; Liu, Shih-Jen
2016-07-28
Synthetic peptides are attractive for cancer immunotherapy because of their safety and flexibility. In this report, we identified a new B cell epitope of tumor-associated antigen L6 (TAL6) that could induce antibody-dependent cellular cytotoxicity (ADCC) in vivo. We incorporated the B cell epitope with a cytotoxic T lymphocyte (CTL) and a helper T (Th) epitope to form a chimeric long peptide. We formulated the chimeric peptide with different adjuvants to immunize HLA-A2 transgenic mice and evaluate their immunogenicity. The chimeric peptide formulated with an emulsion type nanoparticle (PELC) adjuvant and a toll-like receptor 9 agonist (CpG ODN) (PELC/CpG) induced the greatest ADCC and CTL responses. The induced anti-tumor immunity inhibited the growth of TAL6-positive cancer cells. Moreover, we observed that immunization with the chimeric peptide inhibited cancer cell migration in vitro and metastasis in vivo. These data suggest that a chimeric peptide containing both B and T cell epitopes of TAL6 formulated with PELC/CpG adjuvant is feasible for cancer immunotherapy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
-
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.
-
Wu, An-hua; Xiao, Jing; Anker, Lars; Hall, Walter A; Gregerson, Dale S; Cavenee, Webster K; Chen, Wei; Low, Walter C
2006-01-01
The type III variant of the epidermal growth factor receptor (EGFRvIII) mutation is present in 20-25% of patients with glioblastoma multiforme (GBM). EGFRvIII is not expressed in normal tissue and is therefore a suitable candidate antigen for dendritic cell (DC) based immunotherapy of GBM. To identify the antigenic epitope(s) that may serve as targets for EGFRvIII-specific cytotoxic T lymphocytes (CTLs), the peptide sequence of EGFRvIII was screened with two software programs to predict candidate epitopes restricted by the major histocompatibility complex class I subtype HLA-A0201, which is the predominant subtype in most ethnic groups. Three predicted peptides were constructed and loaded to mature human DCs generated from peripheral blood monocytes. Autologous CD8+ T cells were stimulated in vitro with the EGFRvIII peptide-pulsed DCs. One of the three peptides was found to induce EGFRvIII-specific CTLs as demonstrated by IFN-gamma production and cytotoxicity against HLA-A0201+ EGFRvIII transfected U87 glioma cells. These results suggest that vaccination with EGFRvIII peptide-pulsed DCs or adoptive transfer of in vitro elicited EGFRvIII-specific CTLs by EGFRvIII peptide-pulsed DCs are potential approaches to the treatment of glioma patients.
-
Dendritic cells loaded with HeLa-derived exosomes simulate an antitumor immune response.
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 .
-
Ando, Satomi; Hasegawa, Atsuhiko; Murakami, Yuji; Zeng, Na; Takatsuka, Natsuko; Maeda, Yasuhiro; Masuda, Takao; Suehiro, Youko; Kannagi, Mari
2017-02-01
Adult T cell leukemia/lymphoma (ATL), a CD4 + T cell malignancy with a poor prognosis, is caused by human T cell leukemia virus type 1 (HTLV-1) infection. High proviral load (PVL) is a risk factor for the progression to ATL. We previously reported that some asymptomatic carriers had severely reduced functions of CTLs against HTLV-1 Tax, the major target Ag. Furthermore, the CTL responses tended to be inversely correlated with PVL, suggesting that weak HTLV-1-specific CTL responses may be involved in the elevation of PVL. Our previous animal studies indicated that oral HTLV-1 infection, the major route of infection, caused persistent infection with higher PVL in rats compared with other routes. In this study, we found that Tax-specific CD8 + T cells were present, but not functional, in orally infected rats as observed in some human asymptomatic carriers. Even in the infected rats with immune unresponsiveness against Tax, Tax-specific CTL epitope-pulsed dendritic cell (DC) therapy reduced the PVL and induced Tax-specific CD8 + T cells capable of proliferating and producing IFN-γ. Furthermore, we found that monocyte-derived DCs from most infected individuals still had the capacity to stimulate CMV-specific autologous CTLs in vitro, indicating that DC therapy may be applicable to most infected individuals. These data suggest that peptide-pulsed DC immunotherapy will be useful to induce functional HTLV-1-specific CTLs and decrease PVL in infected individuals with high PVL and impaired HTLV-1-specific CTL responses, thereby reducing the risk of the development of ATL. Copyright © 2017 by The American Association of Immunologists, Inc.
-
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
-
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.
-
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
-
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
-
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.
-
Engineering parvovirus-like particles for the induction of B-cell, CD4(+) and CTL responses.
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.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Gogate, N.; Yamabe, Toshio; Verma, L.
1996-04-01
Lack of major histocompatibility class I antigens on neurons has been implicated as a possible mechanism for viral persistence in the brain since these antigens are required for cytotoxic T-lymphocyte recognition of infected cells. In subacute sclerosing panencephalitis (SSPE), measles virus (MV) persists in neurons, resulting in a fatal chronic infection. MHC class I mRNA expression was examined in formalin-fixed brain tissue from 6 SSPE patients by in situ hybridization. In addition MHC class I protein expression in MV-infected neurons was examined in experimental Subacute Measles Encephalitis (SME) by double immunohistochemistry. MHC class I mRNA expression was found to bemore » upregulated in SSPE tissues studied, and in 5 out of 6 cases the expression was definitively seen on neurons. The percentage of neurons expressing MHC class I mRNA ranged between 20 to 84% in infected areas. There was no correlation between the degree of infection and expression of MHC class I molecules on neurons. Importantly, the number of neurons co-expressing MHC class I and MV antigens was markedly low, varying between 2 to 8%. Similar results were obtained in SME where 20 to 30% of the neurons expressed MHC class I but < 8% co-expressed MHC class I and MV antigens. Perivascular infiltrating cells in the infected regions in SME expressed IFN{gamma} immunoreactivity. The results suggest that MV may not be directly involved in the induction of MHC class I on neurons and that cytokines such as IFN{gamma} may play an important role. Furthermore, the paucity of neurons co-expressing MHC class I and MV antigens in SSPE and SME suggests that such cells are either rapidly cleared by cytotoxic T lymphocytes (CTL), or, alternatively, lack of co-expression of MHC class I on MV infected neurons favors MV persistence in these cells by escaping CTL recognition. 33 refs., 3 figs., 3 tabs.« less
-
The HIV hide and seek game: an immunogenomic analysis of the HIV epitope repertoire.
Vider-Shalit, Tal; Almani, Michal; Sarid, Ronit; Louzoun, Yoram
2009-07-17
Viruses employ various means to evade immune detection. One common evasion strategy is the removal of CD8 cytotoxic T-lymphocyte (CTL) epitopes. Here, we use bioinformatic tools to compute the HIV CTL epitope repertoire presented by over 8000 HIV sequences in multiple Human Leukocyte Antigen alleles. We define the 'Size of Immune Repertoire' (SIR) score, which represents the ratio between the number of the predicted epitopes within a protein and their expected number within a scrambled version of the same protein. We show that HIV proteins present less epitopes than expected and that the number of epitopes gradually decreases from SIV to recent HIV sequences. The decrease of the SIR score of HIV is accompanied by a high frequency of replacement mutations within epitopes. The SIR score of the different HIV proteins is not uniform. The regulatory proteins, Tat and Rev, expressed early during cellular infection have a low SIR score, whereas virion-associated genes that are expressed later, such as Env, Pol and Gag, have a higher SIR score. Actually, the SIR score of Gag keeps increasing over time. We hypothesize that our results reflect an HIV immune evasion strategy. This involves the targeting of the CTL immune response to viral structural and enzyme proteins, allowing the virus a time interval to propagate before its host cells are destroyed by CTLs. An efficient anti-HIV CTL response against HIV should thus also target the regulatory genes that HIV seeks to hide from the immune system.
-
Global Manufacturing of CAR T Cell Therapy.
Levine, Bruce L; Miskin, James; Wonnacott, Keith; Keir, Christopher
2017-03-17
Immunotherapy using chimeric antigen receptor-modified T cells has demonstrated high response rates in patients with B cell malignancies, and chimeric antigen receptor T cell therapy is now being investigated in several hematologic and solid tumor types. Chimeric antigen receptor T cells are generated by removing T cells from a patient's blood and engineering the cells to express the chimeric antigen receptor, which reprograms the T cells to target tumor cells. As chimeric antigen receptor T cell therapy moves into later-phase clinical trials and becomes an option for more patients, compliance of the chimeric antigen receptor T cell manufacturing process with global regulatory requirements becomes a topic for extensive discussion. Additionally, the challenges of taking a chimeric antigen receptor T cell manufacturing process from a single institution to a large-scale multi-site manufacturing center must be addressed. We have anticipated such concerns in our experience with the CD19 chimeric antigen receptor T cell therapy CTL019. In this review, we discuss steps involved in the cell processing of the technology, including the use of an optimal vector for consistent cell processing, along with addressing the challenges of expanding chimeric antigen receptor T cell therapy to a global patient population.
-
Zhang, Haihong; Liu, Chenlu; Zhang, Fangfang; Geng, Fei; Xia, Qiu; Lu, Zhenzhen; Xu, Ping; Xie, Yu; Wu, Hui; Yu, Bin; Wu, Jiaxin; Yu, Xianghui; Kong, Wei
2016-05-23
MUC1 and survivin are ideal tumor antigens. Although many cancer vaccines targeting survivin or MUC1 have entered clinical trials, no vaccine combining MUC1 and survivin have been reported. Due to tumor heterogeneity, vaccines containing a combination of antigens may have improved efficacy and coverage of a broader spectrum of cancer targets. Here, cellular responses and anti-tumor activities induced by a combination of DNA vaccine targeting MUC1 and survivin (MS) were evaluated. Results showed that CTL activity and inhibition of tumor growth were obviously enhanced in mice immunized with the combined vaccine in a protection assay. However, in order to enhance the therapeutic effect in the treatment assay, a recombinant adenovirus (rAd) vaccine expressing MUC1 and survivin (Ad-MS) was used as a booster following the DNA vaccine prime. Meanwhile, IL-2 promoting T cell proliferation was used as an immunoadjuvant for the DNA vaccine. Results showed that the CTL activity response to the DNA vaccine was enhanced nearly 200% when boosted by the rAd vaccine and was further enhanced by nearly 60% when combined with the IL-2 adjuvant. Therefore, DNA prime combined with rAd boost and IL-2 (MS/IL2/Ad-MS) adjuvant was considered as the best strategy and further evaluated. Multiple cytokines promoting cellular immune responses were shown to be greatly enhanced in mice immunized with MS/IL2/Ad-MS. Moreover, in the treatment assay, the tumor inhibition rate of MS/IL2/Ad-MS reached up to 50.1%, which may be attributed to the enhancement of immune responses and reduction of immunosuppressive factors in tumor-bearing mice. These results suggested that immunization with the combination vaccine targeting MUC1 and survivin using a DNA prime-rAd boost strategy along with IL-2 adjuvant may be an effective method for breaking through immune tolerance to tumors expressing these antigens with potential therapeutic benefits in melanoma cancer. Copyright © 2016. Published by Elsevier Ltd.
-
Sutmuller, Roger P.M.; van Duivenvoorde, Leonie M.; van Elsas, Andrea; Schumacher, Ton N.M.; Wildenberg, Manon E.; Allison, James P.; Toes, Rene E.M.; Offringa, Rienk; Melief, Cornelis J.M.
2001-01-01
Therapeutic efficacy of a tumor cell–based vaccine against experimental B16 melanoma requires the disruption of either of two immunoregulatory mechanisms that control autoreactive T cell responses: the cytotoxic T lymphocyte–associated antigen (CTLA)-4 pathway or the CD25+ regulatory T (Treg) cells. Combination of CTLA-4 blockade and depletion of CD25+ Treg cells results in maximal tumor rejection. Efficacy of the antitumor therapy correlates with the extent of autoimmune skin depigmentation as well as with the frequency of tyrosinase-related protein 2180–188–specific CTLs detected in the periphery. Furthermore, tumor rejection is dependent on the CD8+ T cell subset. Our data demonstrate that the CTL response against melanoma antigens is an important component of the therapeutic antitumor response and that the reactivity of these CTLs can be augmented through interference with immunoregulatory mechanisms. The synergism in the effects of CTLA-4 blockade and depletion of CD25+ Treg cells indicates that CD25+ Treg cells and CTLA-4 signaling represent two alternative pathways for suppression of autoreactive T cell immunity. Simultaneous intervention with both regulatory mechanisms is therefore a promising concept for the induction of therapeutic antitumor immunity. PMID:11560997
-
C-type lectins: their network and roles in pathogen recognition and immunity.
Mayer, Sabine; Raulf, Marie-Kristin; Lepenies, Bernd
2017-02-01
C-type lectins (CTLs) represent the most complex family of animal/human lectins that comprises 17 different groups. During evolution, CTLs have developed by diversification to cover a broad range of glycan ligands. However, ligand binding by CTLs is not necessarily restricted to glycans as some CTLs also bind to proteins, lipids, inorganic molecules, or ice crystals. CTLs share a common fold that harbors a Ca 2+ for contact to the sugar and about 18 invariant residues in a phylogenetically conserved pattern. In vertebrates, CTLs have numerous functions, including serum glycoprotein homeostasis, pathogen sensing, and the initiation of immune responses. Myeloid CTLs in innate immunity are mainly expressed by antigen-presenting cells and play a prominent role in the recognition of a variety of pathogens such as fungi, bacteria, viruses, and parasites. However, myeloid CTLs such as the macrophage inducible CTL (Mincle) or Clec-9a may also bind to self-antigens and thus contribute to immune homeostasis. While some CTLs induce pro-inflammatory responses and thereby lead to activation of adaptive immune responses, other CTLs act as inhibitory receptors and dampen cellular functions. Since CTLs are key players in pathogen recognition and innate immunity, targeting CTLs may be a promising strategy for cell-specific delivery of drugs or vaccine antigens and to modulate immune responses.
-
Plasmodium parasite as an effective hepatocellular carcinoma antigen glypican-3 delivery vector.
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.
-
Human Immune Response to Dengue Infections
1989-07-31
antigens of all 4 serotypes. These CTL lysed autologous fibroblasts infected with vaccinia-dengue recombinant viruses containing the E, or several non...responses of PBMC from a dengue 4-immune donor to call-free dengue viruses . .. ........... 6 Table 2. Lysis of dengue virus-infected fibroblasts by dengue...4-immune PBMC stimulated with dengue viruses ... ...... 7 Table 3. Inhibition of the lysis of dengue- infected fibroblasts by monoclonal anti-CD8
-
Shen, Dongxu; Wang, Lei; Ji, Jiayue; Liu, Qizhi; An, Chunju
2018-01-01
Abstract C-type lectins (CTLs) are a large family of calcium-dependent carbohydrate-binding proteins. They function primarily in cell adhesion and immunity by recognizing various glycoconjugates. We identified 14 transcripts encoding proteins with one or two CTL domains from the transcriptome from Asian corn borer, Ostrinia furnacalis (Guenée; Lepidoptera: Pyralidae). Among them, five (OfCTL-S1 through S5) only contain one CTL domain, the remaining nine (OfIML-1 through 9) have two tandem CTL domains. Five CTL-Ss and six OfIMLs have a signal peptide are likely extracellular while another two OfIMLs might be cytoplasmic. Phylogenetic analysis indicated that OfCTL-Ss had 1:1 orthologs in Lepidoptera, Diptera, Coleoptera and Hymenoptera species, but OfIMLs only clustered with immulectins (IMLs) from Lepidopteran. Structural modeling revealed that the 22 CTL domains adopt a similar double-loop fold consisting of β-sheets and α-helices. The key residues for calcium-dependent or independent binding of specific carbohydrates by CTL domains were predicted with homology modeling. Expression profiles assay showed distinct expression pattern of 14 CTLs: the expression and induction were related to the developmental stages and infected microorganisms. Overall, our work including the gene identification, sequence alignment, phylogenetic analysis, structural modeling, and expression profile assay would provide a valuable basis for the further functional studies of O. furnacalis CTLs. PMID:29718486
-
Boutwell, Christian L.; Carlson, Jonathan M.; Lin, Tien-Ho; Seese, Aaron; Power, Karen A.; Peng, Jian; Tang, Yanhua; Brumme, Zabrina L.; Heckerman, David; Schneidewind, Arne
2013-01-01
Cytotoxic-T-lymphocyte (CTL) escape mutations undermine the durability of effective human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cell responses. The rate of CTL escape from a given response is largely governed by the net of all escape-associated viral fitness costs and benefits. The observation that CTL escape mutations can carry an associated fitness cost in terms of reduced virus replication capacity (RC) suggests a fitness cost-benefit trade-off that could delay CTL escape and thereby prolong CD8 response effectiveness. However, our understanding of this potential fitness trade-off is limited by the small number of CTL escape mutations for which a fitness cost has been quantified. Here, we quantified the fitness cost of the 29 most common HIV-1B Gag CTL escape mutations using an in vitro RC assay. The majority (20/29) of mutations reduced RC by more than the benchmark M184V antiretroviral drug resistance mutation, with impacts ranging from 8% to 69%. Notably, the reduction in RC was significantly greater for CTL escape mutations associated with protective HLA class I alleles than for those associated with nonprotective alleles. To speed the future evaluation of CTL escape costs, we also developed an in silico approach for inferring the relative impact of a mutation on RC based on its computed impact on protein thermodynamic stability. These data illustrate that the magnitude of CTL escape-associated fitness costs, and thus the barrier to CTL escape, varies widely even in the conserved Gag proteins and suggest that differential escape costs may contribute to the relative efficacy of CD8 responses. PMID:23365420
-
Quantification of simian immunodeficiency virus cytotoxic T lymphocyte escape mutant viruses.
Loh, Liyen; Kent, Stephen J
2008-08-01
Escape from cytotoxic T-lymphocyte (CTL) pressure is common in HIV-1 infection of humans and simian immunodeficiency virus (SIV) infections of macaques. CTL escape typically incurs a fitness cost as reversion back to wild-type can occur upon transmission. We utilized sequence-specific primers and DNA probes with real-time polymerase chain reaction (PCR) to sensitively and specifically track wild-type and escape mutant viremia at the Mane-A*17-restricted SIV Gag(371379) epitope AF9 in pigtail macaques. The generation of minor escape mutant populations is detected by the real-time PCR 2 weeks earlier than observed using standard sequencing techniques. We passaged the AF9 CTL escape mutant virus into two naïve Mane-A*17-negative pigtail macaques and showed that reversion to wild-type was rapid during acute infection and then slowed considerably at later stages of the infection. These data help refine our understanding of how CTL escape mutant viruses evolve.
-
Yuan, Xuefeng; Teng, Xindong; Jing, Yukai; Ma, Jilei; Tian, Maopeng; Yu, Qi; Zhou, Lei; Wang, Ruibo; Wang, Weihua; Li, Li; Fan, Xionglin
2015-12-01
Tuberculosis (TB) remains one of the most menacing infectious diseases, although attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine has been widely used to protect children against primary TB. There are increasing evidences that rapid growing and dormant Mycobacterium tuberculosis (M. tuberculosis) coexist in vivo after infection. However, BCG vaccine only elicits cell-mediated immune responses to secretory antigens expressed by rapid growing pathogen. BCG vaccine is thus unable to thwart the reactivation of latent tuberculosis infection (LTBI), and its protection wanes over age after neonatal immunization. In order to extend its ability for a durable protection, a novel recombinant BCG (rBCG) strain, named rBCG::XB, was constructed by overexpressing immunodominant multistage antigens of Ag85B and HspX, which are expressed by both rapid replicating and dormant M. tuberculosis. Long-term protective effect and immunogenicity of rBCG::XB were compared with the parental BCG in vaccinated C57BL/6 mice. Our results demonstrated that rBCG::XB provided the stronger and long-lasting protection against M. tuberculosis H37Rv intranasal infection than BCG. The rBCG::XB not only elicited the more durable multistage antigen-specific CD4(+)Th1-biased immune responses and specific polyfunctional CD4(+)T cells but also augmented the CD8(+) CTL effects against Ag85B in vivo. In particular, higher levels of CD4(+) TEM and CD8(+) TCM cells, dominated by IL2(+) CD4(+) and CD8(+) TCM cells, were obtained in the spleen of rBCG::XB vaccinated mice. Therefore, our findings indicate that rBCG::XB is a promising candidate to improve the efficacy of BCG.
-
Ito, Yoshinori; Kondo, Eisei; Demachi-Okamura, Ayako; Akatsuka, Yoshiki; Tsujimura, Kunio; Tanimoto, Mitsune; Morishima, Yasuo; Takahashi, Toshitada; Kuzushima, Kiyotaka
2006-01-01
The precise roles of gamma interferon-inducible immunoproteasome-associated molecules in generation of cytotoxic T-lymphocyte (CTL) epitopes have yet to be fully elucidated. We describe here a unique epitope derived from the Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) presented by HLA-A*2402 molecules. Generation of the epitope, designated LMP2A222-230, from the full-length protein requires the immunoproteasome subunit low-molecular-weight protein 7 (ip-LMP7) and the proteasome activator 28-α subunit and is accelerated by ip-LMP2, as revealed by gene expression experiments using an LMP2A222-230-specific CTL clone as a responder in enzyme-linked immunospot assays. The unequivocal involvement of all three components was confirmed by RNA interference gene silencing. Interestingly, the LMP2A222-230 epitope could be efficiently generated from incomplete EBV-LMP2A fragments that were produced by puromycin treatment or gene-engineered shortened EBV-LMP2A lacking some of its hydrophobic domains. In addition, epitope generation was increased by a single amino acid substitution from leucine to alanine immediately flanking the C terminus, this being predicted by a web-accessible program to increase the cleavage strength. Taken together, the data indicate that the generation of LMP2A222-230 is influenced not only by extrinsic factors such as immunoproteasomes but also by intrinsic factors such as the length of the EBV-LMP2A protein and proteasomal cleavage strength at specific positions in the source antigen. PMID:16378990
-
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.
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ordonhez Rigato, Paula; Maciel, Milton; Goldoni, Adriana Leticia
2010-10-10
Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses,more » as measured by the breadth of the Gag peptide-specific IFN-{gamma}, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric LAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory.« less
-
Characterization of choline transporters in the human placenta over gestation.
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
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
-
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.
-
McMurtrey, Curtis P; Lelic, Alina; Piazza, Paolo; Chakrabarti, Ayan K; Yablonsky, Eric J; Wahl, Angela; Bardet, Wilfried; Eckerd, Annette; Cook, Robert L; Hess, Rachael; Buchli, Rico; Loeb, Mark; Rinaldo, Charles R; Bramson, Jonathan; Hildebrand, William H
2008-02-26
Cytotoxic T lymphocytes (CTL) play an important role in the control and elimination of infection by West Nile virus (WNV), yet the class I human leukocyte antigen (HLA)-presented peptide epitopes that enable CTL recognition of WNV-infected cells remain uncharacterized. The goals of this work were first to discover the peptide epitopes that distinguish the class I HLA of WNV-infected cells and then to test the T cell reactivity of newly discovered WNV epitopes. To discover WNV-immune epitopes, class I HLA was harvested from WNV (NY99 strain)-infected and uninfected HeLa cells. Then peptide epitopes were eluted from affinity-purified HLA, and peptide epitopes from infected and uninfected cells were comparatively mapped by mass spectroscopy. Six virus-derived peptides from five different viral proteins (E, NS2b, NS3, NS4b, and NS5) were discovered as unique to HLA-A*0201 of infected cells, demonstrating that the peptides sampled by class I HLA are distributed widely throughout the WNV proteome. When tested with CTL from infected individuals, one dominant WNV target was apparent, two epitopes were subdominant, and three demonstrated little CTL reactivity. Finally, a sequence comparison of these epitopes with the hundreds of viral isolates shows that HLA-A*0201 presents epitopes derived from conserved regions of the virus. Detection and recovery from WNV infection are therefore functions of the ability of class I HLA molecules to reveal conserved WNV epitopes to an intact cellular immune system that subsequently recognizes infected cells.
-
Belyakov, I M; Ahlers, J D
2011-01-01
Mucosal tissues are major sites of HIV entry and initial infection. Induction of a local mucosal cytotoxic T lymphocyte response is considered an important goal in developing an effective HIV vaccine. In addition, activation and recruitment of memory CD4(+) and CD8(+) T cells in systemic lymphoid circulation to mucosal effector sites might provide the firewall needed to prevent virus spread. Therefore a vaccine that generates CD4(+) and CD8(+) responses in both mucosal and systemic tissues might be required for protection against HIV. However, optimal routes and number of vaccinations required for the generation of long lasting CD4(+) and CD8(+) CTL effector and memory responses are not well understood especially for mucosal T cells. A number of studies looking at protective immune responses against diverse mucosal pathogens have shown that mucosal vaccination is necessary to induce a compartmentalized immune response including maximum levels of mucosal high-avidity CD8(+) CTL, antigen specific mucosal antibodies titers (especially sIgA), as well as induction of innate anti-viral factors in mucosa tissue. Immune responses are detectable at mucosal sites after systemic delivery of vaccine, and prime boost regimens can amplify the magnitude of immune responses in mucosal sites and in systemic lymphoid tissues. We believe that the most optimal mucosal and systemic HIV/SIV specific protective immune responses and innate factors might best be achieved by simultaneous mucosal and systemic prime and boost vaccinations. Similar principals of vaccination may be applied for vaccine development against cancer and highly invasive pathogens that lead to chronic infection.
-
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.
-
Evasion of adaptive immunity by HIV through the action of host APOBEC3G/F enzymes.
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.
-
Kim, Yeon-Jeong; Ko, Hyun-Jeong; Kim, Yun-Sun; Kim, Dong-Hyeon; Kang, Seock; Kim, Jong-Mook; Chung, Yeonseok; Kang, Chang-Yuil
2008-06-15
Most of the current tumor vaccines successfully elicit strong protection against tumor but offer little therapeutic effect against existing tumors, highlighting the need for a more effective vaccine strategy. Vaccination with tumor antigen-presenting cells can induce antitumor immune responses. We have previously shown that NKT-licensed B cells prime cytotoxic T lymphocytes (CTLs) with epitope peptide and generate prophylactic/therapeutic antitumor effects. To extend our B cell vaccine approach to the whole antigen, and to overcome the MHC restriction, we used a nonreplicating adenovirus to transduce B cells with antigenic gene. Primary B cells transduced with an adenovirus-encoding truncated Her-2/neu (AdHM) efficiently expressed Her-2/neu. Compared with the moderate antitumor activity induced by vaccination with adenovirus-transduced B cells (B/AdHM), vaccination with alpha-galactosylceramide-loaded B/AdHM (B/AdHM/alpha GalCer) induced significantly stronger antitumor immunity, especially in the tumor-bearing mice. The depletion study showed that CD4(+), CD8(+) and NK cells were all necessary for the therapeutic immunity. Confirming the results of the depletion study, B/AdHM/alpha GalCer vaccination induced cytotoxic NK cell responses but B/AdHM did not. Vaccination with B/AdHM/alpha GalCer generated Her-2/neu-specific antibodies more efficiently than B/AdHM immunization. More importantly, B/AdHM/alpha GalCer could prime Her-2/neu-specific cytotoxic T cells more efficiently and durably than B/AdHM. CD4(+) cells appeared to be necessary for the induction of antibody and CTL responses. Our results demonstrate that, with the help of NKT cells, antigen-transduced B cells efficiently induce innate immunity as well as a wide range of adaptive immunity against the tumor, suggesting that they could be used to develop a novel cellular vaccine. (c) 2008 Wiley-Liss, Inc.
-
[Immune response induced by HIV DNA vaccine combined with recombinant adeno-associated virus].
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.
-
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
-
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.
-
Antibody-peptide-MHC fusion conjugates target non-cognate T cells to kill tumour cells.
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.
-
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.
-
Ross, Peter; Holmes, Jennifer C; Gojanovich, Gregory S; Hess, Paul R
2012-12-15
Identifying immunodominant CTL epitopes is essential for studying CD8+ T-cell responses in populations, but remains difficult, as peptides within antigens typically are too numerous for all to be synthesized and screened. Instead, to facilitate discovery, in silico scanning of proteins for sequences that match the motif, or binding preferences, of the restricting MHC class I allele - the largest determinant of immunodominance - can be used to predict likely candidates. The high false positive rate with this analysis ideally requires binding confirmation, which is obtained routinely by an assay using cell lines such as RMA-S that have defective transporter associated with antigen processing (TAP) machinery, and consequently, few surface class I molecules. The stabilization and resultant increased life-span of peptide-MHC complexes on the cell surface by the addition of true binders validates their identity. To determine whether a similar assay could be developed for dogs, we transfected a prevalent class I allele, DLA-88*50801, into RMA-S. In the BARC3 clone, the recombinant heavy chain was associated with murine β2-microglobulin, and importantly, could differentiate motif-matched and -mismatched peptides by surface MHC stabilization. This work demonstrates the potential to use RMA-S cells transfected with canine alleles as a tool for CTL epitope discovery in this species. Copyright © 2012 Elsevier B.V. All rights reserved.
-
Li, Jianqiang; Ge, Jun; Ren, Sulin; Zhou, Tong; Sun, Ying; Sun, Honglin; Gu, Yue; Huang, Hongying; Xu, Zhenxing; Chen, Xiaoxiao; Xu, Xiaowei; Zhuang, Xiaoqian; Song, Cuiling; Jia, Fangmiao; Xu, Aiguo; Yin, Xiaojin; Du, Sean X
2015-08-20
Hepatitis B virus infection is a non-cytopathic hepatotropic virus which can lead to chronic liver disease and hepatocellular carcinoma. Traditional therapies fail to provide sustained control of viral replication and liver damage in most patients. As an alternative strategy, immunotherapeutic approaches have shown promising efficacy in the treatment of chronic hepatitis B patients. Here, we investigated the efficacy of a novel therapeutic vaccine formulation consisting of two HBV antigens, HBsAg and HBcAg, and CpG adjuvant. This vaccine formulation elicits forceful humoral responses directed against HBsAg/HBcAg, and promotes a Th1/Th2 balance response against HBsAg and a Th1-biased response against HBcAg in both C57BL/6 and HBV transgenic mice. Vigorous cellular immune response was also detected in HBV transgenic mice, for a significantly higher number of HBs/HBc-specific IFN-γ secreting CD4+ and CD8+ T cells was generated. Moreover, vaccinated mice elicited significantly intense in vivo CTL attack, reduced serum HBsAg level without causing liver damage in HBV transgenic mice. In summary, this study demonstrates a novel therapeutic vaccine with the potential to elicit vigorous humoral and cellular response, overcoming tolerance in HBV transgenic mice. Copyright © 2015 Elsevier Ltd. All rights reserved.
-
Designer vaccine nanodiscs for personalized cancer immunotherapy
NASA Astrophysics Data System (ADS)
Kuai, Rui; Ochyl, Lukasz J.; Bahjat, Keith S.; Schwendeman, Anna; Moon, James J.
2017-04-01
Despite the tremendous potential of peptide-based cancer vaccines, their efficacy has been limited in humans. Recent innovations in tumour exome sequencing have signalled the new era of personalized immunotherapy with patient-specific neoantigens, but a general methodology for stimulating strong CD8α+ cytotoxic T-lymphocyte (CTL) responses remains lacking. Here we demonstrate that high-density lipoprotein-mimicking nanodiscs coupled with antigen (Ag) peptides and adjuvants can markedly improve Ag/adjuvant co-delivery to lymphoid organs and sustain Ag presentation on dendritic cells. Strikingly, nanodiscs elicited up to 47-fold greater frequencies of neoantigen-specific CTLs than soluble vaccines and even 31-fold greater than perhaps the strongest adjuvant in clinical trials (that is, CpG in Montanide). Moreover, multi-epitope vaccination generated broad-spectrum T-cell responses that potently inhibited tumour growth. Nanodiscs eliminated established MC-38 and B16F10 tumours when combined with anti-PD-1 and anti-CTLA-4 therapy. These findings represent a new powerful approach for cancer immunotherapy and suggest a general strategy for personalized nanomedicine.
-
Mizuno, Shinji; Sonoda, Masatoshi; Tamura, Yayoi; Nishino, Eisho; Suzuki, Hideyuki; Sato, Takahide; Oizumi, Toshikatsu
2015-11-01
Tendrils are filamentous plant organs that coil on contact with an object, thereby providing mechanical support for climbing to reach more sunlight. Plant tendrils are considered to be modified structure of leaves, stems, or inflorescence, but the origin of cucurbit tendrils is still argued because of the complexity in the axillary organ patterning. We carried out morphological and genetic analyses of the Chiba Tendril-Less (ctl) melon (Cucumis melo) mutant, and found strong evidence that the melon tendril is a modified organ derived from a stem-leaf complex of a lateral shoot. Heterozygous (CTL/ctl) plants showed traits intermediate between tendril and shoot, and ontogenies of wild-type tendrils and mutant modified shoots coincided. We identified the CTL locus in a 200-kb region in melon linkage group IX. A single base deletion in a melon TCP transcription factor gene (CmTCP1) was detected in the mutant ctl sequence, and the expression of CmTCP1 was specifically high in wild-type tendrils. Phylogenetic analysis demonstrated the novelty of the CmTCP1 protein and the unique molecular evolution of its orthologs in the Cucurbitaceae. Our results move us closer to answering the long-standing question of which organ was modified to become the cucurbit tendril, and suggest a novel function of the TCP transcription factor in plant development.
-
Induction of tolerance towards TNP entails down-regulation of an autoimmune attack.
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
-
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
-
Serine Proteases Enhance Immunogenic Antigen Presentation on Lung Cancer Cells.
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; Bernatchez, Chantale; Alatrash, Gheath; Hanash, Samir; Molldrem, Jeffrey J
2017-04-01
Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these reinvigorated 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 TAMs 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 CTLs from healthy donors that had been expanded against select peptides. Finally, CTLs 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. Cancer Immunol Res; 5(4); 319-29. ©2017 AACR . ©2017 American Association for Cancer Research.
-
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.
-
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
-
High Throughput T Epitope Mapping and Vaccine Development
Li Pira, Giuseppina; Ivaldi, Federico; Moretti, Paolo; Manca, Fabrizio
2010-01-01
Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th) and by cytolytic T lymphocytes (CTL) is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP) approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost. PMID:20617148
-
DOE Office of Scientific and Technical Information (OSTI.GOV)
Caivano, Antonella; Doria-Rose, Nicole A.; Dept. of Molecular and Cell Biology, University of Washington, Seattle, WA 98124-6108
2010-11-25
We have constructed stable virus-like particles displaying the HIV-1 Gag(p17) protein as an N-terminal fusion with an engineered protein domain from the Geobacillus stearothermophilus pyruvate dehydrogenase subunit E2. Mice immunized with the Gag(p17)-E2 60-mer scaffold particles mounted a strong and sustained antibody response. Antibodies directed to Gag(p17) were boosted significantly with additional immunizations, while anti-E2 responses reached a plateau. The isotype of the induced antibodies was biased towards IgG1, and the E2-primed CD4+ T cells did not secrete IFN{gamma}. Using transgenic mouse model systems, we demonstrated that CD8+ T cells primed with E2 particles were able to exert lytic activitymore » and produce IFN{gamma}. These results show that the E2 scaffold represents a powerful vaccine delivery system for whole antigenic proteins or polyepitope engineered proteins, evoking antibody production and antigen specific CTL activity even in the absence of IFN{gamma}-producing CD4+ T cells.« less
-
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.
-
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
-
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.
-
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
-
Development of oral CTL vaccine using a CTP-integrated Sabin 1 poliovirus-based vector system.
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.
-
Weihrauch, Martin R; Ansén, Sascha; Jurkiewicz, Elke; Geisen, Caroline; Xia, Zhinan; Anderson, Karen S; Gracien, Edith; Schmidt, Manuel; Wittig, Burghardt; Diehl, Volker; Wolf, Juergen; Bohlen, Heribert; Nadler, Lee M
2005-08-15
We conducted a phase I/II randomized trial to evaluate the clinical and immunologic effect of chemotherapy combined with vaccination in primary metastatic colorectal cancer patients with a carcinoembryonic antigen-derived peptide in the setting of adjuvants granulocyte macrophage colony-stimulating factor, CpG-containing DNA molecules (dSLIM), and dendritic cells. HLA-A2-positive patients with confirmed newly diagnosed metastatic colorectal cancer and elevated serum carcinoembryonic antigen (CEA) were randomized to receive three cycles of standard chemotherapy (irinotecan/high-dose 5-fluorouracil/leucovorin) and vaccinations with CEA-derived CAP-1 peptide admixed with different adjuvants [CAP-1/granulocyte macrophage colony-stimulating factor/interleukin-2 (IL-2), CAP-1/dSLIM/IL-2, and CAP-1/IL-2]. After completion of chemotherapy, patients received weekly vaccinations until progression of disease. Immune assessment was done at baseline and after three cycles of combined chemoimmunotherapy. HLA-A2 tetramers complexed with the peptides CAP-1, human T-cell lymphotrophic virus type I TAX, cytomegalovirus (CMV) pp65, and EBV BMLF-1 were used for phenotypic immune assessment. IFN-gamma intracellular cytokine assays were done to evaluate CTL reactivity. Seventeen metastatic patients were recruited, of whom 12 completed three cycles. Therapy resulted in five complete response, one partial response, five stable disease, and six progressive disease. Six grade 1 local skin reactions and one mild systemic reaction to vaccination treatment were observed. Overall survival after a median observation time of 29 months was 17 months with a survival rate of 35% (6 of 17) at that time. Eight patients (47%) showed elevation of CAP-1-specific CTLs. Neither of the adjuvants provided superiority in eliciting CAP-1-specific immune responses. During three cycles of chemotherapy, EBV/CMV recall antigen-specific CD8+ cells decreased by an average 14%. The presented chemoimmunotherapy is a feasible and safe combination therapy with clinical and immunologic efficacy. Despite concurrent chemotherapy, increases in CAP-1-specific T cells were observed in 47% of patients after vaccination.
-
Wang, Xuan; Zhang, Fang-Cheng; Zhao, Hong-Yang; Lu, Xiao-Ling; Sun, Yun; Xiong, Zhi-Yong; Jiang, Xiao-Bing
2014-08-01
The epidermal growth factor receptor (EGFR) mutant of EGFRvIII is highly expressed in glioma cells, and the EGFRvIII-specific dendritic cell (DC)-induced tumor antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) may hold promise in cancer immunotherapy. Interferon (IFN)-γ-inducible protein (IP)-10 (IP-10) is a potent inhibitor of angiogenesis and can recruit CXCR3(+) T cells, including CD8(+) T cells, which are important for the control of tumor growth. In this study, we assessed if the combination of IP10-EGFRvIIIscFv with DC-induced CTLs would improve the therapeutic antitumor efficacy. IP10-scFv was generated by linking the human IP-10 gene with the DNA fragment for anti-EGFRvIIIscFv with a (Gly4Ser)3 flexible linker, purified by affinity chromatography, and characterized for its anti-EGFRvIII immunoreactivity and chemotactic activity. DCs were isolated from human peripheral blood monocyte cells and pulsed with EGFRvIII-peptide, then co-cultured with autologous CD8(+) T cells. BALB/c-nu mice were inoculated with human glioma U87-EGFRvIII cells in the brain and treated intracranially with IP10-scFv and/or intravenously with DC-induced CTLs for evaluating the therapeutic effect. Treatment with both IP10-scFv and EGFRvIII peptide-pulsed, DC-induced CTL synergistically inhibited the growth of glioma and prolonged the survival of tumor-bearing mice, which was accompanied by the inhibition of tumor angiogenesis and enhancement of cytotoxicity, thereby increasing the numbers of brain-infiltrating lymphocytes (BILs) and prolonging the residence time of CTLs in the tumor.
-
Impact of Pre-adapted HIV Transmission
Carlson, Jonathan M.; Du, Victor Y.; Pfeifer, Nico; Bansal, Anju; Tan, Vincent Y.F.; Power, Karen; Brumme, Chanson J.; Kreimer, Anat; DeZiel, Charles E.; Fusi, Nicolo; Schaefer, Malinda; Brockman, Mark A.; Gilmour, Jill; Price, Matt A.; Kilembe, William; Haubrich, Richard; John, Mina; Mallal, Simon; Shapiro, Roger; Frater, John; Harrigan, P. Richard; Ndung’u, Thumbi; Allen, Susan; Heckerman, David; Sidney, John; Allen, Todd M.; Goulder, Philip J.R.; Brumme, Zabrina L.; Hunter, Eric; Goepfert, Paul A.
2016-01-01
Human Leukocyte Antigen class I (HLA) restricted CD8+ T lymphocyte (CTL) responses are critical to HIV-1 control. Although HIV can evade these responses, the longer-term impact of viral escape mutants remains unclear, since these variants can also reduce intrinsic viral fitness. To address this question, we here develop a metric to determine the degree of HIV adaptation to an HLA profile. We demonstrate that transmission of viruses pre-adapted to the HLA molecules expressed in the recipient is associated with impaired immunogenicity, elevated viral load and accelerated CD4 decline. Furthermore, the extent of pre-adaptation among circulating viruses explains much of the variation in outcomes attributed to expression of certain HLA alleles. Thus, viral pre-adaptation exploits “holes” in the immune response. Accounting for these holes may be critical for vaccine strategies seeking to elicit functional responses from viral variants, and to HIV cure strategies requiring broad CTL responses to achieve successful eradication of HIV reservoirs. PMID:27183217
-
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
-
Shariat, Sheida; Badiee, Ali; Jalali, Seyed Amir; Mansourian, Mercedeh; Yazdani, Mona; Mortazavi, Seyed Alireza; Jaafari, Mahmoud Reza
2014-12-01
Vaccines containing synthetic peptides derived from tumor-associated antigens (TAA) can elicit potent cytotoxic T lymphocyte (CTL) response if they are formulated in an optimal vaccine delivery system. The aim of this study was to develop a simple and effective lipid-based vaccine delivery system using P5 HER2/neu-derived peptide conjugated to Maleimide-PEG2000-DSPE. The conjugated lipid was then incorporated into liposomes composed of DMPC:DMPG:Chol:DOPE containing Monophosphoryl lipid A (MPL) (Lip-DOPE-P5-MPL). Different liposome formulations were prepared and characterized for their physicochemical properties. To evaluate anti-tumoral efficacy, BALB/c mice were immunized subcutaneously 3 times in two-week intervals and the generated immune response was studied. The results demonstrated that Lip-DOPE-P5-MPL induced a significantly higher IFN-γ production by CD8+ T cells intracellularly which represents higher CTL response in comparison with other control formulations. CTL response induced by this formulation caused the lowest tumor size and the longest survival time in a mice model of TUBO tumor. The encouraging results achieved by Lip-DOPE-P5-MPL formulation could make it a promising candidate in developing effective vaccines against Her2 positive breast cancers. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
-
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
-
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.
-
Effector functions of memory CTLs can be affected by signals received during reactivation.
Lv, Yingjun; Mattson, Elliot; Bhadurihauck, Anjuli; Garcia, Karla; Li, Lei; Xiao, Zhengguo
2017-08-01
Memory cytotoxic T lymphocytes (CTLs) are able to provide protections to the host against repeated insults from intracellular pathogens. However, it has not been completely understood how the effector functions of memory CTLs are induced upon antigen challenge, which is directly related to the efficacy of their protection. Third signal cytokines, such as IL-12 and type I interferon, have been suggested to be involved in the protective function of memory CTLs, but direct evidence is warranted. In this report, we found that memory CTLs need to be reactivated to exert effector functions. Infusion of a large population of quiescent memory CTLs did not lead to cancer control in tumor-bearing mice, whereas infusion of a reactivated memory CTL population did. This reactivation of memory CTLs requires cytokines such as IL-12 in addition to antigen but was less dependent upon costimulation and IL-2 compared to naive CTLs. Memory CTLs responded more quickly and with greater strength than their naive counterparts upon stimulation, which is associated with higher upregulation of important transcription factors such as T-bet and phosphorylated STAT4. In addition, memory CTLs underwent less expansion than naive CTLs upon pathogen challenge. In conclusion, effector functions of established memory CTLs may be affected by certain cytokines such as IL-12 and type I IFN. Thus, a pathogen's ability to induce cytokines could contribute to the efficacy of protection of an established memory CTL population.
-
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.
-
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.
-
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.
-
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
-
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.
-
Cobbold, Mark; Khan, Naeem; Pourgheysari, Batoul; Tauro, Sudhir; McDonald, Dorothy; Osman, Husam; Assenmacher, Mario; Billingham, Lucinda; Steward, Colin; Crawley, Charles; Olavarria, Eduardo; Goldman, John; Chakraverty, Ronjon; Mahendra, Premini; Craddock, Charles; Moss, Paul A.H.
2005-01-01
Stem cell transplantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus (CMV) remains a significant cause of morbidity and mortality. Adoptive transfer of donor-derived CMV-specific CD8+ T cell clones has been shown to reduce the rate of viral reactivation; however, the complexity of this approach severely limits its clinical application. We have purified CMV-specific CD8+ T cells from the blood of stem cell transplant donors using staining with HLA–peptide tetramers followed by selection with magnetic beads. CMV-specific CD8+ cells were infused directly into nine patients within 4 h of selection. Median cell dosage was 8.6 × 103/kg with a purity of 98% of all T cells. CMV-specific CD8+ T cells became detectable in all patients within 10 d of infusion, and TCR clonotype analysis showed persistence of infused cells in two patients studied. CMV viremia was reduced in every case and eight patients cleared the infection, including one patient who had a prolonged history of CMV infection that was refractory to antiviral therapy. This novel approach to adoptive transfer has considerable potential for antigen-specific T cell therapy. PMID:16061727
-
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
-
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.
-
Identification of an HLA-A24-restricted OY-TES-1 epitope recognized by cytotoxic T-cells.
Okumura, Hideo; Noguchi, Yuji; Uenaka, Akiko; Aji, Toshiki; Ono, Toshiro; Nakagawa, Kazuhiko; Aoe, Motoi; Shimizu, Nobuyoshi; Nakayama, Eiichi
2005-01-01
OY-TES-1 was identified as a human homologue of the mouse, guinea pig, and pig proacrosin binding protein sp32 precursor. Differential expression levels of OY-TES-1 mRNA between testis and other normal tissues, and its expression in cancers indicated that OY-TES-1 would be classified as a cancer/testis antigen and considered to be a candidate of target antigen for cancer immunotherapy. In this study, we showed identification of HLA-A24-binding OY-TES-1 peptide, TES(401-409) (KTPFVSPLL) recognized by CD8 T-cells. Purified CD8 T-cells from healthy donors stimulated in vitro with the peptide-pulsed autologous DC and PBMC produced IFNgamma in response to the peptide-pulsed PBMC and showed cytotoxicity against the peptide-pulsed autologous EBV-B specifically. Furthermore, cytotoxicity was also observed against an OY-TES-1 mRNA-expressing tumor line, LK79. The retention time of the fraction in HPLC of the acid eluate from LK79 cells that showed positive sensitization against autologous EBV-B cells in recognition by CD8 CTL was the same as that of the fraction of the TES(401-409) peptide itself, suggesting that the TES(401-409) was a naturally processed peptide on LK79.
-
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.
-
Immunogenicity and protective efficacy of DMT liposome-adjuvanted tuberculosis subunit CTT3H vaccine
Teng, Xindong; Tian, Maopeng; Li, Jianrong; Tan, Songwei; Yuan, Xuefeng; Yu, Qi; Jing, Yukai; Zhang, Zhiping; Yue, Tingting; Zhou, Lei; Fan, Xionglin
2015-01-01
Different strategies have been proposed for the development of protein subunit vaccine candidates for tuberculosis (TB), which shows better safety than other types of candidates and the currently used Bacillus Calmette-Guérin (BCG) vaccine. In order to develop more effective protein subunits depending on the mechanism of cell-mediated immunity against TB, a polyprotein CTT3H, based on 5 immunodominant antigens (CFP10, TB10.4, TB8.4, Rv3615c, and HBHA) with CD8+ epitopes of Mycobacterium tuberculosis, was constructed in this study. We vaccinated C57BL/6 mice with a TB subunit CTT3H protein in an adjuvant of dimethyldioctadecylammonium/monophosphoryl lipid A/trehalose 6,6′-dibehenate (DDA/MPL/TDB, DMT) liposome to investigate the immunogenicity and protective efficacy of this novel vaccine. Our results demonstrated that DMT liposome-adjuvanted CTT3H vaccine not only induced an antigen-specific CD4+ Th1 response, but also raised the number of PPD- and CTT3H-specific IFN-γ+ CD8+ T cells and elicited strong CTL responses against TB10.4, which provided more effective protection against a 60 CFU M. tuberculosis aerosol challenge than PBS control and DMT adjuvant alone. Our findings indicate that DMT-liposome is an effective adjuvant to stimulate CD8+ T cell responses and the DMT-adjuvanted subunit CTT3H vaccine is a promising candidate for the next generation of TB vaccine. PMID:25905680
-
Teng, Xindong; Tian, Maopeng; Li, Jianrong; Tan, Songwei; Yuan, Xuefeng; Yu, Qi; Jing, Yukai; Zhang, Zhiping; Yue, Tingting; Zhou, Lei; Fan, Xionglin
2015-01-01
Different strategies have been proposed for the development of protein subunit vaccine candidates for tuberculosis (TB), which shows better safety than other types of candidates and the currently used Bacillus Calmette-Guérin (BCG) vaccine. In order to develop more effective protein subunits depending on the mechanism of cell-mediated immunity against TB, a polyprotein CTT3H, based on 5 immunodominant antigens (CFP10, TB10.4, TB8.4, Rv3615c, and HBHA) with CD8(+) epitopes of Mycobacterium tuberculosis, was constructed in this study. We vaccinated C57BL/6 mice with a TB subunit CTT3H protein in an adjuvant of dimethyldioctadecylammonium/monophosphoryl lipid A/trehalose 6,6'-dibehenate (DDA/MPL/TDB, DMT) liposome to investigate the immunogenicity and protective efficacy of this novel vaccine. Our results demonstrated that DMT liposome-adjuvanted CTT3H vaccine not only induced an antigen-specific CD4(+) Th1 response, but also raised the number of PPD- and CTT3H-specific IFN-γ(+) CD8(+) T cells and elicited strong CTL responses against TB10.4, which provided more effective protection against a 60 CFU M. tuberculosis aerosol challenge than PBS control and DMT adjuvant alone. Our findings indicate that DMT-liposome is an effective adjuvant to stimulate CD8(+) T cell responses and the DMT-adjuvanted subunit CTT3H vaccine is a promising candidate for the next generation of TB vaccine.
-
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.
-
Beloki, Lorea; Ciaurriz, Miriam; Mansilla, Cristina; Zabalza, Amaya; Perez-Valderrama, Estela; Samuel, Edward R; Lowdell, Mark W; Ramirez, Natalia; Olavarria, Eduardo
2014-11-19
Cytomegalovirus (CMV)-specific T cell infusion to immunocompromised patients following allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) is able to induce a successful anti-viral response. These cells have classically been manufactured from steady-state apheresis samples collected from the donor in an additional harvest prior to G-CSF mobilization, treatment that induces hematopoietic stem cell (HSC) mobilization to the periphery. However, two closely-timed cellular collections are not usually available in the unrelated donor setting, which limits the accessibility of anti-viral cells for adoptive immunotherapy. CMV-specific cytotoxic T cell (CTL) manufacture from the same G-CSF mobilized donor stem cell harvest offers great regulatory advantages, but the isolation using MHC-multimers is hampered by the high non-specific binding to myeloid progenitors, which reduces the purity of the cellular product. In the present study we describe an easy and fast method based on plastic adherence to remove myeloid cell subsets from 11 G-CSF mobilized donor samples. CMV-specific CTLs were isolated from the non-adherent fraction using pentamers and purity and yield of the process were compared to products obtained from unmanipulated samples. After the elimination of unwanted cell subtypes, non-specific binding of pentamers was notably reduced. Accordingly, following the isolation process the purity of the obtained cellular product was significantly improved. G-CSF mobilized leukapheresis samples can successfully be used to isolate antigen-specific T cells with MHC-multimers to be adoptively transferred following allo-HSCT, widening the accessibility of this therapy in the unrelated donor setting. The combination of the clinically translatable plastic adherence process to the antigen-specific cell isolation using MHC-multimers improves the quality of the therapeutic cellular product, thereby reducing the clinical negative effects associated with undesired alloreactive cell infusion.
-
Identification of novel HLA-A(*)0201-restricted CTL epitopes from Pokemon.
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.
-
Increased Expression of Fatty-Acid and Calcium Metabolism Genes in Failing Human Heart
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
-
Keller, Salka; Wilson, John T; Patilea, Gabriela I; Kern, Hanna B; Convertine, Anthony J; Stayton, Patrick S
2014-01-01
Synthetic subunit vaccines need to induce CD8+ cytotoxic T-cell (CTL) responses for effective vaccination against intracellular pathogens. Most subunit vaccines primarily generate humoral immune responses, with a weaker than desired CD8+ cytotoxic T-cell response. Here, a neutral, pH-responsive polymer micelle carrier that alters intracellular antigen trafficking was shown to enhance CD8+ T-cell responses with a correlated increase in cytosolic delivery and a decrease in exocytosis. Polymer diblock carriers consisted of a N-(2-hydroxypropyl) methacrylamide corona block with pendant pyridyl disulfide groups for reversible conjugation of thiolated ovalbumin, and a tercopolymer ampholytic core-forming block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The diblock copolymers self-assembled into 25–30 nm diameter micellar nanoparticles. Conjugation of ovalbumin to the micelles significantly enhanced antigen cross-presentation in vitro relative to free ovalbumin, an unconjugated physical mixture of ovalbumin and polymer, and a non pH-responsive micelle-ovalbumin control. Mechanistic studies in a murine dendritic cell line (DC2.4) demonstrated micelle-mediated enhancements in intracellular antigen retention and cytosolic antigen accumulation. Approximately 90% of initially internalized ovalbumin-conjugated micelles were retained in cells after 1.5 h, compared to only ~40% for controls. Furthermore, cells dosed with conjugates displayed 67-fold higher cytosolic antigen levels relative to soluble ovalbumin 4 h post uptake. Subcutaneous immunization of mice with ovalbumin-polymer conjugates significantly enhanced antigen-specific CD8+ T cell responses (0.4 % IFN-γ+ of CD8+) compared to immunization with soluble protein, ovalbumin and polymer mixture, and the control micelle without endosome-releasing activity. Additionally, pH-responsive carrier facilitated antigen delivery to antigen presenting cells in the draining lymph nodes. As early as 90 min post injection ova-micelle conjugates were associated with 28% and 55% of dendritic cells and macrophages, respectively. After 24 h, conjugates preferentially associated with dendritic cells, affording 30-, 3-, and 3-fold enhancements in uptake relative to free protein, physical mixture, and the non pH-responsive conjugate controls, respectively. These results demonstrate the potential of pH-responsive polymeric micelles for use in vaccine applications that rely on CD8+ T cell activation. PMID:24698946
-
Keller, Salka; Wilson, John T; Patilea, Gabriela I; Kern, Hanna B; Convertine, Anthony J; Stayton, Patrick S
2014-10-10
Synthetic subunit vaccines need to induce CD8(+) cytotoxic T cell (CTL) responses for effective vaccination against intracellular pathogens. Most subunit vaccines primarily generate humoral immune responses, with a weaker than desired CD8(+) cytotoxic T cell response. Here, a neutral, pH-responsive polymer micelle carrier that alters intracellular antigen trafficking was shown to enhance CD8(+) T cell responses with a correlated increase in cytosolic delivery and a decrease in exocytosis. Polymer diblock carriers consisted of a N-(2-hydroxypropyl) methacrylamide corona block with pendent pyridyl disulfide groups for reversible conjugation of thiolated ovalbumin, and a tercopolymer ampholytic core-forming block composed of propylacrylic acid (PAA), dimethylaminoethyl methacrylate (DMAEMA), and butyl methacrylate (BMA). The diblock copolymers self-assembled into 25-30nm diameter micellar nanoparticles. Conjugation of ovalbumin to the micelles significantly enhanced antigen cross-presentation in vitro relative to free ovalbumin, an unconjugated physical mixture of ovalbumin and polymer, and a non-pH-responsive micelle-ovalbumin control. Mechanistic studies in a murine dendritic cell line (DC 2.4) demonstrated micelle-mediated enhancements in intracellular antigen retention and cytosolic antigen accumulation. Approximately 90% of initially internalized ovalbumin-conjugated micelles were retained in cells after 1.5h, compared to only ~40% for controls. Furthermore, cells dosed with conjugates displayed 67-fold higher cytosolic antigen levels relative to soluble ovalbumin 4h post uptake. Subcutaneous immunization of mice with ovalbumin-polymer conjugates significantly enhanced antigen-specific CD8(+) T cell responses (0.4% IFN-γ(+) of CD8(+)) compared to immunization with soluble protein, ovalbumin and polymer mixture, and the control micelle without endosome-releasing activity. Additionally, pH-responsive carrier facilitated antigen delivery to antigen presenting cells in the draining lymph nodes. As early as 90min post injection, ova-micelle conjugates were associated with 28% and 55% of dendritic cells and macrophages, respectively. After 24h, conjugates preferentially associated with dendritic cells, affording 30-, 3-, and 3-fold enhancements in uptake relative to free protein, physical mixture, and the non-pH-responsive conjugate controls, respectively. These results demonstrate the potential of pH-responsive polymeric micelles for use in vaccine applications that rely on CD8(+) T cell activation. Copyright © 2014 Elsevier B.V. All rights reserved.
-
Wnt signaling inhibits CTL memory programming
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
-
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.
-
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
-
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
-
Molecular Pathology of Adult T-Cell Leukemia/Lymphoma.
Ohshima, Koichi
2015-01-01
Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell neoplasm of highly pleomorphic lymphoid cells. ATLL is usually widely disseminated, and it is caused by human T-cell leukemia virus type 1 (HTLV-1). It is a disease with a long latency, and affected individuals are usually exposed to the virus very early in life. The cumulative incidence of ATLL is estimated to be 2.5% among HTLV-1 carriers. ATLL cells express CD2, CD3, CD5, CD4, and CD25, as well as CCR4 and FoxP3 of the regulatory T-cell marker. HTLV-1 is causally linked to ATLL, but infection alone is not sufficient to result in neoplastic transformation. A significant finding in this connection is that the Tax viral protein leads to transcriptional activation of many genes, while the HTLV-1 basic leucine zipper factor is thought to be important for T-cell proliferation and oncogenesis. Half of ATLL cases retain the ability to express HTLV-1 Tax, which is a target of HTLV-1-specific cytotoxic T lymphocytes (CTL). An increase in HTLV-1-specific CTL responses is observed in some asymptomatic HTLV-1 carriers. Although HTLV-1-specific CTL are also present in the peripheral blood of ATLL patients, they do not expand sufficiently. We investigated the clinicopathological features and analyzed the staining of Tax-specific CTL and FoxP3. Tax-specific CTL correlated inversely with FoxP3, an increase in the ratio of CD163+ tumor-associated macrophages was associated with worse clinical prognosis, and ATLL cell lines proliferated significantly following direct co-culture with M2 macrophages. Several clinical variants of ATLL have been identified: acute, lymphomatous, chronic, and smoldering. Oligo-array comparative genomic hybridization revealed that genomic loss of 9p21.3 was a significant characteristic of acute-type, but not of chronic-type ATLL. Furthermore, we found that genomic alteration of CD58, which is implicated in immune escape, is more frequently observed in acute than in chronic ATLL. Interestingly, the chronic cases with cell cycle deregulation and disruption of immunosurveillance mechanism were associated with faster progression to acute ATLL. Immune evasion, microenvironment, and genetic alteration are therefore important in the multi-step progression of ATLL lymphomagenesis. © 2015 S. Karger AG, Basel.
-
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
-
Kallert, Sandra M.; Darbre, Stephanie; Bonilla, Weldy V.; Kreutzfeldt, Mario; Page, Nicolas; Müller, Philipp; Kreuzaler, Matthias; Lu, Min; Favre, Stéphanie; Kreppel, Florian; Löhning, Max; Luther, Sanjiv A.; Zippelius, Alfred; Merkler, Doron; Pinschewer, Daniel D.
2017-01-01
Viral infections lead to alarmin release and elicit potent cytotoxic effector T lymphocyte (CTLeff) responses. Conversely, the induction of protective tumour-specific CTLeff and their recruitment into the tumour remain challenging tasks. Here we show that lymphocytic choriomeningitis virus (LCMV) can be engineered to serve as a replication competent, stably-attenuated immunotherapy vector (artLCMV). artLCMV delivers tumour-associated antigens to dendritic cells for efficient CTL priming. Unlike replication-deficient vectors, artLCMV targets also lymphoid tissue stroma cells expressing the alarmin interleukin-33. By triggering interleukin-33 signals, artLCMV elicits CTLeff responses of higher magnitude and functionality than those induced by replication-deficient vectors. Superior anti-tumour efficacy of artLCMV immunotherapy depends on interleukin-33 signalling, and a massive CTLeff influx triggers an inflammatory conversion of the tumour microenvironment. Our observations suggest that replicating viral delivery systems can release alarmins for improved anti-tumour efficacy. These mechanistic insights may outweigh safety concerns around replicating viral vectors in cancer immunotherapy. PMID:28548102
-
Phase 1 clinical study of cyclophilin B peptide vaccine for patients with lung cancer.
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.
-
Kim, Eun-Ha; Choi, Young-Ki; Kim, Chul-Joong; Sung, Moon-Hee; Poo, Haryoung
2015-10-06
The global outbreak of a novel swine-origin strain of the 2009 H1N1 influenza A virus and the sudden, worldwide increase in oseltamivir-resistant H1N1 influenza A viruses highlight the urgent need for novel antiviral therapy. Here, we investigated the antiviral efficacy of poly-gamma glutamate (γ-PGA), a safe and edible biomaterial that is naturally synthesized by Bacillus subtilis, against A/Puerto Rico/8/1934 (PR8) and A/California/04/2009 (CA04) H1N1 influenza A virus infections in C57BL/6 mice. Intranasal administration of γ-PGA for 5 days post-infection improved survival, increased production of antiviral cytokines including interferon-beta (IFN-β) and interleukin-12 (IL-12), and enhanced activation of natural killer (NK) cells and influenza antigen-specific cytotoxic T lymphocytes (CTL) activity. These results suggest that γ-PGA protects mice against H1N1 influenza A virus by enhancing antiviral immune responses.
-
Khedive, A; Norouzi, M; Ramezani, F; Karimzadeh, H; Alavian, S M; Malekzadeh, R; Montazeri, G; Nejatizadeh, A; Ziaee, M; Abedi, F; Ataei, B; Yaran, M; Sayad, B; Somi, M H; Sarizadeh, G; Sanei-Moghaddam, I; Mansour-Ghanaei, F; Rafatpanah, H; Pourhosseingholi, M A; Keyvani, H; Kalantari, E; Saberifiroozi, M; Judaki, M A; Ghamari, S; Daram, M; Mahabadi, M; Fazeli, Z; Goodarzi, Z; Poortahmasebi, V; Jazayeri, S M
2013-07-01
Mutations within the coding region of hepatitis B surface antigen (HBsAg) have been found naturally in chronic carriers. To characterize the mutations of HBsAg from Iranian chronic carriers who were vaccine and/or medication naive. The surface genes from 360 patients were amplified and directly sequenced. The distribution of amino acid substitutions was classified according to different immune epitopes of the surface protein. All isolates belonged to genotype D. 222 (61.6%) of 360 patients contained at least one amino acid substitution. 404 (74.5%) of 542 amino acid changes occurred in different immune epitopes of HBsAg, of which 112 (27.7%) in 32 residues of B-cell epitopes (62 in the 'a' determinant); 111 (27.4%) in 32 residues of T helper; and 197 (48.7%) in 32 residues inside cytotoxic T lymphocyte (CTL) epitopes. One Th (186-197) and two CTL (28-51 and 206-215) epitopes were found to be hotspot motifs for the occurrence of 213 (52.7%) substitutions. 20 stop codons were identified in different epitopes. There was a significant association between amino acid substitutions and anti-HBe seropositivity; however, the correlation between such changes with viral load and ALT levels was not significant. In chronic hepatitis B virus(HBV) carriers, positive selection in particular outside the 'a' determinant appeared to exert influence on the surface proteins. These changes could be immune escape mutations naturally occurring due to the host immune surveillance especially at the T-cell level. © 2013 John Wiley & Sons Ltd.
-
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.
-
Regulatory T cell effects in antitumor laser immunotherapy: a mathematical model and analysis
NASA Astrophysics Data System (ADS)
Dawkins, Bryan A.; Laverty, Sean M.
2016-03-01
Regulatory T cells (Tregs) have tremendous influence on treatment outcomes in patients receiving immunotherapy for cancerous tumors. We present a mathematical model incorporating the primary cellular and molecular components of antitumor laser immunotherapy. We explicitly model developmental classes of dendritic cells (DCs), cytotoxic T cells (CTLs), primary and metastatic tumor cells, and tumor antigen. Regulatory T cells have been shown to kill antigen presenting cells, to influence dendritic cell maturation and migration, to kill activated killer CTLs in the tumor microenvironment, and to influence CTL proliferation. Since Tregs affect explicitly modeled cells, but we do not explicitly model dynamics of Treg themselves, we use model parameters to analyze effects of Treg immunosuppressive activity. We will outline a systematic method for assigning clinical outcomes to model simulations and use this condition to associate simulated patient treatment outcome with Treg activity.
-
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
-
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.
-
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
-
Zhang, Jianhua; Chen, Yong; Qi, Jianxun; Gao, Feng; Liu, Yanjie; Liu, Jun; Zhou, Xuyu; Kaufman, Jim; Xia, Chun; Gao, George F.
2016-01-01
The major histocompatibility complex (MHC) has genetic associations with many diseases, often due to differences in presentation of antigenic peptides by polymorphic MHC molecules to T lymphocytes of the immune system. In chickens, only a single classical class I molecule in each MHC haplotype is expressed well due to co-evolution with the polymorphic transporters associated with antigen presentation (TAPs), which means that resistance and susceptibility to infectious pathogens are particularly easy to observe. Previously, structures of chicken MHC class I molecule BF2*2101 from B21 haplotype showed an unusually large peptide-binding groove that accommodates a broad spectrum of peptides to present as epitopes to cytotoxic T lymphocytes (CTL), explaining the MHC-determined resistance of B21 chickens to Marek's disease. Here, we report the crystal structure of BF2*0401 from the B4 (also known as B13) haplotype, showing a highly positively-charged surface hitherto unobserved in other MHC molecules, as well as a remarkably narrow groove due to the allele-specific residues with bulky side chains. Together, these properties limit the number of epitope peptides that can bind this class I molecule. However, peptide-binding assays show that in vitro BF2*0401 can bind a wider variety of peptides than are found on the surface of B4 cells. Thus, a combination of the specificities of the polymorphic TAP transporter and the MHC results in a very limited set of BF2*0401 peptides with negatively charged anchors to be presented to T lymphocytes. PMID:23041567
-
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
-
Williams, Chad M.; Schonnesen, Alexandra A.; Zhang, Shu-Qi; Ma, Ke-Yue; He, Chenfeng; Yamamoto, Tori; Eckhardt, S. Gail; Klebanoff, Christopher A.; Jiang, Ning
2017-01-01
The discovery of naturally occurring T cell receptors (TCRs) that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC) has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D) system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds) rather than synergy (total CD8 cooperation) alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D) analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our previously established TCR discovery platform using 2D TCR affinity and sequence test would allow for selection of TCRs specific to any given antigen with the desirable attributes of high TCR affinity, CD8 co-receptor independence and functional superiority. Utilizing TCRs with less CD8 contribution could be beneficial for adoptive cell transfer immunotherapies using naturally occurring or genetically engineered T cells against viral or cancer-associated antigens. PMID:28804489
-
Dynamic acquisition of HTLV-1 tax protein by mononuclear phagocytes: Role in neurologic disease.
Matsuura, Eiji; Enose-Akahata, Yoshimi; Yao, Karen; Oh, Unsong; Tanaka, Yuetsu; Takashima, Hiroshi; Jacobson, Steven
2017-03-15
Pathology of HTLV-1 associated myelopathy/Tropical spastic paraparesis (HAM/TSP) is believed to be the result of "bystander damage" involving effector CD8 (+) T lymphocytes (CTLs) killing of virus infected cells. But the specific cellular events leading up to tissue injury are still unclear. Here, we developed the Microscopy Imaging of Cytotoxic T lymphocyte assay with Fluorescence emission (MI-CaFé), an optimized visualization analysis to explore the interactions between CTLs and virus infected or viral antigen presenting target cells. Various cell-to-cell formations can be observed and our results demonstrate elevated frequencies of CTL-target cell conjugates in HAM/TSP patient PBMCs compared to control PBMCs. Furthermore, HTLV-1 Tax protein expression can be localized at the cell-cell junctions and also tracked moving from an infected cell to a CD14 (+) mononuclear phagocyte (MP). Activation of CD14 (+) MPs in HAM/TSP patient PBMCs and antigenic presentation of HTLV-1 Tax by MPs can be inferred by their spontaneous cytotoxicity after 18h of in vitro culture. Given that CD4 (+) T lymphocytes are the primary reservoirs of HTLV-1 and MPs are scavenger cells responsible for pathogen clearance, spontaneous cytotoxicity against MPs in HAM/TSP PBMCs suggests a mechanism of chronic inflammation, secondary to low level of persistent virus infection within the central nervous system. Published by Elsevier B.V.
-
Liu, Chengwen; Lou, Yanyan; Lizée, Gregory; Qin, Hong; Liu, Shujuan; Rabinovich, Brian; Kim, Grace J; Wang, Yi-Hong; Ye, Yang; Sikora, Andrew G; Overwijk, Willem W; Liu, Yong-Jun; Wang, Gang; Hwu, Patrick
2008-03-01
A prerequisite for strong adaptive antiviral immunity is the robust initial activation of the innate immune system, which is frequently mediated by TLR-activated plasmacytoid DCs (pDCs). Natural antitumor immunity is often comparatively weak, potentially due to the lack of TLR-mediated activation signals within the tumor microenvironment. To assess whether pDCs are capable of directly facilitating effective antitumor immune responses, mice bearing established subcutaneous B16 melanoma tumors were administered TLR9-activated pDCs directly into the tumor. We found that TLR9-activated pDCs induced robust, spontaneous CTL cross-priming against multiple B16 tumor antigens, leading to the regression of both treated tumors and untreated tumors at distant contralateral sites. This T cell cross-priming was mediated by conventional DCs (cDCs) and was completely dependent upon the early recruitment and activation of NK cells at the tumor site. NK cell recruitment was mediated by CCR5 via chemokines secreted by pDCs, and optimal IFN-gamma production by NK cells was mediated by OX40L expressed by pDCs. Our data thus demonstrated that activated pDCs are capable of initiating effective and systemic antitumor immunity through the orchestration of an immune cascade involving the sequential activation of NK cells, cDCs, and CD8(+) T cells.
-
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
-
Provenzano, Maurizio; Selleri, Silvia; Jin, Ping; Wang, Ena; Werden, Rosemary; Slezak, Stephanie; Adams, Sharon D; Panelli, Monica C; Leitman, Susan F; Stroncek, David F; Marincola, Francesco M
2007-07-01
Latent membrane protein (LMP)-2 is one of the Epstein-Barr virus (EBV)-encoded proteins consistently expressed by nasopharyngeal carcinoma (NPC). EBV-transformed lymphoblastoid cell lines (LCL) have been used in patients with NPC to induce LMP-2-recognizing T cell lines which have been in turn utilized for protein-wide mapping of T cell epitopes. However, comprehensive mapping of naturally recognized LMP-2 epitopes in non tumor-bearing individuals has not been reported. Here, we applied a low sensitivity epitope-defining technique for the identification of LMP-2 CTL responses detectable ex vivo in EBV-experienced individuals. This screening tool has been previously validated by analyzing memory CTL responses to Flu, cytomegalovirus (CMV), and the melanoma associated antigen gp100/Mel17. Peripheral blood monocytes (PBMC) from ten Caucasian and ten Chinese individuals were stimulated ex vivo with pools of nonamer (9-mer) peptides overlapping in a stepwise fashion each single amino acid of the LMP-2 sequence. No obvious differences were observed between the immune response of the two ethnic groups save for those related to the divergence in the ethnic prevalence of HLA haplotypes. Several novel and known LMP-2 epitopes were identified. Reactivity toward at least one LMP-2 epitope was detected in 18 of the 20 donors but no prevalent human leukocyte antigen (HLA)/epitope combination was observed confirming that LMP-2 reactivity in the context of common HLA alleles is more pleiotropic than that of FLU and CMV. We believe that the usefulness of these epitopes occurring naturally in non-cancer bearing patients as reagents for the immunization of patients with early or advanced stage NPC deserves further evaluation.
-
Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia
Fraietta, Joseph A.; Beckwith, Kyle A.; Patel, Prachi R.; Ruella, Marco; Zheng, Zhaohui; Barrett, David M.; Lacey, Simon F.; Melenhorst, Jan Joseph; McGettigan, Shannon E.; Cook, Danielle R.; Zhang, Changfeng; Xu, Jun; Do, Priscilla; Hulitt, Jessica; Kudchodkar, Sagar B.; Cogdill, Alexandria P.; Gill, Saar; Porter, David L.; Woyach, Jennifer A.; Long, Meixiao; Johnson, Amy J.; Maddocks, Kami; Muthusamy, Natarajan; Levine, Bruce L.; June, Carl H.; Byrd, John C.
2016-01-01
Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749. PMID:26813675
-
Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia.
Fraietta, Joseph A; Beckwith, Kyle A; Patel, Prachi R; Ruella, Marco; Zheng, Zhaohui; Barrett, David M; Lacey, Simon F; Melenhorst, Jan Joseph; McGettigan, Shannon E; Cook, Danielle R; Zhang, Changfeng; Xu, Jun; Do, Priscilla; Hulitt, Jessica; Kudchodkar, Sagar B; Cogdill, Alexandria P; Gill, Saar; Porter, David L; Woyach, Jennifer A; Long, Meixiao; Johnson, Amy J; Maddocks, Kami; Muthusamy, Natarajan; Levine, Bruce L; June, Carl H; Byrd, John C; Maus, Marcela V
2016-03-03
Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is highly promising but requires robust T-cell expansion and engraftment. A T-cell defect in chronic lymphocytic leukemia (CLL) due to disease and/or therapy impairs ex vivo expansion and response to CAR T cells. To evaluate the effect of ibrutinib treatment on the T-cell compartment in CLL as it relates to CAR T-cell generation, we examined the phenotype and function of T cells in a cohort of CLL patients during their course of treatment with ibrutinib. We found that ≥5 cycles of ibrutinib therapy improved the expansion of CD19-directed CAR T cells (CTL019), in association with decreased expression of the immunosuppressive molecule programmed cell death 1 on T cells and of CD200 on B-CLL cells. In support of these findings, we observed that 3 CLL patients who had been treated with ibrutinib for ≥1 year at the time of T-cell collection had improved ex vivo and in vivo CTL019 expansion, which correlated positively together and with clinical response. Lastly, we show that ibrutinib exposure does not impair CAR T-cell function in vitro but does improve CAR T-cell engraftment, tumor clearance, and survival in human xenograft models of resistant acute lymphocytic leukemia and CLL when administered concurrently. Our collective findings indicate that ibrutinib enhances CAR T-cell function and suggest that clinical trials with combination therapy are warranted. Our studies demonstrate that improved T-cell function may also contribute to the efficacy of ibrutinib in CLL. These trials were registered at www.clinicaltrials.gov as #NCT01747486, #NCT01105247, and #NCT01217749. © 2016 by The American Society of Hematology.
-
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.
-
Ohshima, K; Suzumiya, J; Sugihara, M; Nagafuchi, S; Ohga, S; Kikuchi, M
1999-01-01
The Epstein-Barr virus (EBV) induces infectious mononucleosis (IM) and can be associated with chronic active EBV infection (CAEBV). Cytotoxic T lymphocytes (CTL) play an important role in excluding EBV-infected cells. Two cytotoxic mechanisms of CTL have been demonstrated: one perforin/granzyme-based and the other Fas (CD95)/Fas ligand (FasL)-based. To clarify these two pathways in CAEBV, we analyzed six patients with CAEBV and four patients with IM using immunohistochemical staining of the lymph nodes. In both CAEBV and IM, CD8+ T-cells increased in number, but CD56+ natural killer cells were rare. In four of six cases with CAEBV, approximately half the lymphocytes were positive for T cell-restricted intracellular antigens (TIA-1), which were recognized by the cytolytic granules of CTL. In IM, the number of TIA-1 positive cells was smaller than that in CAEBV. Fas-positive lymphocytes were frequently encountered in both CAEBV and IM. However, FasL-positive lymphocytes increased in three of six patients with CAEBV, but not in patients with IM. Except for one case with CAEBV, the number of perforin- and/or granzyme-positive cells was small in number in both CAEBV and IM cases. In double-staining FasL and EBV in situ hybridization, FasL-positive EBV-infected lymphocytes were detected in CAEBV but not in IM. In CAEBV, the Fas/FasL pathway and not perforin pathways appears to play an important role in the pathogenesis. The data suggest that EBV-infected lymphocytes may evade immune attack through the expression of FasL.
-
Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy
Lei, Janet; Osen, Wolfram; Gardyan, Adriane; Hotz-Wagenblatt, Agnes; Wei, Guochao; Gissmann, Lutz; Eichmüller, Stefan; Löchelt, Martin
2015-01-01
The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated in vitro for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy. PMID:26397953
-
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
-
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
-
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.
-
Inventory of File sref_nmb.t03z.pgrb221.ctl.grib2
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
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
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
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
-
Preexisting CD8+ T-cell immunity to the H7N9 influenza A virus varies across ethnicities
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
-
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.
-
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
-
CRTAM determines the CD4+ cytotoxic T lymphocyte lineage
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
-
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.
-
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.
-
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.
-
Dendritic Cell-Based Immunotherapy for Myeloid Leukemias
Schürch, Christian M.; Riether, Carsten; Ochsenbein, Adrian F.
2013-01-01
Acute and chronic myeloid leukemia (AML, CML) are hematologic malignancies arising from oncogene-transformed hematopoietic stem/progenitor cells known as leukemia stem cells (LSCs). LSCs are selectively resistant to various forms of therapy including irradiation or cytotoxic drugs. The introduction of tyrosine kinase inhibitors has dramatically improved disease outcome in patients with CML. For AML, however, prognosis is still quite dismal. Standard treatments have been established more than 20 years ago with only limited advances ever since. Durable remission is achieved in less than 30% of patients. Minimal residual disease (MRD), reflected by the persistence of LSCs below the detection limit by conventional methods, causes a high rate of disease relapses. Therefore, the ultimate goal in the treatment of myeloid leukemia must be the eradication of LSCs. Active immunotherapy, aiming at the generation of leukemia-specific cytotoxic T cells (CTLs), may represent a powerful approach to target LSCs in the MRD situation. To fully activate CTLs, leukemia antigens have to be successfully captured, processed, and presented by mature dendritic cells (DCs). Myeloid progenitors are a prominent source of DCs under homeostatic conditions, and it is now well established that LSCs and leukemic blasts can give rise to “malignant” DCs. These leukemia-derived DCs can express leukemia antigens and may either induce anti-leukemic T cell responses or favor tolerance to the leukemia, depending on co-stimulatory or -inhibitory molecules and cytokines. This review will concentrate on the role of DCs in myeloid leukemia immunotherapy with a special focus on their generation, application, and function and how they could be improved in order to generate highly effective and specific anti-leukemic CTL responses. In addition, we discuss how DC-based immunotherapy may be successfully integrated into current treatment strategies to promote remission and potentially cure myeloid leukemias. PMID:24427158
-
Park, Jung-Sun; Park, Soo-Young; Cho, Hyun-Il; Sohn, Hyun-Jung
2011-01-01
Background Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. Methods To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-γ ELISPOT assay, cytotoxicity assay and tetramer staining. Results DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of CD8+ and CD4+ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. Conclusion Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines. PMID:21860612
-
Sloan, Derek D.; Lam, Chia-Ying Kao; Irrinki, Alivelu; Liu, Liqin; Tsai, Angela; Pace, Craig S.; Kaur, Jasmine; Murry, Jeffrey P.; Balakrishnan, Mini; Moore, Paul A.; Johnson, Syd; Nordstrom, Jeffrey L.; Cihlar, Tomas; Koenig, Scott
2015-01-01
HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs. PMID:26539983
-
Choline transporter-like protein 4 (CTL4) links to non-neuronal acetylcholine synthesis
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
-
Analysis of the role of tripeptidyl peptidase II in MHC class I antigen presentation in vivo1
Kawahara, Masahiro; York, Ian A.; Hearn, Arron; Farfan, Diego; Rock, Kenneth L.
2015-01-01
Previous experiments using enzyme inhibitors and RNAi in cell lysates and cultured cells have suggested that tripeptidyl peptidase II (TPPII) plays a role in creating and destroying MHC class I-presented peptides. However, its precise contribution to these processes has been controversial. To elucidate the importance of TPPII in MHC class I antigen presentation, we analyzed TPPII-deficient gene-trapped mice and cell lines from these animals. In these mice, the expression level of TPPII was reduced by >90% compared to wild-type mice. Thymocytes from TPPII gene-trapped mice displayed more MHC class I on the cell surface, suggesting that TPPII normally limits antigen presentation by destroying peptides overall. TPPII gene-trapped mice responded as well as did wild-type mice to four epitopes from lymphocytic choriomeningitis virus (LCMV). The processing and presentation of peptide precursors with long N-terminal extensions in TPPII gene-trapped embryonic fibroblasts was modestly reduced, but in vivo immunization with recombinant lentiviral or vaccinia virus vectors revealed that such peptide precursors induced an equivalent CD8 T cell response in wild type and TPPII-deficient mice. These data indicate while TPPII contributes to the trimming of peptides with very long N-terminal extensions, TPPII is not essential for generating most MHC class I-presented peptides or for stimulating CTL responses to several antigens in vivo. PMID:19841172
-
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.
-
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
-
Novel Therapeutic Strategies for Solid Tumor Based on Body's Intrinsic Antitumor Immune System.
Duan, Haifeng
2018-05-22
The accumulation of mutated somatic cells due to the incompetency of body's immune system may lead to tumor onset. Therefore, enhancing the ability of the system to eliminate such cells should be the core of tumor therapy. The intrinsic antitumor immunity is triggered by tumor-specific antigens (TSA) or TSA-sensitized dendritic cells (DC). Once initiated, specific anti-tumor antibodies are produced and tumor-specific killer immune cells, including cytotoxic T lymphocytes (CTL), NK cells, and macrophages, are raised or induced. Several strategies may enhance antitumor action of immune system, such as supplying tumor-targeted antibody, activating T cells, enhancing the activity and tumor recognition of NK cells, promoting tumor-targeted phagocytosis of macrophages, and eliminating the immunosuppressive myeloid-derived suppressor cells (MDSCs) and Treg cells. Apart from the immune system, the removal of tumor burden still needs to be assisted by drugs, surgery or radiation. And the body's internal environment and tumor microenvironment should be improved to recover immune cell function and prevent tumor growth. Multiple microenvironment modulatory therapies may be applied, including addressing hypoxia and oxidative stress, correcting metabolic disorders, and controlling chronic inflammation. Finally, to cure tumor and prevent tumor recurrence, repairing or supporting therapy that consist of tissue repair and nutritional supplement should be applied properly. © 2018 The Author(s). Published by S. Karger AG, Basel.
-
Palermo, Belinda; Del Bello, Duilia; Sottini, Alessandra; Serana, Federico; Ghidini, Claudia; Gualtieri, Novella; Ferraresi, Virginia; Catricalà, Caterina; Belardelli, Filippo; Proietti, Enrico; Natali, Pier Giorgio; Imberti, Luisa; Nisticò, Paola
2010-09-15
Combination of chemotherapy and immunotherapy to increase the effectiveness of an antitumor immune response is currently regarded as an attractive antitumor strategy. In a pilot clinical trial, we have recently documented an increase of melanoma antigen A (Melan-A)-specific, tumor-reactive, long-lasting effector-memory CD8(+) T cells after the administration of dacarbazine (DTIC) 1 day before peptide vaccination in melanoma patients. Global transcriptional analysis revealed a DTIC-induced activation of genes involved in the immune response and leukocyte activation. To identify the possible mechanisms underlying this improved immune response, we have compared the endogenous and the treatment-induced anti-Melan-A response at the clonal level in patients treated with the vaccine alone or with DTIC plus vaccine. We report a progressive widening of T-cell receptor (TCR) repertoire diversity, accompanied by high avidity and tumor reactivity, only in Melan-A-specific T-cell clones of patients treated with chemoimmunotherapy, with a trend toward longer survival. Differently, patients treated with vaccine alone showed a tendency to narrowing the TCR repertoire diversity, accompanied by a decrease of tumor lytic activity in one patient. Collectively, our findings indicate that DTIC plus vaccination shapes the TCR repertoire in terms of diversity and antitumor response, suggesting that this combined therapy could be effective in preventing melanoma relapse. ©2010 AACR.
-
Immune-tolerant elastin-like polypeptides (iTEPs) and their application as CTL vaccine carriers.
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.
-
Mukherjee, Pinku; Tinder, Teresa L; Basu, Gargi D; Pathangey, Latha B; Chen, Lieping; Gendler, Sandra J
2004-01-01
To study immunology in breast tumors, we have utilized a mammary gland adenocarcinoma model in which mice develop spontaneous tumors of the mammary gland which are initiated at puberty and express a human tumor antigen, MUC1. MUC1 (CD227) is over-expressed in 90% of human breast cancers and its glycosylation status and pattern of expression in cancer cells is altered. Humoral and cellular responses to MUC1 have been reported in breast cancer patients and therefore, MUC1 is being evaluated as a target for immune intervention. This mouse model of spontaneous breast cancer allows the evaluation of anti-MUC1 immune responses at all stages of the disease. In this report, we review the model as it pertains to a) the development of the tumor, b) MUC1 expression, and the native immune responses against MUC1 as tumors progress, and c) the immune suppressive microenvironment within the developing tumor. Finally, we report our latest findings describing the therapeutic efficacy of adoptively transferred MUC1-specific cytotoxic T lymphocytes (MUC1-CTL) in these mice and discuss ways to increase their effectiveness by agonistic monoclonal antibody against CD137 T cell costimulatory molecule.
-
NFATc2 is an intrinsic regulator of melanoma dedifferentiation.
Perotti, V; Baldassari, P; Molla, A; Vegetti, C; Bersani, I; Maurichi, A; Santinami, M; Anichini, A; Mortarini, R
2016-06-02
Melanoma dedifferentiation, characterized by the loss of MITF and MITF regulated genes and by upregulation of stemness markers as CD271, is implicated in resistance to chemotherapy, target therapy and immunotherapy. The identification of intrinsic mechanisms fostering melanoma dedifferentiation may provide actionable therapeutic targets to improve current treatments. Here, we identify NFATc2 transcription factor as an intrinsic regulator of human melanoma dedifferentiation. In panels of melanoma cell lines, NFATc2 expression correlated inversely with MITF at both mRNA and protein levels. NFATc2(+/Hi) melanoma cell lines were CD271(+) and deficient for expression of melanocyte differentiation antigens (MDAs) MART-1, gp100, tyrosinase and of GPNMB, PGC1-α and Rab27a, all regulated by MITF. Targeting of NFATc2 by small interfering RNA, short hairpin RNA and by an NFATc2 inhibitor upregulated MITF, MDAs, GPNMB, PGC-1α, tyrosinase activity and pigmentation and suppressed CD271. Mechanistically, we found that NFATc2 controls melanoma dedifferentiation by inducing expression in neoplastic cells of membrane-bound tumor necrosis factor-α (mTNF-α) and that melanoma-expressed TNF-α regulates a c-myc-Brn2 axis. Specifically, NFATc2, mTNF-α and expression of TNF receptors were significantly correlated in panels of cell lines. NFATc2 silencing suppressed TNF-α expression, and neutralization of melanoma-expressed TNF-α promoted melanoma differentiation. Moreover, silencing of NFATc2 and TNF-α neutralization downmodulated c-myc and POU3F2/Brn2. Brn2 was strongly expressed in NFATc2(+/Hi) MITF(Lo) cell lines and its silencing upregulated MITF. Targeting of c-myc, by silencing or by a c-myc inhibitor, suppressed Brn2 and upregulated MITF and MART-1 in melanoma cells. The relevance of NFATc2-dependent melanoma dedifferentiation for immune escape was shown by cytolytic T-cell assays. NFATc2(Hi) MITF(Lo) MDA(Lo) HLA-A2.1(+) melanoma cells were poorly recognized by MDA-specific and HLA-A2-restricted CTL lines, but NFATc2 targeting significantly increased CTL-mediated tumor recognition. Taken together, these results suggest that the expression of NFATc2 promotes melanoma dedifferentiation and immune escape.
-
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.
-
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.
-
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.
-
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
-
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
2014-11-01
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. © 2014 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.
-
Cho, Seok-Goo; Kim, Nayoun; Sohn, Hyun-Jung; Lee, Suk Kyeong; Oh, Sang Taek; Lee, Hyun-Joo; Cho, Hyun-Il; Yim, Hyeon Woo; Jung, Seung Eun; Park, Gyeongsin; Oh, Joo Hyun; Choi, Byung-Ock; Kim, Sung Won; Kim, Soo Whan; Chung, Nak Gyun; Lee, Jong Wook; Hong, Young Seon; Kim, Tai-Gyu
2015-08-01
Extranodal NK/T-cell lymphoma (ENKTCL) is associated with latent Epstein-Barr virus (EBV) infection and frequent relapse even after complete response (CR) to intensive chemotherapy and radiotherapy. The expression of EBV proteins in the tumor provides targets for adoptive immunotherapy with antigen-specific cytotoxic T cells (CTL). To evaluate the efficacy and safety of EBV latent membrane protein (LMP)-1 and LMP-2a-specific CTLs (LMP1/2a CTLs) stimulated with LMP1/2a RNA-transferred dendritic cells, we treated 10 ENKTCL patients who showed complete response to induction therapy. Patients who completed and responded to chemotherapy, radiotherapy, and/or high-dose therapy followed by stem cell transplantation (HDT/SCT) were eligible to receive eight doses of 2 × 10(7) LMP1/2a CTLs/m(2). Following infusion, there were no immediate or delayed toxicities. The 4-year overall survival (OS) and progression-free survival (PFS) were 100%, and 90% (95% CI: 71.4 to 100%) respectively with a median follow-up of 55·5 months. Circulating IFN-γ secreting LMP1 and LMP2a-specific T cells within the peripheral blood corresponded with decline in plasma EBV DNA levels in patients. Adoptive transfer of LMP1/2a CTLs in ENKTCL patients is a safe and effective postremission therapeutic approach. Further randomized studies will be needed to define the role of EBV-CTLs in preventing relapse of ENKTCL.
-
Rabinowitz, R; Schlesinger, M
1980-01-01
The nature of the receptors on the surface of cytotoxic T lymphocytes (CTL), which enable these cells to recognize antigens on allogeneic targets, is still a matter of controversy. In the present study various mouse alloantisera were tested for their capacity to inhibit, in the absence of complement, the cytotoxic activity of sensitized peritoneal T lymphocytes. The only antiserum which, even after heat inactivation, consistently inhibited cytotoxic T lymphocytes was an antiserum elicited in (C3H X C57B1/6)F1 mice by immunization with AKR/Cum thymus cells. The serum inhibited the cytotoxic reaction of either AKR/J or AKR/Cum CTL on EL-4 target cells but had no inhibitory activity on the cytotoxic reaction of AKR/J cells against P-815 target cells. Thus the inhibitory activity of the serum could not be attributed to antibodies against Ly-3 determinants present in the serum. This conclusion was strengthened by the finding that the inhibitory activity of the serum could be removed by absorption, not only with AKR/J thymus cells but also with AKR/J bone-marrow cells, a procedure which did not affect the titre of Ly-3 antibodies. The serum failed to exert any inhibition on cytotoxic T lymphocytes of BALB/c and C3H mice reacting against EL-4 target cells, indicating that the inhibitory activity of the antiserum did not result from contamination by antibodies against C57B1 antigenic determinants. It was concluded that the inhibitory activity of the antiserum resulted from the presence of antibodies against idiotypic determinants expressed on AKR/Cum thymus cells reacting against the hybrid hosts. It seems, therefore, that idiotypic determinants expressed on the surface of cytotoxic T lymphocytes may be directly involved in their cytotoxic activity. PMID:6155324
-
Koido, Shigeo; Homma, Sadamu; Okamoto, Masato; Namiki, Yoshihisa; Takakura, Kazuki; Takahara, Akitaka; Odahara, Shunichi; Tsukinaga, Shintaro; Yukawa, Toyokazu; Mitobe, Jimi; Matsudaira, Hiroshi; Nagatsuma, Keisuke; Kajihara, Mikio; Uchiyama, Kan; Arihiro, Seiji; Imazu, Hiroo; Arakawa, Hiroshi; Kan, Shin; Hayashi, Kazumi; Komita, Hideo; Kamata, Yuko; Ito, Masaki; Hara, Eiichi; Ohkusa, Toshifumi; Gong, Jianlin; Tajiri, Hisao
2013-01-01
The therapeutic efficacy of fusion cell (FC)-based cancer vaccine generated with whole tumor cells and dendritic cells (DCs) requires the improved immunogenicity of both cells. Treatment of whole tumor cells with ethanol resulted in blockade of immune-suppressive soluble factors such as transforming growth factor (TGF)-β1, vascular endothelial growth factor, and IL-10 without decreased expression of major histocompatibility complex (MHC) class I and the MUC1 tumor-associated antigen. Moreover, the ethanol-treated tumor cells expressed "eat-me" signals such as calreticulin (CRT) on the cell surface and released immunostimulatory factors such as heat shock protein (HSP)90α and high-mobility group box 1 (HMGB1). A dual stimulation of protein-bound polysaccharides isolated from Coriolus versicolor (TLR2 agonist) and penicillin-inactivated Streptococcus pyogenes (TLR4 agonist) led human monocyte-derived DCs to produce HSP90α and multiple cytokines such as IL-12p70 and IL-10. Interestingly, incorporating ethanol-treated tumor cells and TLRs-stimulated DCs during the fusion process promoted fusion efficiency and up-regulated MHC class II molecules on a per fusion basis. Moreover, fusions of ethanol-treated tumor cells and dual TLRs-stimulated DCs (E-tumor/FCs) inhibited the production of multiple immune-suppressive soluble factors including TGF-β1 and up-regulated the production of IL-12p70 and HSP90α. Most importantly, E-tumor/FCs activated T cells capable of producing high levels of IFN-γ, resulting in augmented MUC1-specific CTL induction. Collectively, our results illustrate the synergy between ethanol-treated whole tumor cells and dual TLRs-stimulated DCs in inducing augmented CTL responses in vitro by FC preparations. The alternative system is simple and may provide a platform for adoptive immunotherapy.
-
Inventory of File sref_nmb.t03z.pgrb212.ctl.grib2
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
-
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
-
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
-
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.
-
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
-
Böttcher, Jan P; Schanz, Oliver; Wohlleber, Dirk; Abdullah, Zeinab; Debey-Pascher, Svenja; Staratschek-Jox, Andrea; Höchst, Bastian; Hegenbarth, Silke; Grell, Jessica; Limmer, Andreas; Atreya, Imke; Neurath, Markus F; Busch, Dirk H; Schmitt, Edgar; van Endert, Peter; Kolanus, Waldemar; Kurts, Christian; Schultze, Joachim L; Diehl, Linda; Knolle, Percy A
2013-03-28
Development of CD8(+) T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1(+) memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.
-
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.
-
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.
-
CD4(+) T-cell help amplifies innate signals for primary CD8(+) T-cell immunity.
Bedoui, Sammy; Heath, William R; Mueller, Scott N
2016-07-01
CD8(+) T cells provide an important component of protection against intracellular infections and cancer. Immune responses by these T cells involve a primary phase of effector expansion and differentiation, followed by a contraction phase leading to memory formation and, if antigen is re-encountered, a secondary expansion phase with more rapid differentiation. Both primary and secondary phases of CD8(+) T-cell immunity have been shown to depend on CD4(+) T-cell help, although during certain infections the primary phase is variable in this requirement. One explanation for such variability relates to the strength of associated inflammatory signals, with weak signals requiring help. Here, we focus on our studies that have dissected the requirements for help in the primary phase of the CTL response to herpes simplex virus, elucidating intricate interactions and communications between CD4(+) T cells, various dendritic cell subsets, and CD8(+) T cells. We place our studies in the context of others and describe a simple model of help where CD40 signaling amplifies innate signals to enable efficient CD8(+) T-cell expansion and differentiation. This model facilitates CTL induction to various different agents, without altering the qualitative innate signals that direct other important arms of immunity. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
-
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.
-
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.
-
Molecular cloning and characterization of a C-type lectin in yellow catfish Tachysurus fulvidraco.
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.
-
Ma, Wenbin; Zhang, Yi; Vigneron, Nathalie; Stroobant, Vincent; Thielemans, Kris; van der Bruggen, Pierre; Van den Eynde, Benoît J
2016-02-15
Cross-presentation enables dendritic cells to present on their MHC class I molecules antigenic peptides derived from exogenous material, through a mechanism that remains partly unclear. It is particularly efficient with long peptides, which are used in cancer vaccines. We studied the mechanism of long-peptide cross-presentation using human dendritic cells and specific CTL clones against melanoma Ags gp100 and Melan-A/MART1. We found that cross-presentation of those long peptides does not depend on the proteasome or the transporter associated with Ag processing, and therefore follows a vacuolar pathway. We also observed that it makes use of newly synthesized MHC class I molecules, through peptide exchange in vesicles distinct from the endoplasmic reticulum and classical secretory pathway, in an SEC22b- and CD74-independent manner. Our results indicate a nonclassical secretion pathway followed by nascent HLA-I molecules that are used for cross-presentation of those long melanoma peptides in the vacuolar pathway. Our results may have implications for the development of vaccines based on long peptides. Copyright © 2016 by The American Association of Immunologists, Inc.
-
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
-
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.
-
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.
-
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
-
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.
-
Pathology during acute infections: contributions of intracellular pathogens and the CTL response.
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.
-
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.
-
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
-
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
-
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.
-
Silva, A L; Rosalia, R A; Sazak, A; Carstens, M G; Ossendorp, F; Oostendorp, J; Jiskoot, W
2013-04-01
Overlapping synthetic long peptides (SLPs) hold great promise for immunotherapy of cancer. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are being developed as delivery systems to improve the potency of peptide-based therapeutic cancer vaccines. Our aim was to optimize PLGA NP for SLP delivery with respect to encapsulation and release, using OVA24, a 24-residue long synthetic antigenic peptide covering a CTL epitope of ovalbumin (SIINFEKL), as a model antigen. Peptide-loaded PLGA NPs were prepared by a double emulsion/solvent evaporation technique. Using standard conditions (acidic inner aqueous phase), we observed that either encapsulation was very low (1-30%), or burst release extremely high (>70%) upon resuspension of NP in physiological buffers. By adjusting formulation and process parameters, we uncovered that the pH of the first emulsion was critical to efficient encapsulation and controlled release. In particular, an alkaline inner aqueous phase resulted in circa 330 nm sized NP with approximately 40% encapsulation efficiency and low (<10%) burst release. These NP showed enhanced MHC class I restricted T cell activation in vitro when compared to high-burst releasing NP and soluble OVA24, proving that efficient entrapment of the antigen is crucial to induce a potent cellular immune response. Copyright © 2012 Elsevier B.V. All rights reserved.
-
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
-
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.
-
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
-
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
-
Granzyme B ELISPOT assay to measure influenza-specific cellular immunity.
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.
-
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.
-
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.
-
Lee, Chih-hung; Kakinuma, Takashi; Wang, Julia; Zhang, Hong; Palmer, Douglas C.; Restifo, Nicholas P.; Hwang, Sam T.
2008-01-01
Expression of the chemokine receptor CXCR4 by tumor cells promotes metastasis, possibly by activating pro-survival signals that render cancer cells resistant to immune attack. Inhibition of CXCR4 with a peptide antagonist, T22, blocks metastatic implantation of CXCR4-transduced B16 (CXCR4-luc-B16) melanoma cells in lung, but not the outgrowth of established metastases, raising the question of how T22 can best be used in a clinical setting. Herein, whereas the treatment of CXCR4-luc-B16 cells in vitro with the CXCR4 ligand CXCL12 did not reduce killing induced by cisplatin or cyclophosphamide, CXCL12 markedly reduced Fas-dependent killing by gp100-specific (pmel-1) CD8+ T cells. T22 pretreatment restored sensitivity of CXCR4-luc-B16 cells to pmel-1 killing, even in the presence of CXCL12. Two immune-augmenting regimens were used in combination with T22 to treat experimental lung metastases. First, low-dose cyclophosphamide treatment (100 mg/kg) on day 5 in combination with T22 (days 4–7) yielded a ~70% reduction of B16 metastatic tumor burden in the lungs compared with cyclophosphamide treatment alone (P < 0.001). Furthermore, whereas anti–CTL antigen 4 (CTLA4) monoclonal antibody (mAb; or T22 treatment) alone had little effect on established B16 metastases, pretreatment with T22 (in combination with anti-CTLA4 mAb) resulted in a 50% reduction in lung tumor burden (P = 0.02). Thus, in vitro, CXCR4 antagonism with T22 renders B16 cells susceptible to killing by antigen-specific T cells. In vivo, T22 synergizes with cyclophosphamide or anti-CTLA4 mAb in the treatment of established lung metastases, suggesting a novel strategy for augmenting the efficacy of immunotherapy. PMID:17041104
-
Das, Krishna; Eisel, David; Lenkl, Clarissa; Goyal, Ashish; Diederichs, Sven; Dickes, Elke; Osen, Wolfram; Eichmüller, Stefan B
2017-01-01
In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a β2m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO) clones did not give rise to tumors in syngeneic mice (C57BL/6N), unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.
-
Hoogendoorn, M; Wolbers, J Olde; Smit, W M; Schaafsma, M R; Barge, R M Y; Willemze, R; Falkenburg, J H F
2004-07-01
Allogeneic stem cell transplantation following reduced-intensity conditioning is being evaluated in patients with advanced B-cell chronic lymphocytic leukemia (B-CLL). The curative potential of this procedure is mediated by donor-derived alloreactive T cells, resulting in a graft-versus-leukemia effect. However, B-CLL may escape T-cell-mediated immune reactivity since these cells lack expression of costimulatory molecules. We examined the most optimal method to transform B-CLL cells into efficient antigen-presenting cells (APC) using activating cytokines, by triggering toll-like receptors (TLRs) using microbial pathogens and by CD40 stimulation with CD40L-transfected fibroblasts. CD40 activation in the presence of IL-4 induced strongest upregulation of costimulatory and adhesion molecules on B-CLL cells and induced the production of high amounts of IL-12 by the leukemic cells. In contrast to primary B-CLL cells as stimulator cells, these malignant APCs were capable of inducing the generation of B-CLL-reactive CD8(+) CTL lines and clones from HLA class I-matched donors. These CTL lines and clones recognized and killed primary B-CLL as well as patient-derived lymphoblasts, but not donor cells. These results show the feasibility of ex vivo generation of B-CLL-reactive CD8(+) CTLs. This opens new perspectives for adoptive immunotherapy, following allogeneic stem cell transplantation in patients with advanced B-CLL.
-
Inhibitors of Glioma Growth that Reveal the Tumour to the Immune System
Nieto-Sampedro, Manuel; Valle-Argos, Beatriz; Gómez-Nicola, Diego; Fernández-Mayoralas, Alfonso; Nieto-Díaz, Manuel
2011-01-01
Treated glioblastoma patients survive from 6 to 14 months. In the first part of this review, we describe glioma origins, cancer stem cells and the genomic alterations that generate dysregulated cell division, with enhanced proliferation and diverse response to radiation and chemotherapy. We review the pathways that mediate tumour cell proliferation, neo-angiogenesis, tumor cell invasion, as well as necrotic and apoptotic cell death. Then, we examine the ability of gliomas to evade and suppress the host immune system, exhibited at the levels of antigen recognition and immune activation, limiting the effective signaling between glioma and host immune cells. The second part of the review presents current therapies and their drawbacks. This is followed by a summary of the work of our laboratory during the past 20 years, on oligosaccharide and glycosphingolipid inhibitors of astroblast and astrocytoma division. Neurostatins, the O-acetylated forms of gangliosides GD1b and GT1b naturally present in mammalian brain, are cytostatic for normal astroblasts, but cytotoxic for rat C6 glioma cells and human astrocytoma grades III and IV, with ID50 values ranging from 200 to 450 nM. The inhibitors do not affect neurons or fibroblasts up to concentrations of 4 μM or higher. At least four different neurostatin-activated, cell-mediated antitumoral processes, lead to tumor destruction: (i) inhibition of tumor neovascularization; (ii) activation of microglia; (iii) activation of natural killer (NK) cells; (iv) activation of cytotoxic lymphocytes (CTL). The enhanced antigenicity of neurostatin-treated glioma cells, could be related to their increased expression of connexin 43. Because neurostatins and their analogues show specific activity and no toxicity for normal cells, a clinical trial would be the logical next step. PMID:22084619
-
A new vesicle trafficking regulator CTL1 plays a crucial role in ion homeostasis.
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
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
-
Clonal predominance of CD8+ T cells in patients with unexplained neutropenia
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
-
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
-
Kasamatsu, Jun; Takahashi, Shojiro; Azuma, Masahiro; Matsumoto, Misako; Morii-Sakai, Akiko; Imamura, Masahiro; Teshima, Takanori; Takahashi, Akari; Hirohashi, Yoshihiko; Torigoe, Toshihiko; Sato, Noriyuki; Seya, Tsukasa
2015-01-01
CD4(+) T cell effectors are crucial for establishing antitumor immunity. Dendritic cell maturation by immune adjuvants appears to facilitate subset-specific CD4(+) T cell proliferation, but the adjuvant effect for CD4 T on induction of cytotoxic T lymphocytes (CTLs) is largely unknown. Self-antigenic determinants with low avidity are usually CD4 epitopes in mutated proteins with tumor-associated class I-antigens (TAAs). In this study, we made a chimeric version of survivin, a target of human CTLs. The chimeric survivin, where human survivin-2B containing a TAA was embedded in the mouse survivin frame (MmSVN2B), was used to immunize HLA-A-2402/K(b)-transgenic (HLA24(b)-Tg) mice. Subcutaneous administration of MmSVN2B or xenogeneic human survivin (control HsSNV2B) to HLA24(b)-Tg mice failed to induce an immune response without co-administration of an RNA adjuvant polyI:C, which was required for effector induction in vivo. Although HLA-A-2402/K(b) presented the survivin-2B peptide in C57BL/6 mice, 2B-specific tetramer assays showed that no CD8(+) T CTLs specific to survivin-2B proliferated above the detection limit in immunized mice, even with polyI:C treatment. However, the CD4(+) T cell response, as monitored by IFN-γ, was significantly increased in mice given polyI:C+MmSVN2B. The Th1 response and antibody production were enhanced in the mice with polyI:C. The CD4 epitope responsible for effector function was not Hs/MmSNV13-27, a nonconserved region between human and mouse survivin, but region 53-67, which was identical between human and mouse survivin. These results suggest that activated, self-reactive CD4(+) helper T cells proliferate in MmSVN2B+polyI:C immunization and contribute to Th1 polarization followed by antibody production, but hardly participate in CTL induction. Copyright © 2014 Elsevier GmbH. All rights reserved.
-
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
-
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
-
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
-
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.
-
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
-
Characterization of CTL Recognized Epitopes on Human Breast Tumors
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
-
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
-
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.
-
Collinge, Mark; Thorn, Mitchell; Peachee, Vanessa; White, Kimber
2013-01-01
Establishing an in vivo cell-mediated immunity (CMI) assay, such as the delayed-type hypersensitivity (DTH) assay, has been identified as an important gap and recommended to receive highest priority for new model development in several workshops on developmental immunotoxicity. A Candida albicans DTH model has recently been developed that has the advantage over other DTH models, which use alternative sensitizing antigens, in that antigen-specific antibodies, which may interfere with the assay, are not produced. In addition, the in vivo C. albicans DTH model was demonstrated to be more sensitive in detecting immunosuppression than DTH models using keyhole limpet hemocyanin (KLH) or sheep red blood cells as antigens, as well as some ex vivo CMI assays. While KLH and sheep red blood cells are non-physiological immunogens, C. albicans is an important human pathogen. The present studies were conducted in order to optimize and validate the C. albicans DTH model for use in developmental immunotoxicity studies using juvenile rats. Three known immunosuppressive compounds with different mechanisms of action were tested in this model, cyclosprorin A (CsA), cyclophosphamide (CPS), and dexamethasone (DEX). Animals were sensitized with formalin-fixed C. albicans on postnatal day (PND) 28 and challenged with chitosan on PND 38. Drug was administered beginning on PND 23 and continued until PND 37. Exposure to each of the three immunotoxicants resulted in statistically significant decreases in the DTH response to C. albicans-derived chitosan. Decreases in footpad swelling were observed at ≥10 mg CsA/kg/day, ≥5 mg CPS/kg/day, and ≥0.03 mg DEX/kg/day. These results demonstrate that the C. albicans DTH model, optimized for use in juvenile rats, can be used to identify immunotoxic compounds, and fills the need for a sensitive in vivo CMI model for assessments of developmental immunotoxicity. Abbreviations Ab, antibody APC, antigen presenting cell BSA, bovine serum albumin C. albicans, Candida albicans CI, challenge interval CMI, cell-mediated immunity CO, challenge only CPS, cyclophosphamide CsA, cyclosporin A CTL, cytotoxic T lymphocyte DEX, dexamethasone DIT, developmental immunotoxicity DTH, delayed-type hypersensitivity ip, intraperitoneal KLH, keyhole limpet hemocyanin MLR, mixed lymphocyte reaction OVA, ovalbumin PBS, phosphate-buffered saline PND, postnatal day sc, subcutaneous SEM, standard error of the mean SRBC, sheep red blood cells.
-
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.
-
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.
-
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
-
Zhang, Peng; Yi, Shuhong; Li, Xi; Liu, Ruilei; Jiang, Hua; Huang, Zenan; Liu, Yu; Wu, Juekun; Huang, Yong
2014-01-01
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) in human immune system. DC-based tumor vaccine has met with some success in specific malignancies, inclusive of breast cancer. In this study, we electrofused MDA-MB-231 breast cancer cell line with day-3 DCs derived from peripheral blood monocytes, and explored the biological characteristics of fusion vaccine and its anti-tumor effects in vitro. Day-3 mature DCs were generated from day-2 immature DCs by adding cocktails composed of TNF-α, IL-1β, IL-6 and PEG2. Day-3 mature DCs were identified and electofused with breast cancer cells to generate fusion vaccine. Phenotype of fusion cells were identified by fluorescence microscope and flow cytometer. The fusion vaccine was evaluated for T cell proliferation, secretion of IL-12 and IFN-γ, and induction of tumor-specific CTL response. Despite differences in morphology, day-3 and day-7 DC expressed similar surface markers. The secretion of IL-12 and IFN-γ in fusion vaccine group was much higher than that in the control group. Compared with control group, DC-tumor fusion vaccine could better stimulate the proliferation of allogeneic T lymphocytes and kill more breast cancer cells (MDA-MB-231) in vitro. Day-3 DCs had the same function as the day-7 DCs, but with a shorter culture period. Our findings suggested that day-3 DCs fused with whole apoptotic breast cancer cells could elicit effective specific antitumor T cell responses in vitro and may be developed into a prospective candidate for adoptivet immunotherapy.
-
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.
-
Ventilation Increases with Lower Extremity Venous Occlusion in Young Adults
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
-
Domingo-Musibay, Evidio; Heun, James M; Nevala, Wendy K; Callstrom, Matthew; Atwell, Thomas; Galanis, Evanthia; Erickson, Lori A; Markovic, Svetomir N
2017-09-01
Percutaneous thermal ablation combined with in situ granulocyte-macrophage colony-stimulating factor cytokine therapy was technically feasible and well tolerated.No significant clinical or immunologic responses were seen. Melanoma tumor-derived heat-shock proteins (HSPs) and HSP-peptide complexes can elicit protective antitumor responses. The granulocyte-macrophage colony-stimulating factor (GM-CSF) chemokine can also promote uptake and processing by professional antigen presenting cells (APCs). On this basis, we designed a pilot study of percutaneous thermal ablation as a means to induce heat-shock protein vaccination plus GM-CSF to determine safety and preliminary antitumor activity of this combination. This study was designed to assess overall safety of percutaneous ablation combined with GM-CSF for unresectable, metastatic melanoma including uveal and mucosal types. All patients received heat-shock therapy (42°C for 30 minutes), then received one of three treatments: (a) intralesional GM-CSF (500 mcg standard dose); (b) radiofrequency ablation (RFA) + GM-CSF; or (c) cryoablation plus GM-CSF. The primary endpoint of the study was the induction of endogenous HSP70 and melanoma-specific cytotoxic T lymphocytes (CTL). Nine patients (three per study arm) were enrolled. No dose-limiting toxicity was observed as specified per protocol. All patients developed progressive disease and went on to receive alternative therapy. Median overall survival (OS) was 8.2 months (95% confidence interval [CI] 2-17.2). The study was not powered to detect a difference in clinical outcome among treatment groups. Percutaneous thermal ablation plus GM-CSF was well tolerated, technically feasible, and demonstrated an acceptable adverse event profile comparable to conventional RFA and cryoablation. While HSP70 was induced following therapy, the degree of HSP70 elevation was not associated with clinical outcome or induced CTL responses. While percutaneous thermal ablation plus GM-CSF combinations including checkpoint inhibitors could be considered in future studies, the use of GM-CSF remains experimental and for use in the context of clinical trials. © AlphaMedPress; the data published online to support this summary is the property of the authors.
-
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
-
Expression of Antigen Processing and Presenting Molecules in Brain Metastasis of Breast Cancer
Liu, Yan; Komohara, Yoshihiro; Domenick, Natalie; Ohno, Masasuke; Ikeura, Maki; Hamilton, Ronald L.; Horbinski, Craig; Wang, Xinhui; Ferrone, Soldano; Okada, Hideho
2012-01-01
Defects in human leukocyte antigen (HLA) class I antigen processing machinery (APM) component expression can have a negative impact on the clinical course of tumors and the response to T-cell-based immunotherapy. Since brain metastases of breast cancer are of increasing clinical significance, the APM component expression levels and CD8+ T-cell infiltration patterns were analyzed in primary breast and metastatic brain lesions of breast cancer by immunohistochemistry. Comparison of unpaired 50 primary and 33 brain metastases showed lower expression of β2-microgloblin, transporter associated with antigen processing (TAP) 1, TAP2 and calnexin in the brain lesions. Although no significant differences were found in APM component scores between primary breast and brain lesions in 15 paired cases, primary breast lesions of which patients eventually developed brain metastases showed lower levels of β2-microgloblin, TAP1 and calnexin compared with breast lesions without known brain metastases. The extent of CD8+ T cell infiltration was significantly higher in the lesions without metastasis compared with the ones with brain metastases, and was positively associated with the expression of TAP1 and calnexin. Furthermore, mouse tumor cells stably transfected with silencing hairpin (sh)RNA for TAP1 demonstrated a decreased susceptibility to cytotoxic T lymphocytes (CTL) in vitro and enhanced spontaneous brain metastasis in vivo. These data support the functional significance of TAP1 expression in tumor cells. Taken together, our data suggest that patients with low or defective TAP1 or calnexin in primary breast cancers may be at higher risks for developing brain metastasis due to the defects in T cell-based immunosurveillance. PMID:22065046
-
Zhang, Xiaoxiao; Truax, Agnieszka D.; Ma, Ruixue; Liu, Ziyu; Lei, Yingfeng; Zhang, Liang; Ye, Wei; Zhang, Fanglin; Xu, Zhikai; Shang, Lei; Liu, Rongrong; Wang, Fang; Wu, Xingan
2016-01-01
Infection of Hantaan virus (HTNV) usually causes hemorrhagic fever with renal syndrome (HFRS). China has the worst epidemic incidence of HFRS as well as high fatality. Inactivated whole virus has been used for HFRS vaccination, however there are still problems such as safety concerns. CD40 ligand (CD40L) and granulocyte macrophage colony-stimulating factor (GM-CSF) are well-known immune stimulating molecules that can enhance antigen presenting, lymphocytes activation and maturation, incorporation of CD40L and GM-CSF to the surface of virus like particles (VLPs) can greatly improve the vaccination effect. We constructed eukaryotic vectors expressing HTNV M segment and S segment, as well as vectors expressing HTNV M segment with CD40L or GM-CSF, our results showed successful production of CD40L or GM-CSF incorporated HTNV VLPs. In vitro stimulation with CD40L or GM-CSF anchored HTNV VLP showed enhanced activation of macrophages and DCs. CD40L/GM-CSF incorporated VLP can induce higher level of HTNV specific antibody and neutralizing antibody in mice. Immunized mice splenocytes showed higher ability of secreting IFN-γ and IL-2, as well as enhancing CTL activity. These results suggest CD40L/GM-CSF incorporated VLP can serve as prospective vaccine candidate. PMID:27542281
-
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.
-
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
-
Direct antigen presentation and gap junction mediated cross-presentation during apoptosis.
Pang, Baoxu; Neijssen, Joost; Qiao, Xiaohang; Janssen, Lennert; Janssen, Hans; Lippuner, Christoph; Neefjes, Jacques
2009-07-15
MHC class I molecules present peptides from endogenous proteins. Ags can also be presented when derived from extracellular sources in the form of apoptotic bodies. Cross-presentation of such Ags by dendritic cells is required for proper CTL responses. The fate of Ags in cells initiated for apoptosis is unclear as is the mechanism of apoptosis-derived Ag transfer into dendritic cells. Here we show that novel Ags can be generated by caspases and be presented by MHC class I molecules of apoptotic cells. Since gap junctions function until apoptotic cells remodel to form apoptotic bodies, transfer and cross-presentation of apoptotic peptides by neighboring and dendritic cells occurs. We thus define a novel phase in classical Ag presentation and cross-presentation by MHC class I molecules: presentation of Ags created by caspase activities in cells in apoptosis.
-
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 ...
-
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.
-
Improved Anti-Treg Vaccination Targeting Foxp3 Efficiently Decreases Regulatory T Cells in Mice.
Mousavi Niri, Neda; Memarnejadian, Arash; Pilehvar-Soltanahmadi, Younes; Agha Sadeghi, Mohammadreza; Mahdavi, Mehdi; Kheshtchin, Nasim; Arab, Samaneh; Namdar, Afshin; Jadidi, Farhad; Zarghami, Nosratollah; Hajati, Jamshid
2016-09-01
The critical role of regulatory T (Treg) cells in dampening immune responses against tumor cells is apparent. Therefore, several methods have been introduced for eliminating Treg. Among them, inducing immune responses against Treg cells expressing Foxp3 transcription factor is a hopeful approach to decrease the frequency of Tregs. In current study, we used the chimeric FoxP3-Fc(IgG) fusion construct/protein to effectively stimulate the immune responses against Treg cells. Previously constructed FoxP3-Fc(IgG) DNA vaccine and its protein counterpart were injected into C57BL/6 mice in a prime/boost regimen. After 2 weeks, the mice were killed to measure the frequency of Tregs in their spleens, as well as analyze their specific cytokine production, T-cell proliferation, and CD8 T-cell cytotoxicity against FoxP3 protein. FACS analysis of FoxP3 CD4 cells in splenocytes revealed the efficiency of FoxP3 DNA-prime protein-boost strategy to decrease the Treg cells and further showed considerable superiority of Fc(IgG) fusion strategy. This significant reduction in Treg frequency was also concomitant with higher FoxP3-specific CTL and Th1 responses in FoxP3-Fc vaccinated animals. Prime/boost vaccination against FoxP3 in addition to enhanced antigen presentation by means of Fc fusion strategy could be successfully considered for Treg depletion studies. Validity of this approach should be experimentally tested in preclinical tumor models.
-
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).
-
Fujizuka, Yuji; Ito, Kazuto; Oki, Ryo; Suzuki, Rie; Sekine, Yoshitaka; Koike, Hidekazu; Matsui, Hiroshi; Shibata, Yasuhiro; Suzuki, Kazuhiro
2017-08-01
To investigate the predictive value of various molecular forms of prostate-specific antigen in men with baseline prostate-specific antigen <2.0 ng/mL. The case cohort comprised 150 men with a baseline prostate-specific antigen level <2.0 ng/mL, and who developed prostate cancer within 10 years. The control cohort was 300 baseline prostate-specific antigen- and age-adjusted men who did not develop prostate cancer. Serum prostate-specific antigen, free prostate-specific antigen, and [-2] proenzyme prostate-specific antigen were measured at baseline and last screening visit. The predictive impact of baseline prostate-specific antigen- and [-2] proenzyme prostate-specific antigen-related indices on developing prostate cancer was investigated. The predictive impact of those indices at last screening visit and velocities from baseline to final screening on tumor aggressiveness were also investigated. The baseline free to total prostate-specific antigen ratio was a significant predictor of prostate cancer development. The odds ratio was 6.08 in the lowest quintile baseline free to total prostate-specific antigen ratio subgroup. No serum indices at diagnosis were associated with tumor aggressiveness. The Prostate Health Index velocity and [-2] proenzyme prostate-specific antigen/free prostate-specific antigen velocity significantly increased in patients with higher risk D'Amico risk groups and higher Gleason scores. Free to total prostate-specific antigen ratio in men with low baseline prostate-specific antigen levels seems to predict the risk of developing prostate cancer, and it could be useful for a more effective individualized screening system. Longitudinal changes in [-2] proenzyme prostate-specific antigen-related indices seem to correlate with tumor aggressiveness, and they could be used as prognostic tool before treatment and during active surveillance. © 2017 The Japanese Urological Association.
-
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.
-
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.
-
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
-
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.
-
Primary Human Immunodeficiency Virus Type 1 (HIV-1) Infection during HIV-1 Gag Vaccination▿
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
-
Iremashvili, Viacheslav; Barney, Shane L; Manoharan, Murugesan; Kava, Bruce R; Parekh, Dipen J; Punnen, Sanoj
2016-04-01
To analyze the association between prediagnostic prostate-specific antigen kinetics and the risk of biopsy progression in prostate cancer patients on active surveillance, and to study the effect of prediagnostic prostate-specific antigen values on the predictive performance of prostate-specific antigen velocity and prostate-specific antigen doubling time. The study included 137 active surveillance patients with two or more prediagnostic prostate-specific antigen levels measured over a period of at least 3 months. Two sets of analyses were carried out. First, the association between prostate-specific antigen kinetics calculated using only the prediagnostic prostate-specific antigen values and the risk of biopsy progression was studied. Second, using the same cohort of patients, the predictive value of prostate-specific antigen kinetics calculated using only post-diagnostic prostate-specific antigens and compared with that of prostate-specific antigen kinetics based on both pre- and post-diagnostic prostate-specific antigen levels was analyzed. Of 137 patients included in the analysis, 37 (27%) had biopsy progression over a median follow-up period of 3.2 years. Prediagnostic prostate-specific antigen velocity of more than 2 ng/mL/year and 3 ng/mL/year was statistically significantly associated with the risk of future biopsy progression. However, after adjustment for baseline prostate-specific antigen density, these associations were no longer significant. None of the tested prostate-specific antigen kinetics based on combined pre- and post-diagnostic prostate-specific antigen values were statistically significantly associated with the risk of biopsy progression. Historical prediagnostic prostate-specific antigens seems to be not clinically useful in patients diagnosed with low-risk prostate cancer on active surveillance. © 2016 The Japanese Urological Association.
-
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.
-
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
-
Villiers, M B; Villiers, C L; Jacquier-Sarlin, M R; Gabert, F M; Journet, A M; Colomb, M G
1996-01-01
Antigen opsonization by the C3b fragment of complement is a significant event in the modulation of cell-mediated immune response, but its mechanism is still largely unknown. The structural characteristics of C3b allow it to act as a bifunctional ligand between antigen and cells via their membrane C3b receptors. It was thus of interest to study the influence of the covalent link between C3b and antigen on the fixation and internalization of this antigen by antigen-presenting cells. Tetanus toxin (TT) was used as antigen, either free or covalently linked to C3b (TT-C3b). The antigen-presenting cells were TT-specific (4.2) or non-specific (BL15) Epstein-Barr virus (EBV)-transformed B cells. C3b was found to play an important role in antigen fixation and internalization by both antigen-specific and antigen non-specific cells. Covalent binding of C3b on TT (1) permitted fixation and internalization of this antigen by non-specific cells via their complement receptors; (2) enhanced antigen fixation and resulted in cross-linking between membrane immunoglobulins and complement receptors on antigen-specific cells. The consequences of covalent C3b binding to TT were analysed using antigen-specific and antigen-nonspecific cells. In both cases, a net increase in antigen fixation was observed. At the intracellular level, covalent C3b binding to TT resulted in a large TT incorporation in endosomes of nonspecific cells, similar to that observed in antigen-specific cells. Thus, C3b covalently linked to antigen enlarges the array of B-cell types capable of presenting antigen, including non-specific cells. Images Figure 2 PMID:8958046
-
Magheli, Ahmed; Hinz, Stefan; Hege, Claudia; Stephan, Carsten; Jung, Klaus; Miller, Kurt; Lein, Michael
2010-01-01
We investigated the value of pretreatment prostate specific antigen density to predict Gleason score upgrading in light of significant changes in grading routine in the last 2 decades. Of 1,061 consecutive men who underwent radical prostatectomy between 1999 and 2004, 843 were eligible for study. Prostate specific antigen density was calculated and a cutoff for highest accuracy to predict Gleason upgrading was determined using ROC curve analysis. The predictive accuracy of prostate specific antigen and prostate specific antigen density to predict Gleason upgrading was evaluated using ROC curve analysis based on predicted probabilities from logistic regression models. Prostate specific antigen and prostate specific antigen density predicted Gleason upgrading on univariate analysis (as continuous variables OR 1.07 and 7.21, each p <0.001) and on multivariate analysis (as continuous variables with prostate specific antigen density adjusted for prostate specific antigen OR 1.07, p <0.001 and OR 4.89, p = 0.037, respectively). When prostate specific antigen density was added to the model including prostate specific antigen and other Gleason upgrading predictors, prostate specific antigen lost its predictive value (OR 1.02, p = 0.423), while prostate specific antigen density remained an independent predictor (OR 4.89, p = 0.037). Prostate specific antigen density was more accurate than prostate specific antigen to predict Gleason upgrading (AUC 0.61 vs 0.57, p = 0.030). Prostate specific antigen density is a significant independent predictor of Gleason upgrading even when accounting for prostate specific antigen. This could be especially important in patients with low risk prostate cancer who seek less invasive therapy such as active surveillance since potentially life threatening disease may be underestimated. Further studies are warranted to help evaluate the role of prostate specific antigen density in Gleason upgrading and its significance for biochemical outcome.
-
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.
-
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
-
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.
-
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
-
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.
-
The use of a magnesium-based bone adhesive for flexor tendon-to-bone healing
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
-
Li, Geng; Ali, Selman A; McArdle, Stephanie E B; Mian, Shahid; Ahmad, Murrium; Miles, Amanda; Rees, Robert C
2005-01-01
During the last decade, a large number of human tumour antigens have been identified. These antigens are classified as tumour-specific shared antigens, tissue-specific differentiation antigens, overexpressed antigens, tumour antigens resulting from mutations, viral antigens and fusion proteins. Antigens recognised by effectors of immune system are potential targets for antigen-specific cancer immunotherapy. However, most tumour antigens are self-proteins and are generally of low immunogenicity and the immune response elicited towards these tumour antigens is not always effective. Strategies to induce and enhance the tumour antigen-specific response are needed. This review will summarise the approaches to discovery of tumour antigens, the current status of tumour antigens, and their potential application to cancer treatment.
-
Chhabra, Arvind; Chakraborty, Nityo G.; Mukherji, Bijay
2008-01-01
Dendritic cells (DC) present antigenic epitopes to and activate T cells. They also polarize the ensuing T cell response to Th1 or Th2 type response, depending on their cytokine production profile. For example, IL-12 producing DC generate Th1 type T cell response whereas IL-10 producing DC is usually tolerogenic. Different strategies -- such as the use of cytokines and anti-cytokine antibodies, dominant negative forms of protein, anti-sense RNA etc. -- have been employed to influence the cytokine synthetic profile of DC as well as to make DC more immunogenic. Utilizing GFP expressing recombinant adenoviruses in association with lipid-mediated transfection of siRNA, we have silenced the endogenous IL-10 gene in DC. We show that IL-10 gene silenced DC produce more IL-12 and also generates a better cytolytic T cell response against the human melanoma associated epitope, MART-127−35, in-vitro. We also show that the GFP expressing adenoviral vector can be used to optimize the parameters for siRNA delivery in primary cells and show that RNA interference methodology can efficiently knock-down virus encoded genes transcribed at very high multiplicity of infection in DC. PMID:18249038
-
Functional analysis of choline transporters in rheumatoid arthritis synovial fibroblasts.
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.
-
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
-
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.
-
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.
-
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.
-
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.
-
Prostate-specific antigen lowering effect of metabolic syndrome is influenced by prostate volume.
Choi, Woo Suk; Heo, Nam Ju; Paick, Jae-Seung; Son, Hwancheol
2016-04-01
To investigate the influence of metabolic syndrome on prostate-specific antigen levels by considering prostate volume and plasma volume. We retrospectively analyzed 4111 men who underwent routine check-ups including prostate-specific antigen and transrectal ultrasonography. The definition of metabolic syndrome was based on the modified Adult Treatment Panel III criteria. Prostate-specific antigen mass density (prostate-specific antigen × plasma volume / prostate volume) was calculated for adjusting plasma volume and prostate volume. We compared prostate-specific antigen and prostate-specific antigen mass density levels of participants with metabolic syndrome (metabolic syndrome group, n = 1242) and without metabolic syndrome (non-prostate-specific antigen metabolic syndrome group, n = 2869). To evaluate the impact of metabolic syndrome on prostate-specific antigen, linear regression analysis for the natural logarithm of prostate-specific antigen was used. Patients in the metabolic syndrome group had significantly older age (P < 0.001), larger prostate volume (P < 0.001), higher plasma volume (P < 0.001) and lower mean serum prostate-specific antigen (non-metabolic syndrome group vs metabolic syndrome group; 1.22 ± 0.91 vs 1.15 ± 0.76 ng/mL, P = 0.006). Prostate-specific antigen mass density in the metabolic syndrome group was still significantly lower than that in the metabolic syndrome group (0.124 ± 0.084 vs 0.115 ± 0.071 μg/mL, P = 0.001). After adjusting for age, prostate volume and plasma volume using linear regression model, the presence of metabolic syndrome was a significant independent factor for lower prostate-specific antigen (prostate-specific antigen decrease by 4.1%, P = 0.046). Prostate-specific antigen levels in patients with metabolic syndrome seem to be lower, and this finding might be affected by the prostate volume. © 2016 The Japanese Urological Association.
-
1981-01-01
A highly specific and reproducible antigen-induced, antigen-specific culture and assay system for antibody production by human peripheral blood B lymphocytes has been developed. The system is clearly T cell and monocyte dependent and is independent of exogenous mitogens. The major factors in our ability to trigger specific antibody production with antigen alone have been the use of extremely low concentrations of antigen in vitro (doses several orders of magnitude below those inducing a peak blastogenic response), careful attention to in vitro cell density and culture vessel geometry, and appreciation of the kinetics of the circulating antigen-inducible B cell repertoire. A dichotomy and overlap between antigen-induced, antigen-specific and antigen-induced, polyclonal responses was observed in the study of doubly immunized individuals. Whereas antibody responses highly specific for the antigen in culture were observed under one set of culture conditions (flat-bottomed vessels, 1.5 x 10(6) cells), switching to another culture system (round-bottomed vessels, 5 x 10(5) cells) resulted in polyclonal responses to antigen. Despite these culture condition-related differences in the induction of antibody synthesis, the suppression of specific antibody production that occurred at high concentrations of antigen was specific only for the antigen in culture. The capability to easily and reproducibly look at truly antigen-induced, antigen specific antibody production should be a major tool in furthering the understanding of human B cell activation and immunoregulation. PMID:6169778
-
Ahn, So-Shin; Jeon, Bo-Young; Park, Seong-Jeong; Choi, Dong-Hoon; Ku, Sun-Hwa; Cho, Sang-Nae; Sung, Young-Chul
2013-06-12
Improvement to the immunogenicity of DNA vaccines was evaluated in a Mycobacterium tuberculosis (MTB) infection mouse model examining the combined effects of nonlytic Fc-fused IL-7 DNA (IL-7-nFc) and Flt3-ligand fused Mtb32 (F-Mtb32) DNA. Mice were treated with conventional chemotherapy for 6 weeks from 4 weeks after aerosol infection of MTB. Following the start of chemotherapy, DNA immunizations were administered five times with 2-week intervals. Coadministration of IL-7-nFc and F-Mtb32 DNA given during chemotherapy synergistically enhanced the magnitude of Mtb32-specific T cell responses and sustained for one-year after the last immunization assessed by IFN-γ ELISPOT assay. After dexamethasone treatment, a significantly reduced MTB reactivation was observed in mice received both IL-7-nFc and F-Mtb32 DNA, compared with F-MTb32 DNA alone or with control mice. In addition, mice treated with IL-7-nFc and F-Mtb32 DNA together showed improved lung pathology and reduced pulmonary inflammation values relative to F-Mtb32 DNA or saline injected mice. Intracellular cytokine staining revealed that the protection levels induced by combination therapy with IL-7-nFc and F-Mtb32 DNA was associated with enhanced Mtb32-specific IFN-γ secreting CD4(+) T cell responses and CD8(+) T cell responses stimulated with CTL epitope peptide in the lungs and spleens. These data suggest that IL-7-nFc as a novel TB adjuvant may facilitate therapeutic TB DNA vaccine to the clinics through significant enhancement of codelivered DNA vaccine-induced T cell immunity. Copyright © 2013 Elsevier Ltd. All rights reserved.
-
Mucin 1-specific immunotherapy in a mouse model of spontaneous breast cancer.
Mukherjee, Pinku; Madsen, Cathy S; Ginardi, Amelia R; Tinder, Teresa L; Jacobs, Fred; Parker, Joanne; Agrawal, Babita; Longenecker, B Michael; Gendler, Sandra J
2003-01-01
Human mucin 1 (MUC1) is an epithelial mucin glycoprotein that is overexpressed in 90% of all adenocarcinomas including breast, lung, pancreas, prostate, stomach, colon, and ovary. MUC1 is a target for immune intervention, because, in patients with solid adenocarcinomas, low-level cellular and humoral immune responses to MUC1 have been observed, which are not sufficiently strong to eradicate the growing tumor. The hypothesis for this study is that enhancing MUC1-specific immunity will result in antitumor immunity. To test this, the authors have developed a clinically relevant breast cancer model that demonstrates peripheral and central tolerance to MUC1 and develops spontaneous tumors of the mammary gland. In these mice, the authors tested a vaccine formulation comprised of liposomal-MUC1 lipopeptide and human recombinant interleukin-2. Results indicate that when compared with untreated mice, immunized mice develop T cells that express intracellular IFN-gamma, are reactive with MHC class I H-2Db/MUC1 tetramer, and are cytotoxic against MUC1-expressing tumor cells in vitro. The presence of MUC1-specific CTL did not translate into a clinical response as measured by time of tumor onset, tumor burden, and survival. The authors demonstrate that some of the immune-evasion mechanisms used by the tumor cells include downregulation of MHC-class I molecule, expression of TGF-beta2, and decrease in IFN-gamma -expressing effector T cells as tumors progress. Finally, utilizing an injectable breast cancer model, the authors show that targeting a single tumor antigen may not be an effective antitumor treatment, but that immunization with dendritic cells fed with whole tumor lysate is effective in breaking tolerance and protecting mice from subsequent tumor challenge. A physiologically relevant spontaneous breast cancer model has been developed to test improved immunotherapeutic approaches.