Sample records for t-lymphocyte ctl activity

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed

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

    2013-04-01

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

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

    PubMed Central

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

    1999-01-01

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

  4. Identification of Dominant Optimal HLA-B60- and HLA-B61-Restricted Cytotoxic T-Lymphocyte (CTL) Epitopes: Rapid Characterization of CTL Responses by Enzyme-Linked Immunospot Assay

    PubMed Central

    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

  5. Identification of dominant optimal HLA-B60- and HLA-B61-restricted cytotoxic T-lymphocyte (CTL) epitopes: rapid characterization of CTL responses by enzyme-linked immunospot assay.

    PubMed

    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.

  6. Inhibition of the activity of cytotoxic murine T lymphocytes by antibodies to idiotypic determinants.

    PubMed Central

    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

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

    PubMed Central

    1993-01-01

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

  8. The by-products generated during sarin synthesis in the Tokyo sarin disaster induced inhibition of natural killer and cytotoxic T lymphocyte activity.

    PubMed

    Li, Q; Hirata, Y; Piao, S; Minami, M

    2000-05-05

    More than 5000 passengers on Tokyo subway trains were injured by the nerve gas, sarin and its by-products. Analysis of phosphor-carrying metabolites of sarin and its by-products in urine samples from the victims suggested that they were exposed not only to sarin, but also by-products generated during sarin synthesis, i.e. diisopropyl methylphosphonate (DIMP) and diethyl methylphosphonate (DEMP). We suspected genetic after-effects due to sarin by-products, thus, we checked the frequency of sister chromatid exchange (SCE) and found that SCE was significantly higher in the victims than in a control group, and that DIMP and DEMP significantly induced human lymphocyte SCE in vitro. In the present study, to explore whether DIMP and DEMP, which induced a high frequency of SCE of lymphocytes, also affected the lymphocyte functions, we examined the effect of DIMP and DEMP on splenic natural killer (NK) and splenic cytotoxic T lymphocyte (CTL) activity in mice, and NK activity of human lymphocytes in vitro. We found that DIMP and DEMP significantly inhibited NK and CTL activity in a dose-dependent manner. The inhibition induced by DIMP was stronger than that by DEMP. The effect of DIMP and DEMP on the splenic NK activity of mice was stronger than on the splenic CTL activity, and the human lymphocytes is more sensitive to DIMP and DEMP than the splenocytes of mice.

  9. Nicotine Inhibits Memory CTL Programming

    PubMed Central

    Sun, Zhifeng; Smyth, Kendra; Garcia, Karla; Mattson, Elliot; Li, Lei; Xiao, Zhengguo

    2013-01-01

    Nicotine is the main tobacco component responsible for tobacco addiction and is used extensively in smoking and smoking cessation therapies. However, little is known about its effects on the immune system. We confirmed that multiple nicotinic receptors are expressed on mouse and human cytotoxic T lymphocytes (CTLs) and demonstrated that nicotinic receptors on mouse CTLs are regulated during activation. Acute nicotine presence during activation increases primary CTL expansion in vitro, but impairs in vivo expansion after transfer and subsequent memory CTL differentiation, which reduces protection against subsequent pathogen challenges. Furthermore, nicotine abolishes the regulatory effect of rapamycin on memory CTL programming, which can be attributed to the fact that rapamycin enhances expression of nicotinic receptors. Interestingly, naïve CTLs from chronic nicotine-treated mice have normal memory programming, which is impaired by nicotine during activation in vitro. In conclusion, simultaneous exposure to nicotine and antigen during CTL activation negatively affects memory development. PMID:23844169

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

    PubMed Central

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

    2000-01-01

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

  11. Casein expression in cytotoxic T lymphocytes.

    PubMed Central

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

    1990-01-01

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

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

    PubMed

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

    2011-10-01

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

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

    PubMed

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

    1982-04-01

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

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

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

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

    1982-04-01

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

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

    PubMed

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

    2014-04-01

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

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

    PubMed Central

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

    2016-01-01

    MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8+ cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was—even after major histocompatibility complex class I upregulation—insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8−CD69+ T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Significance Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for

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

    PubMed

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

    2016-12-01

    : MultiStem cells are clinical-grade multipotent adult bone marrow-derived progenitor cells (MAPCs), with extensive replication potential and broader differentiation capacity compared with mesenchymal stem cells. Human MAPCs suppress T-cell proliferation induced by alloantigens and mutually interact with allogeneic natural killer cells. In this study, the interaction between MultiStem and CD8 + cytotoxic T lymphocytes (CTLs) was addressed for the first time. In an in vitro setting, the immunogenicity of MultiStem, the susceptibility of MultiStem toward CTL-mediated lysis, and its effects on CTL function were investigated. MultiStem was nonimmunogenic for alloreactive CTL induction and was-even after major histocompatibility complex class I upregulation-insensitive to alloantigen-specific CTL-mediated lysis. Furthermore, MultiStem reduced CTL proliferation and significantly decreased perforin expression during the T-cell activation phase. As a consequence, MultiStem dose-dependently impaired the induction of CTL function. These effects of MultiStem were mediated predominantly through contact-dependent mechanisms. Moreover, MultiStem cells considerably influenced the expression of T-cell activation markers CD25, CD69, and human leukocyte antigen-DR. The MultiStem-induced CD8 - CD69 + T-cell population displayed a suppressive effect on the induction of CTL function during a subsequent mixed-lymphocyte culture. Finally, the killer activity of activated antigen-specific CTLs during their cytolytic effector phase was also diminished in the presence of MultiStem. This study confirms that these clinical-grade MAPCs are an immune-modulating population that inhibits CTL activation and effector responses and are, consequently, a highly valuable cell population for adoptive immunosuppressive therapy in diseases where damage is induced by CTLs. Because multipotent adult progenitor cells (MAPCs) are among the noteworthy adult mesenchymal stem cell populations for immune therapy

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

    PubMed

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

    2006-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  1. Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice. Role in suppression of cytotoxic T lymphocyte response and viral persistence

    PubMed Central

    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

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

    PubMed Central

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

    2014-01-01

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

  3. CRTAM determines the CD4+ cytotoxic T lymphocyte lineage

    PubMed Central

    Takeuchi, Arata; Badr, Mohamed El Sherif Gadelhaq; Miyauchi, Kosuke; Ishihara, Chitose; Onishi, Reiko; Guo, Zijin; Sasaki, Yoshiteru; Ike, Hiroshi; Takumi, Akiko; Tsuji, Noriko M.; Murakami, Yoshinori; Katakai, Tomoya; Kubo, Masato

    2016-01-01

    Naive T cells differentiate into various effector T cells, including CD4+ helper T cell subsets and CD8+ cytotoxic T cells (CTL). Although cytotoxic CD4+ T cells (CD4+CTL) also develop from naive T cells, the mechanism of development is elusive. We found that a small fraction of CD4+ T cells that express class I–restricted T cell–associated molecule (CRTAM) upon activation possesses the characteristics of both CD4+ and CD8+ T cells. CRTAM+ CD4+ T cells secrete IFN-γ, express CTL-related genes, such as eomesodermin (Eomes), Granzyme B, and perforin, after cultivation, and exhibit cytotoxic function, suggesting that CRTAM+ T cells are the precursor of CD4+CTL. Indeed, ectopic expression of CRTAM in T cells induced the production of IFN-γ, expression of CTL-related genes, and cytotoxic activity. The induction of CD4+CTL and IFN-γ production requires CRTAM-mediated intracellular signaling. CRTAM+ T cells traffic to mucosal tissues and inflammatory sites and developed into CD4+CTL, which are involved in mediating protection against infection as well as inducing inflammatory response, depending on the circumstances, through IFN-γ secretion and cytotoxic activity. These results reveal that CRTAM is critical to instruct the differentiation of CD4+CTL through the induction of Eomes and CTL-related gene. PMID:26694968

  4. DR3 regulation of apoptosis of naive T-lymphocytes in children with acute infectious mononucleosis.

    PubMed

    Filatova, Elena Nikolaevna; Anisenkova, Elena Viktorovna; Presnyakova, Nataliya Borisovna; Utkin, Oleg Vladimirovich

    2016-09-01

    Acute infectious mononucleosis (AIM) is a widespread viral disease that mostly affects children. Development of AIM is accompanied by a change in the ratio of immune cells. This is provided by means of different biological processes including the regulation of apoptosis of naive T-cells. One of the potential regulators of apoptosis of T-lymphocytes is a death receptor 3 (DR3). We have studied the role of DR3 in the regulation of apoptosis of naive CD4 + (nTh) and CD8 + (nCTL) T-cells in healthy children and children with AIM. In healthy children as well as in children with AIM, the activation of DR3 is accompanied by inhibition of apoptosis of nTh. In healthy children, the stimulation of DR3 resulted in the increase in apoptosis of nCTL. On the contrary, in children with AIM, the level of apoptosis of nCTL decreased after DR3 activation, which is a positive contribution to the antiviral immune response. In children with AIM, nCTL are characterized by reduced level of apoptosis as compared with healthy children. These results indicate that DR3 can be involved in the reduction of sensitivity of nCTL to apoptosis in children with AIM.

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2012-10-01

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

  7. Tumor-targeting CTL expressing a single-chain Fv specific for VEGFR2.

    PubMed

    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.

  8. Mechanism of recovery from acute virus infection: treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that Lyt-2+ T lymphocytes mediate clearance of virus and regulate the antiviral antibody response.

    PubMed Central

    Moskophidis, D; Cobbold, S P; Waldmann, H; Lehmann-Grube, F

    1987-01-01

    After intravenous infection of mice, lymphocytic choriomeningitis virus multiplied in spleens and livers, attaining highest concentrations on days 4 to 6. The subsequent clearance was as rapid, and 8 to 10 days after inoculation, infectivity was usually below detectability. During the effector phase of virus elimination, both cytotoxic T-cell (CTL) activity and the number of cells producing antiviral antibodies were high. Monoclonal antibodies directed against T lymphocytes and T-lymphocyte subsets were inoculated once intravenously 5, 6, or 7 days after infection of the animals, and the effects on antiviral immune responses, as well as on elimination of virus from the organs, were determined. Treatment with anti-Thy-1 and anti-Lyt-2 antibodies blocked elimination of the virus and profoundly diminished the activity of spleen CTLs but reduced the antibody response partially (anti-Thy-1) or increased it (anti-Lyt-2). In contrast, treatment with the anti-L3T4 antibody had essentially no effect on either virus elimination or CTL response but abolished antibody production. We conclude that Lyt-2+ (cytotoxic-suppressive) T lymphocytes are needed for elimination of the virus and also regulate the humoral response but that antiviral antibodies are not essential for control of the infection. PMID:3494855

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed Central

    1986-01-01

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

  12. Defective activity of Epstein-Barr virus (EBV) specific cytotoxic T lymphocytes in children with chronic active EBV infection and in their parents.

    PubMed

    Fujieda, M; Wakiguchi, H; Hisakawa, H; Kubota, H; Kurashige, T

    1993-10-01

    Antibodies of Epstein-Barr virus (EBV), EBV-specific cytotoxic T lymphocyte (EBVCTL) activity and the lymphocyte subset of CTL were examined in 13 Japanese children with chronic active EBV infection (CAEBV) and their parents (eight fathers and 10 mothers). Anti-virus-capsid antigen (VCA)-IgG antibody titers ranged from 1:640 to 1:5120 in the patients with CAEBV and from 1:40 to 1:640 in the parents. While anti-VCA-IgM antibody was detected in three patients, anti-VCA-IgA antibody in five and anti-early-antigen (EA)-IgG antibody in 11, no antibody was detected in the parents except anti-EA antibody, which was positive in the mothers of cases 5 and 13 (1:10 and 1:40). Anti-EBV-associated nuclear antigen (EBNA) antibody was < or = 1:10 in six out of 13 patients with CAEBV and in 10 out of 18 parents tested. Epstein-Barr virus activity was significantly lower (P < 0.005) both in the children with CAEBV and in their parents than in seropositive age-matched controls. Proportions of a CTL subset (CD8+ CD11- lymphocytes) in the patients with CAEBV were significantly higher (P < 0.005) than in controls, while those in the parents were at the same level as in controls. Defective EBVCTL activity and anti-EBNA-antibody responses were frequently observed both in children with CAEBV and in their parents, which may suggest that the abnormal immune response to EBV may be based on a familial disorder, though no familial involvement has been reported in Japanese children with CAEBV.

  13. Effect of immunodepletion of MHC class II-positive cells from pancreatic islets on generation of cytotoxic T-lymphocytes in mixed islet-lymphocyte coculture.

    PubMed

    Stock, P G; Ascher, N L; Platt, J L; Kaufman, D B; Chen, S; Field, M J; Sutherland, D E

    1989-01-01

    In vitro manipulation of pancreatic islets to decrease islet immunogenicity before transplantation has largely been directed at eliminating the major histocompatibility complex (MHC) class II-positive passenger leukocytes from the islets. The mixed islet-lymphocyte coculture (MILC) system was used to quantitate the efficacy of immunodepletion of MHC class II-positive cells from pancreatic islets in terms of reducing immunogenicity. With these experiments we compared the in vitro immunogenicity of MHC class II-depleted islets with untreated islets. B10.BR (H-2k) islets were treated with anti-Iak alloserum followed by complement. This treatment successfully eliminated MHC class II-positive cells from the islets, as demonstrated by indirect immunofluorescence techniques. Depleted islets generated slightly lower amounts of allospecific cytotoxic T-lymphocyte (CTL) activity when exposed to C57BL/6 (H-2b) splenocytes in the MILC than untreated control islets. Although the amount of CTL generated by the depleted islets was slightly less than that generated by untreated islets, there was significant stimulation of CTL by the MHC class II-depleted islets. Therefore, the presence or absence of MHC class II cells within the islet is unlikely to be the decisive factor contributing to islet immunogenicity.

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

    PubMed

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

    1981-05-01

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

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

    PubMed Central

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

    2001-01-01

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

  16. Role of vitamin D in cytotoxic T lymphocyte immunity to pathogens and cancer.

    PubMed

    Sarkar, Surojit; Hewison, Martin; Studzinski, George P; Li, Yan Chun; Kalia, Vandana

    2016-01-01

    The discovery of vitamin D receptor (VDR) expression in immune cells has opened up a new area of research into immunoregulation by vitamin D, a niche that is distinct from its classical role in skeletal health. Today, about three decades since this discovery, numerous cellular and molecular targets of vitamin D in the immune system have been delineated. Moreover, strong clinical associations between vitamin D status and the incidence/severity of many immune-regulated disorders (e.g. infectious diseases, cancers and autoimmunity) have prompted the idea of using vitamin D supplementation to manipulate disease outcome. While much is known about the effects of vitamin D on innate immune responses and helper T (T(H)) cell immunity, there has been relatively limited progress on the frontier of cytotoxic T lymphocyte (CTL) immunity--an arm of host cellular adaptive immunity that is crucial for the control of such intracellular pathogens as human immunodeficiency virus (HIV), tuberculosis (TB), malaria, and hepatitis C virus (HCV). In this review, we discuss the strong historical and clinical link between vitamin D and infectious diseases that involves cytotoxic T lymphocyte (CTL) immunity, present our current understanding as well as critical knowledge gaps in the realm of vitamin D regulation of host CTL responses, and highlight potential regulatory connections between vitamin D and effector and memory CD8 T cell differentiation events during infections.

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

    PubMed

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

    2005-07-20

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

  18. Effect of Aerva lanata on cell-mediated immune responses and cytotoxic T-lymphocyte generation in normal and tumor-bearing mice.

    PubMed

    Siveen, K S; Kuttan, Girija

    2012-01-01

    Cell-mediated immunity offers protection against virus-infected cells and tumor cells, involves activation of natural killer (NK) cells, production of antigen-specific cytotoxic T-lymphocytes, and release of various cytokines in response to an antigen. Administration of an ethanolic extract of Aerva lanata was found to stimulate cell-mediated immunological responses in normal and tumor-bearing BALB/c mice. A significant enhancement in NK cell activity in both normal and tumor-bearing hosts was observed after administration of A. lanata. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were significantly enhanced as well in both sets of treated hosts. In addition, in vivo production of IL-2 and IFNg were each significantly enhanced by extract treatment. The stimulatory effect of A. lanata on cytotoxic T-lymphocyte (CTL) production was determined by Winn's neutralization assay using CTL-sensitive EL4 thymoma cells. A. lanata treatment caused a significant increase in CTL production in both in vivo and in vitro models, in each case as indicated by a significant increase in the life-spans of tumor-injected mice. Taken together, all of these results in the murine model indicate that administration of an ethanolic extract of A. lanata could enhance the cell-mediated anti-tumor response.

  19. Wnt signaling inhibits CTL memory programming

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2000-07-01

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

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  4. Quantification of simian immunodeficiency virus cytotoxic T lymphocyte escape mutant viruses.

    PubMed

    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.

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

    PubMed

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

    2001-11-01

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

  6. Regulated expression of telomerase activity in human T lymphocyte development and activation

    PubMed Central

    1996-01-01

    Telomerase, a ribonucleoprotein that is capable of synthesizing telomeric repeats, is expressed in germline and malignant cells, and is absent in most normal human somatic cells. The selective expression of telomerase has thus been proposed to be a basis for the immortality of the germline and of malignant cells. In the present study, telomerase activity was analyzed in normal human T lymphocytes. It was found that telomerase is expressed at a high level in thymocyte subpopulations, at an intermediate level in tonsil T lymphocytes, and at a low to undetectable level in peripheral blood T lymphocytes. Moreover, telomerase activity is highly inducible in peripheral T lymphocytes by activation through CD3 with or without CD28 costimulation, or by stimulation with phorbol myristate acetate (PMA)/ionomycin. The induction of telomerase by anti-CD3 plus anti-CD28 (anti-CD3/CD28) stimulation required RNA and protein synthesis, and was blocked by herbimycin A, an inhibitor of S pi protein tyrosine kinases. The immunosuppressive drug cyclosporin A selectively inhibited telomerase induction by PMA/ionomycin and by anti-CD3, but not by anti-CD3/CD28. Although telomerase activity in peripheral T lymphocytes was activation dependent and correlated with cell proliferation, it was not cell cycle phase restricted. These results indicate that the expression of telomerase in normal human T lymphocytes is both developmentally regulated and activation induced. Telomerase may thus play a permissive role in T cell development and in determining the capacity of lymphoid cells for cell division and clonal expansion. PMID:8676067

  7. Biophysical Aspects of T Lymphocyte Activation at the Immune Synapse

    PubMed Central

    Hivroz, Claire; Saitakis, Michael

    2016-01-01

    T lymphocyte activation is a pivotal step of the adaptive immune response. It requires the recognition by T-cell receptors (TCR) of peptides presented in the context of major histocompatibility complex molecules (pMHC) present at the surface of antigen-presenting cells (APCs). T lymphocyte activation also involves engagement of costimulatory receptors and adhesion molecules recognizing ligands on the APC. Integration of these different signals requires the formation of a specialized dynamic structure: the immune synapse. While the biochemical and molecular aspects of this cell–cell communication have been extensively studied, its mechanical features have only recently been addressed. Yet, the immune synapse is also the place of exchange of mechanical signals. Receptors engaged on the T lymphocyte surface are submitted to many tensile and traction forces. These forces are generated by various phenomena: membrane undulation/protrusion/retraction, cell mobility or spreading, and dynamic remodeling of the actomyosin cytoskeleton inside the T lymphocyte. Moreover, the TCR can both induce force development, following triggering, and sense and convert forces into biochemical signals, as a bona fide mechanotransducer. Other costimulatory molecules, such as LFA-1, engaged during immune synapse formation, also display these features. Moreover, T lymphocytes themselves are mechanosensitive, since substrate stiffness can modulate their response. In this review, we will summarize recent studies from a biophysical perspective to explain how mechanical cues can affect T lymphocyte activation. We will particularly discuss how forces are generated during immune synapse formation; how these forces affect various aspects of T lymphocyte biology; and what are the key features of T lymphocyte response to stiffness. PMID:26913033

  8. Effect of protease inhibitors on angiotensin-converting enzyme activity in human T-lymphocytes.

    PubMed

    Petrov, V; Fagard, R; Lijnen, P

    2000-05-01

    The purpose of these investigations was to determine whether the aminopeptidase B and leucine aminopeptidase inhibitor bestatin, the chymase inhibitor chymostatin, the calpain inhibitor E-64, and the neutral serine protease inhibitor leupeptin affect the angiotensin converting enzyme (ACE) activity in T-lymphocytes. ACE activity in homogenates of T-lymphocytes or in intact T-lymphocytes in suspension was measured by determining fluorimetrically histidyl-leucine, formed from the conversion of hippuryl-histidyl-leucine, coupled with ophtaldialdehyde. The effect of various concentrations (10(-9) to 10(-3) mol/L) of the angiotensin-converting enzyme inhibitors lisinopril and captopril and of the various protease inhibitors on ACE activity was studied. Lisinopril and captopril reduced the ACE activity in homogenates of T-lymphocytes in a concentration-dependent manner. Lisinopril exhibited a more pronounced inhibition of ACE in T-lymphocytes than did captopril. Chymostatin and E-64 had no effect on the ACE activity in T-lymphocytes, whereas leupeptin inhibited its activity in a dose-dependent fashion. Bestatin, on the contrary, increased the ACE activity in homogenates of T-lymphocytes as well as in intact T-lymphocytes in proportion to the concentration. Our data showed that the ACE activity in T-lymphocytes was stimulated by bestatin and inhibited by leupeptin, whereas chymostatin and E-64 did not affect the ACE activity in T-lymphocytes.

  9. Effect of Vibrio cholerae neuraminidase on the mitogen response of T lymphocytes. I. Enhancement of macrophage T-lymphocyte cooperation in concanavalin-A-induced lymphocyte activation.

    PubMed

    Knop, J

    1980-12-01

    Vibrio cholerae neuraminidase (VCN) enhances the immune response of lymphocytes in various systems, such as antigen- and mitogen-induced blastogenesis, mixed lymphocyte culture (MLC) and tumor-cell response. We used macrophage-depleted and reconstituted murine lymph-node T-cells to investigate the effect of VCN on macrophage-T-lymphocyte co-operation in Con-A-induced lymphocyte activation. In unfractionated lymph-node cells VCN enhanced the Con-A-induced lymphocyte activation as measured by 3H-thymidine (3H-dThd) incorporation. Removing macrophages from the cells resulted in a significantly diminished response. In addition the enhancing effect of VCN was greatly reduced. Reconstitution of the lymphocyte cultures with macrophages in increasing numbers and from various sources rstored the lymphocyte response and the enhancing effect of VCN. VCN proved to be most efficient in cultures reconstituted with normal peritoneal macrophages. Some effect was also observed using bone-marrow-derived (BM) macrophages. However, higher numbers of normal PE macrophages in the presence of VCN inhibited lymphocyte activation, and inhibition by thioglycollate-broth-induced macrophages was considerably increased by VCN. These results suggest that VCN acts by increasing the efficiency of macrophage-T lymphocyte interaction.

  10. CD95 (Fas) ligand expression of Epstein-Barr virus (EBV)-infected lymphocytes: a possible mechanism of immune evasion in chronic active EBV infection.

    PubMed

    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.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2005-01-01

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

  13. Fas Ligand-Mediated Lysis of Self Bystander Targets by Human Papillomavirus-Specific CD8+ Cytotoxic T Lymphocytes

    PubMed Central

    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

  14. Pathology during acute infections: contributions of intracellular pathogens and the CTL response.

    PubMed

    Ganusov, Vitaly V; Antia, Rustom

    2005-06-22

    Previous work has shown how, in the case of cytotoxic T-lymphocyte (CTL) responses to persistent viral infections, pathology may arise as a consequence of cell destruction directly by the virus or indirectly due to the CTL response, leading to maximum pathology at intermediate efficacy of the immune response. We expand these studies to consider pathology arising during acute infections with intracellular pathogens controlled by the CTL response. We show that, in contrast to persistent infections, pathology during acute infections is minimized with increasing efficacy of the immune response. The implications of these results for vaccination are discussed.

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

    PubMed

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

    1999-03-05

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

  16. In vitro reactivity of allospecific cytotoxic T lymphocytes does not explain the taboo phenomenon.

    PubMed

    Stobbe, I; van der Meer-Prins, E; Smits, J M; Doxiadis, I I; Claas, F H

    1999-12-01

    Matching for human leucocyte antigens (HLA) is important for graft survival in kidney transplantation. Nevertheless, most patients receive a kidney graft with multiple HLA mismatches. Some of these mismatches seem to be more harmful than others. By studying the effect of single HLA mismatches in the context of the patients' own HLA, we have previously identified donor/recipient combinations with a significantly higher incidence of early graft failure, the so-called taboo combinations. In the present study we investigated whether a higher cytotoxic T lymphocyte (CTL) response towards taboo mismatches may be involved in this phenomenon. CTL reactivity was determined both in taboo and control combinations by in vitro CTL precursor assays, using peripheral blood mononuclear cells and proximal tubular epithelial cells as target cells. Inhibition studies with CD8-antibody as well as Cyclosporin A were performed to identify high avidity and primed CTLs. Furthermore, in committed CTLp assays indirect recognition of the taboo mismatch was tested using synthetic peptides. The CTL precursor frequencies in taboo combinations were always lower than the CTL precursor frequencies in control combinations. No difference in avidity and activation status of the CTLs could be detected when taboo combinations were compared with the controls. In the committed CTLp assays no reactivity towards any of the synthetic peptides was observed. The significantly poorer graft survival of taboo combinations cannot be explained by a higher number of donor-specific CTLs. Furthermore, the avidity or activation status of these CTLs does not provide a clue to the taboo phenomenon.

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

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

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

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

  18. Protective antitumor activity through dendritic cell immunization is mediated by NK cell as well as CTL activation.

    PubMed

    Kim, K D; Kim, J K; Kim, S J; Choe, I S; Chung, T H; Choe, Y K; Lim, J S

    1999-08-01

    Dendritic cells (DCs) are potent professional antigen-presenting cells (APC) capable of inducing the primary T cell response to antigen. Although tumor cells express target antigens, they are incapable of stimulating a tumor-specific immune response due to a defect in the costimulatory signal that is required for optimal activation of T cells. In this work, we describe a new approach using tumor-DC coculture to improve the antigen presenting capacity of tumor cells, which does not require a source of tumor-associated antigen. Immunization of a weakly immunogenic and progressive tumor cocultured with bone marrow-derived DCs generated an effective tumor vaccine. Immunization with the cocultured DCs was able to induce complete protective immunity against tumor challenges and was effective for the induction of tumor-specific CTL (cytotoxic T lymphocyte) activity. Furthermore, high NK cell activity was observed in mice in which tumors were rejected. In addition, immunization with tumor-pulsed DCs induced delayed tumor growth, but not tumor eradication in tumor-bearing mice. Our results demonstrate that coculture of DCs with tumors generated antitumor immunity due to the NK cell activation as well as tumor-specific T cell. This approach would be useful for designing tumor vaccines using DCs when the information about tumor antigens is limited.

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

    PubMed Central

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

    2000-01-01

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

  20. [Activation of peripheral T lymphocytes in children with epilepsy and production of cytokines].

    PubMed

    Yang, Jie; Hu, Chongkang; Jiang, Xun

    2016-09-01

    Objective To study the state of peripheral T lymphocytes and cytokine levels in children with epilepsy. Methods Twenty children with epilepsy and 20 healthy age-matched children were recruited and their peripheral blood was collected. The activation of T lymphocytes was evaluated by detecting the expressions of CD25, CD69 and cytotoxic T lymphocyte-assicated antigen 4 (CTLA4). The function of T lymphocytes was evaluated by detecting the expressions of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), IL-17A and IL-6. The activation of regulatory T cells (Tregs) was evaluated by detecting the expression of IL-10. Results Children with epilepsy had higher expressions of CD25, CD69 and CTLA-4 in T lymphocytes than the controls did. The expressions of IFN-γ, TNF-α, IL-17A and IL-6 in T lymphocytes of children with epilepsy were higher than those of the controls. Frequency of Tregs producing IL-10 was higher in children with epilepsy as compared with the controls. Conclusion Peripheral T lymphocytes of children with epilepsy are activated and produce cytokines.

  1. Activin receptor-like kinase5 inhibition suppresses mouse melanoma by ubiquitin degradation of Smad4, thereby derepressing eomesodermin in cytotoxic T lymphocytes

    PubMed Central

    Yoon, Jeong-Hwan; Jung, Su Myung; Park, Seok Hee; Kato, Mitsuyasu; Yamashita, Tadashi; Lee, In-Kyu; Sudo, Katsuko; Nakae, Susumu; Han, Jin Soo; Kim, Ok-Hee; Oh, Byung-Chul; Sumida, Takayuki; Kuroda, Masahiko; Ju, Ji-Hyeon; Jung, Kyeong Cheon; Park, Seong Hoe; Kim, Dae-Kee; Mamura, Mizuko

    2013-01-01

    Varieties of transforming growth factor-β (TGF-β) antagonists have been developed to intervene with excessive TGF-β signalling activity in cancer. Activin receptor-like kinase5 (ALK5) inhibitors antagonize TGF-β signalling by blocking TGF-β receptor-activated Smad (R-Smad) phosphorylation. Here we report the novel mechanisms how ALK5 inhibitors exert a therapeutic effect on a mouse B16 melanoma model. Oral treatment with a novel ALK5 inhibitor, EW-7197 (2.5 mg/kg daily) or a representative ALK5 inhibitor, LY-2157299 (75 mg/kg bid) suppressed the progression of melanoma with enhanced cytotoxic T-lymphocyte (CTL) responses. Notably, ALK5 inhibitors not only blocked R-Smad phosphorylation, but also induced ubiquitin-mediated degradation of the common Smad, Smad4 mainly in CD8+ T cells in melanoma-bearing mice. Accordingly, T-cell-specific deletion of Smad4 was sufficient to suppress the progression of melanoma. We further identified eomesodermin (Eomes), the T-box transcription factor regulating CTL functions, as a specific target repressed by TGF-β via Smad4 and Smad3 in CD8+ T cells. Thus, ALK5 inhibition enhances anti-melanoma CTL responses through ubiquitin-mediated degradation of Smad4 in addition to the direct inhibitory effect on R-Smad phosphorylation. PMID:24127404

  2. Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus: clearance of virus and in vitro properties.

    PubMed

    Anderson, J; Byrne, J A; Schreiber, R; Patterson, S; Oldstone, M B

    1985-02-01

    We have generated lymphocytic choriomeningitis virus-specific, H-2-restricted cytotoxic thymus-derived lymphocyte (CTL) clones. By using these reagents in several in vitro assays with infected target cells, we show that CTLs by themselves prevent the release of infectious virus into culture fluids and significantly lower the titers of infectious virus previously produced. This ability of cloned CTLs is not influenced by monensin. However, monensin does abrogate the ability of CTLs from spleens of mice primed 6 to 8 days previously with virus to kill virus-infected syngeneic targets. When tested for the participation of lymphokines in this system, the CTLs proliferate when reacted with syngeneic lymphocytic choriomeningitis virus-infected macrophages but fail to make interleukin-2. These CTLs make gamma interferon when reacted with syngeneic virus-infected targets. However, the production of interferon does not directly correlate with CTL-mediated killing. The number of H-2K and D molecules expressed on the target cell surface is not altered during the course of lymphocytic choriomeningitis virus infection. Electron microscopy shows finger-like projections of the CTL clone thrust into the infected cell and lesions bearing an internal diameter of approximately 15 nm in those membranes, illustrating the lytic process.

  3. The WT hemochromatosis protein HFE inhibits CD8⁺ T-lymphocyte activation.

    PubMed

    Reuben, Alexandre; Phénix, Mikaël; Santos, Manuela M; Lapointe, Réjean

    2014-06-01

    MHC class I (MHC I) antigen presentation is a ubiquitous process by which cells present endogenous proteins to CD8(+) T lymphocytes during immune surveillance and response. Hereditary hemochromatosis protein, HFE, is involved in cellular iron uptake but, while structurally homologous to MHC I, is unable to bind peptides. However, increasing evidence suggests a role for HFE in the immune system. Here, we investigated the impact of HFE on CD8(+) T-lymphocyte activation. Using transient HFE transfection assays in a model of APCs, we show that WT HFE (HFEWT ), but not C282Y-mutated HFE, inhibits secretion of MIP-1β from antigen-specific CD8(+) T lymphocytes. HFEWT expression also resulted in major decreases in CD8(+) T-lymphocyte activation as measured by 4-1BB expression. We further demonstrate that inhibition of CD8(+) T-lymphocyte activation was independent of MHC I surface levels, β2-m competition, HFE interaction with transferrin receptor, antigen origin, or epitope affinity. Finally, we identified the α1-2 domains of HFEWT as being responsible for inhibiting CD8(+) T-lymphocyte activation. Our data imply a new role for HFEWT in altering CD8(+) T-lymphocyte reactivity, which could modulate antigen immunogenicity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. The cytotoxic T lymphocyte immune synapse at a glance.

    PubMed

    Dieckmann, Nele M G; Frazer, Gordon L; Asano, Yukako; Stinchcombe, Jane C; Griffiths, Gillian M

    2016-08-01

    The immune synapse provides an important structure for communication with immune cells. Studies on immune synapses formed by cytotoxic T lymphocytes (CTLs) highlight the dynamic changes and specialised mechanisms required to facilitate focal signalling and polarised secretion in immune cells. In this Cell Science at a Glance article and the accompanying poster, we illustrate the different steps that reveal the specialised mechanisms used to focus secretion at the CTL immune synapse and allow CTLs to be such efficient and precise serial killers. © 2016. Published by The Company of Biologists Ltd.

  5. 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.

  6. Memory CD8+ T Cells Protect Dendritic Cells from CTL Killing1

    PubMed Central

    Watchmaker, Payal B.; Urban, Julie A.; Berk, Erik; Nakamura, Yutaro; Mailliard, Robbie B.; Watkins, Simon C.; van Ham, S. Marieke; Kalinski, Pawel

    2010-01-01

    CD8+ T cells have been shown to be capable of either suppressing or promoting immune responses. To reconcile these contrasting regulatory functions, we compared the ability of human effector and memory CD8+ T cells to regulate survival and functions of dendritic cells (DC). We report that, in sharp contrast to the effector cells (CTLs) that kill DCs in a granzyme B- and perforin-dependent mechanism, memory CD8+ T cells enhance the ability of DCs to produce IL-12 and to induce functional Th1 and CTL responses in naive CD4+ and CD8+ T cell populations. Moreover, memory CD8+ T cells that release the DC-activating factor TNF-α before the release of cytotoxic granules induce DC expression of an endogenous granzyme B inhibitor PI-9 and protect DCs from CTL killing with similar efficacy as CD4+ Th cells. The currently identified DC-protective function of memory CD8+ T cells helps to explain the phenomenon of CD8+ T cell memory, reduced dependence of recall responses on CD4+ T cell help, and the importance of delayed administration of booster doses of vaccines for the optimal outcome of immunization. PMID:18322193

  7. Heparanase promotes tumor infiltration and antitumor activity of CAR-redirected T-lymphocytes

    PubMed Central

    Caruana, Ignazio; Savoldo, Barbara; Hoyos, Valentina; Weber, Gerrit; Liu, Hao; Kim, Eugene S.; Ittmann, Michael M.; Marchetti, Dario; Dotti, Gianpietro

    2015-01-01

    Adoptive transfer of chimeric antigen receptor (CAR)-redirected T lymphocytes (CAR-T cells) has had less striking effects in solid tumors1–3 than in lymphoid malignancies4, 5. Although active tumor-mediated immunosuppression may play a role in limiting efficacy6, functional changes in T lymphocytes following their ex vivo manipulation may also account for cultured CAR-T cells’ reduced ability to penetrate stroma-rich solid tumors. We therefore studied the capacity of human in vitro-cultured CAR-T cells to degrade components of the extracellular matrix (ECM). In contrast to freshly isolated T lymphocytes, we found that in vitro-cultured T lymphocytes lack expression of the enzyme heparanase (HPSE) that degrades heparan sulphate proteoglycans, which are main components of ECM. We found that HPSE mRNA is down regulated in in vitro-expanded T cells, which may be a consequence of p53 binding to the HPSE gene promoter. We therefore engineered CAR-T cells to express HPSE and showed improved capacity to degrade ECM, which promoted tumor T-cell infiltration and antitumor activity. Employing this strategy may enhance the activity of CAR-T cells in individuals with stroma-rich solid tumors. PMID:25849134

  8. Protective Role of Cytotoxic T Lymphocytes in Filovirus Hemorrhagic Fever

    PubMed Central

    Warfield, Kelly Lyn; Olinger, Gene Garrard

    2011-01-01

    Infection with many emerging viruses, such as the hemorrhagic fever disease caused by the filoviruses, Marburg (MARV), and Ebola virus (EBOV), leaves the host with a short timeframe in which to mouse a protective immune response. In lethal cases, uncontrolled viral replication and virus-induced immune dysregulation are too severe to overcome, and mortality is generally associated with a lack of notable immune responses. Vaccination studies in animals have demonstrated an association of IgG and neutralizing antibody responses against the protective glycoprotein antigen with survival from lethal challenge. More recently, studies in animal models of filovirus hemorrhagic fever have established that induction of a strong filovirus-specific cytotoxic T lymphocyte (CTL) response can facilitate complete viral clearance. In this review, we describe assays used to discover CTL responses after vaccination or live filovirus infection in both animal models and human clinical trials. Unfortunately, little data regarding CTL responses have been collected from infected human survivors, primarily due to the low frequency of disease and the inability to perform these studies in the field. Advancements in assays and technologies may allow these studies to occur during future outbreaks. PMID:22253531

  9. Elicitation of anti-Sendai virus cytotoxic T lymphocytes by viral and H-2 antigens incorporated into the same lipid bilayer by membrane fusion and by reconstitution into liposomes.

    PubMed

    Hale, A H; Lyles, D S; Fan, D P

    1980-02-01

    We have investigated the minimal molecular requirements for elicitation of anti-Sendai virus cytotoxic T lymphocytes (CTL), and the minimal molecular requirements for the recognition and lysis processes associated with anti-Sendai virus CTL-target cell interactions. This report demonstrates a) that the hemagglutinin-neuraminidase and/or fusion glycoproteins of Sendai virus can elicit anti-Sendai virus CTL and b) that these glycoproteins and H-2 antigens must be within the same membrane lipid bilayer for effective elicitation of anti-Sendai-virus CTL and for effective recognition and lysis of target cells by anti-Sendai virus CTL.

  10. Mitomycin C-treated or irradiated concanavalin A-activated T cells augment the activation of cytotoxic T cells in vivo.

    PubMed

    Moyers, C; Pottmeyer-Gerber, C; Gerber, M; Buszello, H; Dröge, W

    1984-10-01

    The activation of cytotoxic T lymphocytes (CTL) in vivo after immunization of normal or cyclophosphamide-treated mice with allogeneic cells was strongly augmented by the administration of mitomycin C-treated or irradiated concanavalin A-activated spleen cells (Con A-spl). This effect of the Con A-spl was abrogated by treatment with Anti-Thy 1 antibody plus complement, and was therefore presumably mediated by activated "helper" T cells. (The term "helper" cell is only operationally defined in this context and indicates that the augmenting irradiation resistant T cells are obviously not CTL precursor cells). These observations indicated (i) that even the cytotoxic response against allogeneic stimulator cells suffers in vivo from insufficient "helper" T cell activity, and (ii) that the injection of Con A-spl may serve as a simple procedure to apply this "helper" activity in vivo. This procedure was at least as effective as the repeated injection of interleukin 2 (IL-2)-containing cell supernatants with up to four 30-unit doses of IL-2 per mouse. IL-2-containing cell supernatants were found to mediate similar effects only if injected into the footpads but not intravenously. This was in line with the reported observation that IL-2 has an extremely short half-life in vivo. The injection of Con A-spl was also found to augment the proliferative response in the regional lymph nodes.

  11. 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

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

    PubMed

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

    2012-09-27

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

  13. Transfer of in vitro expanded T lymphocytes after activation with dendritomas prolonged survival of mice challenged with EL4 tumor cells.

    PubMed

    Li, Jinhua; Theofanous, Leigh; Stickel, Sara; Bouton-Verville, Hilary; Burgin, Kelly E; Jakubchak, Susan; Wagner, Thomas E; Wei, Yanzhang

    2007-07-01

    Adoptive T cell transfer after in vitro expansion represents an attractive cancer immunotherapy. The majority of studies so far have been focusing on the expansion of tumor infiltrated lymphocytes (TIL) and some have shown very encouraging results. Recently, we have developed a unique tumor immune response activator, dendritomas, by fusion of dendritic cells and tumor cells. Animal studies and early clinical trials have shown that dendritomas are able to activate tumor specific immune responses. In this study, we hypothesized that naïve T cells can be primed with dendritomas and expanded in vitro to develop an adoptive transfer therapy for patients who do not have solid tumors, such as leukemia. T cells were isolated and purified from lymph nodes of mice. The cells were then incubated with dendritomas made from syngeneic DCs and tumor cells and expanded in vitro using Dynabeads mouse CD3/CD28 T cell expander for approximately three weeks. The in vitro primed and expanded T cells showed tumor cell specific CTL activity and increased secretion of IFN-gamma. Tumor bearing mice receiving the in vitro expanded T cells survived significantly longer than control mice. Furthermore, the depletion of regulator T cells enhanced the survival of the mice that received the adoptive transfer therapy.

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

    PubMed

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

    1999-08-06

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

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

    PubMed Central

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

    1998-01-01

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

  16. Stimulating CTL Towards HER2/neu Overexpressing Breast Cancer

    DTIC Science & Technology

    1998-10-01

    296. 9. Jardetzky, T.S., W.S. Lane, R.A. Robinson, D.R. Madden, and D.C. Wiley, Identification of self peptides bound to purified HLA - B27 . Nature...neu (HN654-662) has been shown to bind to HLA -A2.1 and stimulate cytotoxic T lymphocytes (CTL) that lyse primary tumors from ovarian or breast cancer...demonstrating HN654-662 is an extremely poor HLA -A2.1 binding peptide. In a novel approach, we will make use of biophysical techniques that have recently

  17. Functional Compensation of a Detrimental Amino Acid Substitution in a Cytotoxic-T-Lymphocyte Epitope of Influenza A Viruses by Comutations

    PubMed Central

    Rimmelzwaan, G. F.; Berkhoff, E. G. M.; Nieuwkoop, N. J.; Fouchier, R. A. M.; Osterhaus, A. D. M. E.

    2004-01-01

    Influenza A viruses accumulate amino acid substitutions in cytotoxic-T-lymphocyte (CTL) epitopes, allowing these viruses to escape from CTL immunity. The arginine-to-glycine substitution at position 384 of the viral nucleoprotein is associated with escape from CTLs. Introduction of the R384G substitution in the nucleoprotein gene segment of influenza virus A/Hong Kong/2/68 by site-directed mutagenesis was detrimental to viral fitness. Introduction of one of the comutations associated with R384G, E375G, partially restored viral fitness and nucleoprotein functionality. We hypothesized that influenza A viruses need to overcome functional constraints to accumulate mutations in CTL epitopes and escape from CTLs. PMID:15280506

  18. Helicobacter pylori induces activation of human peripheral γδ+ T lymphocytes.

    PubMed

    Romi, Benedetta; Soldaini, Elisabetta; Pancotto, Laura; Castellino, Flora; Del Giudice, Giuseppe; Schiavetti, Francesca

    2011-04-29

    Helicobacter pylori is a gram-negative bacterium that causes gastric and duodenal diseases in humans. Despite a robust antibody and cellular immune response, H. pylori infection persists chronically. To understand if and how H. pylori could modulate T cell activation, in the present study we investigated in vitro the interaction between H. pylori and human T lymphocytes freshly isolated from peripheral blood of H. pylori-negative donors. A direct interaction of live, but not killed bacteria with purified CD3+ T lymphocytes was observed by microscopy and confirmed by flow cytometry. Live H. pylori activated CD3+ T lymphocytes and predominantly γδ+ T cells bearing the TCR chain Vδ2. Upon interaction with H. pylori, these cells up-regulated the activation molecule CD69 and produced cytokines (such as TNFα, IFNγ) and chemokines (such as MIP-1β, RANTES) in a non-antigen-specific manner. This activation required viable H. pylori and was not exhibited by other gram-negative bacteria. The cytotoxin-associated antigen-A (CagA), was at least partially responsible of this activation. Our results suggest that H. pylori can directly interact with T cells and modulate the response of γδ+ T cells, thereby favouring an inflammatory environment which can contribute to the chronic persistence of the bacteria and eventually to the gastric pathology.

  19. Low Dose Decitabine Treatment Induces CD80 Expression in Cancer Cells and Stimulates Tumor Specific Cytotoxic T Lymphocyte Responses

    PubMed Central

    Zhou, Ji-Hao; Yao, Yu-Shi; Li, Yong-Hui; Xu, Yi-Han; Li, Jing-Xin; Gao, Xiao-Ning; Zhou, Min-Hang; Jiang, Meng-Meng; Gao, Li; Ding, Yi; Lu, Xue-Chun; Shi, Jin-Long; Luo, Xu-Feng; Wang, Jia; Wang, Li-Li; Qu, Chunfeng; Bai, Xue-Feng; Yu, Li

    2013-01-01

    Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8+, but not CD4+ T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy. PMID:23671644

  20. Low dose decitabine treatment induces CD80 expression in cancer cells and stimulates tumor specific cytotoxic T lymphocyte responses.

    PubMed

    Wang, Li-Xin; Mei, Zhen-Yang; Zhou, Ji-Hao; Yao, Yu-Shi; Li, Yong-Hui; Xu, Yi-Han; Li, Jing-Xin; Gao, Xiao-Ning; Zhou, Min-Hang; Jiang, Meng-Meng; Gao, Li; Ding, Yi; Lu, Xue-Chun; Shi, Jin-Long; Luo, Xu-Feng; Wang, Jia; Wang, Li-Li; Qu, Chunfeng; Bai, Xue-Feng; Yu, Li

    2013-01-01

    Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8(+), but not CD4(+) T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy.

  1. Piperine from black pepper inhibits activation-induced proliferation and effector function of T lymphocytes.

    PubMed

    Doucette, Carolyn D; Rodgers, Gemma; Liwski, Robert S; Hoskin, David W

    2015-11-01

    Piperine is a major alkaloid component of black pepper (Piper nigrum Linn), which is a widely consumed spice. Here, we investigated the effect of piperine on mouse T lymphocyte activation. Piperine inhibited polyclonal and antigen-specific T lymphocyte proliferation without affecting cell viability. Piperine also suppressed T lymphocyte entry into the S and G2 /M phases of the cell cycle, and decreased expression of G1 -associated cyclin D3, CDK4, and CDK6. In addition, piperine inhibited CD25 expression, synthesis of interferon-γ, interleukin (IL)-2, IL-4, and IL-17A, and the generation of cytotoxic effector cells. The inhibitory effect of piperine on T lymphocytes was associated with hypophosphorylation of Akt, extracellular signal-regulated kinase, and inhibitor of κBα, but not ZAP-70. The ability of piperine to inhibit several key signaling pathways involved in T lymphocyte activation and the acquisition of effector function suggests that piperine might be useful in the management of T lymphocyte-mediated autoimmune and chronic inflammatory disorders. © 2015 Wiley Periodicals, Inc.

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

    PubMed

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

    2000-02-15

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

  3. Major histocompatibility complex-dependent cytotoxic T lymphocyte repertoire and functional avidity contribute to strain-specific disease susceptibility after murine respiratory syncytial virus infection.

    PubMed

    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.

  4. Human T lymphocytes express N-methyl-D-aspartate receptors functionally active in controlling T cell activation

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

    Miglio, Gianluca; Varsaldi, Federica; Lombardi, Grazia

    2005-12-30

    The aim of this study was to investigate the expression and the functional role of N-methyl-D-aspartate (NMDA) receptors in human T cells. RT-PCR analysis showed that human resting peripheral blood lymphocytes (PBL) and Jurkat T cells express genes encoding for both NR1 and NR2B subunits: phytohemagglutinin (PHA)-activated PBL also expresses both these genes and the NR2A and NR2D genes. Cytofluorimetric analysis showed that NR1 expression increases as a consequence of PHA (10 {mu}g/ml) treatment. D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5), and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [(+)-MK 801], competitive and non-competitive NMDA receptor antagonists, respectively, inhibited PHA-induced T cell proliferation, whereas they did not affect IL-2 (10more » U/ml)-induced proliferation of PHA blasts. These effects were due to the prevention of T cell activation (inhibition of cell aggregate formation and CD25 expression), but not to cell cycle arrest or death. These results demonstrate that human T lymphocytes express NMDA receptors, which are functionally active in controlling cell activation.« less

  5. A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes.

    PubMed

    Schaft, Niels; Dörrie, Jan; Müller, Ina; Beck, Verena; Baumann, Stefanie; Schunder, Tanja; Kämpgen, Eckhart; Schuler, Gerold

    2006-09-01

    Effective T cell receptor (TCR) transfer until now required stable retroviral transduction. However, retroviral transduction poses the threat of irreversible genetic manipulation of autologous cells. We, therefore, used optimized RNA transfection for transient manipulation. The transfection efficiency, using EGFP RNA, was >90%. The electroporation of primary T cells, isolated from blood, with TCR-coding RNA resulted in functional cytotoxic T lymphocytes (CTLs) (>60% killing at an effector to target ratio of 20:1) with the same HLA-A2/gp100-specificity as the parental CTL clone. The TCR-transfected T cells specifically recognized peptide-pulsed T2 cells, or dendritic cells electroporated with gp100-coding RNA, in an IFNgamma-secretion assay and retained this ability, even after cryopreservation, over 3 days. Most importantly, we show here for the first time that the electroporated T cells also displayed cytotoxicity, and specifically lysed peptide-loaded T2 cells and HLA-A2+/gp100+ melanoma cells over a period of at least 72 h. Peptide-titration studies showed that the lytic efficiency of the RNA-transfected T cells was similar to that of retrovirally transduced T cells, and approximated that of the parental CTL clone. Functional TCR transfer by RNA electroporation is now possible without the disadvantages of retroviral transduction, and forms a new strategy for the immunotherapy of cancer.

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

    PubMed

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

    2018-06-01

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

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

    PubMed Central

    Matter, A

    1979-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  9. CD8+ T Lymphocyte Expansion, Proliferation and Activation in Dengue Fever

    PubMed Central

    de Matos, Andréia Manso; Carvalho, Karina Inacio; Rosa, Daniela Santoro; Villas-Boas, Lucy Santos; da Silva, Wanessa Cardoso; Rodrigues, Célia Luiza de Lima; Oliveira, Olímpia Massae Nakasone Peel Furtado; Levi, José Eduardo; Araújo, Evaldo Stanislau Affonso; Pannuti, Claudio Sergio; Luna, Expedito José Albuquerque; Kallas, Esper George

    2015-01-01

    Dengue fever induces a robust immune response, including massive T cell activation. The level of T cell activation may, however, be associated with more severe disease. In this study, we explored the level of CD8+ T lymphocyte activation in the first six days after onset of symptoms during a DENV2 outbreak in early 2010 on the coast of São Paulo State, Brazil. Using flow cytometry we detected a progressive increase in the percentage of CD8+ T cells in 74 dengue fever cases. Peripheral blood mononuclear cells from 30 cases were thawed and evaluated using expanded phenotyping. The expansion of the CD8+ T cells was coupled with increased Ki67 expression. Cell activation was observed later in the course of disease, as determined by the expression of the activation markers CD38 and HLA-DR. This increased CD8+ T lymphocyte activation was observed in all memory subsets, but was more pronounced in the effector memory subset, as defined by higher CD38 expression. Our results show that most CD8+ T cell subsets are expanded during DENV2 infection and that the effector memory subset is the predominantly affected sub population. PMID:25675375

  10. Frequent and Variable Cytotoxic-T-Lymphocyte Escape-Associated Fitness Costs in the Human Immunodeficiency Virus Type 1 Subtype B Gag Proteins

    PubMed Central

    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

  11. Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia

    PubMed Central

    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

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

    PubMed

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

    2016-01-01

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

  13. The production of granulocyte-monocyte colony-stimulating activity by isolated human T lymphocyte subpopulations.

    PubMed

    Hesketh, P J; Sullivan, R; Valeri, C R; McCarroll, L A

    1984-05-01

    Isolated human T lymphocyte subpopulations were obtained by fluorescence-activated cell sorting using the murine monoclonal antibodies, OKT4 and OKT8. The capabilities of the isolated lymphocytes to produce granulocyte-monocyte colony-stimulating activity (CSA) in response to mitogen challenge were assessed by in vitro assays employing light density nonadherent bone marrow cells. Essentially, no CSA production was noted by any isolated T lymphocyte population [OKT4 positive (+) or OKT8 positive (+)] cultured alone or following the addition of 10(4) autologous monocytes/ml. When phytohemagglutinin (PHA) alone was added, OKT4+ lymphocytes elaborated small amounts of CSA. With the addition of concanavalin A (Con-A) alone, both OKT4+ and OKT8+ cells were able to produce modest amounts of CSA. Significantly enhanced CSA production was observed when either OKT4+ or OKT8+ lymphocytes were coincubated with autologous monocytes in the presence of mitogen. We conclude that highly purified T lymphocyte subpopulations, free of monocytes as assessed by nonspecific esterase staining, can elaborate small amounts of CSA in response to PHA or Con-A challenge. A synergistic augmentation of CSA production was noted with coincubation of sorted lymphocytes and autologous monocytes in the presence of mitogen. Finally, our results suggest that the ability of T lymphocytes to make CSA is not exclusively limited to either the OKT4+ or OKT8+ defined subsets.

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

    PubMed

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

    1984-01-01

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

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

    PubMed Central

    2010-01-01

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

  16. An Exosome-Based Vaccine Platform Imparts Cytotoxic T Lymphocyte Immunity Against Viral Antigens.

    PubMed

    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.

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

    PubMed

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

    2009-06-12

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

  18. Induction of protective anti-CTL epitope responses against HER-2-positive breast cancer based on multivalent T7 phage nanoparticles.

    PubMed

    Pouyanfard, Somayeh; Bamdad, Taravat; Hashemi, Hamidreza; Bandehpour, Mojgan; Kazemi, Bahram

    2012-01-01

    We report here the development of multivalent T7 bacteriophage nanoparticles displaying an immunodominant H-2k(d)-restricted CTL epitope derived from the rat HER2/neu oncoprotein. The immunotherapeutic potential of the chimeric T7 nanoparticles as anti-cancer vaccine was investigated in BALB/c mice in an implantable breast tumor model. The results showed that T7 phage nanoparticles confer a high immunogenicity to the HER-2-derived minimal CTL epitope, as shown by inducing robust CTL responses. Furthermore, the chimeric nanoparticles protected mice against HER-2-positive tumor challenge in both prophylactic and therapeutic setting. In conclusion, these results suggest that CTL epitope-carrying T7 phage nanoparticles might be a promising approach for development of T cell epitope-based cancer vaccines.

  19. Induction of Protective Anti-CTL Epitope Responses against HER-2-Positive Breast Cancer Based on Multivalent T7 Phage Nanoparticles

    PubMed Central

    Pouyanfard, Somayeh; Bamdad, Taravat; Hashemi, Hamidreza; Bandehpour, Mojgan; Kazemi, Bahram

    2012-01-01

    We report here the development of multivalent T7 bacteriophage nanoparticles displaying an immunodominant H-2kd-restricted CTL epitope derived from the rat HER2/neu oncoprotein. The immunotherapeutic potential of the chimeric T7 nanoparticles as anti-cancer vaccine was investigated in BALB/c mice in an implantable breast tumor model. The results showed that T7 phage nanoparticles confer a high immunogenicity to the HER-2-derived minimal CTL epitope, as shown by inducing robust CTL responses. Furthermore, the chimeric nanoparticles protected mice against HER-2-positive tumor challenge in both prophylactic and therapeutic setting. In conclusion, these results suggest that CTL epitope-carrying T7 phage nanoparticles might be a promising approach for development of T cell epitope-based cancer vaccines. PMID:23166703

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

    PubMed

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

    2009-02-01

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

  1. Interleukin-6 and vascular endothelial growth factor release by renal cell carcinoma cells impedes lymphocyte-dendritic cell cross-talk.

    PubMed

    Cabillic, F; Bouet-Toussaint, F; Toutirais, O; Rioux-Leclercq, N; Fergelot, P; de la Pintière, C Thomas; Genetet, N; Patard, J-J; Catros-Quemener, V

    2006-12-01

    Anti-tumour T cell response requires antigen presentation via efficient immunological synapse between antigen presenting cells, e.g. dendritic cells (DC), and specific T cells in an adapted Th1 cytokine context. Nine renal cell carcinoma (RCC) primary culture cells were used as sources of tumour antigens which were loaded on DC (DC-Tu) for autologous T cell activation assays. Cytotoxic activity of lymphocytes stimulated with DC-Tu was evaluated against autologous tumour cells. Assays were performed with 75 grays irradiated tumour cells (Tu irr) and with hydrogen peroxide +/- heat shock (Tu H(2)O(2) +/- HS) treated cells. DC-Tu irr failed to enhance cytotoxic activity of autologous lymphocytes in seven of 13 assays. In all these defective assays, irradiated tumour cells displayed high interleukin (IL)-6 and vascular endothelial growth factor (VEGF) release. Conversely, when tumour cells released low IL-6 levels (n = 4), DC-Tu irr efficiently enhanced CTL activity. When assays were performed with the same RCC cells treated with H(2)O(2) + HS, DC-Tu stimulation resulted in improved CTL activity. H(2)O(2) + HS treatment induced post-apoptotic cell necrosis of tumour cells, totally abrogated their cytokine release [IL-6, VEGF, transforming growth factor (TGF)-beta1] and induced HSP70 expression. Taken together, data show that reduction in IL-6 and VEGF release in the environment of the tumour concomitantly to tumour cell HSP expression favours induction of a stronger anti-tumour CTL response.

  2. [Acquirement of autologous murine cytotoxic T lymphocytes via cryopreservation of lymphocytes].

    PubMed

    Wang, Lei; Peng, Na; Hu, Xiaoyan; Liang, Wentao; Liang, Kai; Peng, Guizhu

    2016-11-01

    Objective To evaluate the effects of cryopreservation on the proliferation and killing activity of lymphocytes, and explore a novel protocol of preparing autologous mouse cytotoxic T lymphocytes (CTLs). Methods Mononuclear cells derived from spleen (5.0×10 6 /mL) were cryopreserved in CELLBANKER2 for 6 days and recovered in water bath at 39DegreesCelsius. The fresh lymphocytes and post-cryopreservation lymphocytes were induced by CD3 mAb (100 ng/mL) and recombinant mouse interleukin 2 (rmIL-2, 100 ng/mL) to obtain cytokine-induced killer cells (CIKs). Dendritic cells (DCs) were co-cultured with fresh allogenic lymphocytes and post-cryopreservation autologous lymphocytes to obtain CTLs. The viable cells were counted by trypan blue staining; the percentages of CD3 + T cells and regulatory T cells (Tregs) were determined by flow cytometry; the levels of supernatant IFN-γ were detected through ELISA and the cytotoxicity was evaluated by lactate dehydrogenase (LDH) assay. Results The rate of viable lymphocytes declined to 78% after cryopreservation, and there were no significant differences in the percentages of CD3 + T cells and Tregs between pre-cryopreservation and post-cryopreservation. There were no significant differences in the proliferation of Tregs, the level of IFN-γ and the cytotoxicity between the fresh CIKs and cryopreservation CIKs, and the similar results were get between the autologous CTLs and allogenic CTLs. Conclusion The autologous CTLs acquired via cryopreservation of lymphocytes is equivalent to the allogenic CTLs with the similar proliferation and killing activity in vitro.

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

    PubMed

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

    2009-10-01

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

  4. Dengue virus-specific human T cell clones. Serotype crossreactive proliferation, interferon gamma production, and cytotoxic activity

    PubMed Central

    1989-01-01

    The severe complications of dengue virus infections, hemorrhagic manifestation and shock, are much more commonly observed during secondary infections caused by a different serotype of dengue virus than that which caused the primary infections. It has been speculated, therefore, that dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are caused by serotype crossreactive immunopathological mechanisms. We analyzed clones of dengue serotype crossreactive T lymphocytes derived from the PBMC of a donor who had been infected with dengue 3 virus. These PBMC responded best to dengue 3 antigen, but also responded to dengue 1, 2, and 4 antigens, in bulk culture proliferation assays. 12 dengue antigen-specific clones were established using a limiting dilution technique. All of the clones had CD3+ CD4+ CD8 phenotypes. Eight clones responded to dengue 1, 2, 3, and 4 antigens and are crossreactive, while four other clones responded predominantly to dengue 3 antigen. These results indicate that the serotype crossreactive dengue-specific T lymphocyte proliferation observed in bulk cultures reflects the crossreactive responses detected at the clonal level. Serotype crossreactive clones produced high titers of IFN- gamma after stimulation with dengue 3 antigens, and also produced IFN- gamma to lower levels after stimulation with dengue 1, 2, and 4 antigens. The crossreactive clones lysed autologous lymphoblastoid cell line (LCL) pulsed with dengue antigens, and the crossreactivity of CTL lysis by T cell clones was consistent with the crossreactivity observed in proliferation assays. Epidemiological studies have shown that secondary infections with dengue 2 virus cause DHF/DSS at a higher rate than the other serotypes. We hypothesized that the lysis of dengue virus-infected cells by CTL may lead to DHF/DSS; therefore, the clones were examined for cytotoxic activity against dengue 2 virus-infected LCL. All but one of the serotype crossreactive clones lysed dengue 2 virus

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed Central

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

    2006-01-01

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

  7. T cells in chronic lymphocytic leukemia display dysregulated expression of immune checkpoints and activation markers.

    PubMed

    Palma, Marzia; Gentilcore, Giusy; Heimersson, Kia; Mozaffari, Fariba; Näsman-Glaser, Barbro; Young, Emma; Rosenquist, Richard; Hansson, Lotta; Österborg, Anders; Mellstedt, Håkan

    2017-03-01

    Chronic lymphocytic leukemia is characterized by impaired immune functions largely due to profound T-cell defects. T-cell functions also depend on co-signaling receptors, inhibitory or stimulatory, known as immune checkpoints, including cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1). Here we analyzed the T-cell phenotype focusing on immune checkpoints and activation markers in chronic lymphocytic leukemia patients (n=80) with different clinical characteristics and compared them to healthy controls. In general, patients had higher absolute numbers of CD3 + cells and the CD8 + subset was particularly expanded in previously treated patients. Progressive patients had higher numbers of CD4 + and CD8 + cells expressing PD-1 compared to healthy controls, which was more pronounced in previously treated patients ( P =0.0003 and P =0.001, respectively). A significant increase in antigen-experienced T cells was observed in patients within both the CD4 + and CD8 + subsets, with a significantly higher PD-1 expression. Higher numbers of CD4 + and CD8 + cells with intracellular CTLA-4 were observed in patients, as well as high numbers of proliferating (Ki67 + ) and activated (CD69 + ) CD4 + and CD8 + cells, more pronounced in patients with active disease. The numbers of Th1, Th2, Th17 and regulatory T cells were substantially increased in patients compared to controls ( P <0.05), albeit decreasing to low levels in pre-treated patients. In conclusion, chronic lymphocytic leukemia T cells display increased expression of immune checkpoints, abnormal subset distribution, and a higher proportion of proliferating cells compared to healthy T cells. Disease activity and previous treatment shape the T-cell profile of chronic lymphocytic leukemia patients in different ways. Copyright© Ferrata Storti Foundation.

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

    PubMed

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

    2017-01-01

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

  9. Clonal predominance of CD8+ T cells in patients with unexplained neutropenia

    PubMed Central

    Wlodarski, Marcin Wojciech; Nearman, Zachary; Jiang, Ying; Lichtin, Alan; Maciejewski, Jaroslaw Pawel

    2008-01-01

    Objective T-cell-mediated autoimmunity may be involved in some cases of idiopathic neutropenia. We hypothesized that a precise T-cell receptor repertoire analysis may uncover cytotoxic T-cell (CTL) expansions that are less pronounced than those seen in T large granular lymphocyte leukemia (T-LGL), but are pathophysiologically analogous and thus can serve as markers of a T-cell-mediated process. Materials and Methods Using rational algorithms for T-cell receptor analysis and in vivo tracking of CTL responses previously established in our laboratory, we studied patients with unexplained chronic neutropenia (n = 20), T-LGL (n = 15), and healthy controls (n = 12). We further investigated the involvement of soluble inhibitory factors by coculture assays. To determine the level of immune activation, we studied interferon-g expression in CD8+cells using Taqman polymerase chain reaction. Results Fifteen expanded (immunodominant) CTL clones were detected in 12 of 20 patients. In comparison to LGL leukemia, these clones were less immunodominant, but clearly discernible from subclinical lymphoproliferations in controls. As a surrogate of cytotoxic activity, we found markedly increased production of interferon-γ in most of the neutropenia patients, irrespective of the presence of immunodominant CTL clones. Conclusions These results suggest that, while immunodominant CTL clones are detectable in a proportion of patients only, CTL-mediated pathophysiology may be a general mechanism operating in idiopathic neutropenia. Oligogoclonal CTL expansions in chronic neutropenia may indicate an ongoing autoimmune process, while highly polarized monoclonalities in a subset of neutropenic LGL patients may represent the “extreme” end of the clonal continuum. PMID:18279717

  10. Gag-Positive Reservoir Cells Are Susceptible to HIV-Specific Cytotoxic T Lymphocyte Mediated Clearance In Vitro and Can Be Detected In Vivo

    PubMed Central

    Graf, Erin H.; Pace, Matthew J.; Peterson, Bennett A.; Lynch, Lindsay J.; Chukwulebe, Steve B.; Mexas, Angela M.; Shaheen, Farida; Martin, Jeffrey N.; Deeks, Steven G.; Connors, Mark; Migueles, Stephen A.; O’Doherty, Una

    2013-01-01

    Resting CD4+ T cells infected with HIV persist in the presence of suppressive anti-viral therapy (ART) and are barriers to a cure. One potential curative approach, therapeutic vaccination, is fueled by recognition of the ability of a subset of elite controllers (EC) to control virus without therapy due to robust anti-HIV immune responses. Controllers have low levels of integrated HIV DNA and low levels of replication competent virus, suggesting a small reservoir. As our recent data indicates some reservoir cells can produce HIV proteins (termed GPR cells for Gag-positive reservoir cells), we hypothesized that a fraction of HIV-expressing resting CD4+ T cells could be efficiently targeted and cleared in individuals who control HIV via anti-HIV cytotoxic T lymphocytes (CTL). To test this we examined if superinfected resting CD4+ T cells from EC express HIV Gag without producing infectious virus and the susceptibility of these cells to CTL. We found that resting CD4+ T cells expressed HIV Gag and were cleared by autologous CD8+ T cells from EC. Importantly, we found the extent of CTL clearance in our in vitro assay correlates with in vivo reservoir size and that a population of Gag expressing resting CD4+ T cells exists in vivo in patients well controlled on therapy. PMID:23951263

  11. Visualization of Cytolytic T Cell Differentiation and Granule Exocytosis with T Cells from Mice Expressing Active Fluorescent Granzyme B

    PubMed Central

    Mouchacca, Pierre; Schmitt-Verhulst, Anne-Marie; Boyer, Claude

    2013-01-01

    To evaluate acquisition and activation of cytolytic functions during immune responses we generated knock in (KI) mice expressing Granzyme B (GZMB) as a fusion protein with red fluorescent tdTomato (GZMB-Tom). As for GZMB in wild type (WT) lymphocytes, GZMB-Tom was absent from naïve CD8 and CD4 T cells in GZMB-Tom-KI mice. It was rapidly induced in most CD8 T cells and in a subpopulation of CD4 T cells in response to stimulation with antibodies to CD3/CD28. A fraction of splenic NK cells expressed GZMB-Tom ex vivo with most becoming positive upon culture in IL-2. GZMB-Tom was present in CTL granules and active as a protease when these degranulated into cognate target cells, as shown with target cells expressing a specific FRET reporter construct. Using T cells from mice expressing GZMB-Tom but lacking perforin, we show that the transfer of fluorescent GZMB-Tom into target cells was dependent on perforin, favoring a role for perforin in delivery of GZMB at the target cells’ plasma membranes. Time-lapse video microscopy showed Ca++ signaling in CTL upon interaction with cognate targets, followed by relocalization of GZMB-Tom-containing granules to the synaptic contact zone. A perforin-dependent step was next visualized by the fluorescence signal from the non-permeant dye TO-PRO-3 at the synaptic cleft, minutes before the labeling of the target cell nucleus, characterizing a previously undescribed synaptic event in CTL cytolysis. Transferred OVA-specific GZMB-Tom-expressing CD8 T cells acquired GZMB-Tom expression in Listeria monocytogenes-OVA infected mice as soon as 48h after infection. These GZMB-Tom positive CD8 T cells localized in the splenic T-zone where they interacted with CD11c positive dendritic cells (DC), as shown by GZMB-Tom granule redistribution to the T/DC contact zone. GZMB-Tom-KI mice thus also provide tools to visualize acquisition and activation of cytolytic function in vivo. PMID:23840635

  12. [T and B lymphocytes and serum alpha 1-antitrypsin activity in children with bronchial asthma treated with broncho-vaxom].

    PubMed

    Wartenberg, J; Lewandowska, J; Pilarek, M; Piltz, D

    The aim of this study was to evaluate an effect of Broncho-Vaxom treatment on T and B lymphocytes and serum alpha 1AT in children treated at "Zuch" sanatorium in Szczawno-spa. The trial involved 46 school aged children suffering from infectious or infectious-atopic asthma. The post-Broncho-Vaxom treatment values for T lymphocytes were significantly higher in infectious, and significantly lower for B lymphocytes in infectious-atopic asthma. Serum alpha 1AT activity in children suffering from infectious asthma decreased significantly after the treatment. A correlation between the efficacy of the treatment and the lymphocyte percentage was observed. In children with very effective clinical results of Broncho-Vaxom treatment, the significant increase in T lymphocyte, and decrease in B lymphocyte populations was observed. Changes in T and B lymphocyte percentage were analysed in respect to alpha 1AT pre-treatment activity. In children with high alpha 1AT value, T lymphocytes after the treatment increased significantly in infectious, and B lymphocytes decreased significantly in infectious-atopic group.

  13. Polyclonal activation of human lymphocytes in vitro-II. Reappraisal of T and B cell-specific mitogens.

    PubMed

    Dosch, H M; Schuurman, R K; Gelfand, E W

    1980-08-01

    The capacity of the T cell mitogens phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and Staphylococcus protein A (SpA) to induce B cell proliferation and differentiation was compared with the B cell mitogen, formalinized Staphylococcus aureus (STA). Lymphocyte subpopulations from normal donors and patients with various immunodeficiency diseases were studied. In the presence of the T cell mitogens, irradiated T cells were capable of providing a helper cell activity that enabled co-cultured B lymphocytes to proliferate in response to these mitogens and to differentiate into IgM-secreting (direct) hemolytic plaque-forming cells (PFC). In the PFC response, radioresistant T-helper and radiosensitive T-suppressor cell activities could be demonstrated. T-suppressor cell activity outweighed helper activity only in nonirradiated co-cultures stimulated with Con A. Patients with severe combined immunodeficiency lacked mitogen-induced helper T cells, whereas patients with various forms of humoral immune deficiency were normal in this respect. These findings and the tissue distribution of the helper activity is aquired early in post-thymic T cell differentiation. The data suggest that experiments with cell lineage-specific lymphocyte mitogens should be considered in the context of more complex cell-cell interactions.

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

    PubMed Central

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

    2016-01-01

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

  15. Cytotoxic T cell clones isolated from ovarian tumor-infiltrating lymphocytes recognize multiple antigenic epitopes on autologous tumor cells.

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

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

  17. A mathematical model for CTL effect on a latently infected cell inclusive HIV dynamics and treatment

    NASA Astrophysics Data System (ADS)

    Tarfulea, N. E.

    2017-10-01

    This paper investigates theoretically and numerically the effect of immune effectors, such as the cytotoxic lymphocyte (CTL), in modeling HIV pathogenesis (via a newly developed mathematical model); our results suggest the significant impact of the immune response on the control of the virus during primary infection. Qualitative aspects (including positivity, boundedness, stability, uncertainty, and sensitivity analysis) are addressed. Additionally, by introducing drug therapy, we analyze numerically the model to assess the effect of treatment consisting of a combination of several antiretroviral drugs. Our results show that the inclusion of the CTL compartment produces a higher rebound for an individual's healthy helper T-cell compartment than drug therapy alone. Furthermore, we quantitatively characterize successful drugs or drug combination scenarios.

  18. Differential requirements for activation and growth of unprimed cytotoxic and helper T lymphocytes.

    PubMed

    Gullberg, M; Pobor, G; Bandeira, A; Larsson, E L; Coutinho, A

    1983-09-01

    The requirements for activation and growth of T lymphocytes capable of mediating either cytolytic activity or help to B lymphocytes were studied in unprimed splenic T cell populations. The selectivity of expression of Lyt-2 antigens, the reactivity to soluble concanavalin A (Con A), to partially purified interleukin 2 (IL 2, T cell growth factor[s]) and to lectin-pulsed macrophages (M phi) were used in this analysis. Lectin-dependent cytotoxicity assays and a novel method that allows for the detection of all effector helper cells, regardless of their clonal specificities, were used for the functional identification of the responding T cells. The results show a marked contrast between cytolytic and helper T cells in their growth and activation requirements. Thus, while Lyt-2+ cytotoxic T lymphocyte precursors grow exponentially in IL 2 after a short pulse with soluble Con A in the absence of accessory cells, Lyt-2- helper cell precursors completely fail to proliferate under the same conditions and require the continuous presence of lectin-pulsed M phi for significant growth. Furthermore, addition of IL 2 to M phi-stimulated cultures of Lyt-2- cells has no effect. T cells which produce IL 2 have the same growth characteristics as helper cells. In both cases, effector helper functions could be expanded more than 10-fold on a per cell basis by a 5-day-culture period under those growth supporting conditions. The development of effector helper functions, however, was strongly inhibited by the presence of Lyt-2+ T cells.

  19. Cellular energy metabolism in T-lymphocytes.

    PubMed

    Gaber, Timo; Strehl, Cindy; Sawitzki, Birgit; Hoff, Paula; Buttgereit, Frank

    2015-01-01

    Energy homeostasis is a hallmark of cell survival and maintenance of cell function. Here we focus on the impact of cellular energy metabolism on T-lymphocyte differentiation, activation, and function in health and disease. We describe the role of transcriptional and posttranscriptional regulation of lymphocyte metabolism on immune functions of T cells. We also summarize the current knowledge about T-lymphocyte adaptations to inflammation and hypoxia, and the impact on T-cell behavior of pathophysiological hypoxia (as found in tumor tissue, chronically inflamed joints in rheumatoid arthritis and during bone regeneration). A better understanding of the underlying mechanisms that control immune cell metabolism and immune response may provide therapeutic opportunities to alter the immune response under conditions of either immunosuppression or inflammation, potentially targeting infections, vaccine response, tumor surveillance, autoimmunity, and inflammatory disorders.

  20. Effective Induction of Simian Immunodeficiency Virus-Specific Cytotoxic T Lymphocytes in Macaques by Using a Multiepitope Gene and DNA Prime-Modified Vaccinia Virus Ankara Boost Vaccination Regimen

    PubMed Central

    Hanke, Tomas; Samuel, Rachel V.; Blanchard, Tom J.; Neumann, Veronica C.; Allen, Todd M.; Boyson, Jon E.; Sharpe, Sally A.; Cook, Nicola; Smith, Geoffrey L.; Watkins, David I.; Cranage, Martin P.; McMichael, Andrew J.

    1999-01-01

    DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in humans. Here, by using a multicytotoxic T-lymphocyte (CTL) epitope gene and a DNA prime-MVA boost vaccination regimen, high levels of CTLs specific for a single simian immunodeficiency virus (SIV) gag-derived epitope were elicited in rhesus macaques. These vaccine-induced CTLs were capable of killing SIV-infected cells in vitro. Fluorescence-activated cell sorter analysis using soluble tetrameric major histocompatibility complex-peptide complexes showed that the vaccinated animals had 1 to 5% circulating CD8+ lymphocytes specific for the vaccine epitope, frequencies comparable to those in SIV-infected monkeys. Upon intrarectal challenge with pathogenic SIVmac251, no evidence for protection was observed in at least two of the three vaccinated animals. This study does not attempt to define correlates of protective immunity nor design a protective vaccine against immunodeficiency viruses, but it demonstrates clearly that the DNA prime-MVA boost regimen is an effective protocol for induction of CTLs in macaques. It also shows that powerful tools for studying the role of CTLs in the control of SIV and human immunodeficiency virus infections are now available: epitope-based vaccines, a protocol for an effective induction of CTLs in primates, and a simple and sensitive method for quantitation of epitope-specific T cells. The advantages of the DNA prime-MVA boost regimen as well as the correlations of tetramer staining of peripheral blood lymphocytes with CTL killing in vitro and postchallenge control of viremia are discussed. PMID:10438842

  1. T Lymphocyte Activation Threshold and Membrane Reorganization Perturbations in Unique Culture Model

    NASA Technical Reports Server (NTRS)

    Adams, C. L.; Sams, C. F.

    2000-01-01

    Quantitative activation thresholds and cellular membrane reorganization are mechanisms by which resting T cells modulate their response to activating stimuli. Here we demonstrate perturbations of these cellular processes in a unique culture system that non-invasively inhibits T lymphocyte activation. During clinorotation, the T cell activation threshold is increased 5-fold. This increased threshold involves a mechanism independent of TCR triggering. Recruitment of lipid rafts to the activation site is impaired during clinorotation but does occur with increased stimulation. This study describes a situation in which an individual cell senses a change in its physical environment and alters its cell biological behavior.

  2. Elevated Expression of Immunoreceptor Tyrosine-Based Inhibitory Motif (TIGIT) on T Lymphocytes is Correlated with Disease Activity in Rheumatoid Arthritis.

    PubMed

    Luo, Qing; Deng, Zhen; Xu, Chuxin; Zeng, Lulu; Ye, Jianqing; Li, Xue; Guo, Yang; Huang, Zikun; Li, Junming

    2017-03-10

    BACKGROUND It is well known that lymphocytes play an important role in rheumatoid arthritis (RA). T cell immunoreceptors with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif (TIGIT) have immunosuppressive co-stimulatory molecules that mediate inhibitory effects, but their roles in RA are poorly understood. MATERIAL AND METHODS Were recruited 76 patients with RA and 33 healthy controls (HC). Clinical manifestations, laboratory measurements, physical examination, and medical history of RA patients were recorded. The expression of TIGIT on CD3+ T lymphocytes, B lymphocytes, monocytes, neutrophils, CD3+CD4+ T lymphocytes, and CD3+CD8+ T lymphocytes was determined using flow cytometry. The expression of TIGIT on T lymphocytes in patients with RA was further analyzed to investigate its correlations with markers of autoimmune response, inflammation, and disease activity in RA. RESULTS Compared with HC, the expression levels of TIGIT on CD3+CD4+ T lymphocytes and CD3+CD8+ T lymphocytes were significantly increased in patients with RA (P < 0.01). The frequency of TIGIT-expressing CD3+CD4+ T lymphocytes was positively correlated with RF, increased ACPA, ESR, and CRP levels. The frequency of TIGIT-expressing CD3+CD8+ T lymphocytes was positively correlated with RF and ESR levels. Furthermore, the expression level of TIGIT on CD3+CD4+ T lymphocytes was positively correlated with the DAS28 score in RA. CONCLUSIONS The expression levels of TIGIT on T lymphocytes were elevated and correlated with disease activity in RA.

  3. Th cells promote CTL survival and memory via acquired pMHC-I and endogenous IL-2 and CD40L signaling and by modulating apoptosis-controlling pathways.

    PubMed

    Umeshappa, Channakeshava Sokke; Xie, Yufeng; Xu, Shulin; Nanjundappa, Roopa Hebbandi; Freywald, Andrew; Deng, Yulin; Ma, Hong; Xiang, Jim

    2013-01-01

    Involvement of CD4(+) helper T (Th) cells is crucial for CD8(+) cytotoxic T lymphocyte (CTL)-mediated immunity. However, CD4(+) Th's signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4(+) Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4(+) T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2K(b)/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4(+) Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4(+) Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7) molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4(+) Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy.

  4. Th Cells Promote CTL Survival and Memory via Acquired pMHC-I and Endogenous IL-2 and CD40L Signaling and by Modulating Apoptosis-Controlling Pathways

    PubMed Central

    Umeshappa, Channakeshava Sokke; Xie, Yufeng; Xu, Shulin; Nanjundappa, Roopa Hebbandi; Freywald, Andrew; Deng, Yulin; Ma, Hong; Xiang, Jim

    2013-01-01

    Involvement of CD4+ helper T (Th) cells is crucial for CD8+ cytotoxic T lymphocyte (CTL)-mediated immunity. However, CD4+ Th’s signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4+ Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4+ T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2Kb/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4+ Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4+ Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7) molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4+ Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy. PMID:23785406

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

    PubMed Central

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

    1990-01-01

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

  6. Differential Dynamics of CD4+ and CD8+ T-Lymphocyte Proliferation and Activation in Acute Simian Immunodeficiency Virus Infection

    PubMed Central

    Kaur, Amitinder; Hale, Corrina L.; Ramanujan, Saroja; Jain, Rakesh K.; Johnson, R. Paul

    2000-01-01

    Although lymphocyte turnover in chronic human immunodeficiency virus and simian immunodeficiency virus (SIV) infection has been extensively studied, there is little information on turnover in acute infection. We carried out a prospective kinetic analysis of lymphocyte proliferation in 13 rhesus macaques inoculated with pathogenic SIV. A short-lived dramatic increase in circulating Ki-67+ lymphocytes observed at 1 to 4 weeks was temporally related to the onset of SIV replication. A 5- to 10-fold increase in Ki-67+ CD8+ T lymphocytes and a 2- to 3-fold increase in Ki-67+ CD3− CD8+ natural killer cells accounted for >85% of proliferating lymphocytes at peak proliferation. In contrast, there was little change in the percentage of Ki-67+ CD4+ T lymphocytes during acute infection, although transient increases in Ki-67− and Ki-67+ CD4+ T lymphocytes expressing CD69, Fas, and HLA-DR were observed. A two- to fourfold decline in CD4+ T lymphocytes expressing CD25 and CD69 was seen later in SIV infection. The majority of Ki-67+ CD8+ T lymphocytes were phenotypically CD45RA− CD49dhi Fashi CD25− CD69− CD28− HLA-DR− and persisted at levels twofold above baseline 6 months after SIV infection. Increased CD8+ T-lymphocyte proliferation was associated with cell expansion, paralleled the onset of SIV-specific cytotoxic T-lymphocyte activity, and had an oligoclonal component. Thus, divergent patterns of proliferation and activation are exhibited by CD4+ and CD8+ T lymphocytes in early SIV infection and may determine how these cells are differentially affected in AIDS. PMID:10954541

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

    PubMed

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

    2012-01-01

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

  8. 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

  9. T Lymphocyte Activation Threshold is Increased in Reduced Gravity

    NASA Technical Reports Server (NTRS)

    Adams, Charley L.; Gonzalez, M.; Sams, C. F.

    2000-01-01

    There have been substantial advances in molecular and cellular biology that have provided new insight into the biochemical and genetic basis of lymphocyte recognition, activation and expression of distinct functional phenotypes. It has now become evident that for both T and B cells, stimuli delivered through their receptors can result in either clonal expansion or apoptosis. In the case of T cells, clonal expansion of helper cells is accompanied by differentiation into two major functional subsets which regulate the immune response. The pathways between the membrane and the nucleus and their molecular components are an area of very active investigation. This meeting will draw together scientists working on diverse aspects of this problem, including receptor ligand interactions, intracellular pathways that transmit receptor mediated signals and the effect of such signal transduction pathways on gene regulation. The aim of this meeting is to integrate the information from these various experimental approaches into a new synthesis and molecular explanation of T cell activation, differentiation and death.

  10. MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells

    PubMed Central

    Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

    2015-01-01

    Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach. PMID:25949869

  11. MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells.

    PubMed

    Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

    2015-01-01

    Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach.

  12. T cell priming versus T cell tolerance induced by synthetic peptides

    PubMed Central

    1995-01-01

    It is well known that synthetic peptides are able to both induce and tolerize T cells. We have examined the parameters leading either to priming or tolerance of CD8+ cytotoxic T lymphocytes (CTL) in vivo with a major histocompatibility complex class I (H-2 Db) binding peptide derived from the glycoprotein (GP aa33-41) of lymphocytic choriomeningitis virus (LCMV). By varying dose, route, and frequency of LCMV GP peptide application, we found that a single local subcutaneous injection of 50-500 micrograms peptide emulsified in incomplete Freund's adjuvant protected mice against LCMV infection, whereas repetitive and systemic intraperitoneal application of the same dose caused tolerance of LCMV-specific CTL. The peptide-induced tolerance was transient in euthymic mice but permanent in thymectomized mice. These findings are relevant for a selective use of peptides as a therapeutic approach: peptide-induced priming of T cells for vaccination and peptide-mediated T cell tolerance for intervention in immunopathologies and autoimmune diseases. PMID:7540654

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2007-04-10

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

  15. Immunomodulatory Activity of Ganoderma atrum Polysaccharide on Purified T Lymphocytes through Ca2+/CaN and Mitogen-Activated Protein Kinase Pathway Based on RNA Sequencing.

    PubMed

    Xiang, Quan-Dan; Yu, Qiang; Wang, Hui; Zhao, Ming-Ming; Liu, Shi-Yu; Nie, Shao-Ping; Xie, Ming-Yong

    2017-07-05

    Our previous study has demonstrated that Ganoderma atrum polysaccharide (PSG-1) has immunomodulatory activity on spleen lymphocytes. However, how PSG-1 exerts its effect on purified lymphocytes is still obscure. Thus, this study aimed to investigate the immunomodulatory activity of PSG-1 on purified T lymphocytes and further elucidate the underlying mechanism based on RNA sequencing (RNA-seq). Our results showed that PSG-1 promoted T lymphocytes proliferation and increased the production of IL-2, IFN-γ, and IL-12. Meanwhile, RNA-seq analysis found 394 differentially expressed genes. KEGG pathway analysis identified 20 significant canonical pathways and seven biological functions. Furthermore, PSG-1 elevated intracellular Ca 2+ concentration and calcineurin (CaN) activity and raised the p-ERK, p-JNK, and p-p38 expression levels. T lymphocytes proliferation and the production of IL-2, IFN-γ, and IL-12 were decreased by the inhibitors of calcium channel and mitogen-activated protein kinases (MAPKs). These results indicated that PSG-1 possesses immunomodulatory activity on purified T lymphocytes, in which Ca 2+ /CaN and MAPK pathways play essential roles.

  16. Divergent effects of norepinephrine, dopamine and substance P on the activation, differentiation and effector functions of human cytotoxic T lymphocytes

    PubMed Central

    2009-01-01

    Background Neurotransmitters are important regulators of the immune system, with very distinct and varying effects on different leukocyte subsets. So far little is known about the impact of signals mediated by neurotransmitters on the function of CD8+ T lymphocytes. Therefore, we investigated the influence of norepinephrine, dopamine and substance P on the key tasks of CD8+ T lymphocytes: activation, migration, extravasation and cytotoxicity. Results The activation of naïve CD8+ T lymphocytes by CD3/CD28 cross-linking was inhibited by norepinephrine and dopamine, which was caused by a downregulation of interleukin (IL)-2 expression via Erk1/2 and NF-κB inhibition. Furthermore, all of the investigated neurotransmitters increased the spontaneous migratory activity of naïve CD8+ T lymphocytes with dopamine being the strongest inducer. In contrast, activated CD8+ T lymphocytes showed a reduced migratory activity in the presence of norepinephrine and substance P. With regard to extravasation we found norepinephrine to induce adhesion of activated CD8+ T cells: norepinephrine increased the interleukin-8 release from endothelium, which in turn had effect on the activated CXCR1+ CD8+ T cells. At last, release of cytotoxic granules from activated cells in response to CD3 cross-linking was not influenced by any of the investigated neurotransmitters, as we have analyzed by measuring the β-hexosamidase release. Conclusion Neurotransmitters are specific modulators of CD8+ T lymphocytes not by inducing any new functions, but by fine-tuning their key tasks. The effect can be either stimulatory or suppressive depending on the activation status of the cells. PMID:19968887

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

    PubMed

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

    2002-11-15

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

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

    PubMed

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

    2000-09-01

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

  19. Development of Type 1 Diabetes Mellitus in Nonobese Diabetic Mice Follows Changes in Thymocyte and Peripheral T Lymphocyte Transcriptional Activity

    PubMed Central

    Fornari, Thais A.; Donate, Paula B.; Macedo, Claudia; Sakamoto-Hojo, Elza T.; Donadi, Eduardo A.; Passos, Geraldo A.

    2011-01-01

    As early as one month of age, nonobese diabetic (NOD) mice feature pancreatic infiltration of autoreactive T lymphocytes, which destruct insulin-producing beta cells, producing autoimmune diabetes mellitus (T1D) within eight months. Thus, we hypothesized that during the development of T1D, the transcriptional modulation of immune reactivity genes may occur as thymocytes mature into peripheral T lymphocytes. The transcriptome of thymocytes and peripheral CD3+ T lymphocytes from prediabetic or diabetic mice analyzed through microarray hybridizations identified 2,771 differentially expressed genes. Hierarchical clustering grouped mice according to age/T1D onset and genes according to their transcription profiling. The transcriptional activity of thymocytes developing into peripheral T lymphocytes revealed sequential participation of genes involved with CD4+/CD8+ T-cell differentiation (Themis), tolerance induction by Tregs (Foxp3), and apoptosis (Fasl) soon after T-cell activation (IL4), while the emergence of T1D coincided with the expression of cytotoxicity (Crtam) and inflammatory response genes (Tlr) by peripheral T lymphocytes. PMID:21765850

  20. Cytotoxic T lymphocytes and CD4 epitope mutations in the pre-core/core region of hepatitis B virus in chronic hepatitis B carriers in Northeast Iran.

    PubMed

    Zhand, Sareh; Tabarraei, Alijan; Nazari, Amineh; Moradi, Abdolvahab

    2017-07-01

    Hepatitis B virus (HBV) is vulnerable to many various mutations. Those within epitopes recognized by sensitized T cells may influence the re-emergence of the virus. This study was designed to investigate the mutation in immune epitope regions of HBV pre-core/core among chronic HBV patients of Golestan province, Northeast Iran. In 120 chronic HBV carriers, HBV DNA was extracted from blood plasma samples and PCR was done using specific primers. Direct sequencing and alignment of the pre-core/core region were applied using reference sequence from Gene Bank database (Accession Number AB033559). The study showed 27 inferred amino acid substitutions, 9 of which (33.3%) were in CD4 and 2 (7.4%) in cytotoxic T lymphocytes' (CTL) epitopes and 16 other mutations (59.2%) were observed in other regions. CTL escape mutations were not commonly observed in pre-core/core sequences of chronic HBV carriers in the locale of study. It can be concluded that most of the inferred amino acid substitutions occur in different immune epitopes other than CTL and CD4.

  1. Induction of cytotoxic T lymphocyte response against Mycobacterial antigen using domain I of anthrax edema factor as antigen delivery system

    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

  2. Dynamin2 controls Rap1 activation and integrin clustering in human T lymphocyte adhesion

    PubMed Central

    Eppler, Felix J.

    2017-01-01

    Leukocyte trafficking is crucial to facilitate efficient immune responses. Here, we report that the large GTPase dynamin2, which is generally considered to have a key role in endocytosis and membrane remodeling, is an essential regulator of integrin-dependent human T lymphocyte adhesion and migration. Chemical inhibition or knockdown of dynamin2 expression significantly reduced integrin-dependent T cell adhesion in vitro. This phenotype was not observed when T cells were treated with various chemical inhibitors which abrogate endocytosis or actin polymerization. We furthermore detected dynamin2 in signaling complexes and propose that it controls T cell adhesion via FAK/Pyk2- and RapGEF1-mediated Rap1 activation. In addition, the dynamin2 inhibitor-induced reduction of lymphocyte adhesion can be rescued by Rap1a overexpression. We demonstrate that the dynamin2 effect on T cell adhesion does not involve integrin affinity regulation but instead relies on its ability to modulate integrin valency. Taken together, we suggest a previously unidentified role of dynamin2 in the regulation of integrin-mediated lymphocyte adhesion via a Rap1 signaling pathway. PMID:28273099

  3. Cytotoxic T Lymphocyte Epitopes of HIV-1 Nef

    PubMed Central

    Lucchiari-Hartz, Maria; van Endert, Peter M.; Lauvau, Grégoire; Maier, Reinhard; Meyerhans, Andreas; Mann, Derek; Eichmann, Klaus; Niedermann, Gabriele

    2000-01-01

    Although a pivotal role of proteasomes in the proteolytic generation of epitopes for major histocompatibility complex (MHC) class I presentation is undisputed, their precise function is currently the subject of an active debate: do proteasomes generate many epitopes in definitive form, or do they merely generate the COOH termini, whereas the definitive NH2 termini are cleaved by aminopeptidases? We determined five naturally processed MHC class I ligands derived from HIV-1 Nef. Unexpectedly, the five ligands correspond to only three cytotoxic T lymphocyte (CTL) epitopes, two of which occur in two COOH-terminal length variants. Parallel analyses of proteasomal digests of a Nef fragment encompassing the epitopes revealed that all five ligands are direct products of proteasomes. Moreover, in four of the five ligands, the NH2 termini correspond to major proteasome cleavage sites, and putative NH2-terminally extended precursor fragments were detected for only one of the five ligands. All ligands are transported by the transporter associated with antigen processing (TAP). The combined results from these five ligands provide strong evidence that many definitive MHC class I ligands are precisely cleaved at both ends by proteasomes. Additional evidence supporting this conclusion is discussed, along with contrasting results of others who propose a strong role for NH2-terminal trimming with direct proteasomal epitope generation being a rare event. PMID:10637269

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

    NASA Astrophysics Data System (ADS)

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

    1994-06-01

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

  5. In-vitro responses of T lymphocytes to poly(butylene succinate) based biomaterials.

    PubMed

    Toso, Montree; Patntirapong, Somying; Janvikul, Wanida; Singhatanadgit, Weerachai

    2017-04-01

    Polybutylene succinate (PBSu) and PBSu/β-tricalcium phosphate (TCP) composites are biocompatible and good candidates as bone graft materials. However, little is known about the responses of T lymphocytes to these biomaterials, which play an important role in the success of bone grafting. Activated T lymphocytes were cultured onto 32 mm diameter films (PBSu/TCP films), that had previously been placed in 6-well culture plates, for 8, 24 and 72 hours. A plastic-well culture plate was used as a control surface. The effects of PBSu-based biomaterials on T lymphocytes were examined by the using flow cytometry and reverse-transcription polymerase chain reaction. These biomaterials were non-toxic to T lymphocytes, allowing their normal DNA synthesis and activation. All materials induced only transient activation of T lymphocytes, which existed no longer than 72 hours. Proportions of four main CD4/CD8 T lymphocyte subpopulations were not affected by these biomaterials. Moreover, PBSu and PBSu/TCP significantly suppressed the expression of IL-1β and IL-6 genes by 15-35% and 21-26%, respectively. In contrast, a PBSu/TCP composite (at PBSu:TCP=60:40) significantly stimulated the expression of IL-10 and IL-13 genes by 17% and 19%, respectively. PBSu and PBSu/TCP composites were non-toxic to T lymphocytes and did not induce unfavorable responses of T lymphocytes. The tested biomaterials down-regulated key proinflammatory cytokine genes and up-regulated anti-inflammatory cytokine genes in T lymphocytes. These suggest that the biomaterials studied are good candidates as bone graft materials.

  6. Garlic compounds modulate macrophage and T-lymphocyte functions.

    PubMed

    Lau, B H; Yamasaki, T; Gridley, D S

    1991-06-01

    Organosulfur compounds of garlic have been shown to inhibit growth of animal tumors and to modulate the activity of diverse chemical carcinogens. There is also evidence that garlic may modulate antitumor immunity. In this study, we determined the effects of an aqueous garlic extract and a protein fraction isolated from the extract on the chemiluminescent oxidative burst of the murine J774 macrophage cell line and thioglycollate-elicited peritoneal macrophages obtained from BALB/c mice. T-lymphocyte activity was determined using mouse splenocytes incubated with phytohemagglutinin, labeled with [3H]-thymidine and assayed for lymphoproliferation. Significant dose-related augmentation of oxidative burst was observed with garlic extract and the protein fraction. The protein fraction also enhanced the T-lymphocyte blastogenesis. The data suggest that garlic compounds may serve as biological response modifiers by augmenting macrophage and T-lymphocyte functions.

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

    PubMed Central

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

    2013-01-01

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

  8. Characterization of CD31 expression on murine and human neonatal T lymphocytes during development and activation

    PubMed Central

    Fike, Adam J.; Nguyen, Linda T.; Kumova, Ogan K.; Carey, Alison J.

    2017-01-01

    Background CD31, expressed by the majority of the neonatal T cell pool, is involved in modulation of T-cell Receptor signalling by increasing the threshold for T cell activation. Therefore, CD31 could modulate neonatal tolerance and adaptive immune responses. Methods Lymphocytes were harvested from murine neonates at different ages, human late preterm and term cord blood, and adult peripheral blood. Human samples were activated over a five-day period to simulate acute inflammation. Mice were infected with influenza; lungs and spleens were harvested at days 6 and 9 post-infection and analyzed by flow cytometry. Results CD31 expressing neonatal murine CD4+ and CD8a+ T cells increase over the first week of life. Upon in vitro stimulation, human infants’ CD4+ and CD8a+ T cells shed CD31 faster in comparison to adults. In the context of acute infection, mice infected at 3-days old have an increased number of naive and activated CD31+ T lymphocytes at the site of infection at day 6 and 9 post-infection, as compared to 7-days old; however, the opposite is true in the periphery. Conclusion Differences in trafficking of CD31+ Cytotoxic T Lymphocytes (CTLs) during acute influenza infection could modulate tolerance and contribute to a dampened adaptive immune response in neonates. PMID:28355204

  9. Human T Lymphocytes Are Permissive for Dengue Virus Replication.

    PubMed

    Silveira, Guilherme F; Wowk, Pryscilla F; Cataneo, Allan H D; Dos Santos, Paula F; Delgobo, Murilo; Stimamiglio, Marco A; Lo Sarzi, Maria; Thomazelli, Ana Paula F S; Conchon-Costa, Ivete; Pavanelli, Wander R; Antonelli, Lis R V; Báfica, André; Mansur, Daniel S; Dos Santos, Claudia N Duarte; Bordignon, Juliano

    2018-05-15

    Dengue virus (DV) infection can cause either a self-limiting flu-like disease or a threatening hemorrhage that may evolve to shock and death. A variety of cell types, such as dendritic cells, monocytes, and B cells, can be infected by DV. However, despite the role of T lymphocytes in the control of DV replication, there remains a paucity of information on possible DV-T cell interactions during the disease course. In the present study, we have demonstrated that primary human naive CD4 + and CD8 + T cells are permissive for DV infection. Importantly, both T cell subtypes support viral replication and secrete viable virus particles. DV infection triggers the activation of both CD4 + and CD8 + T lymphocytes, but preactivation of T cells reduces the susceptibility of T cells to DV infection. Interestingly, the cytotoxicity-inducing protein granzyme A is highly secreted by human CD4 + but not CD8 + T cells after exposure to DV in vitro Additionally, using annexin V and polycaspase assays, we have demonstrated that T lymphocytes, in contrast to monocytes, are resistant to DV-induced apoptosis. Strikingly, both CD4 + and CD8 + T cells were found to be infected with DV in acutely infected dengue patients. Together, these results show that T cells are permissive for DV infection in vitro and in vivo , suggesting that this cell population may be a viral reservoir during the acute phase of the disease. IMPORTANCE Infection by dengue virus (DV) causes a flu-like disease that can evolve to severe hemorrhaging and death. T lymphocytes are important cells that regulate antibody secretion by B cells and trigger the death of infected cells. However, little is known about the direct interaction between DV and T lymphocytes. Here, we show that T lymphocytes from healthy donors are susceptible to infection by DV, leading to cell activation. Additionally, T cells seem to be resistant to DV-induced apoptosis, suggesting a potential role as a viral reservoir in humans. Finally, we show

  10. Efficient Processing of the Immunodominant, HLA-A*0201-Restricted Human Immunodeficiency Virus Type 1 Cytotoxic T-Lymphocyte Epitope despite Multiple Variations in the Epitope Flanking Sequences

    PubMed Central

    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

  11. T-dependence of human B lymphocyte proliferative response to mitogens.

    PubMed

    Brochier, J; Samarut, C; Gueho, J P; Revillard, J P

    1976-01-01

    Human peripheral blood and tonsil lymphocytes were fractionated on anti-Ig-coated Sephadex columns or by centrifugation after rosetting with native sheep erythrocytes. Both methods allowed the recovery of B and T-enriched populations the purity of which was checked by fluorescein-labelled anti-Ig serum, E and EAC rosette formation, and heterologous antisera specific for B or T lymphocytes. The proliferative response of T cells to PHA, Con A, PWM, and ALS was not found different from that of unfractionated cells, whereas no response of the B cells could be observed to these mitogens providing that no contaminating T cells were present. Addition of T lymphocytes to these unresponsive B cells allowed them to respond to phytomitogens, but not to ALS. X-irradiated T cells could, to some extent, replace the diving T lymphocytes; no T-replacing factor could be found in cell-free supernatants from T cells, whether or not they had been activated by mitrogens. This model of B-T cooperation appears useful for studying the differentiation and maturation of human B lymphocytes.

  12. Comparison of cytotoxic T lymphocyte responses against pancreatic cancer induced by dendritic cells transfected with total tumor RNA and fusion hybrided with tumor cell

    PubMed Central

    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

  13. Analysis of cytotoxic activity of the CD4+ T lymphocytes generated by local immunotherapy.

    PubMed Central

    Katsumoto, Y.; Monden, T.; Takeda, T.; Haba, A.; Ito, Y.; Wakasugi, E.; Wakasugi, T.; Sekimoto, M.; Kobayashi, T.; Shiozaki, H.; Shimano, T.; Monden, M.

    1996-01-01

    We previously reported that the anti-tumour effect of OK-432 is considerably enhanced by its intratumoral injection together with fibrinogen. In the present study, we generated killer T cells by culturing tumour-infiltrating lymphocytes from thyroid cancer patients who had received this local immunotherapy. Phenotypic analysis revealed that the T cells were positive for CD3+, CD4+, Leu8-, CD45RO+ and T-cell receptor (TCR)alpha beta+, as well as showing strong surface expression of HLA-DR, CD25, LFA-1 and ICAM-1. The generated CD4+ T cells secreted interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, TNF-beta, and interleukin (IL)-6 (but not IL-4), and exhibited a high level of cytolytic activity against several tumour cell lines. The cytolytic activity of these T cells for Daudi cells was inhibited by preincubation with an anti-intercellular adhesion molecule (ICAM)-1 antibody, but not by preincubation with anti-TCR alpha beta, anti-CD2, or anti-LFA-1 antibodies. Pretreatment with anti-ICAM-1 antibody inhibited T-cell cytolytic activity, but not conjugation with target cells. In addition, incubation with immobilised anti-ICAM-1 enhanced the secretion of IFN-gamma by T cells. We conclude that ICAM-1 expressed on the effector cytotoxic CD4+ T lymphocytes delivers regulatory signals that enhance IFN-gamma secretion. PMID:8554971

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

    Science.gov Websites

    006 10 m above ground UGRD analysis U-Component of Wind [m/s] ENS=low-res ctl 007 10 m above ground VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl 008 surface WEASD analysis Water Equivalent of -Component of Wind [m/s] ENS=low-res ctl 021 250 mb VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl

  15. Inventory of File sref_nmb.t03z.pgrb132.ctl.grib2

    Science.gov Websites

    006 10 m above ground UGRD analysis U-Component of Wind [m/s] ENS=low-res ctl 007 10 m above ground VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl 008 surface WEASD analysis Water Equivalent of -Component of Wind [m/s] ENS=low-res ctl 021 250 mb VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl

  16. Inventory of File sref_nmb.t03z.pgrb243.ctl.grib2

    Science.gov Websites

    006 10 m above ground UGRD analysis U-Component of Wind [m/s] ENS=low-res ctl 007 10 m above ground VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl 008 surface WEASD analysis Water Equivalent of -Component of Wind [m/s] ENS=low-res ctl 021 250 mb VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl

  17. Inventory of File sref_nmb.t03z.pgrb216.ctl.grib2

    Science.gov Websites

    006 10 m above ground UGRD analysis U-Component of Wind [m/s] ENS=low-res ctl 007 10 m above ground VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl 008 surface WEASD analysis Water Equivalent of -Component of Wind [m/s] ENS=low-res ctl 021 250 mb VGRD analysis V-Component of Wind [m/s] ENS=low-res ctl

  18. Inhibition of serine-peptidase activity enhances the generation of a survivin-derived HLA-A2-presented CTL epitope in colon-carcinoma cells.

    PubMed

    Preta, G; Marescotti, D; Fortini, C; Carcoforo, P; Castelli, C; Masucci, M; Gavioli, R

    2008-12-01

    Cytotoxic T lymphocytes eliminate tumor cells expressing antigenic peptides in the context of MHC-I molecules. Peptides are generated during protein degradation by the proteasome and resulting products, surviving cytosolic amino-peptidases activity, may be presented by MHC-I molecules. The MHC-I processing pathway is altered in a large number of malignancies and modulation of antigen generation is one strategy employed by cells to evade immune control. In this study we analyzed the generation and presentation of a survivin-derived CTL epitope in HLA-A2-positive colon-carcinoma cells. Although all cell lines expressed the anti-apoptotic protein survivin, some tumors were poorly recognized by ELTLGEFLKL (ELT)-specific CTL cultures. The expression of MHC-I or TAP molecules was similar in all cell lines suggesting that tumors not recognized by CTLs may present defects in the generation of the ELT-epitope which could be due either to lack of generation or to subsequent degradation of the epitope. The cells were analyzed for the expression and the activity of extra-proteasomal peptidases. A significant overexpression and higher activity of TPPII was observed in colon-carcinoma cells which are not killed by ELT-specific CTLs, suggesting a possible role of TPPII in the degradation of the ELT-epitope. To confirm the role of TPPII in the degradation of the ELT-peptide, we showed that treatment of colon-carcinoma cells with a TPPII inhibitor resulted in a dose-dependent increased sensitivity to ELT-specific CTLs. These results suggest that TPPII is involved in degradation of the ELT-peptide, and its overexpression may contribute to the immune escape of colon-carcinoma cells.

  19. Identification of canine T lymphocytes by membrane receptor to peanut agglutinin: T-lymphocyte identification in dogs with lupus-like syndrome.

    PubMed

    Rigal, D; Bendali-Ahcène, S; Monier, J C; Mohana, K; Fournel, C

    1983-09-01

    Canine T lymphocytes were detected, using fluorescent peanut agglutinin (PNA) as a marker. Using a fluorescent technique and cytofluorometry, 70 +/- 11% and 72.4%, respectively, of peripheral blood lymphocytes were bound to PNA. Of thymocytes, 97 +/- 4.5% were detected by fluorescent PNA, but less than 1% were detected for lymphocytes from bone marrow. The T-lymphocyte depletion and enrichment indicated that PNA was bound to lymphocytes recognized by anti-T-lymphocyte heterologous serum. A T-lymphocyte deficiency was detected among 8 dogs with a lupus-like syndrome.

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

    PubMed

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

    2013-09-23

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

  1. T-Cell Therapy Using Interleukin-21-Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results in Long-Term Cell Persistence and Durable Tumor Regression.

    PubMed

    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

  2. Regulation of T lymphocyte apoptotic markers is associated to cell activation during the acute phase of dengue.

    PubMed

    Torrentes-Carvalho, Amanda; Marinho, Cintia Ferreira; de Oliveira-Pinto, Luzia Maria; de Oliveira, Débora Batista; Damasco, Paulo Vieira; Cunha, Rivaldo Venâncio; de Souza, Luiz José; de Azeredo, Elzinandes Leal; Kubelka, Claire Fernandes

    2014-05-01

    Dengue fever, a public health problem in Brazil, may present severe clinical manifestations as result of an increased vascular permeability and coagulation disorders. T cell activation is a critical event for an effective immune response against infection, including the production of cytokines. We aim to reveal mechanisms that modulate the virus-cell interaction, with an emphasis on cell death. Apoptosis is involved in lymphocyte homeostasis, contributes to the clearance of virus-infected cells but also may play a role in the pathogenesis. Phosphatidylserine exposure on CD8T lymphocytes from dengue patients support early apoptotic processes and loss of genomic integrity, observed by DNA fragmentation in T lymphocytes and indicating late apoptosis. These T cells express activation and cytotoxic phenotypes as revealed by CD29 and CD107a upregulation. Higher frequencies of CD95 were detected in T lymphocytes mainly in those with the cytotoxic profile (CD107a+) and lower levels of anti-apoptotic molecule Bcl-2, suggesting that both CD4+ and CD8+ T cell subsets are more susceptible to apoptosis during acute dengue. The analysis of apoptosis-related protein expression profile showed that not only molecules with pro- but also those with anti-apoptotic functions are overexpressed, indicating that survival mechanisms could be possibly protecting cells against apoptosis caused by viral, immune, oxidative and/or genotoxic stresses. These observations led us to propose that in dengue patients there is an association between T cell susceptibility to apoptosis and the activation state. The mechanisms for understanding the immunopathogenesis during dengue infection are discussed. Copyright © 2013 Elsevier GmbH. All rights reserved.

  3. Evidence for the separate human T-lymphocyte subpopulations that collaborate with autologous monocyte/macrophages in the elaboration of colony-stimulating activity and those that suppress this collaboration.

    PubMed

    Verma, D S; Johnston, D A; McCredie, K B

    1983-11-01

    We investigated the interaction of monocyte/macrophages and autologous T lymphocytes in the methanol extraction residue (MER) of BCG-induced production of granulocyte-macrophage colony-stimulating activity (CSA). Coincubation of monocyte/macrophages and T lymphocytes at a 1:3 ratio produces an optimum collaboration; a change to a 1:9 ratio diminished this collaboration. Coincubation of monocyte/macrophages and T lymphocytes primed with lithium carbonate (2 meq/liter) for 40 hr synergistically increased CSA elaboration and prevented the decline in CSA noted for the 1:9 monocyte/macrophage: T lymphocyte ratio. In contrast, concanavalin-A-primed T lymphocytes did not enhance CSA elaboration at any monocyte/macrophage:T lymphocyte ratio except, occasionally, at 1:9. However, this was overcome if the T lymphocytes were primed with both concanavalin-A and lithium carbonate before their coincubation with monocyte/macrophages. Further cell-mixing experiments revealed that concanavalin-A-primed T lymphocytes contained a subpopulation that suppressed monocyte/macrophage and T-lymphocyte collaboration. Activation of suppressor T lymphocytes could be effectively prevented by lithium carbonate and, in a dose-dependent manner, by irradiation. Also, suppressor T lymphocytes not only diminished the elaboration of colony-stimulating factor(s), but also elaborated an inhibitor of granulocyte-macrophage colony-forming cells. We further demonstrated that the respective hemopoietic helper and suppressor T-lymphocyte activities could be enriched with OKT8- (or OKT4+) and OKT8+ subpopulations.

  4. Differential expression of three T lymphocyte-activating CXC chemokines by human atheroma-associated cells

    PubMed Central

    Mach, François; Sauty, Alain; Iarossi, Albert S.; Sukhova, Galina K.; Neote, Kuldeep; Libby, Peter; Luster, Andrew D.

    1999-01-01

    Activated T lymphocytes accumulate early in atheroma formation and persist at sites of lesion growth and rupture, suggesting that they may play an important role in the pathogenesis of atherosclerosis. Moreover, atherosclerotic lesions contain the Th1-type cytokine IFN-γ, a potentiator of atherosclerosis. The present study demonstrates the differential expression of the 3 IFN-γ–inducible CXC chemokines — IFN-inducible protein 10 (IP-10), monokine induced by IFN-γ (Mig), and IFN-inducible T-cell α chemoattractant (I-TAC) — by atheroma-associated cells, as well as the expression of their receptor, CXCR3, by all T lymphocytes within human atherosclerotic lesions in situ. Atheroma-associated endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages (MØ) all expressed IP-10, whereas Mig and I-TAC were mainly expressed in ECs and MØ, as detected by double immunofluorescence staining. ECs of microvessels within lesions also expressed abundant I-TAC. In vitro experiments supported these results and showed that IL-1β, TNF-α, and CD40 ligand potentiated IP-10 expression from IFN-γ–stimulated ECs. In addition, nitric oxide (NO) treatment decreased IFN-γ induction of IP-10. Our findings suggest that the differential expression of IP-10, Mig, and I-TAC by atheroma-associated cells plays a role in the recruitment and retention of activated T lymphocytes observed within vascular wall lesions during atherogenesis. PMID:10525042

  5. Lymphocyte gene expression signatures from patients and mouse models of hereditary hemochromatosis reveal a function of HFE as a negative regulator of CD8+ T-lymphocyte activation and differentiation in vivo.

    PubMed

    Costa, Mónica; Cruz, Eugénia; Oliveira, Susana; Benes, Vladimir; Ivacevic, Tomi; Silva, Maria João; Vieira, Inês; Dias, Francisco; Fonseca, Sónia; Gonçalves, Marta; Lima, Margarida; Leitão, Catarina; Muckenthaler, Martina U; Pinto, Jorge; Porto, Graça

    2015-01-01

    Abnormally low CD8+ T-lymphocyte numbers is characteristic of some patients with hereditary hemochromatosis (HH), a MHC-linked disorder of iron overload. Both environmental and genetic components are known to influence CD8+ T-lymphocyte homeostasis but the role of the HH associated protein HFE is still insufficiently understood. Genome-wide expression profiling was performed in peripheral blood CD8+ T lymphocytes from HH patients selected according to CD8+ T-lymphocyte numbers and from Hfe-/- mice maintained either under normal or high iron diet conditions. In addition, T-lymphocyte apoptosis and cell cycle progression were analyzed by flow cytometry in HH patients. HH patients with low CD8+ T-lymphocyte numbers show a differential expression of genes related to lymphocyte differentiation and maturation namely CCR7, LEF1, ACTN1, NAA50, P2RY8 and FOSL2, whose expression correlates with the relative proportions of naïve, central and effector memory subsets. In addition, expression levels of LEF1 and P2RY8 in memory cells as well as the proportions of CD8+ T cells in G2/M cell cycle phase are significantly different in HH patients compared to controls. Hfe-/- mice do not show alterations in CD8+ T-lymphocyte numbers but differential gene response patterns. We found an increased expression of S100a8 and S100a9 that is most pronounced in high iron diet conditions. Similarly, CD8+ T lymphocytes from HH patients display higher S100a9 expression both at the mRNA and protein level. Altogether, our results support a role for HFE as a negative regulator of CD8+ T-lymphocyte activation. While the activation markers S100a8 and S100a9 are strongly increased in CD8+ T cells from both, Hfe-/- mice and HH patients, a differential profile of genes related to differentiation/maturation of CD8+ T memory cells is evident in HH patients only. This supports the notion that HFE contributes, at least in part, to the generation of low peripheral blood CD8+ T lymphocytes in HH.

  6. Lymphocyte Gene Expression Signatures from Patients and Mouse Models of Hereditary Hemochromatosis Reveal a Function of HFE as a Negative Regulator of CD8+ T-Lymphocyte Activation and Differentiation In Vivo

    PubMed Central

    Costa, Mónica; Cruz, Eugénia; Oliveira, Susana; Benes, Vladimir; Ivacevic, Tomi; Silva, Maria João; Vieira, Inês; Dias, Francisco; Fonseca, Sónia; Gonçalves, Marta; Lima, Margarida; Leitão, Catarina; Muckenthaler, Martina U.; Pinto, Jorge; Porto, Graça

    2015-01-01

    Abnormally low CD8+ T-lymphocyte numbers is characteristic of some patients with hereditary hemochromatosis (HH), a MHC-linked disorder of iron overload. Both environmental and genetic components are known to influence CD8+ T-lymphocyte homeostasis but the role of the HH associated protein HFE is still insufficiently understood. Genome-wide expression profiling was performed in peripheral blood CD8+ T lymphocytes from HH patients selected according to CD8+ T-lymphocyte numbers and from Hfe -/- mice maintained either under normal or high iron diet conditions. In addition, T-lymphocyte apoptosis and cell cycle progression were analyzed by flow cytometry in HH patients. HH patients with low CD8+ T-lymphocyte numbers show a differential expression of genes related to lymphocyte differentiation and maturation namely CCR7, LEF1, ACTN1, NAA50, P2RY8 and FOSL2, whose expression correlates with the relative proportions of naïve, central and effector memory subsets. In addition, expression levels of LEF1 and P2RY8 in memory cells as well as the proportions of CD8+ T cells in G2/M cell cycle phase are significantly different in HH patients compared to controls. Hfe -/- mice do not show alterations in CD8+ T-lymphocyte numbers but differential gene response patterns. We found an increased expression of S100a8 and S100a9 that is most pronounced in high iron diet conditions. Similarly, CD8+ T lymphocytes from HH patients display higher S100a9 expression both at the mRNA and protein level. Altogether, our results support a role for HFE as a negative regulator of CD8+ T-lymphocyte activation. While the activation markers S100a8 and S100a9 are strongly increased in CD8+ T cells from both, Hfe -/- mice and HH patients, a differential profile of genes related to differentiation/maturation of CD8+ T memory cells is evident in HH patients only. This supports the notion that HFE contributes, at least in part, to the generation of low peripheral blood CD8+ T lymphocytes in HH. PMID

  7. Alterations in T lymphocytes and T-lymphocyte subpopulations in patients with syphilis.

    PubMed Central

    Jensen, J R; From, E

    1982-01-01

    The distribution of T-lymphocyte subpopulations was studied in 34 patients with primary or secondary syphilis before and after treatment. An absolute and relative T lymphopenia was found in all patients. In primary syphilis the concentration of helper cells--T cells with Fc receptors for IgM (T mu)--was low whereas in secondary syphilis the suppressor cell concentration--T cells with Fc receptors for IgG (T gamma)--was reduced. Using lymphocytes from healthy subjects this could be imitated in vitro by the addition of serum from patients with secondary syphilis. In many autoimmune diseases a low concentration of T gamma may be a primary factor in the production of autoantibodies. The occurrence of similar changes in patients with secondary syphilis, however, indicates that such fluctuations in the T-cell subpopulations may take place during a strong immune response. PMID:6459815

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

    PubMed

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

    2013-07-01

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

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

    PubMed Central

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

    2011-01-01

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

  10. Metronomic cyclophosphamide eradicates large implanted GL261 gliomas by activating antitumor Cd8+ T-cell responses and immune memory

    PubMed Central

    Wu, Junjie; Waxman, David J

    2015-01-01

    Cancer chemotherapy using cytotoxic drugs can induce immunogenic tumor cell death; however, dosing regimens and schedules that enable single-agent chemotherapy to induce adaptive immune-dependent ablation of large, established tumors with activation of long-term immune memory have not been identified. Here, we investigate this issue in a syngeneic, implanted GL261 glioma model in immune-competent mice given cyclophosphamide on a 6-day repeating metronomic schedule. Two cycles of metronomic cyclophosphamide treatment induced sustained upregulation of tumor-associated CD8+ cytotoxic T lymphocyte (CTL) cells, natural killer (NK) cells, macrophages, and other immune cells. Expression of CTL- and NK–cell-shared effectors peaked on Day 6, and then declined by Day 9 after the second cyclophosphamide injection and correlated inversely with the expression of the regulatory T cell (Treg) marker Foxp3. Sustained tumor regression leading to tumor ablation was achieved after several cyclophosphamide treatment cycles. Tumor ablation required CD8+ T cells, as shown by immunodepletion studies, and was associated with immunity to re-challenge with GL261 glioma cells, but not B16-F10 melanoma or Lewis lung carcinoma cells. Rejection of GL261 tumor re-challenge was associated with elevated CTLs in blood and increased CTL infiltration in tumors, consistent with the induction of long-term, specific CD8+ T-cell anti-GL261 tumor memory. Co-depletion of CD8+ T cells and NK cells did not inhibit tumor regression beyond CD8+ T-cell depletion alone, suggesting that the metronomic cyclophosphamide-activated NK cells function via CD8a+ T cells. Taken together, these findings provide proof-of-concept that single-agent chemotherapy delivered on an optimized metronomic schedule can eradicate large, established tumors and induce long-term immune memory. PMID:26137402

  11. T-Cell Therapy Using Interleukin-21–Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results in Long-Term Cell Persistence and Durable Tumor Regression

    PubMed Central

    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

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

    PubMed

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

    2018-06-01

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

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

    PubMed Central

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

    1996-01-01

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

  14. Tracking in vivo migration and distribution of antigen-specific cytotoxic T lymphocytes by 5,6-carboxyfluorescein diacetate succinimidyl ester staining during cancer immunotherapy.

    PubMed

    Xu, Wei-li; Li, Suo-lin; Wen, Ming; Wen, Jun-ye; Han, Jie; Zhang, Hong-zhen; Gao, Fei; Cai, Jian-hui

    2013-08-01

    Killing of targeted tumors during adoptive cell transfer therapy is associated with cytotoxic T lymphocyte (CTL) numbers, immunophenotype, tumor-specificity, and in vivo residence time, migration, and distribution. Therefore, tracing in vivo persistence, migration, and distribution of CTLs is important for cancer immunotherapy. Optimal staining concentration for CTL proliferation was determined by cell counting kit-8 (CCK-8) assay and killing efficiencies of CTLs or carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled melanoma antigen-specific cytotoxic T lymphocytes (CFSE-CTLs) for malignant melanoma cells in vitro were compared. Additionally, CFSE-CTLs were intravenously transfused to mice receiving B16 melanoma, and their residence time, migration, and distribution in vivo were observed by measuring fluorescence intensities of CFSE-CTLs per gram of tissue (%FI/g) in various tissues and analyzing tumor/non-tumor (T/NT) values. Anti-tumor effects of transferred CTLs and correlation between %FI/g and D-value of tumor size were analyzed. Five-micromolar CFSE was optimal for labeling CTLs with minimal cytotoxicity. No significant difference occurred between CTLs and CFSE-CTLs for tumor cell killing (P = 0.849) or interleukin-2 (P = 0.318) and interferon-γ (P = 0.201) levels. Distribution of CTLs in vivo varied with time. A negative correlation between %FI/g in tumors and D-value of tumor sizes by Spearman correlation analysis was observed. CTLs were recruited to and killed tumors from 6 hours to 3 days after cell infusion. CTLs were observed up to three weeks later in the tumor, liver, kidneys, and spleen; this was related to the abundant blood supply or the nature of immune organs. CCK-8 assay is a novel method to select optimal CFSE staining concentrations. Fluorescence intensity of transferred CTLs reflects their killing efficiency of tumors. CFSE fluorescent markers can trace in vivo CTL persistence, migration, and distribution because of its stability

  15. Mitogenic signal transduction in T lymphocytes in microgravity

    NASA Technical Reports Server (NTRS)

    Cogoli, A.; Bechler, B.; Cogoli-Greuter, M.; Criswell, S. B.; Joller, H.; Joller, P.; Hunzinger, E.; Muller, O.

    1993-01-01

    The activation by concanavalin A Con A of human peripheral blood lymphocytes (PBLs) in the presence of monocytes as accessory cells was investigated in cultures exposed to microgravity conditions in Spacelab. Activation of T cells was measured as incorporation of [3H]thymidine into DNA, secretion of interleukin-2 (IL-2), and interferon-gamma, and expression of IL-2 receptors. Whereas, as discovered in earlier experiments, the activation of resuspended T cells is strongly inhibited, activation of cells attached to microcarrier beads is more than doubled in microgravity. The results suggest that the depression of the activation in resuspended cells may be attributed to a malfunction of monocytes acting as accessory cells. In fact, although the ultrastructure of resuspended monocytes is not altered in microgravity, the secretion of IL-1 is strongly inhibited. Our data suggest that (1) IL-2 is produced independently of IL-1, (2) IL-1 production is triggered only when monocytes (and lymphocytes?) adhere to microcarriers, (3) the expression of IL-2 receptors depends on IL-1, and (4) provided sufficient IL-1 is available, activation is enhanced in microgravity. Finally, cultures of resuspended PBLs and monocytes in microgravity constitute a complete and natural system in which monocytes are not operational. This may be useful for studies of the role of accessory cells and cell-cell interactions in T lymphocyte activation.

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

    PubMed Central

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

    1998-01-01

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

  17. T lymphocyte activation and cytokine expression in periapical granulomas and radicular cysts.

    PubMed

    Ihan Hren, N; Ihan, A

    2009-02-01

    Radicular cysts (RCs) are periapical lesions resulting in jaw bone destruction. The inflammatory dental periapical granuloma (PG) is considered to be the origin of RC formation; however the mechanism of RC development remains unclear. Cell suspension from the surgically extirpated tissue of 27 RCs and 25 PGs was obtained. Bacteriological analysis of the PG tissue samples was performed in order to define two major groups of PG according to the prevailing causative bacterial infection: the streptococcal PG (PG-S, n=10) and the anaerobe PG (PG-A, n=9) group. The inflammatory response of tissue infiltrating lymphocytes was assessed by following T lymphocyte activation (HLA-DR expression) as well as interferon gamma (IFN-gamma) and interleukin 4 (IL-4) production which were evaluated by the flow cytometry. In comparison to RC both types of PG contained a higher proportion of activated T cells (HLA-DR) and lower proportion of IL-4 producing cells. PG-A tissue contained increased percentage of CD3 cells and increased percentage of T helper 1 (Th1) cells in comparison with PG-S. In RC the IFN-gamma production is higher than in streptococcal PG-S but similar as in PG-A. Tissue infiltration by Th2 cells and IL-4 production is likely to play an etiopathogenic role in RC formation.

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

    PubMed

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

    2000-07-01

    Epstein-Barr virus (EBV)-induced lymphoproliferative disease and cytomegalovirus (CMV) infection are major causes of morbidity and mortality in individuals with compromised cellular immunity. Although anti-viral pharmacological agents exist, severe side effects such as myelosuppression often limit the application of these medications. Infusion of ex vivo-expanded, virus-specific cytotoxic T-lymphocytes (CTL) has been proven to be safe and efficacious for the prophylaxis and treatment of EBV and CMV complications. While EBV-specific CTL can be readily and reliably produced with EBV-immortalized B-lymphoblastoid cell lines (BLCL) as stimulators, current protocols for CMV-specific CTL, which use CMV-infected fibroblasts as stimulators, may be associated with alloreactivity and the need for cloning, as well as the potential for exposure to human blood-born infectious agents. Our laboratory has developed a novel system to generate EBV/CMV-bi-specific CTL by co-culturing PBMC with autologous BLCL expressing a CMV protein pp65 (BLCLpp65) (Sun et al., 1999). pp65, an immunodominant CMV antigen, is transduced into BLCL by a recombinant retrovirus MSCVpp65. While low in alloreactivity, BLCLpp65-stimulated CTL are cytolytic to autologous cells infected with EBV or CMV, and this cytotoxicity is mediated by polyclonal, CD8+, MHC Class I-restricted T-cells. Further experiments revealed that retroviral transduction and expression of pp65 do not compromise the capacity of presenting EBV antigens, and T cells stimulated by BLCLpp65 recognize clinical strains of CMV (Sun et al., 2000). These data indicated that BLCLpp65 could substitute for BLCL as antigen presenting cells in adoptive immunotherapy against EBV-LPD, with the benefit of providing protection against CMV reactivation. This protocol is a Phase I/II study to examine the toxicity associated with and the immunologic effects of ex vivo simultaneously expanded EBV- and CMV-specific CTL for prophylaxis against EBV and CMV

  19. Detecting specific cytotoxic T lymphocytes against SARS-coronavirus with DimerX HLA-A2:Ig fusion protein.

    PubMed

    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.

  20. A novel adoptive transfer model of chronic lymphocytic leukemia suggests a key role for T lymphocytes in the disease

    PubMed Central

    Bagnara, Davide; Kaufman, Matthew S.; Calissano, Carlo; Marsilio, Sonia; Patten, Piers E. M.; Simone, Rita; Chum, Philip; Yan, Xiao-Jie; Allen, Steven L.; Kolitz, Jonathan E.; Baskar, Sivasubramanian; Rader, Christoph; Mellstedt, Hakan; Rabbani, Hodjattallah; Lee, Annette; Gregersen, Peter K.; Rai, Kanti R.

    2011-01-01

    Chronic lymphocytic leukemia (CLL) is an incurable adult disease of unknown etiology. Understanding the biology of CLL cells, particularly cell maturation and growth in vivo, has been impeded by lack of a reproducible adoptive transfer model. We report a simple, reproducible system in which primary CLL cells proliferate in nonobese diabetes/severe combined immunodeficiency/γcnull mice under the influence of activated CLL-derived T lymphocytes. By cotransferring autologous T lymphocytes, activated in vivo by alloantigens, the survival and growth of primary CFSE-labeled CLL cells in vivo is achieved and quantified. Using this approach, we have identified key roles for CD4+ T cells in CLL expansion, a direct link between CD38 expression by leukemic B cells and their activation, and support for CLL cells preferentially proliferating in secondary lymphoid tissues. The model should simplify analyzing kinetics of CLL cells in vivo, deciphering involvement of nonleukemic elements and nongenetic factors promoting CLL cell growth, identifying and characterizing potential leukemic stem cells, and permitting preclinical studies of novel therapeutics. Because autologous activated T lymphocytes are 2-edged swords, generating unwanted graph-versus-host and possibly autologous antitumor reactions, the model may also facilitate analyses of T-cell populations involved in immune surveillance relevant to hematopoietic transplantation and tumor cytoxicity. PMID:21385850

  1. Graded versus Intermittent Exercise Effects on Lymphocytes in Chronic Fatigue Syndrome.

    PubMed

    Broadbent, Suzanne; Coutts, Rosanne

    2016-09-01

    There is increasing evidence of immune system dysfunction in chronic fatigue syndrome (CFS), but little is known of the regular exercise effects on immune cell parameters. This pilot study investigated the effects of graded and intermittent exercise on CD4 lymphocyte subset counts and activation compared with usual care. Twenty-four CFS patients (50.2 ± 10 yr) were randomized to graded exercise (GE), intermittent exercise (IE), or usual care (UC) groups; 18 sedentary non-CFS participants (50.6 ± 10 yr) were controls (CTL) for blood and immunological comparisons. Outcome measures were pre- and postintervention flow cytometric analyses of circulating lymphocyte subset cell counts; expression of CD3, CD4, CD25, and CD134; full blood counts; and V˙O2peak. Preintervention, CD3 cell counts, and expression of CD4, CD25, CD134, and CD4CD25CD134 were significantly lower in GE, IE, and UC compared with CTL (P < 0.05). Total lymphocyte concentration was significantly lower in GE and IE groups compared with CTL. There were significant postintervention increases in i) expression of CD4 and CD4CD25CD134 for GE and IE, but CD25 and CD134 for IE only; ii) circulating counts of CD3 and CD4 for GE, and CD3, CD4, CD8, CD3CD4CD8, CD3CD16CD56, CD19, and CD45 for IE; iii) neutrophil concentration for GE; and iv) V˙O2peak and elapsed test time for IE and GE, V˙Epeak for IE. Twelve weeks of GE and IE training significantly improved CD4 lymphocyte activation and aerobic capacity without exacerbating CFS symptoms. IE may be a more effective exercise modality with regard to enhanced CD4 activation in CFS patients.

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

    PubMed Central

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

    2003-01-01

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

  3. TAP, a novel T cell-activating protein involved in the stimulation of MHC-restricted T lymphocytes

    PubMed Central

    1986-01-01

    Five mAbs have been generated and used to characterize TAP (T cell activating protein) a novel, functional murine T cell membrane antigen. The TAP molecule is a 12-kD protein that is synthesized by T cells. By antibody crossblocking, it appears to be closely associated with a 16- kD protein on the T cell membrane also identified with a novel mAb. These molecules are clearly distinct from the major well-characterized murine T cell antigens previously described. Antibody binding to TAP can result in the activation of MHC-restricted, antigen-specific inducer T cell hybridomas that is equivalent in magnitude to maximal antigen or lectin stimulation. This is a direct effect of soluble antibody and does not require accessory cells or other factors. The activating anti-TAP mAbs are also mitogenic for normal heterogeneous T lymphocytes in the presence of accessory cells or IL-1. In addition, these antibodies are observed to modulate specific immune stimulation. Thus, the activating anti-TAP mAbs synergise with antigen-specific stimulation of T cells, while a nonactivating anti-TAP mAb inhibits antigen driven activation. These observations suggest that the TAP molecule may participate in physiologic T cell activation. The possible relationship of TAP to known physiologic triggering structures, the T3- T cell receptor complex, is considered. TAP is expressed on 70% of peripheral T cells and therefore defines a major T cell subset, making it perhaps the first example of a murine subset-specific activating protein. PMID:2418146

  4. Herpes simplex virus antigens directly activate NK cells via TLR2, thus facilitating their presentation to CD4 T lymphocytes.

    PubMed

    Kim, Min; Osborne, Naomi R; Zeng, Weiguang; Donaghy, Heather; McKinnon, Kay; Jackson, David C; Cunningham, Anthony L

    2012-05-01

    NK cells infiltrate human herpetic lesions, but their role has been underexplored. HSV can stimulate innate immune responses via surface TLR2, which is expressed on monocyte-derived dendritic cells (DCs) and NK cells. In this study, UV-inactivated HSV1/2 and immunodominant HSV2 glycoprotein D peptides conjugated to the TLR2 agonist dipalmitoyl-S-glyceryl cysteine stimulated CD4 T lymphocyte IFN-γ responses within PBMCs or in coculture with monocyte-derived DCs. NK cells contributed markedly to the PBMC responses. Furthermore, NK cells alone were activated directly by both Ags, also upregulating HLA-DR and HLA-DQ and then they activated autologous CD4 T lymphocytes. Using Transwells, Ag-stimulated NK cells and CD4 T lymphocytes were shown to interact through both cell-to-cell contact and cytokines, differing in relative importance in different donors. A distinct immunological synapse between Ag-stimulated NK cells and CD4 T lymphocytes was observed, indicating the significance of their cell-to-cell contact. A large proportion (57%) of NK cells was also in contact with CD4 T lymphocytes in the dermal infiltrate of human recurrent herpetic lesions. Thus, NK cells stimulated by TLR2-activating HSV Ags can present Ag alone or augment the role of DCs in vitro and perhaps in herpetic lesions or draining lymph nodes. In addition to DCs, NK cells should be considered as targets for adjuvants during HSV vaccine development.

  5. Investigation of the immunosuppressive activity of Physalin H on T lymphocytes.

    PubMed

    Yu, Youjun; Sun, Lijuan; Ma, Lei; Li, Jiyu; Hu, Lihong; Liu, Jianwen

    2010-03-01

    Physalis angulata is an annual herb widely used in folk medicine. It is mainly used for treating rheumatoid arthritis (RA). Following bioactivity-guided isolation, a representative immunosuppressive compound, Physalin H was been identified from this herb medicine. The purpose of this work was to assess the immunosuppressive activity of Physalin H on T cells and to explore its potential mode of action. The results showed that Physalin H in a dose-dependent manner significantly inhibited the proliferation of T cells induced by concanavalin A (ConA) and by the mixed lymphocyte culture reaction (MLR). This inhibitive activity was mainly due to interfering DNA replication in G1 stages. In vivo experiments showed that, administration of Physalin H dose-dependently suppressed CD4(+) T cell mediated delayed-type hypersensitivity (DTH) reactions, and suppressed antigen-specific T-cell response in ovalbumin (OVA) immunized mice. Further study indicated that Physalin H could modulate Th1/Th2 cytokine balance and induce the production of immune regulation target Heme oxygenase (HO)-1 in T-cells in vitro. In this study, we demonstrated the immunosuppressive effect of Physalin H on T cells both in vitro and in vivo, and the immunosuppressive activity might be attributed to the suppression of T cell activation and proliferation, the modulation of Th1/Th2 cytokine balance and the induction of HO-1 in T cells. Copyright 2009 Elsevier B.V. All rights reserved.

  6. Epstein-Barr virus-transformed B-cells as efficient antigen presenting cells to propagate Aspergillus-specific cytotoxic T-lymphocytes.

    PubMed

    Ramadan, Gamal

    2008-01-01

    To overcome the cytotoxic T-lymphocytes (CTL) expansion limitations imposed by the lack of sufficient dendritic cells (DC) alternative sources of autologous antigen presenting cells (APC) such as Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (BLCL), which are easy to establish in vitro, have been considered and studied in the present work. Non-adherent peripheral blood mononuclear cells of three healthy donors were repeatedly primed with autologous Aspergillus fumigatus commercial culture-filtrate antigen-pulsed fast monocyte-derived DC (Aspf-CFA-DC) alone, Aspf-CFA-pulsed BLCL (Aspf-CFA-BLCL) alone or Aspf-CFA-BLCL after one, two, or three primings with Aspf-CFA-DC (1DC/BLCL, 2DC/BLCL or 3DCIBLCL; respectively). After 5th priming, lines generated by Aspf-CFA-BLCL only showed strong/weak lytic activity for EBV/Aspf; respectively. Aspf-specific lytic activity in all donors was increased by increasing the number of primings with Aspf-CFA-DC before switching to Aspf-CFA-BLCL (18.20 +/- 1.65% versus 35.67 +/- 1.02% and 40.03 +/- 1.41% in bulk cultures generated by 1DC/BLCL versus 2DC/BLCL and 3DC/BLCL, respectively). Bulk cultures generated by Aspf-CFA-BLCL after at least two primings with Aspf-CFA-DC showed approximately the same Aspf-specific lytic activity, effector cell phenotype, expansion level and percentage expression of IFN-gamma, CD69 and CD107a without any significant differences (p > 0.05) as standard bulk cultures generated by only Aspf-CFA-DC. Thus, this study explored the use of a combined DC/BLCL protocol to establish/propagate Aspf-specific CTL for adoptive immunotherapy to prevent or treat invasive pulmonary aspergillosis.

  7. Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

    PubMed

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

    2003-10-01

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

  8. CXCL4-induced migration of activated T lymphocytes is mediated by the chemokine receptor CXCR3.

    PubMed

    Mueller, Anja; Meiser, Andrea; McDonagh, Ellen M; Fox, James M; Petit, Sarah J; Xanthou, Georgina; Williams, Timothy J; Pease, James E

    2008-04-01

    The chemokine CXCL4/platelet factor-4 is released by activated platelets in micromolar concentrations and is a chemoattractant for leukocytes via an unidentified receptor. Recently, a variant of the human chemokine receptor CXCR3 (CXCR3-B) was described, which transduced apoptotic but not chemotactic signals in microvascular endothelial cells following exposure to high concentrations of CXCL4. Here, we show that CXCL4 can induce intracellular calcium release and the migration of activated human T lymphocytes. CXCL4-induced chemotaxis of T lymphocytes was inhibited by a CXCR3 antagonist and pretreatment of cells with pertussis toxin (PTX), suggestive of CXCR3-mediated G-protein signaling via Galphai-sensitive subunits. Specific binding by T lymphocytes of the CXCR3 ligand CXCL10 was not effectively competed by CXCL4, suggesting that the two are allotopic ligands. We subsequently used expression systems to dissect the potential roles of each CXCR3 isoform in mediating CXCL4 function. Transient expression of the CXCR3-A and CXCR3-B isoforms in the murine pre-B cell L1.2 produced cells that migrated in response to CXCL4 in a manner sensitive to PTX and a CXCR3 antagonist. Binding of radiolabeled CXCL4 to L1.2 CXCR3 transfectants was of low affinity and appeared to be mediated chiefly by glycosaminoglycans (GAGs), as no specific CXCL4 binding was observed in GAG-deficient 745-Chinese hamster ovary cells stably expressing CXCR3. We suggest that following platelet activation, the CXCR3/CXCL4 axis may play a role in T lymphocyte recruitment and the subsequent amplification of inflammation observed in diseases such as atherosclerosis. In such a setting, antagonism of the CXCR3/CXCL4 axis may represent a useful, therapeutic intervention.

  9. Cytotoxic T Lymphocyte Trafficking and Survival in an Augmented Fibrin Matrix Carrier

    PubMed Central

    Zou, Zhaoxia; Denny, Erin; Brown, Christine E.; Jensen, Michael C.; Li, Gang; Fujii, Tatsuhiro; Neman, Josh; Jandial, Rahul; Chen, Mike

    2012-01-01

    Cell-based therapies have intriguing potential for the treatment of a variety of neurological disorders. One such example is genetically engineered cytotoxic T lymphocytes (CTLs) that are being investigated in brain tumor clinical trials. The development of methods for CTL delivery is critical to their use in the laboratory and clinical setting. In our study, we determined whether CTLs can migrate through fibrin matrices and if their migration, survival, and function could be modulated by adding chemokines to the matrix. Our results indicated that CTLs can freely migrate through fibrin matrices. As expected, the addition of the monocyte chemotactic protein-1 (MCP-1), also known as chemokine C-C motif ligand 2 (CCL2), to the surrounding media increased egress of the CTLs out of the fibrin clot. Interleukin (IL) -2 and/or IL-15 embedded in the matrix enhanced T cell survival and further promoted T cell migration. The interleukin-13 receptor alpha 2 specific (IL-13R alpha2) T cells that traveled out of the fibrin clot retained the capacity to kill U251 glioma cells. In summary, CTLs can survive and migrate robustly in fibrin matrices. These processes can be influenced by modification of matrix constituents. We conclude that fibrin matrices may be suitable T cell carriers and can be used to facilitate understanding of T cell interaction with the surrounding microenvironment. PMID:22496835

  10. Cytotoxic T lymphocyte trafficking and survival in an augmented fibrin matrix carrier.

    PubMed

    Zou, Zhaoxia; Denny, Erin; Brown, Christine E; Jensen, Michael C; Li, Gang; Fujii, Tatsuhiro; Neman, Josh; Jandial, Rahul; Chen, Mike

    2012-01-01

    Cell-based therapies have intriguing potential for the treatment of a variety of neurological disorders. One such example is genetically engineered cytotoxic T lymphocytes (CTLs) that are being investigated in brain tumor clinical trials. The development of methods for CTL delivery is critical to their use in the laboratory and clinical setting. In our study, we determined whether CTLs can migrate through fibrin matrices and if their migration, survival, and function could be modulated by adding chemokines to the matrix. Our results indicated that CTLs can freely migrate through fibrin matrices. As expected, the addition of the monocyte chemotactic protein-1 (MCP-1), also known as chemokine C-C motif ligand 2 (CCL2), to the surrounding media increased egress of the CTLs out of the fibrin clot. Interleukin (IL) -2 and/or IL-15 embedded in the matrix enhanced T cell survival and further promoted T cell migration. The interleukin-13 receptor alpha 2 specific (IL-13R alpha2) T cells that traveled out of the fibrin clot retained the capacity to kill U251 glioma cells. In summary, CTLs can survive and migrate robustly in fibrin matrices. These processes can be influenced by modification of matrix constituents. We conclude that fibrin matrices may be suitable T cell carriers and can be used to facilitate understanding of T cell interaction with the surrounding microenvironment.

  11. Inventory of File sref_nmb.t03z.pgrb212.ctl.grib2

    Science.gov Websites

    10 m above ground UGRD 3 hour fcst U-Component of Wind [m/s] ENS=low-res ctl 004 10 m above ground VGRD 3 hour fcst V-Component of Wind [m/s] ENS=low-res ctl 005 mean sea level PRMSL 3 hour fcst 250 mb UGRD 3 hour fcst U-Component of Wind [m/s] ENS=low-res ctl 014 500 mb UGRD 3 hour fcst U

  12. T cell chronic lymphocytic leukaemia with suppressor phenotype.

    PubMed Central

    Hofman, F M; Smith, D; Hocking, W

    1982-01-01

    The peripheral blood cells from a patient with T cell chronic lymphocytic leukaemia were examined for surface marker and functional characteristics. Eighty-91% of the peripheral blood cells formed SRBC rosettes and 22-49% possessed Fc receptors; 73% of the peripheral blood cells were reactive with the OKT8 antiserum and 61% expressed DR antigens. Response to PHA stimulation was markedly reduced, whereas allogeneic responsiveness in mixed leucocyte culture was intact. The ability of Con A-stimulated peripheral blood cells to generate suppressor activity in a mixed leucocyte reaction was deficient, whereas suppression of in vitro immunoglobulin synthesis was greater than normal. The leukaemic peripheral blood cell population expressed a T suppressor phenotype. Functional studies suggest that these cells were derived from the subset of T lymphocytes with regulatory activity for immunoglobulin synthesis as opposed to mitogenic responsiveness. PMID:6215199

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

    PubMed Central

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

    2001-01-01

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

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

    PubMed

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

    2003-01-17

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

  15. T-lymphocyte populations following a period of high volume training in female soccer players.

    PubMed

    Brown, F F; Bigley, A B; Ross, J C; LaVoy, E C; Simpson, R J; Galloway, S D R

    2015-12-01

    To investigate the T-lymphocyte response to a period of increased training volume in trained females compared to habitual activity in female controls. Thirteen trained female (19.8 ± 1.9 yrs) soccer players were monitored during a two-week long high volume training period (increased by 39%) and thirteen female untrained (20.5 ± 2.2 yrs) controls were monitored during two-weeks of habitual activity. Blood lymphocytes, collected at rest, were isolated before and after the two-week period. Isolated lymphocytes were assessed for the cell surface expression of the co-receptor CD28, a marker of T-lymphocyte naivety, and CD57 a marker used to identify highly-differentiated T-lymphocytes. Co-expression of these markers was identified on helper CD4(+) and cytotoxic CD8(+) T-lymphocytes. In addition a further population of γδ(+) T-lymphocytes were identified. Plasma was used to determine Cytomegalovirus (CMV) serostatus. No difference was observed in the T-lymphocyte populations following the two-week period of increased volume training. At baseline the number of total CD3(+), cytotoxic CD8(+), naïve (CD8(+) CD28(+) CD57(-)), intermediate (CD8(+) CD28(+) CD57(+)) T-lymphocytes and the number and proportion of γδ(+) T-lymphocytes were greater in the trained compared to the untrained females (p<0.05). The proportion of CD4(+)T-lymphocytes was greater in the untrained compared to the trained (p<0.05), in turn the CD4(+):CD8(+) ratio was also greater in the untrained females (p<0.05). Inclusion of percentage body fat as a covariate removed the main effect of training status in all T-lymphocyte sub-populations, with the exception of the γδ(+) T-lymphocyte population. 8% of the untrained group was defined as positive for CMV whereas 23% of the trained group was positive for CMV. However, CMV was not a significant covariate in the analysis of T-lymphocyte proportions. The period of high volume training had no effect on T-lymphocyte populations in trained females. However

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

    PubMed

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

    2013-12-01

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

  17. Efficient Identification of Novel Hla-A*0201–Presented Cytotoxic T Lymphocyte Epitopes in the Widely Expressed Tumor Antigen Prame by Proteasome-Mediated Digestion Analysis

    PubMed Central

    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

  18. Association of neutrophil-lymphocyte ratio and T lymphocytes with the pathogenesis and progression of HBV-associated primary liver cancer

    PubMed Central

    Han, Junyan; Wang, Lijia; Li, Mengge; Jiang, Yuyong; Wang, Xianbo; Yang, Zhiyun

    2017-01-01

    Background The neutrophil–lymphocyte ratio (NLR) is a new prognostic predictor for patients with liver cancer. The association of NLR and T lymphocytes with the pathogenesis and progression of liver cancer is poorly understood. Methods Seventy-three patients with hepatitis B virus (HBV)-associated primary liver cancer (HBV-PLC), 50 patients with HBV-associated liver cirrhosis (HBV-LC) and 37 patients with chronic HBV infection (CHB) were prospectively enrolled from July 1, 2013 to February 28, 2014 in Beijing Ditan Hospital, Capital Medical University (Beijing, China). The NLR, proportions and concentrations of neutrophils and lymphocytes, concentration of subpopulations of lymphocytes, and the expression of CD31 (index for recent thymic output) and HLA-DR (index for activation of T lymphocytes) of T cells in the peripheral blood samples of the patients were assessed and statistically compared between different groups. Results The NLR was significantly increased from patients with CHB, those with HBV-LC to those with HBV-PLC (P<0.05), along with significant increase of neutrophils and decrease of lymphocytes in the same way (P<0.05). The concentrations of T lymphocytes, natural killer cells, B cells, CD4+ T cells and CD8+ T cells were decreased from patients with CHB, those with HBV-LC to those with HBV-PLC, and were significantly reduced in patients with HBV-PLC compared with those in patients with CHB (P<0.05). The CD31 and HLA-DR expression of naive CD4+ and CD8+ T cells was significantly decreased and increased, respectively in patients with HBV-PLC compared with that in patients with CHB. Conclusions Elevated NLR, resulted from the increase of neutrophils and decrease of lymphocytes, is positively associated with the pathogenesis and progression of HBV-PLC. The reduced thymic output and hyperactivation of T lymphocytes may contribute to the decrease of T lymphocytes, which could be also related to the pathogenesis of HBV-PLC. PMID:28231294

  19. Randomized, controlled trial of TNF-α antagonist in CTL-mediated severe cutaneous adverse reactions.

    PubMed

    Wang, Chuang-Wei; Yang, Lan-Yan; Chen, Chun-Bing; Ho, Hsin-Chun; Hung, Shuen-Iu; Yang, Chih-Hsun; Chang, Chee-Jen; Su, Shih-Chi; Hui, Rosaline Chung-Yee; Chin, See-Wen; Huang, Li-Fang; Lin, Yang Yu-Wei; Chang, Wei-Yang; Fan, Wen-Lang; Yang, Chin-Yi; Ho, Ji-Chen; Chang, Ya-Ching; Lu, Chun-Wei; Chung, Wen-Hung

    2018-03-01

    Cytotoxic T lymphocyte-mediated (CTL-mediated) severe cutaneous adverse reactions (SCARs), including Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), are rare but life-threatening adverse reactions commonly induced by drugs. Although high levels of CTL-associated cytokines, chemokines, or cytotoxic proteins, including TNF-α and granulysin, were observed in SJS-TEN patients in recent studies, the optimal treatment for these diseases remains controversial. We aimed to evaluate the efficacy, safety, and therapeutic mechanism of a TNF-α antagonist in CTL-mediated SCARs. We enrolled 96 patients with SJS-TEN in a randomized trial to compare the effects of the TNF-α antagonist etanercept versus traditional corticosteroids. Etanercept improved clinical outcomes in patients with SJS-TEN. Etanercept decreased the SCORTEN-based predicted mortality rate (predicted and observed rates, 17.7% and 8.3%, respectively). Compared with corticosteroids, etanercept further reduced the skin-healing time in moderate-to-severe SJS-TEN patients (median time for skin healing was 14 and 19 days for etanercept and corticosteroids, respectively; P = 0.010), with a lower incidence of gastrointestinal hemorrhage in all SJS-TEN patients (2.6% for etanercept and 18.2% for corticosteroids; P = 0.03). In the therapeutic mechanism study, etanercept decreased the TNF-α and granulysin secretions in blister fluids and plasma (45.7%-62.5% decrease after treatment; all P < 0.05) and increased the Treg population (2-fold percentage increase after treatment; P = 0.002), which was related to mortality in severe SJS-TEN. The anti-TNF-α biologic agent etanercept serves as an effective alternative for the treatment of CTL-mediated SCARs. ClinicalTrials.gov NCT01276314. Ministry of Science and Technology of Taiwan.

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

    PubMed Central

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

    2015-01-01

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

  1. Oxidized lipids enhance RANKL production by T lymphocytes: implications for lipid-induced bone loss.

    PubMed

    Graham, Lucia S; Parhami, Farhad; Tintut, Yin; Kitchen, Christina M R; Demer, Linda L; Effros, Rita B

    2009-11-01

    Osteoporosis is a systemic disease that is associated with increased morbidity, mortality and health care costs. Whereas osteoclasts and osteoblasts are the main regulators of bone homeostasis, recent studies underscore a key role for the immune system, particularly via activation-induced T lymphocyte production of receptor activator of NFkappaB ligand (RANKL). Well-documented as a mediator of T lymphocyte/dendritic cell interactions, RANKL also stimulates the maturation and activation of bone-resorbing osteoclasts. Given that lipid oxidation products mediate inflammatory and metabolic disorders such as osteoporosis and atherosclerosis, and since oxidized lipids affect several T lymphocyte functions, we hypothesized that RANKL production might also be subject to modulation by oxidized lipids. Here, we show that short term exposure of both unstimulated and activated human T lymphocytes to minimally oxidized low density lipoprotein (LDL), but not native LDL, significantly enhances RANKL production and promotes expression of the lectin-like oxidized LDL receptor-1 (LOX-1). The effect, which is also observed with 8-iso-Prostaglandin E2, an inflammatory isoprostane produced by lipid peroxidation, is mediated via the NFkappaB pathway, and involves increased RANKL mRNA expression. The link between oxidized lipids and T lymphocytes is further reinforced by analysis of hyperlipidemic mice, in which bone loss is associated with increased RANKL mRNA in T lymphocytes and elevated RANKL serum levels. Our results suggest a novel pathway by which T lymphocytes contribute to bone changes, namely, via oxidized lipid enhancement of RANKL production. These findings may help elucidate clinical associations between cardiovascular disease and decreased bone mass, and may also lead to new immune-based approaches to osteoporosis.

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

    PubMed

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

    2006-01-01

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

  3. In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor.

    PubMed

    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.

  4. In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor

    PubMed Central

    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

  5. Clonal Dominance among T-Lymphocyte Infiltrates in Arthritis

    NASA Astrophysics Data System (ADS)

    Stamenkovic, Ivan; Stegagno, Michele; Wright, Kathryn A.; Krane, Stephen M.; Amento, Edward P.; Colvin, Robert B.; Duquesnoy, Rene J.; Kurnick, James T.

    1988-02-01

    Synovial membranes in patients with rheumatoid arthritis as well as other types of chronic destructive inflammatory arthritis contain infiltrates of activated T lymphocytes that probably contribute to the pathogenesis of the disease. In an effort to elucidate the nature of these infiltrates, interleukin 2 (IL-2)-responsive T lymphocytes were grown out of synovial fragments from 14 patients undergoing surgery for advanced destructive inflammatory joint disease. Eleven of the samples examined were from patients with classical rheumatoid arthritis, while three others were obtained from individuals with clinical osteoarthritis. Southern blot analysis of T-cell receptor (TCR) β -chain genes in 13 of 14 cultures showed distinct rearrangements, indicating that each culture was characterized by the predominance of a limited number of clones. T-cell populations from peripheral blood stimulated with a variety of activators and expanded with IL-2 did not demonstrate evidence of similar clonality in long-term culture. These results suggest that a limited number of activated T-cell clones predominate at the site of tissue injury in rheumatoid synovial membranes as well as in other types of destructive inflammatory joint disease. Further characterization of these T-cell clones may aid our understanding of the pathogenesis of these rheumatic disorders.

  6. Clonal dominance among T-lymphocyte infiltrates in arthritis

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

    Stamenkovic, I.; Stegagno, M.; Wright, K.A.

    1988-02-01

    Synovial membranes in patients with rheumatoid arthritis as well as other types of chronic destructive inflammatory arthritis contain infiltrates of activated T lymphocytes that probably contribute to the pathogenesis of the disease. In an effort to elucidate the nature of these infiltrates, interleukin 2 (IL-2)-responsive T lymphocytes were grown out of synovial fragments from 14 patients undergoing surgery for advanced destructive inflammatory joint disease. Eleven of the samples examined were from patients with classical rheumatoid arthritis, while three others were obtained from individuals with clinical osteoarthritis. Southern blot analysis of T-cell receptor (TCR) ..beta..-chain genes in 13 of 14 culturesmore » showed distinct rearrangements, indicating that each culture was characterized by the predominance of a limited number of clones. T-cell populations from peripheral blood stimulated with a variety of activators and expanded with IL-2 did not demonstrate evidence of similar clonality in long-term culture. These results suggest that a limited number of activated T-cell clones predominate at the site of tissue injury in rheumatoid synovial membranes as well as in other types of destructive inflammatory joint disease. Further characterization of these T-cell clones may aid our understanding of the pathogenesis of these rheumatic disorders.« less

  7. Antithymocyte globulins in renal transplantation-from lymphocyte depletion to lymphocyte activation: The doubled-edged sword.

    PubMed

    Bamoulid, Jamal; Crépin, Thomas; Courivaud, Cécile; Rebibou, Jean-Michel; Saas, Philippe; Ducloux, Didier

    2017-07-01

    Compelling data suggest that lymphocyte depletion following T cell depleting therapy may induce prolonged CD4 T cell lymphopenia and trigger lymphocyte activation in some patients. These profound and non-reversible immune changes in T cell pool subsets are the consequence of both impaired thymic renewal and peripheral homeostatic proliferation. Chronic viral challenges by CMV play a major role in these immune alterations. Even when the consequences of CD4 T cell lymphopenia have been now well described, recent studies shed new light on the clinical consequences of immune activation. In this review, we will first focus on the mechanisms involved in T cell pool reconstitution after T cell depletion and further consider the clinical consequences of ATG-induced T cell activation and senescence in renal transplant recipients. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Generation of B-cell chronic lymphocytic leukemia (B-CLL)-reactive T-cell lines and clones from HLA class I-matched donors using modified B-CLL cells as stimulators: implications for adoptive immunotherapy.

    PubMed

    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.

  9. [Activity of non-specific T-suppressors regulating the proliferation of B- and T-lymphocytes in patients with fibroadenomatosis, breast cancer and stomach cancer].

    PubMed

    Grinevich, Iu A; Drannik, G N; Nikol'skiĭ, I S; Kalinina, N A; Litvishchenko, E I

    1984-01-01

    A decrease in proliferative rate of blood-circulating lymphocytes in response to LPS and PHA was registered in patients with fibroadenomatosis and cancer of the breast and stomach cancer. The said cells preincubated with Concanavalin A showed a weak inhibitory action on B-cells. The inhibition of T-cell proliferation by lymphocytes either remained unchanged or became less apparent in stage II breast cancer and slightly increased in stage III gastric cancer. Since no correlation was established between proliferative levels of T- and B-lymphocytes, two separate subpopulations of non-specific lymphocytes (T-T and T-B) were suggested.

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

    PubMed Central

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

    2008-01-01

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

  11. beta2-Microglobulin production by highly purified human T and B lymphocytes in cell culture stimulated with various mitogens.

    PubMed

    Kin, K; Kasahara, T; Itoh, Y; Sakurabayashi, I; Kawai, T; Morita, M

    1979-01-01

    This study attempts to evaluate beta2-microglobulin production by highly purified (greater than 98%) peripheral and tonsil T and B lymphocytes cultured with various mitogens. beta2-Microglobulin was measured by the radioimmunoassay method. It was found that PHA and Con A markedly stimulated beta2-microglobulin production in cultures of T but not B lymphocytes. B lymphocytes were greatly activated, on the other hand, by Staphylococcus aureau Cowan I organisms cSpA), though the level of beta2-microglobulin production was less than that observed in PHA- and Con A-stimulated T lymphocytes. PWM only slightly increased beta2-microglobulin production of T lymphocytes, although the incorporation of [3H]-thymidine was highly enhanced. The highest level of beta2-microglobulin obtained with PHA or Con A was observed when the T/B lymphocyte ratio was between 90/10 and 80/20. These results lead to the conclusion that: (1) SpA is a specific mitogen for B lymphocytes, and its mitogenicity is independent of the presence of T lymphocytes, while PHA, Con A, and PWM are ineffective as stimulants of B lymphocytes; (2) the beta2-microglobulin producing ability of B lymphocytes is less than that of T lymphocytes, even when the lymphocytes are markedly activated; (3) the beta2-microglobulin production and DNA synthesis by T lymphocytes is markedly enhanced by the helper effect of B lymphocytes; (4) the level of beta2-microglobulin production reflects lymphocyte activation, especially in T lymphocytes stimulated with PHA or Con A.

  12. In Utero Exposure to Histological Chorioamnionitis Primes the Exometabolomic Profiles of Preterm CD4+ T Lymphocytes.

    PubMed

    Matta, Poojitha; Sherrod, Stacy D; Marasco, Christina C; Moore, Daniel J; McLean, John A; Weitkamp, Joern-Hendrik

    2017-11-01

    Histological chorioamnionitis (HCA) is an intrauterine inflammatory condition that increases the risk for preterm birth, death, and disability because of persistent systemic and localized inflammation. The immunological mechanisms sustaining this response in the preterm newborn remain unclear. We sought to determine the consequences of HCA exposure on the fetal CD4 + T lymphocyte exometabolome. We cultured naive CD4 + T lymphocytes from HCA-positive and -negative preterm infants matched for gestational age, sex, race, prenatal steroid exposure, and delivery mode. We collected conditioned media samples before and after a 6-h in vitro activation of naive CD4 + T lymphocytes with soluble staphylococcal enterotoxin B and anti-CD28. We analyzed samples by ultraperformance liquid chromatography ion mobility-mass spectrometry. We determined the impact of HCA on the CD4 + T lymphocyte exometabolome and identified potential biomarker metabolites by multivariate statistical analyses. We discovered that: 1) CD4 + T lymphocytes exposed to HCA exhibit divergent exometabolomic profiles in both naive and activated states; 2) ∼30% of detected metabolites differentially expressed in response to activation were unique to HCA-positive CD4 + T lymphocytes; 3) metabolic pathways associated with glutathione detoxification and tryptophan degradation were altered in HCA-positive CD4 + T lymphocytes; and 4) flow cytometry and cytokine analyses suggested a bias toward a T H 1-biased immune response in HCA-positive samples. HCA exposure primes the neonatal adaptive immune processes by inducing changes to the exometabolomic profile of fetal CD4 + T lymphocytes. These exometabolomic changes may link HCA exposure to T H 1 polarization of the neonatal adaptive immune response. Copyright © 2017 by The American Association of Immunologists, Inc.

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

    PubMed Central

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

    2009-01-01

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

  14. Activation of microcarrier-attached lymphocytes in microgravity

    NASA Technical Reports Server (NTRS)

    Bechler, B.; Cogoli, A.; Cogoli-Greuter, M.; Muller, O.; Hunzinger, E.; Criswell, S. B.

    1992-01-01

    A technology has been developed to achieve optimal attachment of adhesion-independent lymphocytes to microcarrier beads. The activation of T-lymphocytes by concanavalin A was tested under microgravity conditions in an experiment carried out in space during the first Spacelab Life Science Mission. Activation, measured as the synthesis of deoxyribonucleic acid (DNA) and the production of interferon-gamma, more than doubled in attached lymphocytes in microgravity. The depression of the activation discovered in previous space experiments is due to an impairment not of the lymphocyte but of the macrophage function. The system described here may be useful for radiobiological investigations on the effect of high-energy particles and for testing the efficiency of the immune system in humans during the long-duration space flight planned in the future. The biotechnological significance of the increased lymphokine production in space remains to be assessed.

  15. An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations[S

    PubMed Central

    Romano, Maria; Di Taranto, Maria Donata; Mirabelli, Peppino; D'Agostino, Maria Nicoletta; Iannuzzi, Arcangelo; Marotta, Gennaro; Gentile, Marco; Raia, Maddalena; Di Noto, Rosa; Del Vecchio, Luigi; Rubba, Paolo; Fortunato, Giuliana

    2011-01-01

    The main causes of familial hypercholesterolemia (FH) are mutations in LDL receptor (LDLR) gene. Functional studies are necessary to demonstrate the LDLR function impairment caused by mutations and would be useful as a diagnostic tool if they allow discrimination between FH patients and controls. In order to identify the best method to detect LDLR activity, we compared continuous Epstein-Barr virus (EBV)-transformed B-lymphocytes and mitogen stimulated T-lymphocytes. In addition, we characterized both novel and known mutations in the LDLR gene. T-lymphocytes and EBV-transformed B-lymphocytes were obtained from peripheral blood of 24 FH patients and 24 control subjects. Functional assays were performed by incubation with fluorescent LDL followed by flow cytometry analysis. Residual LDLR activity was calculated normalizing fluorescence for the mean fluorescence of controls. With stimulated T-lymphocytes we obtained a better discrimination capacity between controls and FH patients compared with EBV-transformed B-lymphocytes as demonstrated by receiver operating characteristic (ROC) curve analysis (the areas under the curve are 1.000 and 0.984 respectively; P < 0.0001 both). The characterization of LDLR activity through T-lymphocytes is more simple and faster than the use of EBV-transformed B-lymphocytes and allows a complete discrimination between controls and FH patients. Therefore the evaluation of residual LDLR activity could be helpful not only for mutation characterization but also for diagnostic purposes. PMID:21865347

  16. Transcutaneous immunization with a novel imiquimod nanoemulsion induces superior T cell responses and virus protection.

    PubMed

    Lopez, Pamela Aranda; Denny, Mark; Hartmann, Ann-Kathrin; Alflen, Astrid; Probst, Hans Christian; von Stebut, Esther; Tenzer, Stefan; Schild, Hansjörg; Stassen, Michael; Langguth, Peter; Radsak, Markus P

    2017-09-01

    Transcutaneous immunization (TCI) is a novel vaccination strategy utilizing the skin associated lymphatic tissue to induce immune responses. TCI using a cytotoxic T lymphocyte (CTL) epitope and the Toll-like receptor 7 (TLR7) agonist imiquimod mounts strong CTL responses by activation and maturation of skin-derived dendritic cells (DCs) and their migration to lymph nodes. However, TCI based on the commercial formulation Aldara only induces transient CTL responses that needs further improvement for the induction of durable therapeutic immune responses. Therefore we aimed to develop a novel imiquimod solid nanoemulsion (IMI-Sol) for TCI with superior vaccination properties suited to induce high quality T cell responses for enhanced protection against infections. TCI was performed by applying a MHC class I or II restricted epitope along with IMI-Sol or Aldara (each containing 5% Imiquimod) on the shaved dorsum of C57BL/6, IL-1R, Myd88, Tlr7 or Ccr7 deficient mice. T cell responses as well as DC migration upon TCI were subsequently analyzed by flow cytometry. To determine in vivo efficacy of TCI induced immune responses, CTL responses and frequency of peptide specific T cells were evaluated on day 8 or 35 post vaccination and protection in a lymphocytic choriomeningitis virus (LCMV) infection model was assessed. TCI with the imiquimod formulation IMI-Sol displayed equal skin penetration of imiquimod compared to Aldara, but elicited superior CD8 + as well as CD4 + T cell responses. The induction of T-cell responses induced by IMI-Sol TCI was dependent on the TLR7/MyD88 pathway and independent of IL-1R. IMI-Sol TCI activated skin-derived DCs in skin-draining lymph nodes more efficiently compared to Aldara leading to enhanced protection in a LCMV infection model. Our data demonstrate that IMI-Sol TCI can overcome current limitations of previous imiquimod based TCI approaches opening new perspectives for transcutaneous vaccination strategies and allowing the use of this

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

    PubMed

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

    2013-10-01

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

  18. Differential Inhibition of T Lymphocyte Proliferation and Cytokine Synthesis by [6]-Gingerol, [8]-Gingerol, and [10]-Gingerol.

    PubMed

    Bernard, Megan; Furlong, Suzanne J; Power Coombs, Melanie R; Hoskin, David W

    2015-11-01

    [6]-Gingerol, [8]-gingerol, and [10]-gingerol are pungent components of fresh ginger, extracts of which inhibit various components of the inflammatory response. Because little is known regarding the effect of gingerols with different unbranched alkyl side chain lengths on the activation and effector function of T lymphocytes, we compared the effects of [6]-gingerol, [8]-gingerol, and [10]-gingerol on murine T lymphocyte proliferation, expression of CD25 and CD69 activation markers, cytokine synthesis, and interleukin (IL)-2 receptor signaling. All three gingerols inhibited DNA synthesis by T lymphocytes, as well as interferon-γ synthesis. In contrast, only [8]-gingerol and [10]-gingerol inhibited CD25 and CD69 expression, and IL-2 synthesis. None of the gingerols affected IL-4 synthesis. Exogenous IL-2 enhanced T lymphocyte proliferation in the presence of [6]-gingerol but did not significantly increase T lymphocyte proliferation in the presence of [8]-gingerol or [10]-gingerol. In line with this finding, [8]-gingerol and [10]-gingerol impaired IL-2-induced proliferation of CTLL-2 cells, but constitutive CD25 expression was unaffected, indicating inhibition of IL-2 receptor signaling. In general, [10]-gingerol and [8]-gingerol were more potent inhibitors of T lymphocytes than [6]-gingerol. Suppression of T lymphocyte responses by gingerols suggests that these phytochemicals may be beneficial in chronic inflammatory conditions associated with excessive or inappropriate T lymphocyte activation. Copyright © 2015 John Wiley & Sons, Ltd.

  19. Expression of alpha-AR subtypes in T lymphocytes and role of the alpha-ARs in mediating modulation of T cell function.

    PubMed

    Bao, Jing-Yin; Huang, Yan; Wang, Feng; Peng, Yu-Ping; Qiu, Yi-Hua

    2007-01-01

    Previous work in our laboratory has shown that alpha-adrenoreceptors (alpha-ARs) and beta-ARs exist on lymphocytes from functional profile, and that the receptors mediate the regulation of lymphocyte function by catecholamines. In the present study, we directly examined the expression of alpha-AR subtypes, alpha(1)-AR and alpha(2)-AR mRNAs, in T lymphocytes and explored the roles of the alpha-AR subtypes and intracellular signal transduction mechanisms linked to the receptors in mediating the modulation of T lymphocyte function. T lymphocytes from mesenteric lymph nodes of rats were purified by using a nylon wool column. Reverse transcription polymerase chain reaction was used to detect the expression of alpha(1)-AR and alpha(2)-AR mRNAs in the freshly isolated T cells and the mitogen concanavalin A (Con A)-activated lymphocytes. Colorimetric methylthiazoletetrazolium assay was employed to measure lymphocyte proliferation induced by Con A. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) levels in the Con A-stimulated lymphocyte culture supernatants were examined by enzyme-linked immunosorbent assay. T cells expressed both alpha(1)-AR and alpha(2)-AR mRNAs. The expression of both alpha(1)-AR and alpha(2)-AR mRNAs was significantly higher in the Con A-activated lymphocytes than in the resting lymphocytes. Phenylephrine, a selective alpha(1)-AR agonist, had no evident effect on lymphocyte proliferation nor on IFN-gamma and IL-4 production induced by Con A. However, the selective alpha(2)-AR agonist clonidine attenuated Con A-induced lymphocyte proliferation as well as IFN-gamma and IL-4 production. The inhibited lymphocyte proliferation and IFN-gamma and IL-4 production by clonidine were blocked by yohimbine, an alpha(2)-AR antagonist. Either phospholipase C inhibitor U-73122 or protein kinase C inhibitor chelerythrine partially prevented the suppressive effect of clonidine on Con A-stimulated lymphocyte proliferation and IL-4 production. T lymphocytes express

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

    PubMed Central

    Sin, Jeong-Im

    2009-01-01

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

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

    PubMed

    Sin, Jeong-Im

    2009-09-01

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

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

    PubMed Central

    Duan, Susu; Thomas, Paul G.

    2016-01-01

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

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

    PubMed

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

    2012-05-01

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

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

    PubMed Central

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

    1997-01-01

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

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

    PubMed

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

    2018-06-05

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

  6. Imaging Polarized Secretory Traffic at the Immune Synapse in Living T Lymphocytes.

    PubMed

    Calvo, Víctor; Izquierdo, Manuel

    2018-01-01

    Immune synapse (IS) formation by T lymphocytes constitutes a crucial event involved in antigen-specific, cellular and humoral immune responses. After IS formation by T lymphocytes and antigen-presenting cells, the convergence of secretory vesicles toward the microtubule-organizing center (MTOC) and MTOC polarization to the IS are involved in polarized secretion at the synaptic cleft. This specialized mechanism appears to specifically provide the immune system with a fine strategy to increase the efficiency of crucial secretory effector functions of T lymphocytes, while minimizing non-specific, cytokine-mediated stimulation of bystander cells, target cell killing and activation-induced cell death. The molecular bases involved in the polarized secretory traffic toward the IS in T lymphocytes have been the focus of interest, thus different models and several imaging strategies have been developed to gain insights into the mechanisms governing directional secretory traffic. In this review, we deal with the most widely used, state-of-the-art approaches to address the molecular mechanisms underlying this crucial, immune secretory response.

  7. Defective immunoregulatory T-cell function in chronic lymphocytic leukemia

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

    Han, T.; Ozer, H.; Henderson, E.S.

    Chronic lymphocytic leukemia (CLL) of B-cell origin results in the malignant proliferation of small immunoglobulin-bearing lymphocytes. There is currently a controversy in the literature regarding both the ability of this leukemic population to differentiate into mature plasma cells, as well as the ability of apparently normal T cells from these patients to regulate allogeneic B-cell differentiation. In the present study we have examined the lymphocytes of CLL patients in various clinical stages of their disease and with different surface phenotypes of their leukemic B-cell population. Our results show that leukemic CLL B cells from all 20 patients (including one patientmore » with a monoclonal IgM paraprotein and another with a monoclonal IgG paraprotein) are incapable of further differentiation even in the absence of suppressor T cells and the presence of helper T lymphocytes. This lack of capacity to differentiate is unaffected by clinical stage, by therapy, or by the phenotype of the malignant population. Since the leukemic B population did not suppress normal allogeneic B-cell differentiation, the maturation deficit is evidently intrinsic to the leukemic clone rather than a result of activity of non-T suppressor cells. T helper function was also variably depressed in the blood of some patients with CLL, and this depression did not correlate with clinical stage, with therapy, or with the degree of lymphocytosis. Dysfunction of radiosensitive T suppressor cells was found to be the most consistent regulatory deficit of CLL T cells. Each of 11 patients whose leukemic cell population was of the ..mu..delta, ..mu cap alpha.., or ..mu.. phenotype had both helper and suppressor cell defects.« less

  8. In Vivo Killing Capacity of Cytotoxic T Cells Is Limited and Involves Dynamic Interactions and T Cell Cooperativity

    PubMed Central

    Halle, Stephan; Keyser, Kirsten Anja; Stahl, Felix Rolf; Busche, Andreas; Marquardt, Anja; Zheng, Xiang; Galla, Melanie; Heissmeyer, Vigo; Heller, Katrin; Boelter, Jasmin; Wagner, Karen; Bischoff, Yvonne; Martens, Rieke; Braun, Asolina; Werth, Kathrin; Uvarovskii, Alexey; Kempf, Harald; Meyer-Hermann, Michael; Arens, Ramon; Kremer, Melanie; Sutter, Gerd; Messerle, Martin; Förster, Reinhold

    2016-01-01

    Summary According to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2–16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8+ T cell immunity. PMID:26872694

  9. The effect of propofol and sevoflurane on cancer cell, natural killer cell, and cytotoxic T lymphocyte function in patients undergoing breast cancer surgery: an in vitro analysis.

    PubMed

    Lim, Jeong-Ae; Oh, Chung-Sik; Yoon, Tae-Gyoon; Lee, Ji Yeon; Lee, Seung-Hyun; Yoo, Young-Bum; Yang, Jung-Hyun; Kim, Seong-Hyop

    2018-02-07

    To clarify the effect of anaesthetic agents on cancer immunity, we evaluated the effects of propofol and sevoflurane on natural killer (NK) cell, cytotoxic T lymphocyte (CTL) counts and apoptosis rate in breast cancer and immune cells co-cultures from patients who underwent breast cancer surgery. Venous blood samples were collected after inducing anaesthesia and at 1 and 24 h postoperatively in patients who had undergone breast cancer surgery. The patients were allocated randomly to the propofol- or sevoflurane-based anaesthesia groups. We counted and detected apoptosis in cancer cell, NK cell and CTL of patients with breast cancer by co-culture with a breast cancer cell line in both groups. We also evaluated changes in the cytokines tumour necrosis factor-alpha, interleukin (IL)-6 and IL-10 during the perioperative period. Forty-four patients were included in the final analysis. No difference in NK cell count, CTL count or apoptosis rate was detected between the groups. Furthermore, the number of breast cancer cells undergoing apoptosis in the breast cancer cell co-cultures was not different between the groups. No changes in cytokines were detected between the groups. Although basic science studies have suggested the potential benefits of propofol over a volatile agent during cancer surgery, propofol was not superior to sevoflurane, on the aspects of NK and CTL cells counts with apoptosis rate including breast cancer cell, during anaesthesia for breast cancer surgery in a clinical environment. NCT02758249 on February 26, 2016.

  10. Designing and Testing Broadly-Protective Filoviral Vaccines Optimized for Cytotoxic T-Lymphocyte Epitope Coverage

    PubMed Central

    Fenimore, Paul W.; Foley, Brian T.; Bakken, Russell R.; Thurmond, James R.; Yusim, Karina; Yoon, Hyejin; Parker, Michael; Hart, Mary Kate; Dye, John M.; Korber, Bette; Kuiken, Carla

    2012-01-01

    We report the rational design and in vivo testing of mosaic proteins for a polyvalent pan-filoviral vaccine using a computational strategy designed for the Human Immunodeficiency Virus type 1 (HIV-1) but also appropriate for Hepatitis C virus (HCV) and potentially other diverse viruses. Mosaics are sets of artificial recombinant proteins that are based on natural proteins. The recombinants are computationally selected using a genetic algorithm to optimize the coverage of potential cytotoxic T lymphocyte (CTL) epitopes. Because evolutionary history differs markedly between HIV-1 and filoviruses, we devised an adapted computational technique that is effective for sparsely sampled taxa; our first significant result is that the mosaic technique is effective in creating high-quality mosaic filovirus proteins. The resulting coverage of potential epitopes across filovirus species is superior to coverage by any natural variants, including current vaccine strains with demonstrated cross-reactivity. The mosaic cocktails are also robust: mosaics substantially outperformed natural strains when computationally tested against poorly sampled species and more variable genes. Furthermore, in a computational comparison of cross-reactive potential a design constructed prior to the Bundibugyo outbreak performed nearly as well against all species as an updated design that included Bundibugyo. These points suggest that the mosaic designs would be more resilient than natural-variant vaccines against future Ebola outbreaks dominated by novel viral variants. We demonstrate in vivo immunogenicity and protection against a heterologous challenge in a mouse model. This design work delineates the likely requirements and limitations on broadly-protective filoviral CTL vaccines. PMID:23056184

  11. Generation of T-cell receptors targeting a genetically stable and immunodominant cytotoxic T-lymphocyte epitope within hepatitis C virus non-structural protein 3.

    PubMed

    Pasetto, Anna; Frelin, Lars; Brass, Anette; Yasmeen, Anila; Koh, Sarene; Lohmann, Volker; Bartenschlager, Ralf; Magalhaes, Isabelle; Maeurer, Markus; Sällberg, Matti; Chen, Margaret

    2012-02-01

    Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell-BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS3(1073-1081) CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS3(1073) peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV.

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

    PubMed

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

    2011-05-01

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

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

    PubMed Central

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

    2011-01-01

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

  14. Signal transduction in primary human T lymphocytes in altered gravity during parabolic flight and clinostat experiments.

    PubMed

    Tauber, Svantje; Hauschild, Swantje; Paulsen, Katrin; Gutewort, Annett; Raig, Christiane; Hürlimann, Eva; Biskup, Josefine; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Pantaleo, Antonella; Cogoli, Augusto; Pippia, Proto; Layer, Liliana E; Thiel, Cora S; Ullrich, Oliver

    2015-01-01

    Several limiting factors for human health and performance in microgravity have been clearly identified arising from the immune system, and substantial research activities are required in order to provide the basic information for appropriate integrated risk management. The gravity-sensitive nature of cells of the immune system renders them an ideal biological model in search for general gravity-sensitive mechanisms and to understand how the architecture and function of human cells is related to the gravitational force and therefore adapted to life on Earth. We investigated the influence of altered gravity in parabolic flight and 2D clinostat experiments on key proteins of activation and signaling in primary T lymphocytes. We quantified components of the signaling cascade 1.) in non-activated T lymphocytes to assess the "basal status" of the cascade and 2.) in the process of activation to assess the signal transduction. We found a rapid decrease of CD3 and IL-2R surface expression and reduced p-LAT after 20 seconds of altered gravity in non-activated primary T lymphocytes during parabolic flight. Furthermore, we observed decreased CD3 surface expression, reduced ZAP-70 abundance and increased histone H3-acetylation in activated T lymphocytes after 5 minutes of clinorotation and a transient downregulation of CD3 and stable downregulation of IL-2R during 60 minutes of clinorotation. CD3 and IL-2R are downregulated in primary T lymphocytes in altered gravity. We assume that a gravity condition around 1g is required for the expression of key surface receptors and appropriate regulation of signal molecules in T lymphocytes. © 2015 S. Karger AG, Basel.

  15. Administration of 6-gingerol greatly enhances the number of tumor-infiltrating lymphocytes in murine tumors.

    PubMed

    Ju, Seong-A; Park, Sang-Min; Lee, Yea-Sol; Bae, Jun-Hyeong; Yu, Rina; An, Won G; Suh, Jae-Hee; Kim, Byung-Sam

    2012-06-01

    Tumor-infiltrating lymphocytes (TILs) play critical roles in host antitumor immune responses. It is known that cancer patients with tumor-reactive lymphocyte infiltration in their tumors have better prognoses, while patients with tumors infiltrated by immunosuppressive cells have worse prognoses. We found that administration of 6-gingerol, which is a component of ginger, inhibited tumor growth in several types of murine tumors, such as B16F1 melanomas, Renca renal cell carcinomas and CT26 colon carcinomas, which were established by inoculating tumor cells on the flanks of mice. However, administration of 6-gingerol did not lead to complete eradication of the tumors. 6-Gingerol treatment of tumor-bearing mice caused massive infiltration of CD4 and CD8 T-cells and B220(+) B-cells, but reduced the number of CD4(+) Foxp3(+) regulatory T-cells. The CD8 tumor-infiltrating T lymphocytes in 6-gingerol-treated mice strongly expressed IFN-γ, a marker of activation of cytotoxic T lymphocytes (CTL) CD107a and chemokine receptors that are expressed on T(H) 1 cells, such as CXCR3 and CCR5. To test whether 6-gingerol could promote infiltration of tumor antigen-specific CD8 T-cells into tumors, we adoptively transferred CFSE-labeled OT-1 CD8 T-cells into EG7 tumor-bearing mice. We found that CD8 T cells isolated from 6-gingerol pretreated OT-1 mice, but not from control OT-1 mice, massively infiltrated tumors and tumor draining lymph nodes and divided several times. Our results strongly suggest that 6-gingerol can be used in tumor immunotherapy to increase the number of TILs. Copyright © 2011 UICC.

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

    PubMed

    Cassell, D; Forman, J

    1991-01-01

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

  17. Vaccination with liposome-coupled glypican-3-derived epitope peptide stimulates cytotoxic T lymphocytes and inhibits GPC3-expressing tumor growth in mice.

    PubMed

    Iwama, Tatsuaki; Uchida, Tetsuya; Sawada, Yu; Tsuchiya, Nobuhiro; Sugai, Shiori; Fujinami, Norihiro; Shimomura, Manami; Yoshikawa, Toshiaki; Zhang, Rong; Uemura, Yasushi; Nakatsura, Tetsuya

    2016-01-01

    Because therapeutic manipulation of immunity can induce tumor regression, anti-cancer immunotherapy is considered a promising treatment modality. We previously reported that glypican-3 (GPC3), an oncofetal antigen overexpressed in hepatocellular carcinoma (HCC), is a useful target for cytotoxic T lymphocyte (CTL)-mediated cancer immunotherapy, and we have performed clinical trials using the GPC3-derived peptide vaccine. Although vaccine-induced GPC3-peptide-specific CTLs were often tumor reactive in vitro and were correlated with overall survival, no complete response was observed. In the current study, we synthesized liposome-coupled GPC3-derived CTL epitope peptide (pGPC3-lipsome) and investigated its antitumor potential. Vaccination with pGPC3-liposome induced peptide-specific CTLs at a lower dose than conventional vaccine emulsified in incomplete Freund's adjuvant. Coupling of pGPC3 to liposomes was essential for effective priming of GPC3-specific CTLs. In addition, immunization with pGPC3-liposome inhibited GPC3-expressing tumor growth. Thus, vaccination with tumor-associated antigen-derived epitope peptides coupled to the surfaces of liposomes may be a novel therapeutic strategy for cancer. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  18. T Lymphocyte Inhibition by Tumor-Infiltrating Dendritic Cells Involves Ectonucleotidase CD39 but Not Arginase-1.

    PubMed

    Trad, Malika; Gautheron, Alexandrine; Fraszczak, Jennifer; Alizadeh, Darya; Larmonier, Claire; LaCasse, Collin J; Centuori, Sara; Audia, Sylvain; Samson, Maxime; Ciudad, Marion; Bonnefoy, Francis; Lemaire-Ewing, Stéphanie; Katsanis, Emmanuel; Perruche, Sylvain; Saas, Philippe; Bonnotte, Bernard

    2015-01-01

    T lymphocytes activated by dendritic cells (DC) which present tumor antigens play a key role in the antitumor immune response. However, in patients suffering from active cancer, DC are not efficient at initiating and supporting immune responses as they participate to T lymphocyte inhibition. DC in the tumor environment are functionally defective and exhibit a characteristic of immature phenotype, different to that of DC present in nonpathological conditions. The mechanistic bases underlying DC dysfunction in cancer responsible for the modulation of T-cell responses and tumor immune escape are still being investigated. Using two different mouse tumor models, we showed that tumor-infiltrating DC (TIDC) are constitutively immunosuppressive, exhibit a semimature phenotype, and impair responder T lymphocyte proliferation and activation by a mechanism involving CD39 ectoenzyme.

  19. Nicotine-mediated signals modulate cell death and survival of T lymphocytes

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

    Oloris, Silvia C.S.; Instituto de Ciencias Exatas e Naturais, Universidade do Estado do Rio Grande do Norte, Mossoro, RN; Frazer-Abel, Ashley A.

    The capacity of nicotine to affect the behavior of non-neuronal cells through neuronal nicotinic acetylcholine receptors (nAChRs) has been the subject of considerable recent attention. Previously, we showed that exposure to nicotine activates the nuclear factor of activated T cells (NFAT) transcription factor in lymphocytes and endothelial cells, leading to alterations in cellular growth and vascular endothelial growth factor production. Here, we extend these studies to document effects of nicotine on lymphocyte survival. The data show that nicotine induces paradoxical effects that might alternatively enforce survival or trigger apoptosis, suggesting that depending on timing and context, nicotine might act bothmore » as a survival factor or as an inducer of apoptosis in normal or transformed lymphocytes, and possibly other non-neuronal cells. In addition, our results show that, while having overlapping functions, low and high affinity nAChRs also transmit signals that promote distinct outcomes in lymphocytes. The sum of our data suggests that selective modulation of nAChRs might be useful to regulate lymphocyte activation and survival in health and disease.« less

  20. Apoptosis of T lymphocytes invading glioblastomas multiforme: a possible tumor defense mechanism.

    PubMed

    Didenko, Vladimir V; Ngo, Hop N; Minchew, Candace; Baskin, David S

    2002-03-01

    The goal of this study was to investigate whether apoptosis occurs in T lymphocytes that invade Fas ligand (FasL)-expressing glioblastomas multiforme (GBMs) and if its induction could be mediated by Fas. Apoptotic T lymphocytes were detected in GBMs by using detection of cell-type markers combined with active caspase-3 immunohistochemical analysis, a recently introduced apoptosis-specific in situ ligation assay, as well as by examining morphological criteria. Apoptotic T cells expressed Fas and were localized in the vicinity or in direct contact with FasL-expressing tumor cells. The T lymphocytes were undergoing apoptosis in spite of Bcl-2 expression. Expression of Bax was also detected in dying T cells, which can explain the absence of the protective effect of Bcl-2. because Bax inhibits Bcl-2 death-repressor activity. On the basis of the data presented in this paper, the authors suggest that GBM cells that express FasL can induce apoptosis in invading immune cells. This phenomenon may play an important role in these tumors' maintenance of immune privilege and evasion of immune attacks. Awareness of this phenomenon should be helpful for the development of novel strategies for treatment of malignant gliomas.

  1. Modulation of cytotoxic T lymphocyte, natural killer cell, antibody-dependent cellular cytotoxicity, and antibody-dependent complement-mediated cytotoxicity by Vernonia cinerea L. and vernolide-A in BALB/c mice via enhanced production of cytokines IL-2 and IFN-γ.

    PubMed

    Pratheeshkumar, Poyil; Kuttan, Girija

    2012-02-01

    Effect of Vernonia cinerea L. and vernolide-A on cell-mediated immune (CMI) response was studied in normal as well as tumor-bearing BALB/c mice. Administration of V. cinerea and vernolide-A significantly enhanced natural killer (NK) cell activity in both normal as well as tumor-bearing animals, and the activity was observed earlier than in tumor-bearing control animals. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were also enhanced significantly in both normal as well as tumor-bearing animals after V. cinerea and vernolide-A administration compared with untreated control tumor-bearing animals. Extract and vernolide-A showed a significant increase in cytotoxic T lymphocyte (CTL) production in both the in vivo and in vitro models. The level of cytokines such as interleukin (IL)-2 and interferon (IFN)-γ were also enhanced by the treatment of V. cinerea and vernolide-A in both normal as well as tumor-bearing animals. This study demonstrated that V. cinerea extract and vernolide-A stimulate the CTL, NK cell, ADCC, and ADCC through enhanced secretion of IL-2 and IFN-γ.

  2. Semi-allogeneic dendritic cells can induce antigen-specific T-cell activation, which is not enhanced by concurrent alloreactivity.

    PubMed

    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.

  3. The immunometabolite S-2-hydroxyglutarate regulates CD8+ T-lymphocyte fate

    PubMed Central

    Tyrakis, Petros A.; Palazon, Asis; Macias, David; Lee, Kian. L.; Phan, Anthony. T.; Veliça, Pedro; You, Jia; Chia, Grace S.; Sim, Jingwei; Doedens, Andrew; Abelanet, Alice; Evans, Colin E.; Griffiths, John R.; Poellinger, Lorenz; Goldrath, Ananda. W.; Johnson, Randall S.

    2016-01-01

    R-2-hydroxyglutarate accumulates to millimolar levels in cancers with gain-of-function isocitrate dehydrogenase 1/2 mutations. These levels of R-2-hydroxyglutarate affect 2-oxoglutarate-dependent dioxygenases. Both R- and S-2-hydroxyglutarate, the other enantiomer of this metabolite, are detectible in healthy individuals, yet their physiological function remains elusive. Here we show that CD8+ T-lymphocytes accumulate 2-hydroxyglutarate in response to T-cell receptor triggering. This increases to millimolar levels in physiological oxygen conditions, via a hypoxia inducible factor 1 alpha-dependent mechanism. S-2-hydroxyglutarate predominates over R-2-hydroxyglutarate in activated T cells, and we demonstrate alterations in markers of CD8+ T-lymphocyte differentiation in response to this metabolite. Modulation of histone and DNA demethylation as well as hypoxia inducible factor 1 alpha stability mediate these effects. S-2-hydroxyglutarate treatment greatly enhances the in vivo proliferation, persistence and anti-tumour capacity of adoptively transferred CD8+ T-lymphocytes. Thus S-2-hydroxyglutarate acts as an immunometabolite that links environmental context, via a metabolic-epigenetic axis, to immune fate and function. PMID:27798602

  4. Basal shuttle of NF-κB/IκBα in resting T lymphocytes regulates HIV-1 LTR dependent expression

    PubMed Central

    Coiras, Mayte; López-Huertas, María Rosa; Rullas, Joaquín; Mittelbrunn, Maria; Alcamí, José

    2007-01-01

    Background In HIV-infected T lymphocytes, NF-κB/Rel transcription factors are major elements involved in the activation of LTR-dependent transcription from latency. Most NF-κB heterodimer p65/p50 is sequestered as an inactive form in the cytoplasm of resting T lymphocytes via its interaction with IκB inhibitors. In these cells, both absolute HIV latency and low level ongoing HIV replication have been described. These situations could be related to differences in the balance between NF-κB and IκBα ratio. Actually, control of IκBα by cellular factors such as Murr-1 plays a critical role in maintaining HIV latency in unstimulated T lymphocytes. Formerly, our group demonstrated the presence of nuclear IκBα in T cells after PMA activation. Now we attempt to determine the dynamics of NF-κB/IκBα nucleocytosolic transport in absence of activation as a mechanism to explain both the maintenance of latency and the existence of low level ongoing HIV replication in resting CD4+ T lymphocytes. Results and conclusion We show that the inhibition of the nuclear export by leptomycin B in resting CD4+ T cells resulted in nuclear accumulation of both IκBα and p65/RelA, as well as formation of NF-κB/IκBα complexes. This proves the existence of a rapid shuttling of IκBα between nucleus and cytosol even in absence of cellular activation. The nuclear accumulation of IκBα in resting CD4+ T lymphocytes results in inhibition of HIV-LTR dependent transcription as well as restrains HIV replication in CD4+ T lymphocytes. On the other hand, basal NF-κB activity detected in resting CD4+ T lymphocytes was related to low level HIV replication in these cells. PMID:17686171

  5. NAADP Activates Two-Pore Channels on T Cell Cytolytic Granules to Stimulate Exocytosis and Killing

    PubMed Central

    Davis, Lianne C.; Morgan, Anthony J.; Chen, Ji-Li; Snead, Charlotte M.; Bloor-Young, Duncan; Shenderov, Eugene; Stanton-Humphreys, Megan N.; Conway, Stuart J.; Churchill, Grant C.; Parrington, John; Cerundolo, Vincenzo; Galione, Antony

    2012-01-01

    Summary A cytotoxic T lymphocyte (CTL) kills an infected or tumorigenic cell by Ca2+-dependent exocytosis of cytolytic granules at the immunological synapse formed between the two cells. Although inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum activates the store-operated Ca2+-influx pathway that is necessary for exocytosis, it is not a sufficient stimulus [1–4]. Here we identify the Ca2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and its recently identified molecular target, two-pore channels (TPCs) [5–7], as being important for T cell receptor signaling in CTLs. We demonstrate that cytolytic granules are not only reservoirs of cytolytic proteins but are also the acidic Ca2+ stores mobilized by NAADP via TPC channels on the granules themselves, so that TPCs migrate to the immunological synapse upon CTL activation. Moreover, NAADP activates TPCs to drive exocytosis in a way that is not mimicked by global Ca2+ signals induced by IP3 or ionomycin, suggesting that critical, local Ca2+ nanodomains around TPCs stimulate granule exocytosis. Hence, by virtue of the NAADP/TPC pathway, cytolytic granules generate Ca2+ signals that lead to their own exocytosis and to cell killing. This study highlights a selective role for NAADP in stimulating exocytosis crucial for immune cell function and may impact on stimulus-secretion coupling in wider cellular contexts. PMID:23177477

  6. Control of T lymphocyte morphology by the GTPase Rho

    NASA Technical Reports Server (NTRS)

    Woodside, Darren G.; Wooten, David K.; Teague, T. Kent; Miyamoto, Yuko J.; Caudell, Eva G.; Udagawa, Taturo; Andruss, Bernard F.; McIntyre, Bradley W.

    2003-01-01

    BACKGROUND: Rho family GTPase regulation of the actin cytoskeleton governs a variety of cell responses. In this report, we have analyzed the role of the GTPase Rho in maintenance of the T lymphocyte actin cytoskeleton. RESULTS: Inactivation of the GTPase Rho in the human T lymphocytic cell line HPB-ALL does not inhibit constitutively high adhesion to the integrin beta1 substrate fibronectin. It did however result in the aberrant extension of finger-like dendritic processes on the substrates VCAM-1, Fn, and mAb specific to beta1 integrins. Time-lapse video microscopy demonstrated that C3 induced extensions were primarily the result of an altered pseudopod elongation rather than retraction. Once the stellate pseudopodia extended, none retracted, and cells became completely immobile. Filipodial structures were absent and the dendritic-like processes in C3 treated cells were rich in filamentous actin. Immunolocalization of RhoA in untreated HPB-ALL cells spreading on fibronectin demonstrated a diffuse staining pattern within the pseudopodia. In C3 treated cells, clusters of RhoA were pronounced and localized within the altered extensions. CONCLUSIONS: GTPase Rho is actively involved in the regulation of T lymphocyte morphology and motility.

  7. Vaticaffinol, a resveratrol tetramer, exerts more preferable immunosuppressive activity than its precursor in vitro and in vivo through multiple aspects against activated T lymphocytes

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

    Feng, Li-Li; Wu, Xue-Feng; Liu, Hai-Liang

    2013-03-01

    In the present study, we aimed to investigate the immunosuppressive activity of vaticaffinol, a resveratrol tetramer isolated from Vatica mangachapoi, on T lymphocytes both in vitro and in vivo, and further explored its potential molecular mechanism. Resveratrol had a wide spectrum of healthy beneficial effects with multiple targets. Interestingly, its tetramer, vaticaffinol, exerted more intensive immunosuppressive activity than resveratrol. Vaticaffinol significantly inhibited T cells proliferation activated by concanavalin A (Con A) or anti-CD3 plus anti-CD28 in a dose- and time-dependent manner. It also induced Con A-activated T cells undergoing apoptosis through mitochondrial pathway. Moreover, this compound prevented cells from enteringmore » S phase and G2/M phase during T cells activation. In addition, vaticaffinol inhibited ERK and AKT signaling pathways in Con A-activated T cells. Furthermore, vaticaffinol significantly ameliorated ear swelling in a mouse model of picryl chloride-induced ear contact dermatitis in vivo. In most of the aforementioned experiments, however, resveratrol had only slight effects on the inhibition of T lymphocytes compared with vaticaffinol. Taken together, our findings suggest that vaticaffinol exerts more preferable immunosuppressive activity than its precursor resveratrol both in vitro and in vivo by affecting multiple targets against activated T cells. - Graphical abstract: Vaticaffinol, a resveratrol tetramer isolated from Vatica mangachapoi, exerts more intensive immunosuppressive activity than its precursor resveratrol does in vitro and in vivo. Its mechanism may involve multiple effects against activated T cells: regulation of signalings involved in cell proliferation, G0/G1 arrest of T cells, as well as an apoptosis induction in activated effector T cells. Highlights: ► Vaticaffinol, a resveratrol tetramer, exerts more potent activity than its precursor. ► It inhibited T cells proliferation and prevented them from

  8. Calreticulin is expressed on the cell surface of activated human peripheral blood T lymphocytes in association with major histocompatibility complex class I molecules.

    PubMed

    Arosa, F A; de Jesus, O; Porto, G; Carmo, A M; de Sousa, M

    1999-06-11

    Calreticulin is an endoplasmic reticulum resident molecule known to be involved in the folding and assembly of major histocompatibility complex (MHC) class I molecules. In the present study, expression of calreticulin was analyzed in human peripheral blood T lymphocytes. Pulse-chase experiments in [35S]methionine-labeled T cell blasts showed that calreticulin was associated with several proteins in the endoplasmic reticulum and suggested that it was expressed at the cell surface. Indeed, the 60-kDa calreticulin was labeled by cell surface biotinylation and precipitated from the surface of activated T cells together with a protein with an apparent molecular mass of 46 kDa. Cell surface expression of calreticulin by activated T lymphocytes was further confirmed by immunofluorescence and flow cytometry, studies that showed that both CD8+ and CD4+ T cells expressed calreticulin in the plasma membrane. Low amounts of cell surface calreticulin were detected in resting T lymphocytes. By sequential immunoprecipitation using the conformation independent monoclonal antibody HC-10, we provided evidence that the cell surface 46-kDa protein co-precipitated with calreticulin is unfolded MHC I. These results show for the first time that after T cell activation, significant amounts of calreticulin are expressed on the T cell surface, where they are found in physical association with a pool of beta2-free MHC class I molecules.

  9. Detection and functional analysis of tumor infiltrating T-lymphocytes (TIL) in liver metastases from colorectal cancer.

    PubMed

    Wagner, Philipp; Koch, Moritz; Nummer, Daniel; Palm, Sylvia; Galindo, Luis; Autenrieth, Daniel; Rahbari, Nuh; Schmitz-Winnenthal, Friedrich H; Schirrmacher, Volker; Büchler, Markus W; Beckhove, Philipp; Weitz, Jürgen

    2008-08-01

    Tumor-infiltrating T lymphocytes (TIL) play an important role in primary colorectal cancer, but their activity in liver metastases has not yet been investigated. The aim of this study was to examine whether tumor-selective infiltration, activation, and cytotoxic activity of TIL can be demonstrated in situ in colorectal liver metastases. TIL were obtained from liver metastases and corresponding normal liver tissue of 16 patients with colorectal liver metastases. Characterization of TIL in situ was performed by multicolor flowcytometric analysis. Presence of tumor antigen-reactive T cells was evaluated by interferon gamma Elispot analysis. TIL in colorectal liver metastases responding against tumor antigens were present in most patients. Although the proportions of CD3(+) T cells were comparable in liver metastasis and normal liver tissue, metastases contained significantly enhanced proportions of CD4(+) cells (49% vs. 22%, P < .001). Among all CD4(+) T helper cells, the proportion of activated (CD4(+)CD25(+)) effector cells was significantly increased in liver metastases (15.0% vs. 7.8%, P = .003). Metastases showed significantly higher proportions of activated (CD69(+) [70.1% vs. 49.8%, P = .02] and CD25(+) [4.1% vs. .6%, P = .06]) and cytotoxically active (CD107a(+)) CD8(+) TIL (3.2% vs. 1.3%, P = .03). Importantly, the presence of activated T helper cells correlated with the frequencies of cytotoxic T lymphocytes that exerted cytotoxic activity in situ (P = .02). CD4(+) and CD8(+) TIL are selectively activated in liver metastases, and cytotoxic T lymphocytes exert tumor-selective cytotoxic activity in situ in the presence of activated T helper cells, suggesting the requirement of in-situ-activated T helper cells for efficient cytotoxic T lymphocytes effector function.

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

    PubMed Central

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

    2013-01-01

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

  11. Differential Impact of In Vivo CD8+ T Lymphocyte Depletion in Controller versus Progressor Simian Immunodeficiency Virus-Infected Macaques.

    PubMed

    Chowdhury, Ankita; Hayes, Timothy L; Bosinger, Steven E; Lawson, Benton O; Vanderford, Thomas; Schmitz, Joern E; Paiardini, Mirko; Betts, Michael; Chahroudi, Ann; Estes, Jacob D; Silvestri, Guido

    2015-09-01

    Numerous studies have demonstrated that CD8(+) T lymphocytes suppress virus replication during human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection. However, the mechanisms underlying this activity of T cells remain incompletely understood. Here, we conducted CD8(+) T lymphocyte depletion in 15 rhesus macaques (RMs) infected intravenously (i.v.) with SIVmac239. At day 70 postinfection, the animals (10 progressors with high viremia and 5 controllers with low viremia) were CD8 depleted by i.v. administration of the antibody M-T807R1. As expected, CD8 depletion resulted in increased virus replication, more prominently in controllers than progressors, which correlated inversely with predepletion viremia. Of note, the feature of CD8(+) T lymphocyte predepletion that correlated best with the increase in viremia postdepletion was the level of CD8(+) T-bet(+) lymphocytes. We next found that CD8 depletion resulted in a homogenous increase of SIV RNA in superficial and mesenteric lymph nodes, spleen, and the gastrointestinal tract of both controllers and progressors. Interestingly, the level of SIV DNA increased postdepletion in both CD4(+) central memory T lymphocytes (TCM) and CD4(+) effector memory T lymphocytes (TEM) in progressor RMs but decreased in the CD4(+) TCM of 4 out of 5 controllers. Finally, we found that CD8 depletion is associated with a greater increase in CD4(+) T lymphocyte activation (measured by Ki-67 expression) in controllers than in progressors. Overall, these data reveal a differential impact of CD8(+) T lymphocyte depletion between controller and progressor SIV-infected RMs, emphasizing the complexity of the in vivo antiviral role of CD8(+) T lymphocytes. In this study, we further dissect the impact of CD8(+) T lymphocytes on HIV/SIV replication during SIV infection. CD8(+) T lymphocyte depletion leads to a relatively homogenous increase in viral replication in peripheral blood and tissues. CD8(+) T lymphocyte depletion

  12. Differential Impact of In Vivo CD8+ T Lymphocyte Depletion in Controller versus Progressor Simian Immunodeficiency Virus-Infected Macaques

    PubMed Central

    Chowdhury, Ankita; Hayes, Timothy L.; Bosinger, Steven E.; Lawson, Benton O.; Vanderford, Thomas; Schmitz, Joern E.; Paiardini, Mirko; Betts, Michael; Chahroudi, Ann; Estes, Jacob D.

    2015-01-01

    ABSTRACT Numerous studies have demonstrated that CD8+ T lymphocytes suppress virus replication during human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection. However, the mechanisms underlying this activity of T cells remain incompletely understood. Here, we conducted CD8+ T lymphocyte depletion in 15 rhesus macaques (RMs) infected intravenously (i.v.) with SIVmac239. At day 70 postinfection, the animals (10 progressors with high viremia and 5 controllers with low viremia) were CD8 depleted by i.v. administration of the antibody M-T807R1. As expected, CD8 depletion resulted in increased virus replication, more prominently in controllers than progressors, which correlated inversely with predepletion viremia. Of note, the feature of CD8+ T lymphocyte predepletion that correlated best with the increase in viremia postdepletion was the level of CD8+ T-bet+ lymphocytes. We next found that CD8 depletion resulted in a homogenous increase of SIV RNA in superficial and mesenteric lymph nodes, spleen, and the gastrointestinal tract of both controllers and progressors. Interestingly, the level of SIV DNA increased postdepletion in both CD4+ central memory T lymphocytes (TCM) and CD4+ effector memory T lymphocytes (TEM) in progressor RMs but decreased in the CD4+ TCM of 4 out of 5 controllers. Finally, we found that CD8 depletion is associated with a greater increase in CD4+ T lymphocyte activation (measured by Ki-67 expression) in controllers than in progressors. Overall, these data reveal a differential impact of CD8+ T lymphocyte depletion between controller and progressor SIV-infected RMs, emphasizing the complexity of the in vivo antiviral role of CD8+ T lymphocytes. IMPORTANCE In this study, we further dissect the impact of CD8+ T lymphocytes on HIV/SIV replication during SIV infection. CD8+ T lymphocyte depletion leads to a relatively homogenous increase in viral replication in peripheral blood and tissues. CD8+ T lymphocyte depletion resulted

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

    PubMed Central

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

    2009-01-01

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

  14. T Lymphocyte Inhibition by Tumor-Infiltrating Dendritic Cells Involves Ectonucleotidase CD39 but Not Arginase-1

    PubMed Central

    Trad, Malika; Gautheron, Alexandrine; Fraszczak, Jennifer; Larmonier, Claire; LaCasse, Collin J.; Centuori, Sara; Audia, Sylvain; Samson, Maxime; Ciudad, Marion; Bonnefoy, Francis; Lemaire-Ewing, Stéphanie; Katsanis, Emmanuel; Perruche, Sylvain; Saas, Philippe; Bonnotte, Bernard

    2015-01-01

    T lymphocytes activated by dendritic cells (DC) which present tumor antigens play a key role in the antitumor immune response. However, in patients suffering from active cancer, DC are not efficient at initiating and supporting immune responses as they participate to T lymphocyte inhibition. DC in the tumor environment are functionally defective and exhibit a characteristic of immature phenotype, different to that of DC present in nonpathological conditions. The mechanistic bases underlying DC dysfunction in cancer responsible for the modulation of T-cell responses and tumor immune escape are still being investigated. Using two different mouse tumor models, we showed that tumor-infiltrating DC (TIDC) are constitutively immunosuppressive, exhibit a semimature phenotype, and impair responder T lymphocyte proliferation and activation by a mechanism involving CD39 ectoenzyme. PMID:26491691

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

    PubMed Central

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

    2013-01-01

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

  16. Leukemia -- Chronic T-Cell Lymphocytic

    MedlinePlus

    ... social workers, and patient advocates. Cancer.Net Guide Leukemia - Chronic T-Cell Lymphocytic Introduction Statistics Risk Factors Symptoms and Signs Diagnosis Stages Treatment Options About Clinical Trials Latest Research ...

  17. Anomalies of the CD8+ T cell pool in haemochromatosis: HLA-A3-linked expansions of CD8+CD28- T cells.

    PubMed

    Arosa, F A; Oliveira, L; Porto, G; da Silva, B M; Kruijer, W; Veltman, J; de Sousa, M

    1997-03-01

    The present study consists of a phenotypic and functional characterization of peripheral blood T lymphocytes in a group of 21 patients with hereditary haemochromatosis (HH), an MHC class I-linked genetic disease resulting in iron overload, and a group of 30 healthy individuals, both HLA-phenotyped. The HH patients studied showed an increased percentage of CD8+ CD28- T cells with a corresponding reduction in the percentage of CD8+ CD28+ T cells in peripheral blood relative to healthy blood donors. No anomalies of CD28 expression were found in the CD4+ subset. The presence of the HLA-A3 antigen but not age accounted for these imbalances. Thus, an apparent failure of the CD8+ CD28+ T cell population 'to expand', coinciding with an 'expansion' of CD8+ CD28- T cells in peripheral blood of HLA-A3+ but not HLA-A3- HH patients was observed when compared with the respective HLA-A3-matched control group. A significantly higher percentage of HLA-DR+ but not CD45RO+ cells was also found within the peripheral CD8+ T cell subset in HH patients relative to controls. Phytohaemagglutinin (PHA) stimulation of peripheral blood mononuclear cells (PBMC) for 5 days showed: (i) that CD8+ CD28+ T cells both in controls and HH were able to expand in vitro; (ii) that CD8+ CD28- T cells decreased markedly after activation in controls but not in HH patients. Moreover, functional studies showed that CD8+ cytotoxic T lymphocytes (CTL) from HH patients exhibited a diminished cytotoxic activity (approx. two-fold) in standard 51Cr-release assays when compared with CD8+ CTL from healthy controls. The present results provide additional evidence for the existence of phenotypic and functional anomalies of the peripheral CD8+ T cell pool that may underlie the clinical heterogeneity of this iron overload disease. They are of particular relevance given the recent discovery of a novel mutated MHC class I-like gene in HH.

  18. Intranasal delivery of recombinant parvovirus-like particles elicits cytotoxic T-cell and neutralizing antibody responses.

    PubMed

    Sedlik, C; Dridi, A; Deriaud, E; Saron, M F; Rueda, P; Sarraseca, J; Casal, J I; Leclerc, C

    1999-04-01

    We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4(+) and CD8(+) T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8(+) T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8(+) T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8(+) T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration.

  19. Intranasal Delivery of Recombinant Parvovirus-Like Particles Elicits Cytotoxic T-Cell and Neutralizing Antibody Responses

    PubMed Central

    Sedlik, C.; Dridi, A.; Deriaud, E.; Saron, M. F.; Rueda, P.; Sarraseca, J.; Casal, J. I.; Leclerc, C.

    1999-01-01

    We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4+ and CD8+ T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8+ T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8+ T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8+ T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration. PMID:10074120

  20. Glycogen Synthase Kinase 3 Inactivation Drives T-bet-Mediated Downregulation of Co-receptor PD-1 to Enhance CD8+ Cytolytic T Cell Responses

    PubMed Central

    Taylor, Alison; Harker, James A.; Chanthong, Kittiphat; Stevenson, Philip G.; Zuniga, Elina I.; Rudd, Christopher E.

    2016-01-01

    Summary Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway that regulates PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoforms α and β) is a serine-threonine kinase implicated in cellular processes. Here, we identified GSK-3 as a key upstream kinase that regulated PD-1 expression in CD8+ T cells. GSK-3 siRNA downregulation, or inhibition by small molecules, blocked PD-1 expression, resulting in increased CD8+ cytotoxic T lymphocyte (CTL) function. Mechanistically, GSK-3 inactivation increased Tbx21 transcription, promoting enhanced T-bet expression and subsequent suppression of Pdcd1 (encodes PD-1) transcription in CD8+ CTLs. Injection of GSK-3 inhibitors in mice increased in vivo CD8+ OT-I CTL function and the clearance of murine gamma-herpesvirus 68 and lymphocytic choriomeningitis clone 13 and reversed T cell exhaustion. Our findings identify GSK-3 as a regulator of PD-1 expression and demonstrate the applicability of GSK-3 inhibitors in the modulation of PD-1 in immunotherapy. PMID:26885856

  1. NK cells modulate the cytotoxic activity generated by Mycobacterium leprae-hsp65 in leprosy patients: role of IL-18 and IL-13

    PubMed Central

    DE LA BARRERA, S; FINIASZ, M; FINK, S; ILARREGUI, J; ALEMÁN, M; OLIVARES, L; FRANCO, M C; PIZZARIELLO, G; DEL CARMEN SASIAIN, M

    2004-01-01

    Protection against intracellular pathogens such as Mycobacterium leprae is critically dependent on the function of NK cells at early stages of the immune response and on Th1 cells at later stages. In the present report we evaluated the role of IL-18 and IL-13, two cytokines that can influence NK cell activity, in the generation of M. leprae-derived hsp65-cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) of leprosy patients. We demonstrated that IL-18 modulates hsp65-induced CTL generation and collaborates with IL-12 for this effect. In paucibacillary (PB) patients and normal controls (N) depletion of NK cells reduces the cytolytic activity. Under these conditions, IL-12 cannot up-regulate this CTL generation, while, in contrast, IL-18 increases the cytotoxic activity both in the presence or absence of NK cells. IL-13 down-regulates the hsp65-induced CTL generation and counteracts the positive effect of IL-18. The negative effect of IL-13 is observed in the early stages of the response, suggesting that this cytokine affects IFNγ production by NK cells. mRNA coding for IFNγ is induced by IL-18 and reduced in the presence of IL-13, when PBMC from N or PB patients are stimulated with hsp65. Neutralization of IL-13 in PBMC from multibacillary (MB) leprosy patients induces the production of IFNγ protein by lymphocytes. A modulatory role on the generation of hsp65 induced CTL is demonstrated for IL-18 and IL-13 and this effect takes place through the production of IFNγ. PMID:14678270

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-07-01

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

  4. Anomalies of the CD8+ T cell pool in haemochromatosis: HLA-A3-linked expansions of CD8+CD28− T cells

    PubMed Central

    AROSA, F A; OLIVEIRA, L; PORTO, G; DA SILVA, B M; KRUIJER, W; VELTMAN, J; DE SOUSA, M

    1997-01-01

    The present study consists of a phenotypic and functional characterization of peripheral blood T lymphocytes in a group of 21 patients with hereditary haemochromatosis (HH), an MHC class I-linked genetic disease resulting in iron overload, and a group of 30 healthy individuals, both HLA-phenotyped. The HH patients studied showed an increased percentage of CD8+ CD28− T cells with a corresponding reduction in the percentage of CD8+ CD28+ T cells in peripheral blood relative to healthy blood donors. No anomalies of CD28 expression were found in the CD4+ subset. The presence of the HLA-A3 antigen but not age accounted for these imbalances. Thus, an apparent failure of the CD8+ CD28+ T cell population ‘to expand’, coinciding with an ‘expansion’ of CD8+ CD28− T cells in peripheral blood of HLA-A3+ but not HLA-A3− HH patients was observed when compared with the respective HLA-A3-matched control group. A significantly higher percentage of HLA-DR+ but not CD45RO+ cells was also found within the peripheral CD8+ T cell subset in HH patients relative to controls. Phytohaemagglutinin (PHA) stimulation of peripheral blood mononuclear cells (PBMC) for 5 days showed: (i) that CD8+ CD28+ T cells both in controls and HH were able to expand in vitro; (ii) that CD8+ CD28− T cells decreased markedly after activation in controls but not in HH patients. Moreover, functional studies showed that CD8+ cytotoxic T lymphocytes (CTL) from HH patients exhibited a diminished cytotoxic activity (approx. two-fold) in standard 51Cr-release assays when compared with CD8+ CTL from healthy controls. The present results provide additional evidence for the existence of phenotypic and functional anomalies of the peripheral CD8+ T cell pool that may underlie the clinical heterogeneity of this iron overload disease. They are of particular relevance given the recent discovery of a novel mutated MHC class I-like gene in HH. PMID:9067531

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  7. Antitumor activity of cytotoxic T lymphocytes engineered to target vascular endothelial growth factor receptors

    NASA Astrophysics Data System (ADS)

    Niederman, Thomas M. J.; Ghogawala, Zoher; Carter, Bob S.; Tompkins, Hillary S.; Russell, Margaret M.; Mulligan, Richard C.

    2002-05-01

    The demonstration that angiogenesis is required for the growth of solid tumors has fueled an intense interest in the development of new therapeutic strategies that target the tumor vasculature. Here we report the development of an immune-based antiangiogenic strategy that is based on the generation of T lymphocytes that possess a killing specificity for cells expressing vascular endothelial growth factor receptors (VEGFRs). To target VEGFR-expressing cells, recombinant retroviral vectors were generated that encoded a chimeric T cell receptor comprised of VEGF sequences linked to intracellular signaling sequences derived from the chain of the T cell receptor. After transduction of primary murine CD8 lymphocytes by such vectors, the transduced cells were shown to possess an efficient killing specificity for cells expressing the VEGF receptor, Flk-1, as measured by in vitro cytotoxicity assays. After adoptive transfer into tumor-bearing mice, the genetically modified cytotoxic T lymphocytes strongly inhibited the growth of a variety of syngeneic murine tumors and human tumor xenografts. An increased effect on in vivo tumor growth inhibition was seen when this therapy was combined with the systemic administration of TNP-470, a conventional angiogenesis inhibitor. The utilization of the immune system to target angiogenic markers expressed on tumor vasculature may prove to be a powerful means for controlling tumor growth.

  8. Psychosocial factors and T lymphocyte counts in Brazilian peacekeepers.

    PubMed

    Silva, Angela M Monteiro da; Speranza, Francisco A B; Ishii, Solange Kiyoko; Hirata, Raphael; Mattos-Guaraldi, Ana Luíza; Milagres, Lucimar Gonçalves

    2015-02-01

    To investigate the associations between psychosocial factors and peripheral blood CD4 and CD8 T lymphocyte numbers in Brazilian peacekeepers. Venous blood was collected from 759 peacekeepers who had just returned from a peace mission in Haiti. Among the 759 soldiers, 642 individuals completed the psychosocial measures. CD4 and CD8 T lymphocyte counts were measured by flow cytometry using a commercially available kit. Psychosocial factors, including military peace force stressors, clinical stress, anxiety and depression, were recorded. As a reference for T lymphocyte numbers, we measured T lymphocyte counts in 75 blood donors from the Instituto de Biologia do Exército, Rio de Janeiro. The median numbers of CD4 and CD8 T lymphocytes in the blood donors were 819 cells/µl and 496 cells/µl, respectively, with a CD4:CD8 ratio of 1.6. Significantly (p<0.05) lower CD4 T cell counts (759 cells/µl) were recorded for peacekeepers, with similar CD8 levels (548 cells/µl) and smaller CD4:CD8 ratios (1.3, p<0.001) compared to blood donors. These differences were due to a group of 14 military personnel with CD4 and CD8 medians of 308 and 266 cells/µl, respectively. Only one (7.1%) of these 14 individuals was diagnosed with clinical stress compared with 13.5% of the individuals with normal levels of CD4 T lymphocytes. One individual out of 628 (0.16%) had a Lipp's Stress Symptom Inventory score of 3, indicating near exhaustion. The prevalence of psychological disorders was low and there were no associations with CD4 or CD8 T cell numbers.

  9. Decreased circulating T regulatory lymphocytes in obese patients undergoing bariatric surgery.

    PubMed

    Agabiti-Rosei, Claudia; Trapletti, Valentina; Piantoni, Silvia; Airò, Paolo; Tincani, Angela; De Ciuceis, Carolina; Rossini, Claudia; Mittempergher, Francesco; Titi, Amin; Portolani, Nazario; Caletti, Stefano; Coschignano, Maria Antonietta; Porteri, Enzo; Tiberio, Guido A M; Pileri, Paola; Solaini, Leonardo; Kumar, Rajesh; Ministrini, Silvia; Agabiti Rosei, Enrico; Rizzoni, Damiano

    2018-01-01

    It has been previously demonstrated that T lymphocytes may be involved in the development of hypertension and microvascular remodeling, and that circulating T effector lymphocytes may be increased in hypertension. In particular, Th1 and Th 17 lymphocytes may contribute to the progression of hypertension and microvascular damage while T-regulatory (Treg) lymphocytes seem to be protective in this regard. However, no data is available about patients with severe obesity, in which pronounced microvascular alterations were observed. We have investigated 32 severely obese patients undergoing bariatric surgery, as well as 24 normotensive lean subjects and 12 hypertensive lean subjects undergoing an elective surgical intervention. A peripheral blood sample was obtained before surgery for assessment of CD4+ T lymphocyte subpopulations. Lymphocyte phenotype was evaluated by flow cytometry in order to assess T-effector and Treg lymphocytes. A marked reduction of several Treg subpopulations was observed in obese patients compared with controls, together with an increased in CD4+ effector memory T-effector cells. In severely obese patients, Treg lymphocytes are clearly reduced and CD4+ effector memory cells are increased. It may be hypothesized that they might contribute to the development of marked microvascular alterations previously observed in these patients.

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

    PubMed Central

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

    2018-01-01

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

  11. Fluoxetine suppresses calcium signaling in human T lymphocytes through depletion of intracellular calcium stores.

    PubMed

    Gobin, V; De Bock, M; Broeckx, B J G; Kiselinova, M; De Spiegelaere, W; Vandekerckhove, L; Van Steendam, K; Leybaert, L; Deforce, D

    2015-09-01

    Selective serotonin reuptake inhibitors, such as fluoxetine, have recently been shown to exert anti-inflammatory and immunosuppressive effects. Although the effects on cytokine secretion, proliferation and viability of T lymphocytes have been extensively characterized, little is known about the mechanism behind these effects. It is well known that Ca(2+) signaling is an important step in the signaling transduction pathway following T cell receptor activation. Therefore, we investigated if fluoxetine interferes with Ca(2+) signaling in Jurkat T lymphocytes. Fluoxetine was found to suppress Ca(2+) signaling in response to T cell receptor activation. Moreover, fluoxetine was found to deplete intracellular Ca(2+) stores, thereby leaving less Ca(2+) available for release upon IP3- and ryanodine-receptor activation. The Ca(2+)-modifying effects of fluoxetine are not related to its capability to block the serotonin transporter, as even a large excess of 5HT did not abolish the effects. In conclusion, these data show that fluoxetine decreases IP3- and ryanodine-receptor mediated Ca(2+) release in Jurkat T lymphocytes, an effect likely to be at the basis of the observed immunosuppression. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Peptides of a major histocompatibility complex class I (Kb) molecule cause prolongation of skin graft survival and induce specific down-regulatory T cells demonstrable in the mixed lymphocyte reaction.

    PubMed Central

    Brondz, B D; Kazansky, D B; Chernyshova, A D; Ivanov, V S

    1995-01-01

    Six individual peptides of the major histocompatibility complex (MHC) class I molecule H-2Kb were synthesized. Intravenous injection of peptide 6 into mice prolonged the survival of Kb (BL/6 or B10.MBR) skin grafts on allogeneic R101 and B10.AKM mice, respectively. This was specific, as control skin grafts from Kk (B10.BR) or Kd (DBA/2) donors, respectively, were rejected at the same time in both control and peptide-treated mice. The optimal doses for peptide 6, which is from the alpha 2 domain, were defined. The test system was the inhibition of proliferation in vitro of naive lymph node cells by syngeneic mitomycin c-treated spleen cells from R101 mice preimmunized with irradiated stimulator splenocytes of Kb (BL/6) origin. Down-regulation was specific, as proliferation in response to third-party allogeneic stimulator Kk (B10.BR) splenocytes was not inhibited. Of the six peptides of H-2Kb tested, potent down-regulatory cells were induced by peptides 2 (alpha 1 domain) and 5 and 6 (alpha 2 domain). The greatest down-regulatory activity was obtained by giving peptide 2 to mice that had already been immunized against H-2Kb by injecting EL4 cells. Under the same conditions, injecting peptide 2 did not induce any cytotoxic T cells. In contrast, specific cytotoxic lymphocytes (CTL) were induced when cells from primed mice were incubated for 4 days with heated stimulator cells from BL/6 mice. The data suggest that peptides from MHC class I molecules activate precursors of down-regulatory T cells, but not of CTL, and this may explain their ability to prolong skin allograft survival. PMID:7490121

  13. Psychosocial factors and T lymphocyte counts in Brazilian peacekeepers

    PubMed Central

    Monteiro da Silva, Angela M; Speranza, Francisco A B; Ishii, Solange Kiyoko; Hirata, Raphael; Mattos-Guaraldi, Ana Luíza; Milagres, Lucimar Gonçalves

    2015-01-01

    OBJECTIVE: To investigate the associations between psychosocial factors and peripheral blood CD4 and CD8 T lymphocyte numbers in Brazilian peacekeepers. METHODS: Venous blood was collected from 759 peacekeepers who had just returned from a peace mission in Haiti. Among the 759 soldiers, 642 individuals completed the psychosocial measures. CD4 and CD8 T lymphocyte counts were measured by flow cytometry using a commercially available kit. Psychosocial factors, including military peace force stressors, clinical stress, anxiety and depression, were recorded. As a reference for T lymphocyte numbers, we measured T lymphocyte counts in 75 blood donors from the Instituto de Biologia do Exército, Rio de Janeiro. RESULTS: The median numbers of CD4 and CD8 T lymphocytes in the blood donors were 819 cells/µl and 496 cells/µl, respectively, with a CD4:CD8 ratio of 1.6. Significantly (p<0.05) lower CD4 T cell counts (759 cells/µl) were recorded for peacekeepers, with similar CD8 levels (548 cells/µl) and smaller CD4:CD8 ratios (1.3, p<0.001) compared to blood donors. These differences were due to a group of 14 military personnel with CD4 and CD8 medians of 308 and 266 cells/µl, respectively. Only one (7.1%) of these 14 individuals was diagnosed with clinical stress compared with 13.5% of the individuals with normal levels of CD4 T lymphocytes. One individual out of 628 (0.16%) had a Lipp's Stress Symptom Inventory score of 3, indicating near exhaustion. CONCLUSION: The prevalence of psychological disorders was low and there were no associations with CD4 or CD8 T cell numbers. PMID:25789525

  14. Human IP10-scFv and DC-induced CTL synergistically inhibit the growth of glioma in a xenograft model.

    PubMed

    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.

  15. Search for biomarkers of asbestos exposure and asbestos-induced cancers in investigations of the immunological effects of asbestos.

    PubMed

    Matsuzaki, Hidenori; Kumagai-Takei, Naoko; Lee, Suni; Maeda, Megumi; Sada, Nagisa; Hatayama, Tamayo; Yamamoto, Shoko; Ikeda, Miho; Yoshitome, Kei; Min, Yu; Nishimura, Yasumitsu; Otsuki, Takemi

    2017-06-09

    The immunological effects of asbestos exposure on various lymphocytes such as the regulatory T cell (Treg), responder CD4+ T helper cell (Tresp), CD8+ cytotoxic T lymphocytes (CTL), and natural killer (NK) cells were investigated. Results show that asbestos exposure impairs antitumor immunity through enhancement of regulatory T cell function and volume, reduction of CXCR3 chemokine receptor in responder CD4+ T helper cells, and impairment of the killing activities of CD8+ cytotoxic T lymphocytes (CTL) and NK cells. These findings were used to explore biological markers associated with asbestos exposure and asbestos-induced cancers and suggested the usefulness of serum/plasma IL-10 and TGF-β, surface CXCR3 expression in Tresp, the secreting potential of IFN-γ in Tresp, intracellular perforin level in CTL, and surface expression NKp46 in NK cells. Although other unexplored cytokines in serum/plasma and molecules in these immunological cells, including Th17, should be investigated by experimental procedures in addition to a comprehensive analysis of screening methods, biomarkers based on immunological alterations may be helpful in clinical situations to screen the high-risk population exposed to asbestos and susceptible to asbestos-related cancers such as mesothelioma.

  16. NITRIC OXIDE, MITOCHONDRIAL HYPERPOLARIZATION AND T-CELL ACTIVATION

    PubMed Central

    Nagy, Gyorgy; Koncz, Agnes; Fernandez, David; Perl, Andras

    2007-01-01

    T lymphocyte activation is associated with nitric oxide (NO) production that plays an essential role in multiple T cell functions. NO acts as a messenger, activating soluble guanyl cyclase and participating in the transduction signaling pathways involving cyclic GMP. NO modulates mitochondrial events that are involved in apoptosis and regulates mitochondrial membrane potential and mitochondrial biogenesis in many cell types, including lymphocytes. Mitochondrial hyperpolarization (MHP), an early and reversible event during both T lymphocyte activation and apoptosis, is regulated by NO. Here, we discuss recent evidence that NO-induced MHP represents a molecular switch in multiple T cell signaling pathways. Overproduction of NO in systemic lupus erythematosus (SLE) induces mitochondrial biogenesis and alters Ca2+ signaling. Thus, while NO plays a physiological role in lymphocyte cell signaling, its overproduction may disturb normal T cell function, contributing to the pathogenesis of autoimmunity. PMID:17462531

  17. Signaling via the CD2 receptor enhances HTLV-1 replication in T lymphocytes.

    PubMed

    Guyot, D J; Newbound, G C; Lairmore, M D

    1997-07-21

    Human T lymphotropic virus type 1 (HTLV-1) is considered the etiologic agent of adult T cell leukemia/lymphoma and several chronic progressive immune-mediated diseases. Approximately 1-4% of infected individuals develop disease, generally decades following infection. Increased proviral transcription, mediated by the viral 40-kDa trans-activating protein, Tax, has been implicated in the pathogenesis of HTLV-1-associated diseases. Since the HTLV-1 promoter contains sequences responsive to cyclic AMP and protein kinase C, we hypothesized that lymphocyte activation signals initiated through the TCR/CD3 complex or CD2 receptor promote viral replication in HTLV-1-infected lymphocytes. We demonstrate that mAbs directed against the CD2, but not the CD3 receptor increase viral p24 capsid protein 1.5- to 5.7-fold in CD2/CD3+ HTLV-1-infected cell culture supernatants. Northern blot analysis demonstrated a 2.5- to 4-fold increase in all species of viral mRNA following CD2 cross-linking of OSP2/4 cells, an immortalized HTLV-1 cell line. Consistent with transcriptional regulation, reporter gene activity increased approximately 11-fold in CD2-stimulated Jurkat T cells cotransfected with a Tax-expressing plasmid and a CAT reporter gene construct under control of the HTLV-1 promoter. These data suggest a possible physiologic mechanism, whereby CD2-mediated cell adhesion and lymphocyte activation may promote viral transcription in infected lymphocytes.

  18. Effect of heated naringenin on immunomodulatory properties and cellular antioxidant activity.

    PubMed

    Maatouk, Mouna; Elgueder, Dorra; Mustapha, Nadia; Chaaban, Hind; Bzéouich, Imen Mokdad; Loannou, Irina; Kilani, Soumaya; Ghoul, Mohamed; Ghedira, Kamel; Chekir-Ghedira, Leila

    2016-11-01

    Naringenin is one of the most popular flavonoids derived from citrus. It has been reported to be an effective anti-inflammatory compound. Citrus fruit may be used raw, cooked, stewed, or boiled. The present study was conducted to investigate the effect of thermal processes on naringenin in its immunomodulatory and cellular antioxidant activities. The effects of flavonoids on B and T cell proliferation were assessed on splenocytes stimulated or not with mitogens. However, their effects on cytotoxic T lymphocyte (CTL) and natural killer (NK) activities were assessed in splenocytes co-incubated with target cells. The amount of nitric oxide production and the lysosomal enzyme activity were evaluated in vitro on mouse peritoneal macrophages. Cellular antioxidant activity in splenocytes and macrophages was determined by measuring the fluorescence of the dichlorofluorescin (DCF). Our findings revealed that naringenin induces B cell proliferation and enhances NK activity. The highest concentration of native naringenin exhibits a significant proliferation of T cells, induces CTL activity, and inhibits cellular oxidation in macrophages. Conversely, it was observed that when heat-processed, naringenin improves the cellular antioxidant activity in splenocytes, increases the cytotoxic activity of NK cells, and suppresses the cytotoxicity of T cells. However, heat treatment maintains the anti-inflammatory potency of naringenin.

  19. Co-Stimulation through 4-1BB/CD137 Improves the Expansion and Function of CD8+ Melanoma Tumor-Infiltrating Lymphocytes for Adoptive T-Cell Therapy

    PubMed Central

    Chacon, Jessica Ann; Wu, Richard C.; Sukhumalchandra, Pariya; Molldrem, Jeffrey J.; Sarnaik, Amod; Pilon-Thomas, Shari; Weber, Jeffrey; Hwu, Patrick; Radvanyi, Laszlo

    2013-01-01

    Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) can induce tumor regression in up to 50% or more of patients with unresectable metastatic melanoma. However, current methods to expand melanoma TIL, especially the “rapid expansion protocol” (REP) were not designed to enhance the generation of optimal effector-memory CD8+ T cells for infusion. One approach to this problem is to manipulate specific co-stimulatory signaling pathways to enhance CD8+ effector-memory T-cell expansion. In this study, we determined the effects of activating the TNF-R family member 4-1BB/CD137, specifically induced in activated CD8+ T cells, on the yield, phenotype, and functional activity of expanded CD8+ T cells during the REP. We found that CD8+ TIL up-regulate 4-1BB expression early during the REP after initial TCR stimulation, but neither the PBMC feeder cells in the REP or the activated TIL expressed 4-1BB ligand. However, addition of an exogenous agonistic anti-4-1BB IgG4 (BMS 663513) to the REP significantly enhanced the frequency and total yield of CD8+ T cells as well as their maintenance of CD28 and increased their anti-tumor CTL activity. Gene expression analysis found an increase in bcl-2 and survivin expression induced by 4-1BB that was associated with an enhanced survival capability of CD8+ post-REP TIL when re-cultured in the absence or presence of cytokines. Our findings suggest that adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8+ T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients. PMID:23560068

  20. Immunogenicity of Recombinant Classic Swine Fever Virus CD8+ T Lymphocyte Epitope and Porcine Parvovirus VP2 Antigen Coexpressed by Lactobacillus casei in Swine via Oral Vaccination ▿

    PubMed Central

    Xu, Yigang; Cui, Lichun; Tian, Changyong; Zhang, Guocai; Huo, Guicheng; Tang, Lijie; Li, Yijing

    2011-01-01

    Classical swine fever virus (CSFV) and porcine parvovirus (PPV) are highly contagious pathogens, resulting in enormous economic losses in pig industries worldwide. Because vaccines play an important role in disease control, researchers are seeking improved vaccines that could induce antiviral immune responses against CSFV and PPV at the mucosal and systemic levels simultaneously. In this study, a genetically engineered Lactobacillus strain coexpressing the CSFV-specific cytotoxic T lymphocyte (CTL) epitope 290 and the VP2 antigen of PPV was developed, and its immunopotentiating capacity as an oral vaccine in pigs was analyzed. The data demonstrated that in the absence of any adjuvant, the recombinant Lactobacillus strain can efficiently stimulate mucosal and systemic CSFV-specific CD8+ CTL responses to protect pigs against CSFV challenge. Moreover, anti-PPV-VP2 serum IgG and mucosal IgA were induced in pigs immunized orally with the recombinant Lactobacillus strain, showing a neutralizing effect on PPV infection. The results suggest that the recombinant Lactobacillus microecological agent may be a valuable component of a strategy for development of a vaccine against CSFV and PPV. PMID:21940406

  1. Suppressor cell hyperactivity relative to allogeneic lymphocyte proliferation as a manifestation of defective T-T-cell interactions in systemic lupus erythematosus

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

    Stenina, M.A.; Potapova, A.A.; Biryukov, A.V.

    1987-01-01

    The authors study the state of immunoregulatory process in patients with systemic lupus erythematosus at the T-T-cell interaction level and seek to test the possibility of the pharmacological modulation of this process. The proliferative activity of mononuclear lymphocytes, extracted from the blood of ten lupus patients, was assessed by measuring the incorporation of tritiated thymidine into cultures stimulated by phytohemagglutinin, concanavalin, and theophylline. The comparative effects of each of these agents on the immunoregulatory and proliferative activity of the lymphocytes are reported.

  2. Quantifying T Lymphocyte Turnover

    PubMed Central

    De Boer, Rob J.; Perelson, Alan S.

    2013-01-01

    Peripheral T cell populations are maintained by production of naive T cells in the thymus, clonal expansion of activated cells, cellular self-renewal (or homeostatic proliferation), and density dependent cell life spans. A variety of experimental techniques have been employed to quantify the relative contributions of these processes. In modern studies lymphocytes are typically labeled with 5-bromo-2′-deoxyuridine (BrdU), deuterium, or the fluorescent dye carboxy-fluorescein diacetate succinimidyl ester (CFSE), their division history has been studied by monitoring telomere shortening and the dilution of T cell receptor excision circles (TRECs) or the dye CFSE, and clonal expansion has been documented by recording changes in the population densities of antigen specific cells. Proper interpretation of such data in terms of the underlying rates of T cell production, division, and death has proven to be notoriously difficult and involves mathematical modeling. We review the various models that have been developed for each of these techniques, discuss which models seem most appropriate for what type of data, reveal open problems that require better models, and pinpoint how the assumptions underlying a mathematical model may influence the interpretation of data. Elaborating various successful cases where modeling has delivered new insights in T cell population dynamics, this review provides quantitative estimates of several processes involved in the maintenance of naive and memory, CD4+ and CD8+ T cell pools in mice and men. PMID:23313150

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

    PubMed

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

    2013-06-01

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

  4. Cetuximab-activated natural killer (NK) and dendritic cells (DC) collaborate to trigger tumor antigen-specific T cell immunity in head and neck cancer patients

    PubMed Central

    Srivastava, Raghvendra M.; Lee, Steve C.; Filho, Pedro A. Andrade; Lord, Christopher A.; Jie, Hyun-bae; Davidson, H. Carter; López-Albaitero, Andrés; Gibson, Sandra P.; Gooding, William E.; Ferrone, Soldano; Ferris, Robert L.

    2013-01-01

    Purpose Tumor antigen (TA)-specific monoclonal antibodies (mAb) block oncogenic signaling and induce Fcγ receptor (FcγR)-mediated cytotoxicity. However, the role of CD8+ cytotoxic T lymphocyte (CTL) and FcγR in initiating innate and adaptive immune responses in mAb-treated human cancer patients is still emerging. Experimental Design FcγRIIIa codon 158 polymorphism was correlated with survival in 107 cetuximab-treated head and neck cancer (HNC) patients. Flow cytometry was performed to quantify EGFR-specific T cells in cetuximab-treated HNC patients. The effect of cetuximab on NK cell, dendritic cell (DC), and T cell activation was measured using IFN-γ release assays and flow cytometry. Results FcγR IIIa polymorphism did not predict clinical outcome in cetuximab-treated HNC patients, however elevated circulating EGFR -specific CD8+ 853-861 T cells were found in cetuximab-treated HNC patients (p<0.005). Cetuximab promoted EGFR-specific cellular immunity through the interaction of EGFR+ tumor cells and FcγRIIIa on NK cells, but not on the polymorphism per se. Cetuximab-activated NK cells induced IFN-γ dependent expression of DC maturation markers, antigen presentation machinery (APM) components such as TAP-1/2, and Th1 chemokines through NKG2D/MICA binding. Cetuximab initiated adaptive immune responses via NK-cell induced DC maturation, which enhanced cross-presentation to CTL specific for EGFR as well as another TA, MAGE-3. Conclusion Cetuximab-activated NK cells promote DC maturation and CD8+ T cell priming, leading to TA spreading and Th1 cytokine release through ‘NK-DC cross-talk.’ FcγRIIIa polymorphism did not predict clinical response to cetuximab, but was necessary for NK-DC interaction and mAb induced cross-presentation. EGFR-specific T cells in cetuximab treated HNC patients may contribute to clinical response. PMID:23444227

  5. Changes in T and B blood lymphocytes after splenectomy.

    PubMed Central

    Millard, R E; Banerjee, D K

    1979-01-01

    The blood lymphocytes of 37 splenectomised patients were analysed by means of T and B lymphocyte surface markers. Sixteen patients had had a splenectomy for non-haematological and 21 for haematological reasons. The results show that 15 had normal numbers of T and B cells; decreased T cells were found in two patients, raised B cells in seven, raised T and B cells in eight, and raised T cells in five patients. Increased numbers of 'null' cells were observed in some patients, especially in those with raised B cells. Follow-up studies indicate that raised levels of T and B cells can be established by one to three months post-splenectomy and may persist, although in some patients the cells fall to normal levels. The lymphocyte proliferative response to phytohaemagglutinin and Concanavalin A in vitro was normal in eight out of nine patients with raised T cells and was depressed in one patient, possibly due to an intrinsic cell defect. PMID:316436

  6. Regulation of Asymmetric Division and CD8+ T Lymphocyte Fate Specification by PKCζ and PKCλ/ι

    PubMed Central

    Metz, Patrick J.; Arsenio, Janilyn; Kakaradov, Boyko; Kim, Stephanie H.; Remedios, Kelly A.; Oakley, Katherine; Akimoto, Kazunori; Ohno, Shigeo; Yeo, Gene W.; Chang, John T.

    2015-01-01

    During an immune response against a microbial pathogen, activated naïve T lymphocytes give rise to effector cells that provide acute host defense and memory cells that provide long-lived immunity. It has been shown that T lymphocytes can undergo asymmetric division, enabling the daughter cells to inherit unequal amounts of fate-determining proteins and thereby acquire distinct fates from their inception. Here, we show that the absence of the atypical protein kinase C (aPKC) isoforms, PKCζ and PKCλ/ι, disrupts asymmetric CD8+ T lymphocyte division. These alterations were associated with aberrant acquisition of a ‘pre-effector’ transcriptional program, detected by single-cell gene expression analyses, in lymphocytes that had undergone their first division in vivo and enhanced differentiation toward effector fates at the expense of memory fates. Together, these results demonstrate a role for aPKC in regulating asymmetric division and the specification of divergent CD8+ T lymphocyte fates early during an immune response. PMID:25617472

  7. CHARACTERIZATION OF NORMAL HUMAN LUNG LYMPHOCYTES AND INTERLEUKIN-2-INDUCED LUNG T CELL LINES

    EPA Science Inventory

    Lymphocytes from the lower respiratory tract were obtained by bronchoalveolar lavage of healthy, non-smoking individuals. arious monoclonal antibodies characterizing activated T cells, helper-inducer and suppressor-inducer T cell subsets, and naive versus memory cells were used t...

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

    PubMed Central

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

    2009-01-01

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

  9. Deficiency of the autologous mixed lymphocyte reactions of non-T/T and T/T type in intravenous drug abusers infected by the human immunodeficiency virus (HIV).

    PubMed

    Puppo, F; Pierri, I; Rogna, S; Pattarini, R; Piovano, P L; Catellani, S; Varnier, O E; Indiveri, F

    1987-01-01

    In the present study both responsiveness and stimulatory capacity in autologous mixed lymphocyte reactions (AMLRs) of non-T/T and T/T type, as well as in allogeneic mixed lymphocyte reaction (MLR), were evaluated in 30 intravenous drug abusers (IDAs) infected by the human immunodeficiency virus (HIV) and in 10 HIV-negative IDAs. The production of interleukin 2 (IL2), and the expression of HLA Class II antigens and IL2 receptors by PHA-activated T lymphocytes were also evaluated. A severe impairment of both responsiveness and stimulatory capacity in MLR and AMLRs was found in the HIV-positive IDAs and not in the HIV-negative IDAs. The HIV-positive IDAs showed also a defective expression of HLA Class II antigens, whereas the IL2 production and the IL2 receptor expression were in the normal range. The present data are consistent with similar observations in male homosexuals with AIDS-related complex and confirm that the HIV infection induces a broad spectrum of immunological abnormalities leading to a progressive derangement of the immunocompetence.

  10. Ebola Virus Binding to Tim-1 on T Lymphocytes Induces a Cytokine Storm

    PubMed Central

    Younan, Patrick; Iampietro, Mathieu; Nishida, Andrew; Ramanathan, Palaniappan; Santos, Rodrigo I.; Dutta, Mukta; Lubaki, Ndongala Michel; Koup, Richard A.; Katze, Michael G.

    2017-01-01

    ABSTRACT Ebola virus (EBOV) disease (EVD) results from an exacerbated immunological response that is highlighted by a burst in the production of inflammatory mediators known as a “cytokine storm.” Previous reports have suggested that nonspecific activation of T lymphocytes may play a central role in this phenomenon. T-cell immunoglobulin and mucin domain-containing protein 1 (Tim-1) has recently been shown to interact with virion-associated phosphatidylserine to promote infection. Here, we demonstrate the central role of Tim-1 in EBOV pathogenesis, as Tim-1−/− mice exhibited increased survival rates and reduced disease severity; surprisingly, only a limited decrease in viremia was detected. Tim-1−/− mice exhibited a modified inflammatory response as evidenced by changes in serum cytokines and activation of T helper subsets. A series of in vitro assays based on the Tim-1 expression profile on T cells demonstrated that despite the apparent absence of detectable viral replication in T lymphocytes, EBOV directly binds to isolated T lymphocytes in a phosphatidylserine–Tim-1-dependent manner. Exposure to EBOV resulted in the rapid development of a CD4Hi CD3Low population, non-antigen-specific activation, and cytokine production. Transcriptome and Western blot analysis of EBOV-stimulated CD4+ T cells confirmed the induction of the Tim-1 signaling pathway. Furthermore, comparative analysis of transcriptome data and cytokine/chemokine analysis of supernatants highlight the similarities associated with EBOV-stimulated T cells and the onset of a cytokine storm. Flow cytometry revealed virtually exclusive binding and activation of central memory CD4+ T cells. These findings provide evidence for the role of Tim-1 in the induction of a cytokine storm phenomenon and the pathogenesis of EVD. PMID:28951472

  11. Moderate physical activity of music aerobic exercise increases lymphocyte counts, specific subsets, and differentiation.

    PubMed

    Yeh, Shu-Hui; Lai, Hsiu-Ling; Hsiao, Chiu-Yueh; Lin, Li-Wei; Chuang, Yu-Kuan; Yang, Yu-Yeng; Yang, Kuender D

    2014-09-01

    Moderate physical activity has been shown to promote immunity. Different moderate physical activities may have different effects on immunity. This study investigated the impacts of a 12-week regular music aerobic exercise (MAE) program on leukocyte distribution, lymphocyte subsets, and lymphocyte polarization. The study used a case-control design with pretest and posttest. Forty-seven middle-age women were recruited for this study. Three participants dropped out, 22 completed the 12-week MAE program, and the other 22 participants who had heat-intolerance or limited schedule eligibility were enrolled as the control group without the MAE exercise. Results showed that the MAE exercise for 12 weeks didn't change red blood cells or total leukocytes but increased lymphocyte counts. The women in MAE group revealed significant increases (P ≤ 0.01) of CD3CD4, CD3CD8, and CD4CD25 cells, associated with Treg polarization showing enhanced FoxP3 but not T-bet, Gata-3, or RORγT expression (P < .01). The control group without exercise revealed insignificant change of lymphocyte subsets or lymphocyte polarization. This study shows that MAE increases specific lymphocyte subsets and enhances Treg cell differentiation. It is suggested to encourage moderate physical activity of music aerobic exercise to enhance lymphocyte function of middle-aged women.

  12. Chimeric Antigen Receptor–Modified T Cells for Acute Lymphoid Leukemia

    PubMed Central

    Barrett, David; Aplenc, Richard; Porter, David L.; Rheingold, Susan R.; Teachey, David T.; Chew, Anne; Hauck, Bernd; Wright, J. Fraser; Milone, Michael C.; Levine, Bruce L.; June, Carl H.

    2014-01-01

    Summary Chimeric antigen receptor–modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre–B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×106 to 1.2×107 CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce anti-leukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor–modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL. PMID:23527958

  13. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.

    PubMed

    Grupp, Stephan A; Kalos, Michael; Barrett, David; Aplenc, Richard; Porter, David L; Rheingold, Susan R; Teachey, David T; Chew, Anne; Hauck, Bernd; Wright, J Fraser; Milone, Michael C; Levine, Bruce L; June, Carl H

    2013-04-18

    Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.

  14. Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes.

    PubMed

    Tsai, Wei-Jern; Chang, Chu-Ting; Wang, Guei-Jane; Lee, Tzong-Huei; Chang, Shwu-Fen; Lu, Shao-Chun; Kuo, Yuh-Chi

    2011-03-25

    Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT.

  15. Cytotoxic-T-lymphocyte antigen 4 receptor signaling for lymphocyte adhesion is mediated by C3G and Rap1.

    PubMed

    Kloog, Yoel; Mor, Adam

    2014-03-01

    T-lymphocyte adhesion plays a critical role in both inflammatory and autoimmune responses. The small GTPase Rap1 is the key coordinator mediating T-cell adhesion to endothelial cells, antigen-presenting cells, and virus-infected cells. We describe a signaling pathway, downstream of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor, leading to Rap1-mediated adhesion. We identified a role for the Rap1 guanine nucleotide exchange factor C3G in the regulation of T-cell adhesion and showed that this factor is required for both T-cell receptor (TCR)-mediated and CTLA-4-mediated T-cell adhesion. Our data indicated that C3G translocates to the plasma membrane downstream of TCR signaling, where it regulates activation of Rap1. We also showed that CTLA-4 receptor signaling mediates tyrosine phosphorylation in the C3G protein, and that this is required for augmented activation of Rap1 and increased adhesion mediated by leukocyte function-associated antigen type 1 (LFA-1). Zap70 is required for C3G translocation to the plasma membrane, whereas the Src family member Hck facilitates C3G phosphorylation. These findings point to C3G and Hck as promising potential therapeutic targets for the treatment of T-cell-dependent autoimmune disorders.

  16. Cytotoxic-T-Lymphocyte Antigen 4 Receptor Signaling for Lymphocyte Adhesion Is Mediated by C3G and Rap1

    PubMed Central

    Kloog, Yoel

    2014-01-01

    T-lymphocyte adhesion plays a critical role in both inflammatory and autoimmune responses. The small GTPase Rap1 is the key coordinator mediating T-cell adhesion to endothelial cells, antigen-presenting cells, and virus-infected cells. We describe a signaling pathway, downstream of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor, leading to Rap1-mediated adhesion. We identified a role for the Rap1 guanine nucleotide exchange factor C3G in the regulation of T-cell adhesion and showed that this factor is required for both T-cell receptor (TCR)-mediated and CTLA-4-mediated T-cell adhesion. Our data indicated that C3G translocates to the plasma membrane downstream of TCR signaling, where it regulates activation of Rap1. We also showed that CTLA-4 receptor signaling mediates tyrosine phosphorylation in the C3G protein, and that this is required for augmented activation of Rap1 and increased adhesion mediated by leukocyte function-associated antigen type 1 (LFA-1). Zap70 is required for C3G translocation to the plasma membrane, whereas the Src family member Hck facilitates C3G phosphorylation. These findings point to C3G and Hck as promising potential therapeutic targets for the treatment of T-cell-dependent autoimmune disorders. PMID:24396067

  17. 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.

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

    PubMed

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

    2013-12-18

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

  19. microRNA regulation of T lymphocyte immunity: modulation of molecular networks responsible for T cell activation, differentiation and development

    PubMed Central

    Podshivalova, Katie; Salomon, Daniel R.

    2014-01-01

    MicroRNAs (miRNA) are a class of small non-coding RNAs that constitute an essential and evolutionarily conserved mechanism for post-transcriptional gene regulation. Multiple miRNAs have been described to play key roles in T lymphocyte development, differentiation and function. In this review we highlight the current literature regarding the differential expression of miRNAs in various models of mouse and human T cell biology and emphasize mechanistic understandings of miRNA regulation of thymocyte development, T cell activation, and differentiation into effector and memory subsets. We describe the participation of miRNAs in complex regulatory circuits shaping T cell proteomes in a context-dependent manner. It is striking that some miRNAs regulate multiple processes, while others only appear in limited functional contexts. It is also evident that the expression and function of specific miRNAs can differ between mouse and human systems. Ultimately, it is not always correct to simplify the complex events of T cell biology into a model driven by only one or two master regulator miRNAs. In reality, T cell activation and differentiation involves the expression of multiple miRNAs with many mRNA targets and thus, the true extent of miRNA regulation of T cell biology is likely far more vast than currently appreciated. PMID:24099302

  20. Immunostimulation by cytomegalovirus (CMV): helper T cell-dependent activation of immunoglobulin production in vitro by lymphocytes from CMV-immune donors

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

    Yachie, A.; Tosato, G.; Straus, S.E.

    1985-08-01

    Cytomegalovirus (CMV) is the cause of a number of different diseases ranging from self-limited benign infections in healthy adults to life threatening illnesses among immunocompromised hosts and newborns. Suppression of cell-mediated immunity is often found in cases of acute CMV infection, and in addition, the virus may also be a potent stimulant of lymphoid cells in vivo. The authors studied cellular proliferation and immunoglobulin (Ig) production induced by CMV to determine its effect on human lymphocytes in vitro. The CMV that was added to cultures of lymphocytes from CMV-seronegative donors failed to induce either significant cellular proliferation or Ig production.more » By contrast, CMV-stimulated cultures from CMV-seropositive donors induced both prominent cellular proliferation and Ig production. B cell differentiation into Ig-secreting cells required the presence of T cells, and this T cell help was sensitive to irradiation with 2000 rad and to treatment with cyclosporin A. When T cells were depleted of OKT4+ cells with monoclonal antibody and complement, the co-cultured B cells failed to produce Ig, whereas the depletion of OKT8+ cells had no effect on the Ig-secreting cell response. Inactivation of CMV before culture did not result in a reduction of either cellular proliferation or Ig production. Thus, infection of target cells is not required for in vitro lymphocyte activation by CMV. These results demonstrate that CMV is a potent activator of B cells inducing Ig production in vitro, and that this process requires the presence of virus-specific memory T cells.« less

  1. Cytotoxic T cells use mechanical force to potentiate target cell killing

    PubMed Central

    Basu, Roshni; Whitlock, Benjamin M.; Husson, Julien; Le Floc’h, Audrey; Jin, Weiyang; Oyler-Yaniv, Alon; Dotiwala, Farokh; Giannone, Gregory; Hivroz, Claire; Biais, Nicolas; Lieberman, Judy; Kam, Lance C.; Huse, Morgan

    2016-01-01

    SUMMARY The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals. PMID:26924577

  2. Cytotoxic T Cells Use Mechanical Force to Potentiate Target Cell Killing.

    PubMed

    Basu, Roshni; Whitlock, Benjamin M; Husson, Julien; Le Floc'h, Audrey; Jin, Weiyang; Oyler-Yaniv, Alon; Dotiwala, Farokh; Giannone, Gregory; Hivroz, Claire; Biais, Nicolas; Lieberman, Judy; Kam, Lance C; Huse, Morgan

    2016-03-24

    The immunological synapse formed between a cytotoxic T lymphocyte (CTL) and an infected or transformed target cell is a physically active structure capable of exerting mechanical force. Here, we investigated whether synaptic forces promote the destruction of target cells. CTLs kill by secreting toxic proteases and the pore forming protein perforin into the synapse. Biophysical experiments revealed a striking correlation between the magnitude of force exertion across the synapse and the speed of perforin pore formation on the target cell, implying that force potentiates cytotoxicity by enhancing perforin activity. Consistent with this interpretation, we found that increasing target cell tension augmented pore formation by perforin and killing by CTLs. Our data also indicate that CTLs coordinate perforin release and force exertion in space and time. These results reveal an unappreciated physical dimension to lymphocyte function and demonstrate that cells use mechanical forces to control the activity of outgoing chemical signals. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. TRALI ASSOCIATED HNA-3a ANTIBODIES RECOGNIZE COMPLEX DETERMINANTS ON CHOLINE TRANSPORTER-LIKE PROTEIN 2 (CTL2)

    PubMed Central

    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

  4. Effect Of Simulated Microgravity On Activated T Cell Gene Transcription

    NASA Technical Reports Server (NTRS)

    Morrow, Maureen A.

    2003-01-01

    Studies of T lymphocytes under the shear stress environment of clinorotation have demonstrated an inhibition of activation in response to TCR mediated signaling. These results mimic those observed during space flight. This work investigates the molecular signaling events of T lymphocyte activation with clinorotation. Purified human T lymphocytes and the T cell clone Jurkat exhibit an uncoupling of signaling as mediated through the TCR. Activation of the transcription factor AP-1 is inhibited while activation of NFAT occurs. NFAT dephosphorylation and activation is dependent on sustained Ca(++) influx. Alternatively, AP-1, which consists of two transcription factors, jun and fos, is activated by PKC and Ras mediated pathways. TCR signaling is known to be dependent on cytoskeletal rearrangements, in particular, raft aggregation is critical. Raft aggregation, as mediated through GM, crosslinking, overcomes the inhibition of T lymphocyte activation with clinorotation, indicating that the block is occurring upstream of raft aggregation. Clinorotation is shown to have an effect similar to a weak TCR signal.

  5. Reprogramming T cell Lymphocytes to Induced Pluripotent Stem Cells

    NASA Astrophysics Data System (ADS)

    Bared, Kalia

    The discovery of induced pluripotent stem cells (iPSC) provided a novel technology for the study of development and pharmacology and complement embryonic stem cells (ES) for cell therapy applications. Though iPSC are derived from adult tissue they are comparable to ES cells in their behavior; multi-lineage differentiation and self-renewal. This makes iPSC research appealing because they can be studied in great detail and expanded in culture broadly. Fibroblasts were the first cell type reprogrammed to an iPSC using a retrovirus vector, since then alternative cell types including lymphocytes have been used to generate iPSC. Different types of vectors have also been developed to enhance iPSC formation and quality. However, specific T lymphocyte subsets have not been shown to reprogram to a pluripotent state to date. Here, we proposed to derive iPSC from peripheral blood effector and central memory T cells, reasoning that the resultant iPSC will maintain the epigenetic memory of a T lymphocyte, including the T cell receptor (TCR) gene rearrangement. This epigenetic memory will enable the differentiation and expansion of T cell iPSC into professional T cells containing a specific TCR. These could then be used for cell therapy to target specific antigens, as well as to improve culture techniques to expand T cells in vitro. We studied different gene delivery methods to derive iPSC from different types of T lymphocytes. We assessed the viability of viral transduction using flow cytometry to detect green fluorescent marker contained in the viral construct and quantitative real time polymerase chain reaction (qRT-PCR) to detect Oct4, Klf4, Sox2, and c-Myc gene expression. Our results demonstrate that the Sendai virus construct is the most feasible platform to reprogram T lymphocytes. We anticipate that this platform will provide an efficient and safe approach to derive iPSC from different T cell subsets, including memory T cells.

  6. Extracts of chronic lymphocytic leukemia lymphocytes have a high level of DNA repair activity for O/sup 6/-methylguanine

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

    Waldstein, E.A.; Cao, E.H.; Miller, M.E.

    Extracts of peripheral lymphocytes from six individuals with chronic lymphocytic leukemia (CLL) were assayed for the ability to remove O/sup 6/-methylguanine (O/sup 6/MeGua) from exogenous DNA. The O/sup 6/MeGua-removing activity in CLL lymphocytes, predominantly B cells, was approximately 7-fold higher than in B lymphocytes of normal individuals and about 2-fold higher than in the unstimulated T type cells of normal persons. The activity measured in extracts of lymphocytes from three blood relatives was in the upper range of the normal distribution. Over 80% of the removal of O/sup 6/MeGua was accomplished by the transfer of the methyl group to cysteinemore » moieties of acceptor proteins in a stoichiometric reaction. If one assumes one acceptor group per acceptor protein, the calculated number of acceptor molecules per CLL lymphocyte falls between 91,000 and 220,000. Thus CLL lymphocytes do not show lower O/sup 6/MeGua-removing activity, in contrast to many tumor cell strains or transformed cell lines, which are reported to have a deficient methyl excision repair phenotype (Mer/sup -/). Instead, the CLL lymphocytes act as if they have a super-Mer/sup +/ phenotype.« less

  7. Molecular Pathology of Adult T-Cell Leukemia/Lymphoma.

    PubMed

    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

  8. Priming of Anti-Human Immunodeficiency Virus (HIV) CD8^+ Cytotoxic T Cells in vivo by Carrier-Free HIV Synthetic Peptides

    NASA Astrophysics Data System (ADS)

    Hart, Mary Kate; Weinhold, Kent J.; Scearce, Richard M.; Washburn, Eileen M.; Clark, Cynthia A.; Palker, Thomas J.; Haynes, Barton F.

    1991-11-01

    The generation of antiviral cytotoxic T lymphocytes (CTLs) is a critical component of the immune response to viral infections. A safe and nontoxic vaccine for AIDS would optimally use a carrier-free synthetic peptide immunogen containing only components of HIV necessary for induction of protective immune responses. We report that hybrid synthetic peptides containing either a HIV envelope gp120 T-cell determinant (T1) or the envelope gp41 fusion domain (F) N-terminal to HIV CTL determinants are capable of priming murine CD8^+, major histocompatibility complex class I-restricted anti-HIV CTLs in vivo. These data demonstrate that carrier-free, nonderivatized synthetic peptides can be used in vivo to induce anti-HIV CTL responses.

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

    PubMed Central

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

    2017-01-01

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

  10. Double-stranded RNA promotes CTL-independent tumor cytolysis mediated by CD11b+Ly6G+ intratumor myeloid cells through the TICAM-1 signaling pathway

    PubMed Central

    Shime, Hiroaki; Matsumoto, Misako; Seya, Tsukasa

    2017-01-01

    PolyI:C, a synthetic double-stranded RNA analog, acts as an immune-enhancing adjuvant that regresses tumors in cytotoxic T lymphocyte (CTL)-dependent and CTL-independent manner, the latter of which remains largely unknown. Tumors contain CD11b+Ly6G+ cells, known as granulocytic myeloid-derived suppressor cells (G-MDSCs) or tumor-associated neutrophils (TANs) that play a critical role in tumor progression and development. Here, we demonstrate that CD11b+Ly6G+ cells respond to polyI:C and exhibit tumoricidal activity in an EL4 tumor implant model. PolyI:C-induced inhibition of tumor growth was attributed to caspase-8/3 cascade activation in tumor cells that occurred independently of CD8α+/CD103+ dendritic cells (DCs) and CTLs. CD11b+Ly6G+ cells was essential for the antitumor effect because depletion of CD11b+Ly6G+ cells totally abrogated tumor regression and caspase activation after polyI:C treatment. CD11b+Ly6G+ cells that had been activated with polyI:C showed cytotoxicity and inhibited tumor growth through the production of reactive oxygen species (ROS)/reactive nitrogen species (RNS). These responses were abolished in either Toll/interleukin-1 receptor domain-containing adaptor molecule-1 (TICAM-1)−/− or interferon (IFN)-αβ receptor 1 (IFNAR1)−/− mice. Thus, our results suggest that polyI:C activates the TLR3/TICAM-1 and IFNAR signaling pathways in CD11b+Ly6G+ cells in tumors, thereby eliciting their antitumor activity, independent of those in CD8α+/CD103+ DCs that prime CTLs. PMID:27834952

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

    PubMed

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

    2017-11-06

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

  12. Interaction between human mature adipocytes and lymphocytes induces T-cell proliferation.

    PubMed

    Poloni, Antonella; Maurizi, Giulia; Ciarlantini, Marco; Medici, Martina; Mattiucci, Domenico; Mancini, Stefania; Maurizi, Angela; Falconi, Massimo; Olivieri, Attilio; Leoni, Pietro

    2015-09-01

    Adipose tissue is a critical organ that plays a major role in energy balance regulation and the immune response through intricate signals. We report on the inter-relation between mature adipocytes and lymphocytes in terms of adipocyte-derived T-cell chemo-attractants and adipocyte metabolic effects on lymphocytes. During the culture time, mature adipocytes changed their structural and functional properties into de-differentiated cells. Isolated mature adipocytes expressed significantly higher levels of CIITA, major histocompatibility complex II (human leukocyte antigen [HLA]-DR) and costimulatory signal molecule CD80 compared with adipocytes after the de-differentiation process. Moreover, human leukocyte antigen-G, which may prevent the immune responses of mesenchymal stromal cells, was expressed at lower level in mature adipocytes compared with de-differentiated adipocytes. In line with these molecular data, functional results showed different immunoregulatory properties between adipocytes before and after the de-differentiation process. Mature adipocytes stimulated the proliferation of total lymphocytes and immunoselected cell populations CD3+, CD4+ and CD8+ in a direct contact-dependent way that involved the major histocompatibility complex I and II pathways. Moreover, adipocytes secreted potential chemo-attractant factors, but data showed that adipocyte-derived culture medium was not sufficient to activate lymphocyte proliferation, suggesting that a direct contact between adipocytes and immune cells was needed. However, specific mature adipocyte cytokines enhanced lymphocyte proliferation in a mixed lymphocyte reaction. In conclusion, cross-talk occurs between adipocytes and lymphocytes within adipose tissue involving T-cell chemo-attraction by mature adipocytes. Our findings, together with current observations in the field, provide a rationale to identify adipocyte-lymphocyte cross-talk that instigates adipose inflammation. Copyright © 2015 International

  13. The lymphocytic cholinergic system and its contribution to the regulation of immune activity.

    PubMed

    Kawashima, Koichiro; Fujii, Takeshi

    2003-12-26

    Lymphocytes express most of the cholinergic components found in the nervous system, including acetylcholine (ACh), choline acetyltransferase (ChAT), high affinity choline transporter, muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), and acetylcholinesterase. Stimulation of T and B cells with ACh or another mAChR agonist elicits intracellular Ca2+ signaling, up-regulation of c-fos expression, increased nitric oxide synthesis and IL-2-induced signal transduction, probably via M3 and M5 mAChR-mediated pathways. Acute stimulation of nAChRs with ACh or nicotine causes rapid and transient Ca2+ signaling in T and B cells, probably via alpha7 nAChR subunit-mediated pathways. Chronic nicotine stimulation, by contrast, down-regulates nAChR expression and suppresses T cell activity. Activation of T cells with phytohemagglutinin or antibodies against cell surface molecules enhances lymphocytic cholinergic transmission by activating expression of ChAT and M5 mAChR, which is suggestive of local cholinergic regulation of immune system activity. This idea is supported by the facts that lymphocytic cholinergic activity reflects well the changes in immune system function seen in animal models of immune deficiency and immune acceleration. Collectively, these data provide a compelling picture in which lymphocytes constitute a cholinergic system that is independent of cholinergic nerves, and which is involved in the regulation of immune function.

  14. Analysis of the K+ current in human CD4+ T lymphocytes in hypercholesterolemic state.

    PubMed

    Somodi, Sándor; Balajthy, András; Szilágyi, Orsolya; Pethő, Zoltán; Harangi, Mariann; Paragh, György; Panyi, György; Hajdu, Péter

    2013-01-01

    Atherosclerosis involves immune mechanisms: T lymphocytes are found in atherosclerotic plaques, suggesting their activation during atherogenesis. The predominant voltage-gated potassium channel of T cells, Kv1.3 is a key regulator of the Ca(2+)-dependent activation pathway. In the present experiments we studied the proliferation capacity and functional changes of Kv1.3 channels in T cells from healthy and hypercholestaeremic patients. By means of CFSE-assay (carboxyfluorescein succinimidyl ester) we showed that spontaneous activation rate of lymphocytes in hypercholesterolemia was elevated and the antiCD3/antiCD28 co-stimulation was less effective as compared to the healthy group. Using whole-cell patch-clamping we obtained that the activation and deactivation kinetics of Kv1.3 channels were faster in hypercholesterolemic state but no change in other parameters of Kv1.3 were found (inactivation kinetics, steady-state activation, expression level). We suppose that incorporation of oxLDL species via its raft-rupturing effect can modify proliferative rate of T cells as well as the gating of Kv1.3 channels. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes

    PubMed Central

    2011-01-01

    Background Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Methods Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. Results AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. Conclusion AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT. PMID:21435270

  16. Transcription factor NF-kappaB regulates inducible CD83 gene expression in activated T lymphocytes.

    PubMed

    McKinsey, T A; Chu, Z; Tedder, T F; Ballard, D W

    2000-01-01

    The immunoglobulin superfamily member CD83 is expressed on the surface of mature dendritic cells that present processed antigens to T lymphocytes. In addition, T cells acquire CD83 expression following mitogenic stimulation in vitro. Here we report two lines of evidence demonstrating that this inducible lymphocyte response is genetically programmed by transcription factor NF-kappaB and contingent upon proteolytic breakdown of its cytoplasmic inhibitor IkappaBalpha. First, signal-dependent induction of CD83 mRNA expression is blocked in both transformed and primary T cells harboring a degradation-resistant mutant of IkappaBalpha that constitutively represses NF-kappaB. Second, as revealed in gel retardation assays, the IkappaBalpha constitutive repressor prevents the inducible interaction of NF-kappaB with consensus recognition sites identified in the CD83 promoter. Given that IkappaBalpha is functionally coupled to the T-cell antigen receptor, these findings suggest that the downstream transcription unit for CD83 is triggered by NF-kappaB during an adaptive immune response.

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

    PubMed

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

    1997-12-01

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

  18. 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.

  19. Sensitivity of T-Lymphocytes to Hormones of the Anterior Pituitary Gland.

    PubMed

    Tishevskaya, N V; Gevorkyan, N M; Kozlova, N I

    2017-01-01

    The review provides information about the features of the sensitivity of thymocytes, lymphoid organs' cells and T-lymphocytes of peripheral blood to the hormones secreted by anterior pituitary gland's cells: growth hormone, thyrotropin, adrenocorticotropic hormone, prolactin and β-endorphin. Some aspects of the T-lymphocytes's response to humoral signals from the hypophysis are shown in the article. Also the pituitary hormones' role in the regulation of proliferation, differentiation, and cytokine production of T-lymphocytes in normal and pathological conditions of the organism being discussed.

  20. [Novel therapy for malignant lymphoma: adoptive immuno-gene therapy using chimeric antigen receptor(CAR)-expressing T lymphocytes].

    PubMed

    Ozawa, Keiya

    2014-03-01

    Adoptive T-cell therapy using chimeric antigen receptor (CAR) technology is a novel approach to cancer immuno-gene therapy. CARs are hybrid proteins consisting of target-antigen-specific single-chain antibody fragment fused to intracellular T-cell activation domains (CD28 or CD137/CD3 zeta receptor). CAR-expressing engineered T lymphocytes can directly recognize and kill tumor cells in an HLA independent manner. In the United States, promising results have been obtained in the clinical trials of adoptive immuno-gene therapy using CD19-CAR-T lymphocytes for the treatment of refractory B-cell malignancies, including chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In this review article, CD19-CAR-T gene therapy for refractory B-cell non-Hodgkin lymphoma is discussed.

  1. Immunomodulatory potencies of isolated compounds from Crataegus azarolus through their antioxidant activities.

    PubMed

    Mustapha, Nadia; Mokdad-Bzéouich, Imèn; Sassi, Aicha; Abed, Besma; Ghedira, Kamel; Hennebelle, Thierry; Chekir-Ghedira, Leila

    2016-06-01

    The search of natural immunomodulatory agents has become an area of great interest in order to reduce damage to the human body. In this study, the immunomodulatory potential of Crataegus azarolus and its isolated hyperoside on mouse lymphocytes and macrophages in vitro was assessed. The effect of C. azarolus natural compounds on splenocytes proliferation, natural killer (NK) and cytotoxic T lymphocytes (CTL) activities, and on macrophage-mediated cytotoxicity were assessed by MTT test. Phagocytic activity and inhibition of nitric oxide (NO) release by macrophages were also evaluated. The antioxidant capacity of these products was evaluated by determining their cellular antioxidant activity (CAA) in splenocytes and macrophages. Depending on the concentrations, both ethyl acetate (EA) extract and hyperoside (Hyp) from C. azarolus affect macrophage functions by modulating their lysosomal enzyme activity and nitric oxide release. Whereas, the above-mentioned products significantly promote LPS and lectin-stimulated splenocyte proliferation, implying a potential activation of lymphocytes B and T enhancing humoral and cellular immune responses. Moreover, EA extract and Hyp could enhance the activity of NK and T lymphocytes cells, as well as the macrophages-mediated cytotoxicity against B16F10 cells. The anti-inflammatory activity was concomitant with the cellular antioxidant effect of the tested compounds against macrophages and splenocytes. Collectively, C. azarolus and its isolated hyperoside exhibited an immunomodulatory effect through their antioxidant activity. These findings suggest that C. azarolus should be explored as a novel potential immunomodulatory agent for the treatment of inflammatory diseases.

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

    PubMed Central

    1996-01-01

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

  3. Three Immunoproteasome-Associated Subunits Cooperatively Generate a Cytotoxic T-Lymphocyte Epitope of Epstein-Barr Virus LMP2A by Overcoming Specific Structures Resistant to Epitope Liberation

    PubMed Central

    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

  4. Group I mGlu receptor stimulation inhibits activation-induced cell death of human T lymphocytes

    PubMed Central

    Chiocchetti, Annalisa; Miglio, Gianluca; Mesturini, Riccardo; Varsaldi, Federica; Mocellin, Marco; Orilieri, Elisabetta; Dianzani, Chiara; Fantozzi, Roberto; Dianzani, Umberto; Lombardi, Grazia

    2006-01-01

    The effects of L-glutamate on activation-induced cell death (AICD) of human activated (1 μg ml−1 phytohemagglutinin plus 2 U ml−1 interleukin-2; 8 days) T lymphocytes were studied by measuring anti-CD3 monoclonal antibody (10 μg ml−1; 18 h)-induced cell apoptosis (Annexin V and propidium iodide staining). L-Glutamate (1 × 10−8–1 × 10−4 M) significantly (P⩽0.01) inhibited AICD in a concentration-dependent manner (EC50=6.3 × 10−8 M; maximum inhibition 54.8±6.3% at 1 × 10−6 M). The L-glutamate inhibitory effect was pharmacologically characterized as mediated by group I mGlu receptors, since mGlu receptor agonists reproduced this effect. The EC50 values were: 3.2 × 10−7 M for (1S,3R)-ACPD; 4.5 × 10−8 M for quisqualate; 1.0 × 10−6 M for (S)-3,5-DHPG; 2.0 × 10−5 M for CHPG. Group I mGlu receptor antagonists inhibited the effects of quisqualate 1.0 × 10−6 M. The IC50 values calculated were: 8.7 × 10−5, 4.3 × 10−6 and 6.3 × 10−7 M for AIDA, LY 367385 and MPEP, respectively. L-Glutamate (1 × 10−6 M; 18 h) significantly (P⩽0.05) inhibited FasL expression (40.8±11.3%) (cytofluorimetric analysis), whereas it did not affect Fas signalling. Expression of both mGlu1 and mGlu5 receptor mRNA by T lymphocytes and T-cell lines, as demonstrated by reverse transcriptase–PCR analysis, suggests that L-glutamate-mediated inhibition of AICD was exerted on T cells. These data depict a novel role for L-glutamate in the regulation of the immune response through group I mGlu receptor-mediated mechanisms. PMID:16751798

  5. Recombinant parvovirus-like particles as an antigen carrier: A novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells

    PubMed Central

    Sedlik, C.; Saron, M.-F.; Sarraseca, J.; Casal, I.; Leclerc, C.

    1997-01-01

    To develop a strategy that promotes efficient antiviral immunity, hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus VP2 capsid protein carrying a CD8+ T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated- or virus-infected-target cells. This CD8+ class I-restricted cytotoxic activity persisted in vivo for at least 9 months. Furthermore, the hybrid parvovirus-like particles were able to induce a complete protection of mice against a lethal lymphocytic choriomeningitis virus infection. To our knowledge, this study represents the first demonstration that hybrid nonreplicative VLP carrying a single viral CTL epitope can induce protection against a viral lethal challenge, in the absence of any adjuvant. These recombinant particles containing a single type of protein are easily produced by the baculovirus expression system and, therefore, represent a promising and safe strategy to induce strong CTL responses for the elimination of virus-infected cells. PMID:9207121

  6. Immunostimulant activity of noni (Morinda citrifolia) on T and B lymphocytes.

    PubMed

    Nayak, Smita; Mengi, Sushma

    2010-07-01

    Morinda citrifolia Linn (Rubiaceae) is a traditional medicinal herb that has been purported to be beneficial in the treatment of infections due to its immune enhancing properties. However, detailed studies highlighting the effect of different compounds isolated from the plant on the immune system are lacking. In this study, the stimulatory effects of the extracts and fractions of M. citrifolia fruits on important components of the adaptive immune system such as T lymphocytes and B lymphocytes were studied. The effects of the plant extracts on lymphocytes were assessed by in vitro (MTT assay) and in vivo (cell mediated immune response) techniques. Results of the MTT study indicated that the hydroalcoholic (0.5 and 1.0 mg/mL) and aqueous extracts (0.5 and 1.0 mg/mL) significantly (p < 0.05) increased in vitro splenocyte proliferation to the extent of 43.6, 54.5, 32.7, and 36.4%, respectively. Moreover, the hydroalcoholic (200 mg/kg) and the aqueous (200 mg/kg) extracts significantly (p < 0.05) increased the cell-mediated immune response to the extent of 33.52 and 18.56%, respectively. The fractions F I, F II, and F III failed to elicit a significant stimulatory effect on lymphocytes in the in vitro and in vivo studies. The effect of the extractives of M. citrifolia fruits on B-cells was measured by the delayed type hypersensitivity method. The study revealed that the hydroalcoholic extract (200 mg/kg) and fraction F I (40 mg/kg) significantly increased the humoral response to the extent of 33.33 and 35.12%, respectively. The results of this study confirm the cellular and humoral immunostimulant properties of M. citrifolia fruits and justify its usage in traditional medicine.

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

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

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

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

  8. Activated natural killer cell-mediated immunity is required for the inhibition of tumor metastasis by dendritic cell vaccination.

    PubMed

    Kim, Aeyung; Noh, Young-Woock; Kim, Kwang Dong; Jang, Yong-Suk; Choe, Yong-Kyung; Lim, Jong-Seok

    2004-10-31

    Immunization with dendritic cells (DCs) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL), which is responsible for tumor protection and regression. In this study, we examined whether DCs pulsed with necrotic tumor lysates can efficiently prevent malignant melanoma tumor cell metastasis to the lung. DCs derived from mouse bone marrow were found to produce remarkably elevated levels of IL-12 after being pulsed with the tumor lysates. Moreover, immunization with these DCs induced CTL activation and protected mice from metastasis development by intravenously inoculated tumor cells. In addition, these DCs activated NK cells in vitro in a contact-dependent manner, and induced NK activities in vivo. Furthermore, NK cell depletion before DC vaccination significantly reduced the tumor-specific CTL activity, IFN-gamma production, and IFN-gamma- inducible gene expression, and eventually interfered with the antitumor effect of tumor-pulsed DCs. Finally, similar findings with respect to NK cell dependency were obtained in the C57BL/ 6J-bg/bg mice, which have severe deficiency in cytolytic activity of NK cells. These data suggest that the antitumor effect elicited by DC vaccination, at least in a B16 melanoma model, requires the participation of both cytolytic NK and CD8(+) T cells. The findings of this study would provide important data for the effective design of DC vaccines for cancer immunotherapy.

  9. Positive regulation of deoxycytidine kinase activity by phosphorylation of Ser-74 in B-cell chronic lymphocytic leukaemia lymphocytes.

    PubMed

    Smal, Caroline; Van Den Neste, Eric; Maerevoet, Marie; Poiré, Xavier; Théate, Ivan; Bontemps, Françoise

    2007-08-08

    Deoxycytidine kinase (dCK) activates several antileukaemic nucleoside analogues. We have recently reported that the activity of dCK, overexpressed in HEK 293T cells, correlates with its phosphorylation level on Ser-74. Here, we show that dCK from B-cell chronic lymphocytic leukaemia (B-CLL) lymphocytes can be detected by an anti-phospho-Ser-74 antibody and that interindividual variability in dCK activity is related to its phosphorylation level on Ser-74. Moreover, pharmacological intervention modified Ser-74 phosphorylation, in close parallel with changes in dCK activity. These results suggest that activation of dCK via phosphorylation of Ser-74 might constitute a new therapeutic strategy to enhance activation and efficacy of nucleoside analogues.

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

    PubMed

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

    2007-02-09

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

  11. An alternative mode of CD43 signal transduction activates pro-survival pathways of T lymphocytes.

    PubMed

    Bravo-Adame, Maria Elena; Vera-Estrella, Rosario; Barkla, Bronwyn J; Martínez-Campos, Cecilia; Flores-Alcantar, Angel; Ocelotl-Oviedo, Jose Pablo; Pedraza-Alva, Gustavo; Rosenstein, Yvonne

    2017-01-01

    CD43 is one of the most abundant co-stimulatory molecules on a T-cell surface; it transduces activation signals through its cytoplasmic domain, contributing to modulation of the outcome of T-cell responses. The aim of this study was to uncover new signalling pathways regulated by this sialomucin. Analysis of changes in protein abundance allowed us to identify pyruvate kinase isozyme M2 (PKM2), an enzyme of the glycolytic pathway, as an element potentially participating in the signalling cascade resulting from the engagement of CD43 and the T-cell receptor (TCR). We found that the glycolytic activity of this enzyme was not significantly increased in response to TCR+CD43 co-stimulation, but that PKM2 was tyrosine phosphorylated, suggesting that it was performing moonlight functions. We report that phosphorylation of both Y 105 of PKM2 and of Y 705 of signal transducer and activator of transcription 3 was induced in response to TCR+CD43 co-stimulation, resulting in activation of the mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) pathway. ERK5 and the cAMP response element binding protein (CREB) were activated, and c-Myc and nuclear factor-κB (p65) nuclear localization, as well as Bad phosphorylation, were augmented. Consistent with this, expression of human CD43 in a murine T-cell hybridoma favoured cell survival. Altogether, our data highlight novel signalling pathways for the CD43 molecule in T lymphocytes, and underscore a role for CD43 in promoting cell survival through non-glycolytic functions of metabolic enzymes. © 2016 John Wiley & Sons Ltd.

  12. Inorganic arsenic represses interleukin-17A expression in human activated Th17 lymphocytes

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

    Morzadec, Claudie; Macoch, Mélinda; Robineau, Marc

    2012-08-01

    Trivalent inorganic arsenic [As(III)] is an efficient anticancer agent used to treat patients suffering from acute promyelocytic leukemia. Recently, experimental studies have clearly demonstrated that this metalloid can also cure lymphoproliferative and/or pro-inflammatory syndromes in different murine models of chronic immune-mediated diseases. T helper (Th) 1 and Th17 lymphocytes play a central role in development of these diseases, in mice and humans, especially by secreting the potent pro-inflammatory cytokine interferon-γ and IL-17A, respectively. As(III) impairs basic functions of human T cells but its ability to modulate secretion of pro-inflammatory cytokines by differentiated Th lymphocytes is unknown. In the present study,more » we demonstrate that As(III), used at concentrations clinically achievable in plasma of patients, has no effect on the secretion of interferon-γ from Th1 cells but almost totally blocks the expression and the release of IL-17A from human Th17 lymphocytes co-stimulated for five days with anti-CD3 and anti-CD28 antibodies, in the presence of differentiating cytokines. In addition, As(III) specifically reduces mRNA levels of the retinoic-related orphan receptor (ROR)C gene which encodes RORγt, a key transcription factor controlling optimal IL-17 expression in fully differentiated Th17 cells. The metalloid also blocks initial expression of IL-17 gene induced by the co-stimulation, probably in part by impairing activation of the JNK/c-Jun pathway. In conclusion, our results demonstrate that As(III) represses expression of the major pro-inflammatory cytokine IL-17A produced by human Th17 lymphocytes, thus strengthening the idea that As(III) may be useful to treat inflammatory immune-mediated diseases in humans. -- Highlights: ► Arsenic inhibits secretion of IL-17A from human naïve and memory Th17 lymphocytes. ► Arsenic represses early expression of IL-17A gene in human activated T lymphocytes. ► Arsenic interferes with

  13. Lymphocyte and macrophage phenotypes in chronic hepatitis C infection. Correlation with disease activity.

    PubMed Central

    Khakoo, S. I.; Soni, P. N.; Savage, K.; Brown, D.; Dhillon, A. P.; Poulter, L. W.; Dusheiko, G. M.

    1997-01-01

    The pathogenesis of chronic hepatitis C and the mechanisms underlying progressive liver disease in patients with chronic hepatitis C infection are poorly understood. To demonstrate which inflammatory cells might be responsible for the necroinflammatory damage in chronic hepatitis C infection, we have correlated the phenotype of the intrahepatic lymphocytes and macrophages with histological activity in liver biopsy and explant specimens from 19 patients with chronic hepatitis C infection. In all stages of disease, more CD8+ than CD4+ lymphocytes were found. However, histologically active versus histologically mild hepatitis was associated with a trend toward greater parenchymal concentrations of CD4+ lymphocytes (0.71 +/- 0.27 per 10(4) microns 2 versus 0.35 +/- 0.15; not significant), significantly less parenchymal CD8+ lymphocytes (0.90 +/- 0.1 versus 1.70 +/- 0.3; t = 2.32, P = 0.03) and a greater parenchymal CD4/CD8 ratio (4.1 +/- 2.8 versus 0.91 +/- 0.3; t = 1.65, P = 0.07). No difference was found in the number of cells containing cytotoxic granules between the two groups. Greater numbers of CD4+ lymphocytes were found in liver biopsy specimens with little or no staining for hepatitis C virus antigen (1.47 +/- 0.88 versus 0.27 +/- 0.27; t = 2.28, P < 0.05). No significant differences were found in the macrophage subsets between the three stages of disease. Our data suggest that active histological disease in chronic hepatitis C infection may be associated with an increase in CD4+ lymphocytes and suggest that CD4+ T cells may play an important role in the hepatic injury in these patients. Images Figure 2 PMID:9060834

  14. Human T-Lymphotropic Virus Type 1-Induced Overexpression of Activated Leukocyte Cell Adhesion Molecule (ALCAM) Facilitates Trafficking of Infected Lymphocytes through the Blood-Brain Barrier

    PubMed Central

    Curis, Céline; Percher, Florent; Jeannin, Patricia; Montange, Thomas; Chevalier, Sébastien A.; Seilhean, Danielle; Cartier, Luis; Couraud, Pierre-Olivier; Gout, Olivier; Gessain, Antoine; Ceccaldi, Pierre-Emmanuel

    2016-01-01

    ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease develops upon infiltration of HTLV-1-infected lymphocytes into the central nervous system, mostly the thoracic spinal cord. The central nervous system is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. In this study, we investigated the role of activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily, in the crossing of the BBB by HTLV-1-infected lymphocytes. We demonstrated that ALCAM is overexpressed on the surface of HTLV-1-infected lymphocytes, both in chronically infected cell lines and in primary infected CD4+ T lymphocytes. ALCAM overexpression results from the activation of the canonical NF-κB pathway by the viral transactivator Tax. In contrast, staining of spinal cord sections of HAM/TSP patients showed that ALCAM expression is not altered on the BBB endothelium in the context of HTLV-1 infection. ALCAM blockade or downregulation of ALCAM levels significantly reduced the migration of HTLV-1-infected lymphocytes across a monolayer of human BBB endothelial cells. This study suggests a potential role for ALCAM in HAM/TSP pathogenesis. IMPORTANCE Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease is the consequence of the infiltration of HTLV-1-infected lymphocytes into the central nervous system (CNS), mostly the thoracic spinal cord. The CNS is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. The mechanism of migration of lymphocytes into

  15. Human T-Lymphotropic Virus Type 1-Induced Overexpression of Activated Leukocyte Cell Adhesion Molecule (ALCAM) Facilitates Trafficking of Infected Lymphocytes through the Blood-Brain Barrier.

    PubMed

    Curis, Céline; Percher, Florent; Jeannin, Patricia; Montange, Thomas; Chevalier, Sébastien A; Seilhean, Danielle; Cartier, Luis; Couraud, Pierre-Olivier; Gout, Olivier; Gessain, Antoine; Ceccaldi, Pierre-Emmanuel; Afonso, Philippe V

    2016-08-15

    Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease develops upon infiltration of HTLV-1-infected lymphocytes into the central nervous system, mostly the thoracic spinal cord. The central nervous system is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. In this study, we investigated the role of activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily, in the crossing of the BBB by HTLV-1-infected lymphocytes. We demonstrated that ALCAM is overexpressed on the surface of HTLV-1-infected lymphocytes, both in chronically infected cell lines and in primary infected CD4(+) T lymphocytes. ALCAM overexpression results from the activation of the canonical NF-κB pathway by the viral transactivator Tax. In contrast, staining of spinal cord sections of HAM/TSP patients showed that ALCAM expression is not altered on the BBB endothelium in the context of HTLV-1 infection. ALCAM blockade or downregulation of ALCAM levels significantly reduced the migration of HTLV-1-infected lymphocytes across a monolayer of human BBB endothelial cells. This study suggests a potential role for ALCAM in HAM/TSP pathogenesis. Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease is the consequence of the infiltration of HTLV-1-infected lymphocytes into the central nervous system (CNS), mostly the thoracic spinal cord. The CNS is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. The mechanism of migration of lymphocytes into the CNS is unclear

  16. Suppressive effects of fisetin on mice T lymphocytes in vitro and in vivo.

    PubMed

    Song, Bocui; Guan, Shuang; Lu, Jing; Chen, Zhibao; Huang, Guoren; Li, Gen; Xiong, Ying; Zhang, Shuang; Yue, Zhanpeng; Deng, Xuming

    2013-11-01

    Most of the immunosuppressive drugs have satisfactory therapeutic effects on organ transplantation and autoimmune disease. However, their clinical application is limited by side effects. Therefore, new and safe immunosuppressive drugs against acute and chronic rejections are eagerly awaited. Fisetin, a flavonoid present in various types of vegetables and fruits, has few side effects and low level of toxicity, which would be a desirable clinical feature. In the present study, we investigated the immunosuppressive effects and underlying mechanisms of fisetin against T-cell activation in vitro and in vivo. We measured the effect of fisetin on T-lymphocyte proliferation, T-cell subsets, cell cycle progression, cytokine production, and nuclear factor activation in vitro, as well as its influence on T cell-mediated delayed-type hypersensitivity reaction in vivo. In vitro, the results showed that fisetin significantly suppressed mouse splenocytes proliferation, Th1 and Th2 cytokine production, cell cycle and the ratio of CD4(+)/CD8(+) T cells. Furthermore, fisetin exerts an immunosuppressive effect in mouse T lymphocytes through the suppression of nuclear factor kappa B activation and nuclear factor of activated T cells signaling in a dose-dependent manner. In vivo, fisetin treatment also significantly inhibited the dinitrofluorobenzene-induced delayed-type hypersensitivity reactions in mice. Fisetin had strong immunosuppressive activity in vitro and in vivo, suggesting a potential role for fisetin as an immunosuppressive agent. Copyright © 2013. Published by Elsevier Inc.

  17. Expression of K2P5.1 potassium channels on CD4+ T lymphocytes correlates with disease activity in rheumatoid arthritis patients.

    PubMed

    Bittner, Stefan; Bobak, Nicole; Feuchtenberger, Martin; Herrmann, Alexander M; Göbel, Kerstin; Kinne, Raimund W; Hansen, Anker J; Budde, Thomas; Kleinschnitz, Christoph; Frey, Oliver; Tony, Hans-Peter; Wiendl, Heinz; Meuth, Sven G

    2011-02-11

    CD4+ T cells express K(2P)5.1 (TWIK-related acid-sensitive potassium channel 2 (TASK2); KCNK5), a member of the two-pore domain potassium channel family, which has been shown to influence T cell effector functions. Recently, it was shown that K(2P)5.1 is upregulated upon (autoimmune) T cell stimulation. The aim of this study was to correlate expression levels of K(2P)5.1 on T cells from patients with rheumatoid arthritis (RA) to disease activity in these patients. Expression levels of K(2P)5.1 were measured by RT-PCR in the peripheral blood of 58 patients with RA and correlated with disease activity parameters (C-reactive protein levels, erythrocyte sedimentation rates, disease activity score (DAS28) scores). Twenty patients undergoing therapy change were followed-up for six months. Additionally, synovial fluid and synovial biopsies were investigated for T lymphocytes expressing K(2P)5.1. K(2P)5.1 expression levels in CD4+ T cells show a strong correlation to DAS28 scores in RA patients. Similar correlations were found for serological inflammatory parameters (erythrocyte sedimentation rate, C-reactive protein). In addition, K(2P)5.1 expression levels of synovial fluid-derived T cells are higher compared to peripheral blood T cells. Prospective data in individual patients show a parallel behaviour of K(2P)5.1 expression to disease activity parameters during a longitudinal follow-up for six months. Disease activity in RA patients correlates strongly with K(2P)5.1 expression levels in CD4+ T lymphocytes in the peripheral blood in cross-sectional as well as in longitudinal observations. Further studies are needed to investigate the exact pathophysiological mechanisms and to evaluate the possible use of K(2P)5.1 as a potential biomarker for disease activity and differential diagnosis.

  18. New insights into Blimp-1 in T lymphocytes: a divergent regulator of cell destiny and effector function.

    PubMed

    Fu, Shin-Huei; Yeh, Li-Tzu; Chu, Chin-Chen; Yen, B Lin-Ju; Sytwu, Huey-Kang

    2017-07-21

    B lymphocyte-induced maturation protein-1 (Blimp-1) serves as a master regulator of the development and function of antibody-producing B cells. Given that its function in T lymphocytes has been identified within the past decade, we review recent findings with emphasis on its role in coordinated control of gene expression during the development, differentiation, and function of T cells. Expression of Blimp-1 is mainly confined to activated T cells and is essential for the production of interleukin (IL)-10 by a subset of forkhead box (Fox)p3 + regulatory T cells with an effector phenotype. Blimp-1 is also required to induce cell elimination in the thymus and critically modulates peripheral T cell activation and proliferation. In addition, Blimp-1 promotes T helper (Th) 2 lineage commitment and limits Th1, Th17 and follicular helper T cell differentiation. Furthermore, Blimp-1 coordinates with other transcription factors to regulate expression of IL-2, IL-21 and IL-10 in effector T lymphocytes. In CD8 + T cells, Blimp-1 expression is distinct in heterogeneous populations at the stages of clonal expansion, differentiation, contraction and memory formation when they encounter antigens. Moreover, Blimp-1 plays a fundamental role in coordinating cytokine receptor signaling networks and transcriptional programs to regulate diverse aspects of the formation and function of effector and memory CD8 + T cells and their exhaustion. Blimp-1 also functions as a gatekeeper of T cell activation and suppression to prevent or dampen autoimmune disease, antiviral responses and antitumor immunity. In this review, we discuss the emerging roles of Blimp-1 in the complex regulation of gene networks that regulate the destiny and effector function of T cells and provide a Blimp-1-dominated transcriptional framework for T lymphocyte homeostasis.

  19. The clinical implications of mixed lymphocyte reaction with leukemic cells.

    PubMed

    Kim, Hee-Je; Kim, Tai-Gyu; Cho, Hyun Il; Han, Hoon; Min, Woo-Sung; Kim, Chun-Choo

    2002-11-01

    To evaluate the clinical implications of a mixed lymphocyte reaction between leukemic cells and lymphocytes from HLA-matched sibling donors, we attempted to generate donor-derived, graft-versus-leukemia-effective cells and to define their characteristics. We studied 8 patients with chronic myelogenous leukemia (CML), including 5 patients in the chronic phase (CP), 3 patients in the accelerated phase (AP), and 2 patients with acute myelogenous leukemia (AML) in their first complete remission. Cells from these patients were used as stimulators in a mixed lymphocyte reaction.The effects of natural killer (NK) cells and cytotoxic T-lymphocytes (CTLs) were separated by observing tests for cytotoxicity to target cells, including K562 cells, the patient's leukemic cells, and phytohemagglutinin (PHA) blasts. Donor-derived antileukemic CTLs againstthe patient's own leukemic cells are productive in vitro. The efficacy of generating CTLs against leukemic target cells was (in decreasing order) AML, CML-CP, and CML-AP. Cytotoxic activity against leukemic targets was prominent in 4 cases--2 CML-CP and the 2 AML cases. On the contrary, the 3 cases of CML-AP showed low CTL activity. In cases showing 1 positive result among 3 targets (K562 cells, the patient's leukemic cells, and PHA blasts), the relapse rate was significantly lower (P = .022) on follow-up (median, 33 months; 7-40 months) after hematopoietic stem cell transplantation. By a combined analysis of the cytotoxicity effects for all 3 target cells, we were able to demonstrate a correlation between leukemic relapse and the variable degree of the cytotoxicity test results. Although the total sample numbers for this study were low, we speculate that these results may come from differences in the individual characteristics of the leukemic cells that are in line with their clinical disease status.

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

    PubMed

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

    2018-06-01

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

  1. Cerebroside D, a glycoceramide compound, improves experimental colitis in mice with multiple targets against activated T lymphocytes

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

    Wu, Xue-Feng; Wu, Xing-Xin; Guo, Wen-Jie

    2012-09-15

    In the present paper, we aimed to examine the novel effects of cerebroside D, a glycoceramide compound, on murine experimental colitis. Cerebroside D significantly reduced the weight loss, mortality rate and alleviated the macroscopic and microscopic appearances of colitis induced by dexran sulfate sodium. This compound also decreased the levels of TNF-α, IFN-γ and IL-1β in intestinal tissue of mice with experimental colitis in a concentration-dependent manner, accompanied with markedly increased serum level of IL-10. Cerebroside D inhibited proliferation and induced apoptosis of T cells activated by concanavalin A or anti-CD3 plus anti-CD28 antibodies. The compound did not show anmore » effect on naive lymphocytes but prevented cells from entering S phase and G2/M phase during T cells activation. Moreover, the treatment of cerebroside D led to apoptosis of activated T cells with the cleavage of caspase 3, 9, 12 and PARP. These results showed multiple effects of cerebroside D against activated T cells for a novel approach to treatment of colonic inflammation. Highlights: ► Cerebroside D, a glycoceramide compound, alleviated DSS induced colitis. ► The mechanism of the compound involved multiple effects against activated T cells. ► It regulated cytokine profiles in mice with experimental colitis. ► It prevented T cells from entering S and G2/M phases during activation. ► It led to apoptosis of activated T cells with the cleavage of caspases and PARP.« less

  2. Suppression of antigen-specific lymphocyte activation in modeled microgravity

    NASA Technical Reports Server (NTRS)

    Cooper, D.; Pride, M. W.; Brown, E. L.; Risin, D.; Pellis, N. R.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Various parameters of immune suppression are observed in lymphocytes from astronauts during and after a space flight. It is difficult to ascribe this suppression to microgravity effects on immune cells in crew specimens, due to the complex physiological response to space flight and the resultant effect on in vitro immune performance. Use of isolated immune cells in true and modeled microgravity in immune performance tests, suggests a direct effect of microgravity on in vitro cellular function. Specifically, polyclonal activation of T-cells is severely suppressed in true and modeled microgravity. These recent findings suggest a potential suppression of oligoclonal antigen-specific lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors as an analog of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction, as a model for a primary immune response, a tetanus toxoid response and a Borrelia burgdorferi response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.

  3. High affinity soluble ILT2 receptor: a potent inhibitor of CD8(+) T cell activation.

    PubMed

    Moysey, Ruth K; Li, Yi; Paston, Samantha J; Baston, Emma E; Sami, Malkit S; Cameron, Brian J; Gavarret, Jessie; Todorov, Penio; Vuidepot, Annelise; Dunn, Steven M; Pumphrey, Nicholas J; Adams, Katherine J; Yuan, Fang; Dennis, Rebecca E; Sutton, Deborah H; Johnson, Andy D; Brewer, Joanna E; Ashfield, Rebecca; Lissin, Nikolai M; Jakobsen, Bent K

    2010-12-01

    Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.

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

    PubMed Central

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

    2009-01-01

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

  5. Multipotent adult germ-line stem cells, like other pluripotent stem cells, can be killed by cytotoxic T lymphocytes despite low expression of major histocompatibility complex class I molecules

    PubMed Central

    Dressel, Ralf; Guan, Kaomei; Nolte, Jessica; Elsner, Leslie; Monecke, Sebastian; Nayernia, Karim; Hasenfuss, Gerd; Engel, Wolfgang

    2009-01-01

    Background Multipotent adult germ-line stem cells (maGSCs) represent a new pluripotent cell type that can be derived without genetic manipulation from spermatogonial stem cells (SSCs) present in adult testis. Similarly to induced pluripotent stem cells (iPSCs), they could provide a source of cellular grafts for new transplantation therapies of a broad variety of diseases. To test whether these stem cells can be rejected by the recipients, we have analyzed whether maGSCs and iPSCs can become targets for cytotoxic T lymphocytes (CTL) or whether they are protected, as previously proposed for embryonic stem cells (ESCs). Results We have observed that maGSCs can be maintained in prolonged culture with or without leukemia inhibitory factor and/or feeder cells and still retain the capacity to form teratomas in immunodeficient recipients. They were, however, rejected in immunocompetent allogeneic recipients, and the immune response controlled teratoma growth. We analyzed the susceptibility of three maGSC lines to CTL in comparison to ESCs, iPSCs, and F9 teratocarcinoma cells. Major histocompatibility complex (MHC) class I molecules were not detectable by flow cytometry on these stem cell lines, apart from low levels on one maGSC line (maGSC Stra8 SSC5). However, using a quantitative real time PCR analysis H2K and B2m transcripts were detected in all pluripotent stem cell lines. All pluripotent stem cell lines were killed in a peptide-dependent manner by activated CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the SIINFEKL peptide. Conclusion Pluripotent stem cells, including maGSCs, ESCs, and iPSCs can become targets for CTLs, even if the expression level of MHC class I molecules is below the detection limit of flow cytometry. Thus they are not protected against CTL-mediated cytotoxicity. Therefore, pluripotent cells might be rejected after transplantation by this mechanism if specific antigens are presented and if specific

  6. Suppression of Antigen-Specific Lymphocyte Activation in Simulated Microgravity

    NASA Technical Reports Server (NTRS)

    Cooper, David; Pride, Michael W.; Brown, Eric L.; Risin, Diana; Pellis, Neal R.

    1999-01-01

    Various parameters of immune suppression are observed in astronauts during and after spaceflight, and in isolated immune cells in true and simulated microgravity. Specifically, polyclonal activation of T cells is severely suppressed in true and simulated microgravity. These recent findings with various polyclonal activators suggests a suppression of oligoclonal lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors that simulate aspects of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction (MLR), as a model for a primary immune response; a tetanus toxoid (TT) response and a B. burgdorferi (Bb) response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.

  7. Different roles of CD4, CD8 and γδ T-lymphocytes in naive and vaccinated chickens during Salmonella Enteritidis infection.

    PubMed

    Sekelova, Zuzana; Polansky, Ondrej; Stepanova, Hana; Fedr, Radek; Faldynova, Marcela; Rychlik, Ivan; Vlasatikova, Lenka

    2017-07-01

    Lymphocytes represent the key antigen-specific leukocyte subpopulation. Despite their importance in mounting an immune response, an unbiased description of proteins expressed by chicken lymphocytes has not been presented. In this study, we therefore intravenously infected chickens with Salmonella Enteritidis, sorted CD4, CD8 and γδ T-lymphocytes from the spleen by flow cytometry and determined the proteome of each population by LC-MS/MS. CD4 T-lymphocyte characteristic proteins included ubiquitin SUMO-like domain and BAR domain containing proteins. CD8 T-lymphocyte specific proteins were characterized by purine ribonucleoside triphosphate binding and were involved in cell differentiation, cell activation and regulation of programmed cell death. γδ T-lymphocyte specific proteins exhibited enrichment of small GTPase of Rab type and GTP binding. Following infection, inducible proteins in CD4 lymphocytes included ribosomal proteins and downregulated proteins localized to the lysosome. CD8 T-lymphocytes induced MCM complex proteins, proteins required for DNA replication and machinery for protein processing in the endoplasmic reticulum. Proteins inducible in γδ T-lymphocytes belonged to immune system response, oxidative phosphorylation and the spliceosome. In this study, we predicted the likely events in lymphocyte response to systemic bacterial infection and identified proteins which can be used as markers specific for each lymphocyte subpopulation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Curcumin serves as a human kv1.3 blocker to inhibit effector memory T lymphocyte activities.

    PubMed

    Lian, Yi-Tian; Yang, Xiao-Fang; Wang, Zhao-Hui; Yang, Yong; Yang, Ying; Shu, Yan-Wen; Cheng, Long-Xian; Liu, Kun

    2013-09-01

    Curcumin, the principal active component of turmeric, has long been used to treat various diseases in India and China. Recent studies show that curcumin can serve as a therapeutic agent for autoimmune diseases via a variety of mechanisms. Effector memory T cells (T(EM), CCR7⁻ CD45RO⁺ T lymphocyte) have been demonstrated to play a crucial role in the pathogenesis of T cell-mediated autoimmune diseases, such as multiple sclerosis (MS) or rheumatoid arthritis (RA). Kv1.3 channels are predominantly expressed in T(EM) cells and control T(EM) activities. In the present study, we examined the effect of curcumin on human Kv1.3 (hKv1.3) channels stably expressed in HEK-293 cells and its ability to inhibit proliferation and cytokine secretion of T(EM) cells isolated from patients with MS or RA. Curcumin exhibited a direct blockage of hKv1.3 channels in a time-dependent and concentration-dependent manner. Moreover, the activation curve was shifted to a more positive potential, which was consistent with an open-channel blockade. Paralleling hKv1.3 inhibition, curcumin significantly inhibited proliferation and interferon-γ secretion of T(EM) cells. Our findings demonstrate that curcumin is able to inhibit proliferation and proinflammatory cytokine secretion of T(EM) cells probably through inhibition of hKv1.3 channels, which contributes to the potency of curcumin for the treatment of autoimmune diseases. This is probably one of pharmacological mechanisms of curcumin used to treat autoimmune diseases. Copyright © 2012 John Wiley & Sons, Ltd.

  9. The effect of stem cell from human exfoliated deciduous teeth on T lymphocyte proliferation.

    PubMed

    Alipour, Razieh; Adib, Minoo; Hashemi-Beni, Batool; Sadeghi, Farzaneh

    2014-01-01

    Mesenchymal stem cells (MSC), a specific type of adult tissue stem cell; have the immunosuppressive effects that make them valuable targets for regenerative medicine and treatment of many human illnesses. Hence, MSC have been the subject of numerous studies. The classical source of MSC is adult bone marrow (BM). Due to many shortcomings of harvesting MSC from BM, finding the alternative sources for MSC is an urgent. Stem cells from human exfoliated deciduous teeth (SHED) are relative new MSC populations that fulfill these criteria but their potential immunosuppressive effect has not been studied enough yet. Thus, in this work the effect of SHED on the proliferation of in vitro activated T lymphocytes were explored. In this study, both mitogen and alloantigen activated T cells were cultured in the presence of different numbers of SHED. In some co-cultures, activated T cells were in direct contact to MSCs and in other co-cultures; they were separated from SHED by a permeable membrane. In all co-cultures, the proliferation of T cells was measured by ELISA Bromodeoxyuridine proliferation assay. In general, our results showed that SHED significantly suppress the proliferation of activated T cells in a dose-dependent manner. Moreover, the suppression was slightly stronger when MSCs were in physical contact to activated T cells. This study showed that SHED likewise other MSC populations can suppress the activation of T lymphocytes, which can be used instead of BM derived MSCs in many investigational and clinical applications.

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

    PubMed

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

    2009-12-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2008-01-01

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

  13. Therapeutic efficacy of MUC1-specific cytotoxic T lymphocytes and CD137 co-stimulation in a spontaneous breast cancer model.

    PubMed

    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.

  14. Red blood cells inhibit activation-induced cell death and oxidative stress in human peripheral blood T lymphocytes.

    PubMed

    Fonseca, A M; Porto, G; Uchida, K; Arosa, F A

    2001-05-15

    Red blood cells (RBCs) are known to perform one prominent function: to carry and deliver oxygen to the tissues. Earlier studies, however, suggested a role for RBCs in potentiating T-cell proliferation in vitro. Here it is shown that the presence of RBCs in cultures of stimulated human peripheral blood lymphocytes strengthens T-cell proliferation and survival. Analysis of phosphatidylserine externalization and DNA fragmentation showed that RBCs inhibit T-cell apoptosis. This inhibition correlated with a reduction in CD71 but not CD95 expression. RBCs enhanced T-cell proliferation and survival upon activation with phytohemagglutinin and with OKT3 antibodies. Studies aimed at characterizing the cellular and molecular basis of the protection afforded to T cells by RBCs showed that (1) optimal protection required intact RBCs and red cell/T-cell contact but not monocytes; (2) RBCs markedly reduced the level of intracellular reactive oxygen species; and (3) RBCs inhibited the formation of protein-bound acrolein, a peroxidation adduct in biologic systems. Overall, these data indicate that human RBCs protect T cells from activation-induced cell death, at least in part by reducing the pro-oxidant state, and suggest a role for RBCs as conceivable modulators of T-cell homeostasis.

  15. Hepatitis B virus surface protein mutations clustered mainly in CTL immune epitopes in chronic carriers: results of an Iranian nationwide study.

    PubMed

    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.

  16. Asymmetric T lymphocyte division in the initiation of adaptive immune responses.

    PubMed

    Chang, John T; Palanivel, Vikram R; Kinjyo, Ichiko; Schambach, Felix; Intlekofer, Andrew M; Banerjee, Arnob; Longworth, Sarah A; Vinup, Kristine E; Mrass, Paul; Oliaro, Jane; Killeen, Nigel; Orange, Jordan S; Russell, Sarah M; Weninger, Wolfgang; Reiner, Steven L

    2007-03-23

    A hallmark of mammalian immunity is the heterogeneity of cell fate that exists among pathogen-experienced lymphocytes. We show that a dividing T lymphocyte initially responding to a microbe exhibits unequal partitioning of proteins that mediate signaling, cell fate specification, and asymmetric cell division. Asymmetric segregation of determinants appears to be coordinated by prolonged interaction between the T cell and its antigen-presenting cell before division. Additionally, the first two daughter T cells displayed phenotypic and functional indicators of being differentially fated toward effector and memory lineages. These results suggest a mechanism by which a single lymphocyte can apportion diverse cell fates necessary for adaptive immunity.

  17. AKT/SGK-sensitive phosphorylation of GSK3 in the regulation of L-selectin and perforin expression as well as activation induced cell death of T-lymphocytes

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

    Bhavsar, Shefalee K.; Merches, Katja; Bobbala, Diwakar

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer Akt/SGK dependent phosphorylation of GSK3{alpha},{beta} regulates T lymphocytes. Black-Right-Pointing-Pointer T cells from mice expressing Akt/SGK insensitive GSK3{alpha},{beta} (gsk3{sup KI}) release less IL-2. Black-Right-Pointing-Pointer CD4{sup +} cells from gsk3{sup KI} mice express less CD62L. Black-Right-Pointing-Pointer CD8{sup +} cells from gsk3{sup KI} mice are relatively resistant to activation induced cell death. Black-Right-Pointing-Pointer Perforin expression is enhanced in gsk3{sup KI} T cells. -- Abstract: Survival and function of T-lymphocytes critically depends on phosphoinositide (PI) 3 kinase. PI3 kinase signaling includes the PKB/Akt and SGK dependent phosphorylation and thus inhibition of glycogen synthase kinase GSK3{alpha},{beta}. Lithium, a known unspecific GSK3 inhibitor protectsmore » against experimental autoimmune encephalomyelitis. The present study explored, whether Akt/SGK-dependent regulation of GSK3 activity is a determinant of T cell survival and function. Experiments were performed in mutant mice in which Akt/SGK-dependent GSK3{alpha},{beta} inhibition was disrupted by replacement of the serine residue in the respective SGK/Akt-phosphorylation consensus sequence by alanine (gsk3{sup KI}). T cells from gsk3{sup KI} mice were compared to T cells from corresponding wild type mice (gsk3{sup WT}). As a result, in gsk3{sup KI} CD4{sup +} cells surface CD62L (L-selectin) was significantly less abundant than in gsk3{sup WT} CD4{sup +} cells. Upon activation in vitro T cells from gsk3{sup KI} mice reacted with enhanced perforin production and reduced activation induced cell death. Cytokine production was rather reduced in gsk3{sup KI} T cells, suggesting that GSK3 induces effector function in CD8{sup +} T cells. In conclusion, PKB/Akt and SGK sensitive phosphorylation of GSK3{alpha},{beta} is a potent regulator of perforin expression and activation induced cell death in T lymphocytes.« less

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

    PubMed

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

    2013-06-01

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

  19. T Lymphocyte Migration: An Action Movie Starring the Actin and Associated Actors.

    PubMed

    Dupré, Loïc; Houmadi, Raïssa; Tang, Catherine; Rey-Barroso, Javier

    2015-01-01

    The actin cytoskeleton is composed of a dynamic filament meshwork that builds the architecture of the cell to sustain its fundamental properties. This physical structure is characterized by a continuous remodeling, which allows cells to accomplish complex motility steps such as directed migration, crossing of biological barriers, and interaction with other cells. T lymphocytes excel in these motility steps to ensure their immune surveillance duties. In particular, actin cytoskeleton remodeling is a key to facilitate the journey of T lymphocytes through distinct tissue environments and to tune their stop and go behavior during the scanning of antigen-presenting cells. The molecular mechanisms controlling actin cytoskeleton remodeling during T lymphocyte motility have been only partially unraveled, since the function of many actin regulators has not yet been assessed in these cells. Our review aims to integrate the current knowledge into a comprehensive picture of how the actin cytoskeleton drives T lymphocyte migration. We will present the molecular actors that control actin cytoskeleton remodeling, as well as their role in the different T lymphocyte motile steps. We will also highlight which challenges remain to be addressed experimentally and which approaches appear promising to tackle them.

  20. Altered lipid raft–associated signaling and ganglioside expression in T lymphocytes from patients with systemic lupus erythematosus

    PubMed Central

    Jury, Elizabeth C.; Kabouridis, Panagiotis S.; Flores-Borja, Fabian; Mageed, Rizgar A.; Isenberg, David A.

    2004-01-01

    Systemic lupus erythematosus (SLE) is characterized by abnormalities in T lymphocyte receptor–mediated signal transduction pathways. Our previous studies have established that lymphocyte-specific protein tyrosine kinase (LCK) is reduced in T lymphocytes from patients with SLE and that this reduction is associated with disease activity and parallels an increase in LCK ubiquitination independent of T cell activation. This study investigated the expression of molecules that regulate LCK homeostasis, such as CD45, C-terminal Src kinase (CSK), and c-Cbl, in lipid raft domains from SLE T cells and investigated the localization of these proteins during T cell receptor (TCR) triggering. Our results indicate that the expression of raft-associated ganglioside, GM1, is increased in T cells from SLE patients and LCK may be differentially regulated due to an alteration in the association of CD45 with lipid raft domains. CD45 tyrosine phosphatase, which regulates LCK activity, was differentially expressed and its localization into lipid rafts was increased in T cells from patients with SLE. Furthermore, T cells allowed to “rest” in vitro showed a reversal of the changes in LCK, CD45, and GM1 expression. The results also revealed that alterations in the level of GM1 expression and lipid raft occupancy cannot be induced by serum factors from patients with SLE but indicated that cell-cell contact, activating aberrant proximal signaling pathways, may be important in influencing abnormalities in T cell signaling and, therefore, function in patients with SLE. PMID:15085197

  1. Natural killer cells facilitate PRAME-specific T-cell reactivity against neuroblastoma

    PubMed Central

    Spel, Lotte; Boelens, Jaap-Jan; van der Steen, Dirk M.; Blokland, Nina J.G.; van Noesel, Max M.; Molenaar, Jan J.; Heemskerk, Mirjam H.M.

    2015-01-01

    Neuroblastoma is the most common solid tumor in children with an estimated 5-year progression free survival of 20–40% in stage 4 disease. Neuroblastoma actively avoids recognition by natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Although immunotherapy has gained traction for neuroblastoma treatment, these immune escape mechanisms restrain clinical results. Therefore, we aimed to improve neuroblastoma immunogenicity to further the development of antigen-specific immunotherapy against neuroblastoma. We found that neuroblastoma cells significantly increase surface expression of MHC I upon exposure to active NK cells which thereby readily sensitize neuroblastoma cells for recognition by CTLs. We show that oncoprotein PRAME serves as an immunodominant antigen for neuroblastoma as NK-modulated neuroblastoma cells are recognized by PRAMESLLQHLIGL/A2-specific CTL clones. Furthermore, NK cells induce MHC I upregulation in neuroblastoma through contact-dependent secretion of IFNγ. Our results demonstrate remarkable plasticity in the peptide/MHC I surface expression of neuroblastoma cells, which is reversed when neuroblastoma cells experience innate immune attack by sensitized NK cells. These findings support the exploration of NK cells as adjuvant therapy to enforce neuroblastoma-specific CTL responses. PMID:26452036

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

    PubMed Central

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

    2016-01-01

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

  3. Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia

    PubMed Central

    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

  4. Nucleolar organizer regions activity in lymphocytes of patients with laryngeal carcinoma.

    PubMed

    Maione, S; Lamberti, L

    1993-12-01

    The activity of nucleolar organizer regions (Ag-NORs) and the frequency of NOR associations in chromosomes of phytohemagglutinin-stimulated lymphocytes from 12 patients with laryngeal carcinoma and 12 healthy subjects were studied using the gelatine silver staining technique. This study was undertaken to examine whether any disease associated changes occur in NOR activity. A lower mean number of Ag-NORs per metaphase (t test, 0.05 > p > 0.02) was found in patients compared to controls. This difference was not due to any specific group of acrocentric chromosomes (D or G). The mean number of NOR associations per metaphase was also found to be markedly lower (t test, 0.01 > p > 0.001) in patients than in controls. This difference was principally due to the significant decrease in the associations between 2 chromosomes (t test, 0.02 > p > 0.01), and in particular to the decrease in the D-G type associations (t test, 0.05 > p > 0.02). These findings are discussed in relation to existing data on the nucleolar activity of lymphocytes in a variety of solid tumours and leukemias.

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

    PubMed Central

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

    2017-01-01

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

  6. Effects of Mutations on Replicative Fitness and Major Histocompatibility Complex Class I Binding Affinity Are Among the Determinants Underlying Cytotoxic-T-Lymphocyte Escape of HIV-1 Gag Epitopes.

    PubMed

    Du, Yushen; Zhang, Tian-Hao; Dai, Lei; Zheng, Xiaojuan; Gorin, Aleksandr M; Oishi, John; Wu, Ting-Ting; Yoshizawa, Janice M; Li, Xinmin; Yang, Otto O; Martinez-Maza, Otoniel; Detels, Roger; Sun, Ren

    2017-11-28

    Certain "protective" major histocompatibility complex class I (MHC-I) alleles, such as B*57 and B*27, are associated with long-term control of HIV-1 in vivo mediated by the CD8 + cytotoxic-T-lymphocyte (CTL) response. However, the mechanism of such superior protection is not fully understood. Here we combined high-throughput fitness profiling of mutations in HIV-1 Gag, in silico prediction of MHC-peptide binding affinity, and analysis of intraperson virus evolution to systematically compare differences with respect to CTL escape mutations between epitopes targeted by protective MHC-I alleles and those targeted by nonprotective MHC-I alleles. We observed that the effects of mutations on both viral replication and MHC-I binding affinity are among the determinants of CTL escape. Mutations in Gag epitopes presented by protective MHC-I alleles are associated with significantly higher fitness cost and lower reductions in binding affinity with respect to MHC-I. A linear regression model accounting for the effect of mutations on both viral replicative capacity and MHC-I binding can explain the protective efficacy of MHC-I alleles. Finally, we found a consistent pattern in the evolution of Gag epitopes in long-term nonprogressors versus progressors. Overall, our results suggest that certain protective MHC-I alleles allow superior control of HIV-1 by targeting epitopes where mutations typically incur high fitness costs and small reductions in MHC-I binding affinity. IMPORTANCE Understanding the mechanism of viral control achieved in long-term nonprogressors with protective HLA alleles provides insights for developing functional cure of HIV infection. Through the characterization of CTL escape mutations in infected persons, previous researchers hypothesized that protective alleles target epitopes where escape mutations significantly reduce viral replicative capacity. However, these studies were usually limited to a few mutations observed in vivo Here we utilized our recently

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

    PubMed

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

    1997-09-15

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

  8. Sp1 transcriptional activity is up-regulated by phosphatase 2A in dividing T lymphocytes.

    PubMed

    Lacroix, Isabelle; Lipcey, Carol; Imbert, Jean; Kahn-Perlès, Brigitte

    2002-03-15

    We have followed Sp1 expression in primary human T lymphocytes induced, via CD2 plus CD28 costimulation, to sustained proliferation and subsequent return to quiescence. Binding of Sp1 to wheat germ agglutinin lectin was not modified following activation, indicating that the overall glycosylation of the protein was unchanged. Sp1 underwent, instead, a major dephosphorylation that correlated with cyclin A expression and, thus, with cell cycle progression. A similar change was observed in T cells that re-entered cell cycle following secondary interleukin-2 stimulation, as well as in serum-induced proliferating NIH/3T3 fibroblasts. Phosphatase 2A (PP2A) appears involved because 1) treatment of dividing cells with okadaic acid or cantharidin inhibited Sp1 dephosphorylation and 2) PP2A dephosphorylated Sp1 in vitro and strongly interacted with Sp1 in vivo. Sp1 dephosphorylation is likely to increase its transcriptional activity because PP2A overexpression potentiated Sp1 site-driven chloramphenicol acetyltransferase expression in dividing Kit225 T cells and okadaic acid reversed this effect. This increase might be mediated by a stronger affinity of dephosphorylated Sp1 for DNA, as illustrated by the reduced DNA occupancy by hyperphosphorylated Sp factors from cantharidin- or nocodazole-treated cells. Finally, Sp1 dephosphorylation appears to occur throughout cell cycle except for mitosis, a likely common feature to all cycling cells.

  9. Functional and quantitative alterations in T lymphocyte subpopulations in acute toxoplasmosis.

    PubMed

    Luft, B J; Kansas, G; Engleman, E G; Remington, J S

    1984-11-01

    The cellular immune response to Toxoplasma gondii has been studied in 23 patients with acute toxoplasma infection. Abnormalities of T cell subpopulations included a marked and significant elevation in suppressor (Leu 2) T cells in patients with prolonged symptoms due to acute infection and either a decrease in the number of T helper cells or an increase in the number of suppressor cells--or both--in patients with asymptomatic lymphadenopathy. There was no significant difference in lymphocyte proliferation to phytohemagglutinin or pokeweed mitogen among the various groups tested. The peak lymphocyte response to toxoplasma antigen, however, was significantly depressed in patients with acute infection compared with that in chronically infected control patients. The kinetics of the depression were consistent with the induction of a non-Leu 2 suppressor cell. These results demonstrate marked quantitative alterations in T lymphocyte subpopulations and functional alterations of T cells to toxoplasma antigen during infection with T. gondii.

  10. ALS patients’ regulatory T lymphocytes are dysfunctional, and correlate with disease progression rate and severity

    PubMed Central

    Beers, David R.; Zhao, Weihua; Wang, Jinghong; Zhang, Xiujun; Wen, Shixiang; Neal, Dan; Thonhoff, Jason R.; Alsuliman, Abdullah S.; Shpall, Elizabeth J.; Rezvani, Katy

    2017-01-01

    Neuroinflammation is a pathological hallmark of ALS in both transgenic rodent models and patients, and is characterized by proinflammatory T lymphocytes and activated macrophages/microglia. In ALS mouse models, decreased regulatory T lymphocytes (Tregs) exacerbate the neuroinflammatory process, leading to accelerated motoneuron death and shortened survival; passive transfer of Tregs suppresses the neuroinflammation and prolongs survival. Treg numbers and FOXP3 expression are also decreased in rapidly progressing ALS patients. A key question is whether the marked neuroinflammation in ALS can be attributed to the impaired suppressive function of ALS Tregs in addition to their decreased numbers. To address this question, T lymphocyte proliferation assays were performed. Compared with control Tregs, ALS Tregs were less effective in suppressing responder T lymphocyte proliferation. Although both slowly and rapidly progressing ALS patients had dysfunctional Tregs, the greater the clinically assessed disease burden or the more rapidly progressing the patient, the greater the Treg dysfunction. Epigenetically, the percentage methylation of the Treg-specific demethylated region was greater in ALS Tregs. After in vitro expansion, ALS Tregs regained suppressive abilities to the levels of control Tregs, suggesting that autologous passive transfer of expanded Tregs might offer a novel cellular therapy to slow disease progression. PMID:28289705

  11. ALS patients' regulatory T lymphocytes are dysfunctional, and correlate with disease progression rate and severity.

    PubMed

    Beers, David R; Zhao, Weihua; Wang, Jinghong; Zhang, Xiujun; Wen, Shixiang; Neal, Dan; Thonhoff, Jason R; Alsuliman, Abdullah S; Shpall, Elizabeth J; Rezvani, Katy; Appel, Stanley H

    2017-03-09

    Neuroinflammation is a pathological hallmark of ALS in both transgenic rodent models and patients, and is characterized by proinflammatory T lymphocytes and activated macrophages/microglia. In ALS mouse models, decreased regulatory T lymphocytes (Tregs) exacerbate the neuroinflammatory process, leading to accelerated motoneuron death and shortened survival; passive transfer of Tregs suppresses the neuroinflammation and prolongs survival. Treg numbers and FOXP3 expression are also decreased in rapidly progressing ALS patients. A key question is whether the marked neuroinflammation in ALS can be attributed to the impaired suppressive function of ALS Tregs in addition to their decreased numbers. To address this question, T lymphocyte proliferation assays were performed. Compared with control Tregs, ALS Tregs were less effective in suppressing responder T lymphocyte proliferation. Although both slowly and rapidly progressing ALS patients had dysfunctional Tregs, the greater the clinically assessed disease burden or the more rapidly progressing the patient, the greater the Treg dysfunction. Epigenetically, the percentage methylation of the Treg-specific demethylated region was greater in ALS Tregs. After in vitro expansion, ALS Tregs regained suppressive abilities to the levels of control Tregs, suggesting that autologous passive transfer of expanded Tregs might offer a novel cellular therapy to slow disease progression.

  12. Long term persistence of herpes simplex virus-specific CD8+ CTL in persons with frequently recurring genital herpes.

    PubMed

    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.

  13. A primary cell model of HIV-1 latency that uses activation through the T cell receptor and return to quiescence to establish latent infection

    PubMed Central

    Kim, Michelle; Hosmane, Nina N.; Bullen, C. Korin; Capoferri, Adam; Yang, Hung-Chih; Siliciano, Janet D.; Siliciano, Robert F.

    2015-01-01

    A mechanistic understanding of HIV-1 latency depends upon a model system that recapitulates the in vivo condition of latently infected, resting CD4+ T lymphocytes. Latency appears to be established after activated CD4+ T cells, the principal targets of HIV-1 infection, become productively infected and survive long enough to return to a resting memory state in which viral expression is inhibited by changes in the cellular environment. This protocol describes an ex vivo primary cell system that is generated under conditions that reflect the in vivo establishment of latency. Creation of these latency model cells takes 12 weeks and, once established, the cells can be maintained and used for several months. The resulting cell population contains both uninfected and latently infected cells. This primary cell model can be used to perform drug screens, study CTL responses to HIV-1, compare viral alleles, or to expand the ex vivo lifespan of cells from HIV-1 infected individuals for extended study. PMID:25375990

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

    PubMed

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

    2017-06-01

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

  15. The effect of stem cell from human exfoliated deciduous teeth on T lymphocyte proliferation

    PubMed Central

    Alipour, Razieh; Adib, Minoo; Hashemi-Beni, Batool; Sadeghi, Farzaneh

    2014-01-01

    Background: Mesenchymal stem cells (MSC), a specific type of adult tissue stem cell; have the immunosuppressive effects that make them valuable targets for regenerative medicine and treatment of many human illnesses. Hence, MSC have been the subject of numerous studies. The classical source of MSC is adult bone marrow (BM). Due to many shortcomings of harvesting MSC from BM, finding the alternative sources for MSC is an urgent. Stem cells from human exfoliated deciduous teeth (SHED) are relative new MSC populations that fulfill these criteria but their potential immunosuppressive effect has not been studied enough yet. Thus, in this work the effect of SHED on the proliferation of in vitro activated T lymphocytes were explored. Materials and Methods: In this study, both mitogen and alloantigen activated T cells were cultured in the presence of different numbers of SHED. In some co-cultures, activated T cells were in direct contact to MSCs and in other co-cultures; they were separated from SHED by a permeable membrane. In all co-cultures, the proliferation of T cells was measured by ELISA Bromodeoxyuridine proliferation assay. Results: In general, our results showed that SHED significantly suppress the proliferation of activated T cells in a dose-dependent manner. Moreover, the suppression was slightly stronger when MSCs were in physical contact to activated T cells. Conclusion: This study showed that SHED likewise other MSC populations can suppress the activation of T lymphocytes, which can be used instead of BM derived MSCs in many investigational and clinical applications. PMID:25337532

  16. High Throughput T Epitope Mapping and Vaccine Development

    PubMed Central

    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

  17. Ultrasound molecular imaging of acute cardiac transplantation rejection using nanobubbles targeted to T lymphocytes.

    PubMed

    Liu, Jinfeng; Chen, Yihan; Wang, Guohua; Lv, Qing; Yang, Yali; Wang, Jing; Zhang, Pingyu; Liu, Jie; Xie, Yu; Zhang, Li; Xie, Mingxing

    2018-04-01

    Clinical surveillance of acute heart transplantation rejection requires repeated invasive endomyocardial biopsies and noninvasive diagnostic techniques are desperately needed. It is acknowledged that T lymphocyte infiltration is the central process of acute rejection. We hypothesized that ultrasound molecular imaging with T lymphocyte-targeted nanobubbles could be used to detect acute rejection in heart transplantation. In this study, nanobubbles bearing anti-CD3 antibody (NB CD3 ) or isotype antibody (NB con ) were prepared and characterized. There was significant adhesion of NB CD3 to T lymphocytes compared with NB con in vitro. The signal intensity of the adherent NB CD3 was significantly higher than that of the NB con in allograft rats, but not significantly different in isograft rats. Furthermore, the signal intensity of NB CD3 in allograft rats was significantly higher than that in isograft rats, indicating more T lymphocyte infiltration in allograft rats compared with isograft rats. These results were further confirmed by immunohistochemistry examination, and the signal intensity of NB CD3 was positively correlated with the number of T lymphocytes in allograft rats. In summary, ultrasound molecular imaging with T lymphocyte-targeted nanobubbles can detect T lymphocyte infiltration in acute rejection and could be used as a noninvasive method in acute rejection detection after cardiac transplantation. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  18. Sezary syndrome cells unlike normal circulating T lymphocytes fail to migrate following engagement of NT1 receptor.

    PubMed

    Magazin, Marilyn; Poszepczynska-Guigné, Ewa; Bagot, Martine; Boumsell, Laurence; Pruvost, Christelle; Chalon, Pascale; Culouscou, Jean-Michel; Ferrara, Pascual; Bensussan, Armand

    2004-01-01

    Circulating malignant Sezary cells are a clonal proliferation of CD4+CD45RO+ T lymphocytes primarily involving the skin. To study the biology of these malignant T lymphocytes, we tested their ability to migrate in chemotaxis assays. Previously, we had shown that the neuropeptide neurotensin (NT) binds to freshly isolated Sezary malignant cells and induces through NT1 receptors the cell migration of the cutaneous T cell lymphoma cell line Cou-L. Here, we report that peripheral blood Sezary cells as well as the Sezary cell line Pno fail to migrate in response to neurotensin although they are capable of migrating to the chemokine stromal-cell-derived factor 1 alpha. This is in contrast with normal circulating CD4+ or CD8+ lymphocytes, which respond to both types of chemoattractants except after ex vivo short-time anti-CD3 monoclonal antibody activation, which abrogates the neurotensin-induced lymphocyte migration. Furthermore, we demonstrate that neurotensin-responsive T lymphocytes express the functional NT1 receptor responsible for chemotaxis. In these cells, but not in Sezary cells, neurotensin induces recruitment of phosphatidylinositol-3 kinase, and redistribution of phosphorylated cytoplasmic tyrosine kinase focal adhesion kinase and filamentous actin. Taken together, these results, which show functional distinctions between normal circulating lymphocytes and Sezary syndrome cells, contribute to further understanding of the physiopathology of these atypical cells.

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

    PubMed Central

    1992-01-01

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

  20. [Adoptive transfer of Epstein-Barr virus-specific T-lymphocytes in chronic active Epstein-Barr infection].

    PubMed

    Babel, N; Hammer, M H; Reinke, P

    2003-03-14

    A 27-year-old man was admitted because of intermittent fever, fatigue, lymphadenopathy and splenomegaly for 20 years. Chronic administration of 6 - 8 g aspirin per day (self-prescribed) resulted in limited control of symptoms and in the development of analgesic nephropathy. The patient had prominent splenomegaly and lymphadenopathy without histological signs of malignancy. Monocytosis and T-lymphopenia were also present. Infectious disease testing revealed IgG+/IgM- EBV serology and EA-EBV-mRNA nested PCR clearly demonstrated the presence of lytic EBV-proteins in PBMCs. As chronic active Epstein-Barr virus infection (CAEBV) was highly probable, treatment with aciclovir, gancilovir and steroids was started. Because treatment failed adoptive T-cell transfer with autologous EBV-specific T-cells was performed. After three consecutive infusions the patient responded with a complete remission of fever, fatigue, lymphadenopathy and splenomegaly without adverse effects. Retrospective real-time PCR analysis showed a decrease in viral load from 62847 copies/ microg DNA to 45 - 250 copies after treatment. The patient remains in stable remission without signs of CAEBV (> 4 years). Adoptive transfer of autologous, EBV-specific T-lymphocytes is a promising treatment in CAEBV.

  1. Lymphocyte changes in beta-thalassaemia major.

    PubMed Central

    Musumeci, S; Schiliro, G; Romeo, M A; Sciotto, A; Rosalba, A; Pizzarelli, G

    1979-01-01

    Lymphocyte subpopulations were studied in 20 hypertransfused patients with beta-thalassaemia major, some of whom had been splenectomised. B-lymphocytes were normal but T-lymphocytes were decreased in all patients. The T-cell count was lower in the splenectomised patients than in the nonsplenectomised ones. In the former, the active rosette-forming lymphocytes were also diminished, but the difference was not significant. In all patients the percentage of null cells was greater and the activity of K-cells increased compared with controls. PMID:316991

  2. Reactivity of inducer cell subsets and T8-cell activation during the human autologous mixed lymphocyte reaction.

    PubMed

    Romain, P L; Morimoto, C; Daley, J F; Palley, L S; Reinherz, E L; Schlossman, S F

    1984-01-01

    To characterize the responding T cells in the autologous mixed lymphocyte reaction (AMLR), T cells were fractionated into purified subpopulations employing monoclonal antibodies and a variety of separation techniques including fluorescence-activated cell sorting. It was found that isolated T4 cells, but not T8 cells, proliferated in response to autologous non-T cells. More importantly, within the T4 subset, the autoreactive population was greatly enriched in a fraction reactive with an autoantibody from patients with juvenile chronic arthritis (JRA) or the monoclonal antibody anti-TQ1. Although T8 cells themselves were unable to proliferate in the AMLR, they could be induced to respond in the presence of either T4 cells or exogenous IL-2 containing medium. This was demonstrated by direct measurement of tritiated thymidine uptake by T8 cells during the course of the AMLR as well as by analysis of their relative DNA content. Taken together, these data indicate that the AMLR represents a complex pattern of immune responsiveness distinct from that observed in response to soluble antigen or alloantigen. The precise function of this T-cell circuit remains to be determined.

  3. Mesenchymal stem cells promote the sustained expression of CD69 on activated T lymphocytes: roles of canonical and non-canonical NF-κB signalling

    PubMed Central

    Saldanha-Araujo, Felipe; Haddad, Rodrigo; de Farias, Kelen C R Malmegrim; Souza, Alessandra de Paula Alves; Palma, Patrícia V; Araujo, Amélia G; Orellana, Maristela D; Voltarelli, Julio C; Covas, Dimas T; Zago, Marco A; Panepucci, Rodrigo A

    2012-01-01

    Abstract Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T cells into regulatory T cells in vitro. The marker CD69 is a target of canonical nuclear factor kappa-B (NF-κB) signalling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mechanisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3+ T cells were activated and cultured in the presence or absence of MSCs. CD4+ cell mRNA expression was then characterized by microarray analysis. The drug BAY11-7082 (BAY) and a siRNA against v-rel reticuloendotheliosis viral oncogene homolog B (RELB) were used to explore the differential roles of canonical and non-canonical NF-κB signalling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF-κB subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co-cultured with MSCs. The frequency of CD69+ cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69+ cells increased significantly following activation in lymphocytes co-cultured with MSCs. Inhibition of canonical NF-κB signalling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF-κB signalling on the third day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF-κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF-κB signalling. PMID:21777379

  4. Ebola Virus Binding to Tim-1 on T Lymphocytes Induces a Cytokine Storm.

    PubMed

    Younan, Patrick; Iampietro, Mathieu; Nishida, Andrew; Ramanathan, Palaniappan; Santos, Rodrigo I; Dutta, Mukta; Lubaki, Ndongala Michel; Koup, Richard A; Katze, Michael G; Bukreyev, Alexander

    2017-09-26

    Ebola virus (EBOV) disease (EVD) results from an exacerbated immunological response that is highlighted by a burst in the production of inflammatory mediators known as a "cytokine storm." Previous reports have suggested that nonspecific activation of T lymphocytes may play a central role in this phenomenon. T-cell immunoglobulin and mucin domain-containing protein 1 (Tim-1) has recently been shown to interact with virion-associated phosphatidylserine to promote infection. Here, we demonstrate the central role of Tim-1 in EBOV pathogenesis, as Tim-1 -/- mice exhibited increased survival rates and reduced disease severity; surprisingly, only a limited decrease in viremia was detected. Tim-1 -/- mice exhibited a modified inflammatory response as evidenced by changes in serum cytokines and activation of T helper subsets. A series of in vitro assays based on the Tim-1 expression profile on T cells demonstrated that despite the apparent absence of detectable viral replication in T lymphocytes, EBOV directly binds to isolated T lymphocytes in a phosphatidylserine-Tim-1-dependent manner. Exposure to EBOV resulted in the rapid development of a CD4 Hi CD3 Low population, non-antigen-specific activation, and cytokine production. Transcriptome and Western blot analysis of EBOV-stimulated CD4 + T cells confirmed the induction of the Tim-1 signaling pathway. Furthermore, comparative analysis of transcriptome data and cytokine/chemokine analysis of supernatants highlight the similarities associated with EBOV-stimulated T cells and the onset of a cytokine storm. Flow cytometry revealed virtually exclusive binding and activation of central memory CD4 + T cells. These findings provide evidence for the role of Tim-1 in the induction of a cytokine storm phenomenon and the pathogenesis of EVD. IMPORTANCE Ebola virus infection is characterized by a massive release of inflammatory mediators, which has come to be known as a cytokine storm. The severity of the cytokine storm is

  5. Vpr Promotes Macrophage-Dependent HIV-1 Infection of CD4+ T Lymphocytes

    PubMed Central

    Collins, David R.; Lubow, Jay; Lukic, Zana; Mashiba, Michael; Collins, Kathleen L.

    2015-01-01

    Vpr is a conserved primate lentiviral protein that promotes infection of T lymphocytes in vivo by an unknown mechanism. Here we demonstrate that Vpr and its cellular co-factor, DCAF1, are necessary for efficient cell-to-cell spread of HIV-1 from macrophages to CD4+ T lymphocytes when there is inadequate cell-free virus to support direct T lymphocyte infection. Remarkably, Vpr functioned to counteract a macrophage-specific intrinsic antiviral pathway that targeted Env-containing virions to LAMP1+ lysosomal compartments. This restriction of Env also impaired virological synapses formed through interactions between HIV-1 Env on infected macrophages and CD4 on T lymphocytes. Treatment of infected macrophages with exogenous interferon-alpha induced virion degradation and blocked synapse formation, overcoming the effects of Vpr. These results provide a mechanism that helps explain the in vivo requirement for Vpr and suggests that a macrophage-dependent stage of HIV-1 infection drives the evolutionary conservation of Vpr. PMID:26186441

  6. B and T lymphocyte attenuator restricts the protective immune response against experimental malaria.

    PubMed

    Adler, Guido; Steeg, Christiane; Pfeffer, Klaus; Murphy, Theresa L; Murphy, Kenneth M; Langhorne, Jean; Jacobs, Thomas

    2011-11-15

    The immune response against the blood stage of malaria has to be tightly regulated to allow for vigorous antiplasmodial activity while restraining potentially lethal immunopathologic damage to the host like cerebral malaria. Coinhibitory cell surface receptors are important modulators of immune activation. B and T lymphocyte attenuator (BTLA) (CD272) is a coinhibitory receptor expressed by most leukocytes, with the highest expression levels on T and B cells, and is involved in the maintenance of peripheral tolerance by dampening the activation of lymphocytes. The function of BTLA is described in several models of inflammatory disorders and autoimmunity, but its function in infectious diseases is less well characterized. Also, little is known about the influence of BTLA on non-T cells. In this study, we analyzed the function of BTLA during blood-stage malaria infection with the nonlethal Plasmodium yoelii strain 17NL. We show that BTLA knockout mice exhibit strongly reduced parasitemia and clear the infection earlier compared with wild-type mice. This increased resistance was seen before the onset of adaptive immune mechanisms and even in the absence of T and B cells but was more pronounced at later time points when activation of T and B cells was observed. We demonstrate that BTLA regulates production of proinflammatory cytokines in a T cell-intrinsic way and B cell intrinsically regulates the production of P. yoelii 17NL-specific Abs. These results indicate that the coinhibitory receptor BTLA plays a critical role during experimental malaria and attenuates the innate as well as the subsequent adaptive immune response.

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

    PubMed Central

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

    2017-01-01

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

  8. Alterations in circulating T-cell lymphocyte populations in children with obstructive sleep apnea.

    PubMed

    Tan, Hui-Leng; Gozal, David; Wang, Yang; Bandla, Hari P R; Bhattacharjee, Rakesh; Kulkarni, Richa; Kheirandish-Gozal, Leila

    2013-06-01

    Changes in lymphocyte phenotype and functionality have been described in adult patients with obstructive sleep apnea (OSA). We hypothesized that OSA is associated with T lymphocyte alterations in children, particularly in T regulatory lymphocytes (T regs), and aimed to characterize circulating T lymphocyte subsets in children with OSA. Cross-sectional. Kosair Children's Hospital (Louisville, KY, USA) and Comer Children's Hospital (Chicago, IL, USA). Consecutively recruited children being evaluated for habitual snoring. N/A. Overnight polysomnography (PSG) was performed and a fasting blood sample was obtained from the patients. Flow cytometry was performed on peripheral blood mononuclear cells stained for CD3, CD4, CD8, CD25, FOXP3, interleukin-4 (IL-4), interferon-γ (IFN-γ), and IL-17. Patients were divided into three groups based on their PSG: controls (apnea-hypopnea indices [AHI] < 1/h total sleep time [TST]), mild OSA (1 ≤ AHI < 5/hTST), moderate-severe OSA (AHI ≥ 5/h TST). The percentage of CD4+ and T reg lymphocytes differed across groups. Children with moderate-severe OSA had significantly reduced T reg than control children (median [interquartile range] 4.8 [3.8-5.7% CD4+] versus 7.8 [7.0-9.2% CD4+]; P < 0.001). There were also significant differences in the percentage of T helper 1 (Th1) lymphocytes and in Th1:Th2 ratios between groups. Children with moderate-severe OSA had increased Th1 cells (P = 0.001) and Th1:Th2 ratios (P = 0.0026) compared with children with mild OSA and control children. Associations between AHI and T reg (P = 0.0003; r = -0.46), CD4+ lymphocytes (P = 0.0047; r = -0.37), and Th1:Th2 ratios (P = 0.0009; r = 0.43) emerged. In addition, the percentage of T reg was inversely correlated with Th1:Th2 ratios (P = 0.029; r = -0.29). Pediatric OSA is associated with reduced T reg population and altered Th1:Th2 balance toward Th1 predominance, suggesting a shift to a proinflammatory state. The changes in lymphocytic phenotypes

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

    NASA Technical Reports Server (NTRS)

    Sastry, Jagannadha K.

    1998-01-01

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

  10. Effect of melatonin on monochromatic light-induced T-lymphocyte proliferation in the thymus of chickens.

    PubMed

    Chen, Fuju; Reheman, Aikebaier; Cao, Jing; Wang, Zixu; Dong, Yulan; Zhang, Yuxian; Chen, Yaoxing

    2016-08-01

    A total of 360 post-hatching day 0 (P0) Arbor Acre male broilers, including intact, sham operation and pinealectomy groups, were exposed to white light (WL), red light (RL), green light (GL) and blue light (BL) from a light-emitting diode (LED) system until for P14. We studied the effects of melatonin and its receptors on monochromatic light-induced T-lymphocyte proliferation in the thymus of broilers. The density of proliferating cell nuclear antigen (PCNA) cells and the proliferation of T-lymphocytes in response to Concanavalin A (ConA) in GL significantly increased both in vivo and in vitro (from 9.57% to 32.03% and from 34.30% to 50.53%, respectively) compared with other lights (p<0.005) and was strongly correlated with melatonin levels in plasma (p<0.005). Pinealectomy reduced the levels of circulatory melatonin and the proliferation of T-lymphocytes and eliminated the differences between GL and other lights (p<0.005). However, exogenous melatonin (10(-9)M) significantly increased the proliferative activity of T-lymphocyte by 9.64% (p=0.002). In addition, GL significantly increased mRNA expression levels of Mel1a, Mel1b and Mel1c receptors from 21.09% to 32.57%, and protein expression levels from 24.43% to 42.92% compared with RL (p<0.05). However, these effects were blocked after pinealectomy. Furthermore, 4P-PDOT (a selective Mel1b antagonist) and prazosin (a selective Mel1c antagonist) attenuated GL-induced T-lymphocyte proliferation in response to ConA (p=0.000). Luzindole (a nonselective Mel1a/Mel1b antagonist), however, did not induce these effects (p=0.334). These results suggest that melatonin may mediate GL-induced T-lymphocyte proliferation via the Mel1b and Mel1c receptors but not via the Mel1a receptor. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    USDA-ARS?s Scientific Manuscript database

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

  12. Human T-lymphotropic virus type I-associated myelopathy and tax gene expression in CD4+ T lymphocytes.

    PubMed

    Moritoyo, T; Reinhart, T A; Moritoyo, H; Sato, E; Izumo, S; Osame, M; Haase, A T

    1996-07-01

    Infection by human T-lymphotropic virus type I (HTLV-I) is associated with adult T-cell leukemia and a slowly progressive disease of the central nervous system (CNS), HTLV-I-associated myelopathy/tropical spastic paraparesis, characterized pathologically by inflammation and white matter degeneration in the spinal cord. One of the explanations for the tissue destruction is that HTLV-I infects cells in the CNS, or HTLV-I-infected CD4+ T lymphocytes enter the CNS, and this drives local expansion of virus-specific CD8+ cytotoxic T lymphocytes, which along with cytokines cause the pathological changes. Because both in the circulation and in the cerebrospinal fluid, CD8+ cytotoxic T lymphocytes are primarily reactive to the product of the HTLV-I tax gene, we sought evidence of expression of this gene within cells in the inflammatory lesions. After using double-label in situ hybridization techniques, we now report definitive localization of HTLV-I tax gene expression in CD4+ T lymphocytes in areas of inflammation and white matter destruction. These findings lend support to a hypothetical scheme of neuropathogenesis in which HTLV-I tax gene expression provokes and sustains an immunopathological process that progressively destroys myelin and axons in the spinal cord.

  13. A photosensitizer delivered by bispecific antibody redirected T lymphocytes enhances cytotoxicity against EpCAM-expressing carcinoma cells upon light irradiation.

    PubMed

    Blaudszun, André-René; Moldenhauer, Gerhard; Schneider, Marc; Philippi, Anja

    2015-01-10

    Recently conducted clinical trials have provided impressive evidence that chemotherapy resistant metastatic melanoma and several hematological malignancies can be cured using adoptive T cell therapy or T cell-recruiting bispecific antibodies. However, a significant fraction of patients did not benefit from these treatments. Here we have evaluated the feasibility of a novel combination therapy which aims to further enhance the killing potential of bispecific antibody-redirected T lymphocytes by using these cells as targeted delivery system for photosensitizing agents. For a first in vitro proof-of-concept study, ex vivo activated human donor T cells were loaded with a poly(styrene sulfonate) (PSS)-complex of the model photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP). In the absence of light and when loading with the water-soluble PSS/mTHPP-complex occurred at a tolerable concentration, viability and cytotoxic function of loaded T lymphocytes were not impaired. When "drug-enhanced" T cells were co-cultivated with EpCAM-expressing human carcinoma cells, mTHPP was transferred to target cells. Notably, in the presence of a bispecific antibody, which cross-links effector and target cells thereby inducing the cytolytic activity of cytotoxic T lymphocytes, significantly more photosensitizer was transferred. Consequently, upon irradiation of co-cultures, redirected drug-loaded T cells were more effective in killing A549 lung and SKOV-3 ovarian carcinoma cells than retargeted unloaded T lymphocytes. Particularly, the additive approach using redirected unloaded T cells in combination with appropriate amounts of separately applied PSS/mTHPP was less efficient as well. Thus, by loading T lymphocytes with a stimulus-sensitive anti-cancer drug, we were able to enhance the cytotoxic capacity of carrier cells. Photosensitizer boosted T cells could open new perspectives for adoptive T cell therapy as well as targeted photodynamic therapy. Copyright © 2014

  14. Hapten-specific T-Cell Unresponsiveness Induced by Benzylpenicilloyl Autologous Gamma Globulin Conjugates in Human Lymphocytes In Vitro

    PubMed Central

    Geha, Raif S.; Fruchter, Lazar; Borel, Yves

    1980-01-01

    The aim of these studies was to determine whether unresponsiveness to the main determinant of penicillin, benzylpenicilloyl, can be induced in human peripheral lymphocytes in vitro by conjugates of benzylpenicilloyl (BPO) autologous gamma globulin (HGG). Initially it was shown that conjugates of BPO-keyhole limpet hemocyanin (KLH) elicited lymphocyte proliferation in the peripheral blood lymphocytes of six out of nine adult individuals in vitro. In contrast, conjugates of dinitrophenylated KLH and of BPO-HGG and the carriers HGG and KLH alone failed to do so. Similarly, release of the non-specific helper factor, lymphocyte mitogenic factor (LMF) occurred only after BPO-KLH stimulation. LMF activity was measured by B-cell proliferation and incorporation of radioactive amino acids into secreted immunoglobulin. Treatment with BPO-HGG for 24 h in vitro inhibited BPO-KLH-induced lymphocyte proliferation and LMF release. Treatment with either HGG, dinitrophenylated HGG, BPO-KLH, or BPO-human serum albumin failed to abrogate T-cell lymphocyte proliferation of human lymphocytes in vitro. The antigen specificity of the reduced immunologic responsiveness was further demonstrated by the observation that lymphocytes treated with BPO-HGG for 24 h in vitro responded normally to tetanus toxoid antigen. The data suggest that conjugates of BPO-HGG induce hapten-specific helper T-cell unresponsiveness in vitro. PMID:6157701

  15. ICAMs Redistributed by Chemokines to Cellular Uropods as a Mechanism for Recruitment of T Lymphocytes

    PubMed Central

    del Pozo, Miguel Angel; Cabañas, Carlos; Montoya, María C.; Ager, Ann; Sánchez-Mateos, Paloma; Sánchez-Madrid, Francisco

    1997-01-01

    The recruitment of leukocytes from the bloodstream is a key step in the inflammatory reaction, and chemokines are among the main regulators of this process. During lymphocyte–endothelial interaction, chemokines induce the polarization of T lymphocytes, with the formation of a cytoplasmic projection (uropod) and redistribution of several adhesion molecules (ICAM-1,-3, CD43, CD44) to this structure. Although it has been reported that these cytokines regulate the adhesive state of integrins in leukocytes, their precise mechanisms of chemoattraction remain to be elucidated. We have herein studied the functional role of the lymphocyte uropod. Confocal microscopy studies clearly showed that cell uropods project away from the cell bodies of adhered lymphocytes and that polarized T cells contact other T cells through the uropod structure. Time-lapse videomicroscopy studies revealed that uropod-bearing T cells were able, through this cellular projection, to contact, capture, and transport additional bystander T cells. Quantitative analysis revealed that the induction of uropods results in a 5–10-fold increase in cell recruitment. Uropod-mediated cell recruitment seems to have physiological relevance, since it was promoted by both CD45R0+ peripheral blood memory T cells as well as by in vivo activated lymphocytes. Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process. To determine whether the increment in cell recruitment mediated by uropod may affect the transendothelial migration of T cells, we carried out chemotaxis assays through confluent monolayers of endothelial cells specialized in lymphocyte extravasation. An enhancement of T cell migration was observed under conditions of uropod formation, and this

  16. T cell immunoregulation in active ocular toxoplasmosis.

    PubMed

    Cordeiro, Cynthia A; Vieira, Erica L M; Castro, Vinicius M; Dutra, Walderez O; Costa, Rogerio A; Orefice, Juliana L; Campos, Wesley R; Orefice, Fernando; Young, Lucy H; Teixeira, Antonio Lucio

    2017-04-01

    Toxoplasma gondii infection is an important cause of infectious ocular disease. The physiopathology of retinochoroidal lesions associated with this infection is not completely understood. The present study was undertaken to investigate cytokine production by T cells from individuals with active toxoplasmic retinochoroiditis (TR) comparing with controls. Eighteen patients with active TR and 15 healthy controls (6 controls IgG + to Toxoplasma and 9 negative controls) were included in the study. Peripheral blood mononuclear cells were incubated in the presence or absence of T. gondii antigen (STAg), and stained against CD4, CD8, TNF, IL-10 and IFN-γ. Baseline expression of cytokines was higher in TR/IgG + patients in comparison with controls. Cytokine expression was not increased by STAg in vitro stimulation in controls. After stimulation, TR/IgG + patients' lymphocytes increased cytokine as compared to cultures from both controls. While T cells were the main source of IL-10, but also IFN-γ and TNF, other lymphocyte populations were relevant source of inflammatory cytokines. Interestingly, it was observed a negative correlation between ocular lesion size and IL-10 expression by CD4 + lymphocytes. This study showed that T cells are the main lymphocyte populations expressing IL-10 in patients with TR. Moreover, expression of IL-10 plays a protective role in active TR. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

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

    PubMed

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

    2018-05-15

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

  18. Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in leukemia.

    PubMed

    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.

  19. Profound CD4+/CCR5+ T cell expansion is induced by CD8+ lymphocyte depletion but does not account for accelerated SIV pathogenesis.

    PubMed

    Okoye, Afam; Park, Haesun; Rohankhedkar, Mukta; Coyne-Johnson, Lia; Lum, Richard; Walker, Joshua M; Planer, Shannon L; Legasse, Alfred W; Sylwester, Andrew W; Piatak, Michael; Lifson, Jeffrey D; Sodora, Donald L; Villinger, Francois; Axthelm, Michael K; Schmitz, Joern E; Picker, Louis J

    2009-07-06

    Depletion of CD8(+) lymphocytes during acute simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) results in irreversible prolongation of peak-level viral replication and rapid disease progression, consistent with a major role for CD8(+) lymphocytes in determining postacute-phase viral replication set points. However, we report that CD8(+) lymphocyte depletion is also associated with a dramatic induction of proliferation among CD4(+) effector memory T (T(EM)) cells and, to a lesser extent, transitional memory T (T(TrM)) cells, raising the question of whether an increased availability of optimal (activated/proliferating), CD4(+)/CCR5(+) SIV "target" cells contributes to this accelerated pathogenesis. In keeping with this, depletion of CD8(+) lymphocytes in SIV(-) RMs led to a sustained increase in the number of potential CD4(+) SIV targets, whereas such depletion in acute SIV infection led to increased target cell consumption. However, we found that the excess CD4(+) T(EM) cell proliferation of CD8(+) lymphocyte-depleted, acutely SIV-infected RMs was completely inhibited by interleukin (IL)-15 neutralization, and that this inhibition did not abrogate the rapidly progressive infection in these RMs. Moreover, although administration of IL-15 during acute infection induced robust CD4(+) T(EM) and T(TrM) cell proliferation, it did not recapitulate the viral dynamics of CD8(+) lymphocyte depletion. These data suggest that CD8(+) lymphocyte function has a larger impact on the outcome of acute SIV infection than the number and/or activation status of target cells available for infection and viral production.

  20. Terminally differentiated CD8+ T cells and CD57−FOXP3+CD8+ T cells are highly associated with the efficacy of immunotherapy using activated autologous lymphocytes

    PubMed Central

    Akagi, Junji; Baba, Hideo; Sekine, Teruaki; Ogawa, Kenji

    2018-01-01

    Treatment with activated autologous lymphocytes (AALs) has demonstrated mixed results for cancer treatment. Preliminary results revealed that the proportion of cluster of differentiation (CD)8+CD57+ T cells is significantly increased in AALs, indicating that they are able to determine treatment outcome. Therefore, the role of CD8+CD57+ T cells in AAL efficacy was investigated. T lymphocytes were isolated from 35 patients with stage IV gastric carcinomas (17 men and 18 women; aged 41–84 years) receiving immunotherapy using AALs (IAAL). Using fluorescence activated cell sorting, CD8, CD27, CD57, and forkhead box protein 3 (FOXP3) expression was investigated on CD8+ T cell populations in CD8+ T cell differentiation prior to and following in vitro culture. The association between these populations and progression-free survival (PFS) was analyzed using Cox univariate, and multivariate analyses and Kaplan-Meier survival analysis. CD57 expression was negative in early-differentiated CD8+ T cells (CD27+CD8+CD57−), and positive in intermediate- (CD27+CD8+CD57+) and terminal- (CD27−CD8+CD57+) differentiated CD8+ T cells. Univariate analysis revealed a significant association between terminal-CD8+ T cells and longer PFS times (P=0.035), whereas CD57−FOXP3+CD8+ T cells were associated with shorter PFS times. Multivariate analysis revealed that CD57−FOXP3+CD8+ T cells was an independent poor prognostic factor, whereas CD57+FOXP3+CD8+ T cells were not associated with PFS. Although IAAL increased the proportion of terminal-CD8+ T cells relative to the pre-culture proportions, patients with a high CD57−FOXP3+CD8+ T cell percentage exhibited repressed terminal-CD8+ T cell induction, leading to poor patient prognosis. Terminally differentiated CD27−CD8+CD57+ T cells were responsible for the effectiveness of AALs; however, CD57−FOXP3+CD8+ T cells abrogated their efficacy, possibly by inhibiting their induction.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-12-01

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

  3. HDAC6 regulates the dynamics of lytic granules in cytotoxic T lymphocytes

    PubMed Central

    Núñez-Andrade, Norman; Iborra, Salvador; Trullo, Antonio; Moreno-Gonzalo, Olga; Calvo, Enrique; Catalán, Elena; Menasche, Gaël; Sancho, David; Vázquez, Jesús; Yao, Tso-Pang

    2016-01-01

    HDAC6 is a tubulin deacetylase involved in many cellular functions related to cytoskeleton dynamics including cell migration and autophagy. In addition, HDAC6 affects antigen-dependent CD4+ T cell activation. In this study, we show that HDAC6 contributes to the cytotoxic function of CD8+ T cells. Immunization studies revealed defective cytotoxic activity in vivo in the absence of HDAC6. Adoptive transfer of wild-type or Hdac6-/- CD8+ T cells to Rag1-/- mice demonstrated specific impairment in CD8+ T cell responses against vaccinia infection. Mechanistically, HDAC6-deficient cytotoxic T lymphocytes (CTLs) showed defective in vitro cytolytic activity related to altered dynamics of lytic granules, inhibited kinesin 1 – dynactin mediated terminal transport of lytic granules to the immune synapse and deficient exocytosis, but not to target cell recognition, T cell receptor (TCR) activation or interferon (IFNγ) production. Our results establish HDAC6 as an effector of the immune cytotoxic response that acts by affecting the dynamics, transport and secretion of lytic granules by CTLs. PMID:26869226

  4. Umbilical Cord Tissue-Derived Mesenchymal Stem Cells Induce T Lymphocyte Apoptosis and Cell Cycle Arrest by Expression of Indoleamine 2, 3-Dioxygenase

    PubMed Central

    Li, Xiuying; Xu, Zhuo; Bai, Jinping; Yang, Shuyuan; Zhao, Shuli; Zhang, Yingjie; Chen, Xiaodong

    2016-01-01

    It has been reported that human mesenchymal stem cells are able to inhibit T lymphocyte activation; however, the discrepancy among different sources of MSCs is not well documented. In this study, we have compared the MSCs from bone marrow (BM), adipose tissue (AT), placenta (PL), and umbilical cord (UC) to determine which one displayed the most efficient immunosuppressive effects on phytohemagglutinin-induced T cell proliferation. Among them we found that hUC-MSC has the strongest effects on inhibiting T cell proliferation and is chosen to do the further study. We observed that T lymphocyte spontaneously released abundant IFN-γ. And IFN-γ secreted by T lymphocyte could induce the expression of indoleamine 2, 3-dioxygenase (IDO) in hUC-MSCs. IDO was previously reported to induce T lymphocyte apoptosis and cell cycle arrest in S phase. When cocultured with hUC-MSCs, T lymphocyte expression of caspase 3 was significantly increased, while Bcl2 and CDK4 mRNA expression decreased dramatically. Addition of 1-methyl tryptophan (1-MT), an IDO inhibitor, restored T lymphocyte proliferation, reduced apoptosis, and induced resumption of the cell cycle. In addition, the changes in caspase 3, CDK4, and Bcl2 expression were reversed by 1-MT. These findings demonstrate that hUC-MSCs induce T lymphocyte apoptosis and cell cycle arrest by expressing abundant IDO and provide an explanation for some of the immunomodulatory effects of MSCs. PMID:27418932

  5. Equine interferon gamma synthesis in lymphocytes after in vivo infection and in vitro stimulation with EHV-1.

    PubMed

    Paillot, R; Daly, J M; Juillard, V; Minke, J M; Hannant, D; Kydd, J H

    2005-08-22

    Equine cytotoxic T lymphocyte (CTL) responses to equine herpesvirus-1 (EHV-1) are well characterised but little is known about the cytokine response after infection or vaccination. EHV-1 is common in horses and infects lymphocytes in vivo. This virus was used as a model to measure the synthesis of interferon gamma (IFN-gamma) by equine peripheral blood mononuclear cells (PBMC) after in vivo infection and/or in vitro stimulation with EHV-1. Both flow cytometry and ELISPOT assays were used to quantify equine IFN-gamma using a mouse anti-bovine IFN-gamma monoclonal antibody (clone CC302; shown to cross-react with recombinant equine IFN-gamma) and a rabbit anti-canine IFN-gamma polyclonal antibody. The percentage of PBMC synthesising IFN-gamma after in vitro stimulation with EHV-1 increased with age. In yearlings infected experimentally with EHV-1, PBMC showed two peaks of IFN-gamma synthesis, 11 and 56 days after infection. The IFN-gamma synthesis was principally associated with CD8(+) cells. The patterns of IFN-gamma synthesis detected by intracellular IFN-gamma staining or ELISPOT were compared with CTL data and shown to be similar. These methods were also applied successfully to frozen samples of PBMC. Measurement of equine IFN-gamma using these simple techniques can now be applied to future studies on protective cellular immune responses following virus infection and/or vaccination of horses.

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

    PubMed

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

    2004-04-01

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

  7. Chromosome aberrations in T lymphocytes carrying adult T-cell leukemia-associated antigens (ATLA) from healthy adults.

    PubMed

    Fukuhara, S; Hinuma, Y; Gotoh, Y I; Uchino, H

    1983-01-01

    Chromosomes were studied in cultured T lymphocytes carrying adult T-cell leukemia-associated antigens (ATLA) that were obtained from five Japanese anti-ATLA seropositive healthy adults. Chromosomally abnormal cells were observed in three of the five healthy adults, and these cells were clonal in two subjects. All cells examined in one subject had rearrangements of chromosome nos. 7 and 14. Clonal cells from the second had a minute chromosome of unknown origin. A few cells in the third had nonclonal rearrangements of chromosomes. Thus, ATLA-positive T lymphocytes in some anti-ATLA seropositive healthy people have chromosome aberrations.

  8. Regulation of Asymmetric Division by Atypical Protein Kinase C Influences Early Specification of CD8+ T Lymphocyte Fates

    PubMed Central

    Metz, Patrick J.; Lopez, Justine; Kim, Stephanie H.; Akimoto, Kazunori; Ohno, Shigeo; Chang, John T.

    2016-01-01

    Naïve CD8+ T lymphocytes responding to microbial pathogens give rise to effector T cells that provide acute defense and memory T cells that provide long-lived immunity. Upon activation, CD8+ T lymphocytes can undergo asymmetric division, unequally distributing factors to the nascent daughter cells that influence their eventual fate towards the effector or memory lineages. Individual loss of either atypical protein kinase C (aPKC) isoform, PKCζ or PKCλ/ι, partially impairs asymmetric divisions and increases CD8+ T lymphocyte differentiation toward a long-lived effector fate at the expense of memory T cell formation. Here, we show that deletion of both aPKC isoforms resulted in a deficit in asymmetric divisions, increasing the proportion of daughter cells that inherit high amounts of effector fate-associated molecules, IL-2Rα, T-bet, IFNγR, and interferon regulatory factor 4 (IRF4). However, unlike CD8+ T cells deficient in only one aPKC isoform, complete loss of aPKC unexpectedly increased CD8+ T cell differentiation toward a short-lived, terminal effector fate, as evidenced by increased rates of apoptosis and decreased expression of Eomes and Bcl2 early during the immune response. Together, these results provide evidence for an important role for asymmetric division in CD8+ T lymphocyte fate specification by regulating the balance between effector and memory precursors at the initiation of the adaptive immune response. PMID:26765121

  9. ETP-46321, a dual p110α/δ class IA phosphoinositide 3-kinase inhibitor modulates T lymphocyte activation and collagen-induced arthritis.

    PubMed

    Aragoneses-Fenoll, L; Montes-Casado, M; Ojeda, G; Acosta, Y Y; Herranz, J; Martínez, S; Blanco-Aparicio, C; Criado, G; Pastor, J; Dianzani, U; Portolés, P; Rojo, J M

    2016-04-15

    Class IA phosphoinositide 3-kinases (PI3Ks) are essential to function of normal and tumor cells, and to modulate immune responses. T lymphocytes express high levels of p110α and p110δ class IA PI3K. Whereas the functioning of PI3K p110δ in immune and autoimmune reactions is well established, the role of p110α is less well understood. Here, a novel dual p110α/δ inhibitor (ETP-46321) and highly specific p110α (A66) or p110δ (IC87114) inhibitors have been compared concerning T cell activation in vitro, as well as the effect on responses to protein antigen and collagen-induced arthritis in vivo. In vitro activation of naive CD4(+) T lymphocytes by anti-CD3 and anti-CD28 was inhibited more effectively by the p110δ inhibitor than by the p110α inhibitor as measured by cytokine secretion (IL-2, IL-10, and IFN-γ), T-bet expression and NFAT activation. In activated CD4(+) T cells re-stimulated through CD3 and ICOS, IC87114 inhibited Akt and Erk activation, and the secretion of IL-2, IL-4, IL-17A, and IFN-γ better than A66. The p110α/δ inhibitor ETP-46321, or p110α plus p110δ inhibitors also inhibited IL-21 secretion by differentiated CD4(+) T follicular (Tfh) or IL-17-producing (Th17) helper cells. In vivo, therapeutic administration of ETP-46321 significantly inhibited responses to protein antigen as well as collagen-induced arthritis, as measured by antigen-specific antibody responses, secretion of IL-10, IL-17A or IFN-γ, or clinical symptoms. Hence, p110α as well as p110δ Class IA PI3Ks are important to immune regulation; inhibition of both subunits may be an effective therapeutic approach in inflammatory autoimmune diseases like rheumatoid arthritis. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Occurrence of primary lymphocytic hypophysitis in two horses and presence of scattered T-lymphocytes in the normal equine pituitary gland.

    PubMed

    Grau-Roma, Llorenç; Peckham, Robert; Paton, Jacqui; Stahel, Anina; de Brot, Simone

    2017-01-01

    The postmortem examination of a 14-y-old Appaloosa gelding with clinically diagnosed pituitary pars intermedia dysfunction showed a unique finding of moderate multifocal lymphocytic hypophysitis (LH). The pituitary glands of 24 horses submitted for postmortem examination were examined grossly and examined histologically for the presence of lymphocytes. Of these 23 horses, 1 additional case suffered from moderate LH. The 2 cases with LH tested negative for Equid herpesvirus 1 and 4 by polymerase chain reaction and immunohistochemistry (IHC), and no viral particles were observed by electron microscopy in 1 case examined. The cause of LH remains unknown, but based on the T-lymphocytic nature of the inflammation and the human literature, an immune-mediated origin is hypothesized. In addition, the review of 24 cases revealed that 10 horses had few and small multifocal lymphocytic infiltrates within the pituitary gland; the remaining 12 horses showed no evident lymphocytes when examined by hematoxylin and eosin. IHC for CD3 showed the presence of a small number of individual T-lymphocytes scattered through the gland in all examined horses, which appears therefore to be a normal feature of the pituitary gland in horses.

  11. Cyclic adenosine 5'-monophosphate and calcium induce CD152 (CTLA-4) up-regulation in resting CD4+ T lymphocytes.

    PubMed

    Vendetti, Silvia; Riccomi, Antonella; Sacchi, Alessandra; Gatta, Lucia; Pioli, Claudio; De Magistris, Maria Teresa

    2002-12-01

    The CTLA-4 (CD152) molecule is up-regulated upon T cell activation and proliferation, and plays a critical role in the inhibition of immune responses. We show in this study that cAMP induces up-regulation of CD152 in human CD4(+) T lymphocytes. This effect occurs in the absence of the up-regulation of CD69 and CD25 activation markers and T cell proliferation. In addition, we found that the Ca(2+) ionophore ionomycin also up-regulates CD152, and that the combination of a cAMP analog or cAMP inducers with ionomycin further enhances the expression of CD152 in resting CD4(+) T lymphocytes. However, cyclosporin A, which inhibits Ca(2+)/calcineurin signaling pathway, fully prevented the ionomycin- but not the cAMP-induced up-regulation of CD152. The effects of cAMP and ionomycin involve increase of both CD152 mRNA transcripts, coding for the membrane and the soluble forms of CD152. Furthermore, we show that CD152 molecules are translocated to the membrane and are functional, as their engagement by specific mAbs prevented NF-kappaB activation by anti-CD3/CD28 stimulation. These findings demonstrate that at least two novel signal pathways regulate CTLA-4 gene expression and CD152 molecule up-regulation in human CD4(+) T lymphocytes, in the absence of full T cell activation.

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

    PubMed Central

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

    2002-01-01

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

  13. T-lymphocyte and cytokine expression in human inflammatory periapical lesions.

    PubMed

    de Brito, Luciana Carla Neves; Teles, Flávia Rocha Fonseca; Teles, Ricardo Palmier; Totola, Antônio Helvécio; Vieira, Leda Quércia; Sobrinho, Antônio Paulino Ribeiro

    2012-04-01

    Lymphocytes, among many cells, express different sets of cytokines, chemokines, and receptors, which are considered important mediators of periapical immune response to infection. The aim of this study was to evaluate the mRNA expression of CD4(+)CD28(+) and CD8(+) T genes and the gene expression of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-17A, IL-10, CCL2/MCP-1, CCL4, CCL5, CXCR4, CCR5, and receptor activator for nuclear factor kappa B ligand (RANKL) in periapical interstitial fluid from human root canal infections. The samples were collected immediately after root canal cleaning and 7 days later (restrained root canal bacterial load) to characterize those gene expressions. Real-time polymerase chain reaction demonstrated significantly higher levels of CD4(+)CD28(+) and CD8(+) T-cell markers in the former root canal condition and an increase of IL-10 and CXCR4, followed by a decrease of proinflammatory cytokines such as RANKL, interferon-γ, IL-1β, and CCL5. Analyses of T-lymphocyte and cytokine expression in periapical area were able to show that distinct root canal conditions might play regulatory roles in controlling local immune/inflammatory processes. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  14. DNA damage in B and T lymphocytes of farmers during one pesticide spraying season.

    PubMed

    Lebailly, Pierre; Mirey, Gladys; Herin, Fabrice; Lecluse, Yannick; Salles, Bernard; Boutet-Robinet, Elisa

    2015-10-01

    The effect of one pesticide spraying season on DNA damage was measured on B and T lymphocytes among open-field farmers and controls. At least two peripheral blood samples were collected from each individual: one in a period without any pesticide application, several weeks after the last use (January, at period P0), and another in the intensive pesticide spraying period (May or June, at period P4). DNA damage was studied by alkaline comet assay on isolated B or T lymphocytes. Longitudinal comparison of DNA damage observed at both P0 and P4 periods revealed a statistically significant genotoxic effect of the pesticide spraying season in both B (P = 0.02) and T lymphocytes (P = 0.02) in exposed farmers. In contrast, non-farmers did not show any significant modifications. DNA damage levels in B and T lymphocytes were significantly higher in farmers than in non-farmers during the P4 period (P = 0.003 and P = 0.001 for B and T lymphocytes, respectively) but not during the P0 period. The seasonal effect observed among farmers was not correlated with either total farm area, farm area devoted to crops or recent solar exposure. On average, farmers used pesticides for 21 days between P0 and P4. Between the two time points studied, there was a tendency for a potential effect of the number of days of fungicide treatments (r (2) = 0.43; P = 0.11) on T lymphocyte DNA damage. A genotoxic effect was found in lymphocytes of farmers exposed to pesticides, suggesting in particular the possible implication of fungicides.

  15. Opinion: Interactions of innate and adaptive lymphocytes

    PubMed Central

    Gasteiger, Georg; Rudensky, Alexander Y.

    2015-01-01

    Innate lymphocytes, including natural killer (NK) cells and the recently discovered innate lymphoid cells (ILCs) have crucial roles during infection, tissue injury and inflammation. Innate signals regulate the activation and homeostasis of innate lymphocytes. Less well understood is the contribution of the adaptive immune system to the orchestration of innate lymphocyte responses. We review our current understanding of the interactions between adaptive and innate lymphocytes, and propose a model in which adaptive T cells function as antigen-specific sensors for the activation of innate lymphocytes to amplify and instruct local immune responses. We highlight the potential role of regulatory and helper T cells in these processes and discuss major questions in the emerging area of crosstalk between adaptive and innate lymphocytes. PMID:25132095

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

    PubMed

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

    2017-10-01

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

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

    PubMed

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

    2018-07-01

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

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

    PubMed Central

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

    2017-01-01

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

  19. Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

    PubMed Central

    Datta, Antara; Silverman, Lee; Phipps, Andrew J; Hiraragi, Hajime; Ratner, Lee; Lairmore, Michael D

    2007-01-01

    Background Human T-lymphotropic virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation. Results Herein, we analyzed the role of p30 in cell cycle regulation. Jurkat T-cells transduced with a p30 expressing lentivirus vector accumulated in the G2-M phase of cell cycle. We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C), had enhanced checkpoint kinase 1 (Chk1) serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1), diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198. Finally, primary human lymphocyte derived cell lines immortalized by a HTLV-1 proviral clone defective in p30 expression were more susceptible to camptothecin induced apoptosis. Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes. Conclusion Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell survival. PMID:17634129

  20. Physical association and functional interaction between beta1 integrin and CD98 on human T lymphocytes

    NASA Technical Reports Server (NTRS)

    Miyamoto, Yuko J.; Mitchell, Jason S.; McIntyre, Bradley W.

    2003-01-01

    CD98 is a cell surface protein previously characterized as a cell activation marker, an amino acid transporter, and has recently been implicated in integrin-related functions. Integrins are cell surface proteins, important for homotypic cell aggregation, cell adhesion, and coactivation of T lymphocytes. We have previously shown that the anti-CD98 mAb 80A10, when coimmobilized with anti-CD3 mAb OKT3, is able to mediate human T cell coactivation that is inhibited by anti-beta1 integrin specific mAb 18D3. These results indicated a functional association of CD98 and beta1 integrin signaling but left open the question of a physical association. We now show the induction of homotypic aggregation through CD98 among human T cells and this aggregation was inhibited by anti-beta1 integrin mAb. Therefore, CD98-dependent lymphocyte proliferation and adhesion may involve integrins. Competitive binding assays and fluorescence colocalization analysis suggested that CD98 and beta1 integrin were physically associated. Differential extraction techniques and immunoprecipitations provided the first evidence that the alpha4beta1 integrin and CD98 are specifically associated on human T lymphocytes.

  1. Fish oil feeding enhances lymphocyte proliferation but impairs virus-specific T lymphocyte cytotoxicity in mice following challenge with influenza virus

    PubMed Central

    Byleveld, M; Pang, G T; Clancy, R L; Roberts, D C K

    2000-01-01

    The effect of a fish oil diet on virus-specific cytotoxicity and lymphocyte proliferation was investigated. Mice were fed fish oil (17 g fish oil and 3 g sunflower/100 g) or beef tallow (17 g tallow and 3 g sunflower/100 g) diets for 14 days before intranasal challenge with influenza virus. At day 5 after infection, lung virus-specific T lymphocyte, but not macrophage or natural killer (NK) cell, cytotoxicity was significantly lower in mice fed fish oil, while bronchial lymph node cell proliferation to virus was significantly higher. In mice fed fish oil, spleen cell proliferation to virus was also significantly higher following immunization. The results showed that, despite improved lymphocyte proliferation, fish oil impairs primary virus-specific T lymphocyte cytotoxicity. This impairment may explain the delayed virus clearance that we have previously reported in infected mice fed the fish oil diet. PMID:10632664

  2. Fish oil feeding enhances lymphocyte proliferation but impairs virus-specific T lymphocyte cytotoxicity in mice following challenge with influenza virus.

    PubMed

    Byleveld, M; Pang, G T; Clancy, R L; Roberts, D C

    2000-02-01

    The effect of a fish oil diet on virus-specific cytotoxicity and lymphocyte proliferation was investigated. Mice were fed fish oil (17 g fish oil and 3 g sunflower/100 g) or beef tallow (17 g tallow and 3 g sunflower/100 g) diets for 14 days before intranasal challenge with influenza virus. At day 5 after infection, lung virus-specific T lymphocyte, but not macrophage or natural killer (NK) cell, cytotoxicity was significantly lower in mice fed fish oil, while bronchial lymph node cell proliferation to virus was significantly higher. In mice fed fish oil, spleen cell proliferation to virus was also significantly higher following immunization. The results showed that, despite improved lymphocyte proliferation, fish oil impairs primary virus-specific T lymphocyte cytotoxicity. This impairment may explain the delayed virus clearance that we have previously reported in infected mice fed the fish oil diet.

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

    PubMed Central

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

    2013-01-01

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

  4. Effects of interval and continuous exercise training on CD4 lymphocyte apoptotic and autophagic responses to hypoxic stress in sedentary men.

    PubMed

    Weng, Tzu-Pin; Huang, Shu-Chun; Chuang, Yu-Fen; Wang, Jong-Shyan

    2013-01-01

    Exercise is linked with the type/intensity-dependent adaptive immune responses, whereas hypoxic stress facilitates the programmed death of CD4 lymphocytes. This study investigated how high intensity-interval (HIT) and moderate intensity-continuous (MCT) exercise training influence hypoxia-induced apoptosis and autophagy of CD4 lymphocytes in sedentary men. Thirty healthy sedentary males were randomized to engage either HIT (3-minute intervals at 40% and 80%VO2max, n=10) or MCT (sustained 60%VO2max, n=10) for 30 minutes/day, 5 days/week for 5 weeks, or to a control group that did not received exercise intervention (CTL, n=10). CD4 lymphocyte apoptotic and autophagic responses to hypoxic exercise (HE, 100 W under 12%O2 for 30 minutes) were determined before and after various regimens. The results demonstrated that HIT exhibited higher enhancements of pulmonary ventilation, cardiac output, and VO2 at ventilatory threshold and peak performance than MCT did. Before the intervention, HE significantly down-regulated autophagy by decreased beclin-1, Atg-1, LC3-II, Atg-12, and LAMP-2 expressions and acridine orange staining, and simultaneously enhanced apoptosis by increased phospho-Bcl-2 and active caspase-9/-3 levels and phosphotidylserine exposure in CD4 lymphocytes. However, five weeks of HIT and MCT, but not CTL, reduced the extents of declined autophagy and potentiated apoptosis in CD4 lymphocytes caused by HE. Furthermore, both HIT and MCT regimens manifestly lowered plasma myeloperoxidase and interleukin-4 levels and elevated the ratio of interleukin-4 to interferon-γ at rest and following HE. Therefore, we conclude that HIT is superior to MCT for enhancing aerobic fitness. Moreover, either HIT or MCT effectively depresses apoptosis and promotes autophagy in CD4 lymphocytes and is accompanied by increased interleukin-4/interferon-γ ratio and decreased peroxide production during HE.

  5. T Lymphocyte Maturation Is Impaired in Healthy Young Individuals Carrying Trisomy 21 (Down Syndrome)

    ERIC Educational Resources Information Center

    Guazzarotti, Laura; Trabattoni, Daria; Castelletti, Eleonora; Boldrighini, Benedetta; Piacentini, Luca; Duca, Piergiorgio; Beretta, Silvia; Pacei, Michela; Caprio, Cristiana; Vigano, Alessandra; di Natale, Berardo; Zuccotti, Gian Vincenzo; Clerici, Mario

    2009-01-01

    Cytokine production, immune activation, T lymphocytes maturation, and serum IL-7 concentration were examined in 24 youngsters with Down syndrome and no acquired diseases (healthy Down syndrome [12 prepubertal, 13 pubertal]) and 42 age- and gender-matched controls (20 prepubertal, 22 pubertal). Results showed that a complex immune and impairment is…

  6. Activated alveolar macrophage and lymphocyte alveolitis in extrathoracic sarcoidosis without radiological mediastinopulmonary involvement

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

    Wallaert, B.; Ramon, P.; Fournier, E.C.

    1986-01-01

    Cellular characteristics of BAL were investigated in 18 patients with proved extrathoracic sarcoidosis (that is, sarcoidosis that affected the skin, eyes, parotid glands, stomach, nose, kidneys, or meninges) without clinical or radiological mediastinopulmonary involvement. Computed tomography of the thorax was performed on five patients: four patients were normal, and one had enlarged lymph nodes (these enlargements were not detectable on the patient's chest roentgenogram). The results of pulmonary function tests were normal in all patients. The total BAL cell count did not differ significantly between controls and patients. Abnormal percentages of alveolar lymphocytes (from 18 to 87%) were noted inmore » 15 out of 18 patients. SACE levels were normal in 15 patients. No pulmonary gallium uptake was detected. The chemiluminescence of AM's, whether spontaneous or PMA induced, was increased in five out of seven patients. The percentages of T3+ lymphocytes in sarcoidosis patients did not significantly differ from those in controls. The T4+:T8+ ratio was normal in four patients and slightly increased in one. Follow-up of patients showed that alveolar lymphocytosis is as lasting as extrathoracic involvement. Our data demonstrate increased percentages of lymphocytes and activated AM's in the BAL of patients with extrathoracic sarcoidosis. This may be due to the initial involvement of the respiratory tract in extrathoracic sarcoidosis or to the diffusion of activated macrophages and lymphocytes from an extrathoracic site into the lung.« less

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

    PubMed

    Yamaji, Yoshiaki; Nakayama, Tetsuo

    2014-07-31

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

  8. Immunomodulatory and cellular anti-oxidant activities of caffeic, ferulic, and p-coumaric phenolic acids: a structure-activity relationship study.

    PubMed

    Kilani-Jaziri, Soumaya; Mokdad-Bzeouich, Imen; Krifa, Mounira; Nasr, Nouha; Ghedira, Kamel; Chekir-Ghedira, Leila

    2017-10-01

    Many studies have been performed to assess the potential utility of natural products as immunomodulatory agents to enhance host responses and to reduce damage to the human body. To determine whether phenolic compounds (caffeic, ferulic, and p-coumaric acids) have immunomodulatory effects and clarify which types of immune effector cells are stimulated in vitro, we evaluated their effect on splenocyte proliferation and lysosomal enzyme activity. We also investigated the activity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL). In addition, induction of the cellular antioxidant activity in splenocytes, macrophages, and red blood cells was determined by measuring the fluorescence of the DCF product. The study first results indicated that caffeic, ferulic, and p-coumaric acids significantly promote LPS-stimulated splenocyte proliferation, suggesting a potential activation of B cells, and enhanced humoral immune response in hosts treated by the tested natural products. Phenolic acids significantly enhanced the killing activity of isolated NK and CTL cells but had negligible effects on mitogen-induced proliferation of splenic T cells. We showed that caffeic acid enhances lysosomal enzyme activity in murine peritoneal macrophages, suggesting a potential role in activating such cells. Immunomodulatory activity was concomitant with the cellular antioxidant effect in macrophages and splenocytes of caffeic and ferulic acids. We conclude from this study that caffeic, ferulic, and p-coumaric acids exhibited an immunomodulatory effect which could be ascribed, in part, to their cytoprotective effect via their antioxidant capacity. Furthermore, these results suggest that these natural products could be potentially used to modulate immune cell functions in physiological and pathological conditions.

  9. Comparison of Phenotypic and Functional Characteristics Between Canine Non-B, Non-T Natural Killer Lymphocytes and CD3+CD5dimCD21- Cytotoxic Large Granular Lymphocytes.

    PubMed

    Lee, Soo-Hyeon; Shin, Dong-Jun; Kim, Yoseop; Kim, Cheol-Jung; Lee, Je-Jung; Yoon, Mee Sun; Uong, Tung Nguyen Thanh; Yu, Dohyeon; Jung, Ji-Youn; Cho, Duck; Jung, Bock-Gie; Kim, Sang-Ki; Suh, Guk-Hyun

    2018-01-01

    Natural killer (NK) cells play a pivotal role in the immune response against infections and malignant transformation, and adopted transfer of NK cells is thought to be a promising therapeutic approach for cancer patients. Previous reports describing the phenotypic features of canine NK cells have produced inconsistent results. Canine NK cells are still defined as non-B and non-T (CD3 - CD21 - ) large granular lymphocytes. However, a few reports have demonstrated that canine NK cells share the phenotypic characteristics of T lymphocytes, and that CD3 + CD5 dim CD21 - lymphocytes are putative canine NK cells. Based on our previous reports, we hypothesized that phenotypic modulation could occur between these two populations during activation. In this study, we investigated the phenotypic and functional differences between CD3 + CD5 dim CD21 - (cytotoxic large granular lymphocytes) and CD3 - CD5 - CD21 - NK lymphocytes before and after culture of peripheral blood mononuclear cells isolated from normal dogs. The results of this study show that CD3 + CD5 dim CD21 - lymphocytes can be differentiated into non-B, non-T NK (CD3 - CD5 - CD21 - TCRαβ - TCRγδ - GranzymeB + ) lymphocytes through phenotypic modulation in response to cytokine stimulation. In vitro studies of purified CD3 + CD5 dim CD21 - cells showed that CD3 - CD5 - CD21 - cells are derived from CD3 + CD5 dim CD21 - cells through phenotypic modulation. CD3 + CD5 dim CD21 - cells share more NK cell functional characteristics compared with CD3 - CD5 - CD21 - cells, including the expression of T-box transcription factors (Eomes, T-bet), the production of granzyme B and interferon-γ, and the expression of NK cell-related molecular receptors such as NKG2D and NKp30. In conclusion, the results of this study suggest that CD3 + CD5 dim CD21 - and CD3 - CD5 - CD21 - cells both contain a subset of putative NK cells, and the difference between the two populations may be due to the degree of maturation.

  10. Clostridium perfringens enterotoxin is a superantigen reactive with human T cell receptors V beta 6.9 and V beta 22

    PubMed Central

    1992-01-01

    Candidate superantigens were screened for their ability to induce lysis of human histocompatibility leukocyte antigen class II-positive targets by human CD8+ influenza-specific cytotoxic T cell (CTL) lines. Clostridium perfringens enterotoxin (CPET) induced major histocompatibility complex unrestricted killing by some but not all CTL lines. Using "anchored" polymerase chain reactions, CPET was shown to selectively stimulate peripheral blood lymphocytes bearing T cell receptor V beta 6.9 and V beta 22 in five healthy donors. V beta 24, V beta 21, V beta 18, V beta 5, and V beta 6.1-5 appeared to be weakly stimulated. Antigen processing was not required for CPET to induce proliferation. Like the staphylococcal enterotoxins, CPET is a major cause of food poisoning. These data suggest that superantigenic and enterotoxigenic properties may be closely linked. PMID:1512551

  11. Monoclonal antibodies to the equine CD2 T lymphocyte marker, to a pan-granulocyte/monocyte marker and to a unique pan-B lymphocyte marker.

    PubMed

    Tumas, D B; Brassfield, A L; Travenor, A S; Hines, M T; Davis, W C; McGuire, T C

    1994-12-01

    Murine monoclonal antibodies, HB88A, B29A and DH59B separately identify the CD2 T lymphocyte molecule, a unique pan-B lymphocyte surface marker and a pan-granulocyte/monocyte surface molecule, respectively, in the horse. Specificity was shown by two-color immunofluorescent flow cytometry and immunofluorescent microscopy. MAb HB88A reacted with a 52 kDa pan-T lymphocyte molecule present on 75% +/- 7 of peripheral blood lymphocytes (PBL) (n = 15 horses). It also reacted with lymphocytes restricted to T lymphocyte dependent areas of lymph node and spleen. Specificity of mAb HB88A to CD2 was demonstrated by its reactivity to COS7 cells which expressed a transfected 1.5 kb equine lymphocyte c-DNA clone having 77.5% overall sequence homology with human CD2 c-DNA. MAb B29A reacted with a pan-B lymphocyte specific cell surface complex, 143, 72, 50, 40, 27 and 14.5 kDa, present on 19% +/- 7 of PBL (n = 15 horses). This complex has not been described in the horse or other species. MAb DH59B reacted with a 96 kDa pan-granulocyte/monocyte specific surface protein and identified macrophages and Kupffer cells in equine tissue sections. Together these mAbs can be used to identify and quantitate the major constituents of equine leukocytes.

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

    PubMed Central

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

    2016-01-01

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

  13. T lymphocyte subpopulations diverge in commercially raised chickens

    PubMed Central

    Bridle, Byram W.; Julian, Richard; Shewen, Patricia E.; Vaillancourt, Jean-Pierre

    2006-01-01

    Abstract To evaluate immunocompetence in commercially raised chickens, we immunophenotyped Dekalb Delta and H&N White Leghorn (WLH) hybrids, 20 chickens in each of 3 age groups (9 wk [juvenile], 25 wk [young adult], and 79 or 80 wk [adult]), for circulating CD3+, CD4+, CD8+, TCR1+, TCR2+, and TCR3+ lymphocytes. The proportion of CD3+ T cells, including CD4+ and CD8+ subsets, was increased in the hybrids as compared with published values for laboratory-raised outbred WLH chickens. The proportion of the TCR2+ (Vβ1) T cell subpopulation was also increased. An age-related decrease in the proportion of TCR1+ (γδ) T cells was noted in both hybrids. Further, a remarkably low CD4:CD8 ratio was evident in all age groups of both hybrids, indicating decreased immunocompetence. Overall, these experiments provide age-related proportions of various peripheral-blood T lymphocyte subpopulations in commercially raised Dekalb Delta and H&N chickens that diverge from the proportions in laboratory-raised outbred WLH chickens and suggest reduced immunocompetence. Such a decline in immunocompetence, including humoral immune capacity, could be attributed to genetic selection for production traits, environmental factors associated with commercial operations, and intense immunization. PMID:16850940

  14. Reduced Activated T Lymphocytes (CD4+CD25+) and Plasma Levels of Cytokines in Parkinson's Disease.

    PubMed

    Rocha, Natalia Pessoa; Assis, Frankcinéia; Scalzo, Paula Luciana; Vieira, Érica Leandro Marciano; Barbosa, Izabela Guimarães; de Souza, Mariana Soares; Christo, Paulo Pereira; Reis, Helton José; Teixeira, Antonio Lucio

    2018-02-01

    Parkinson's disease (PD) is the second most common neurodegenerative disease. The cause of neurodegeneration in PD is not completely understood, and evidence has shown that inflammatory/immune changes may be involved in PD pathophysiology. Herein, we aimed to determine the profile of the peripheral immune system in patients with PD in comparison with controls. Forty patients with PD and 25 age- and gender-matched controls were enrolled in this study. From these, 23 PD patients and 21 controls were included in the immunophenotyping analyses. Peripheral blood was drawn on the same day of the clinical assessment and submitted to plasma separation for enzyme-linked immunosorbent assay or cytometric bead array. Immunophenotyping analyses of the peripheral blood were performed by flow cytometry. We found that patients with PD presented peripheral immune changes evidenced by decreased percentage of T lymphocytes (CD3+ cells), especially activated T lymphocytes (CD4+CD25+ cells), when compared with controls. In line with these results, we also found decreased plasma levels of the cytokines IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A in the PD group. In vitro experiments demonstrated that the production of cytokines by peripheral blood mononuclear cells harvested from healthy young donors was reduced after exposure to the anti-parkinsonian drugs levodopa and pramipexole. Our data corroborate the hypothesis that immunological mechanisms are involved in PD. It is not clear whether the differences that we have found are due to adaptive mechanisms or to changes associated with PD, including pharmacological treatment, or even directly related to the disease pathophysiology. Future studies are needed in this regard.

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

    PubMed

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

    2004-01-01

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

  16. Two cases of chronic active Epstein-Barr virus infection in which EBV-specific cytotoxic T lymphocyte was induced after allogeneic bone marrow transplantation.

    PubMed

    Miyamura, Takako; Chayama, Kousuke; Wada, Tomoaki; Yamaguchi, Kazunari; Yamashita, Nobuko; Ishida, Toshiaki; Washio, Kana; Morishita, Naoto; Manki, Akira; Oda, Megumi; Morishima, Tsuneo

    2008-08-01

    CAEBV is a high mortality and morbidity disease with life-threatening complications. Nevertheless, the treatment regimens for CAEBV have not yet been established. Although some reports have described CAEBV therapy involving treatments such as antiviral drugs, immunomodulatory agents, and immunochemotherapy, none of these treatments have been demonstrated to be effective. The only treatment reported to be effective is allogeneic SCT. However, the complications of SCT are severe, so treatment results have been poor. Recently, immunotherapy has been devised, but this is still in the developmental stage. In this report, two cases of CAEBV in which allogeneic SCT was performed soon after diagnosis are reported. In both cases, a high EBV genome titer in the peripheral blood was detected at onset. After SCT, the EBV genome titer decreased as CTL activity gradually increased. This fact suggested that not only high-dose chemotherapy as a preconditioning treatment of SCT but also increased CTL activity which could eliminate virus-infected cells might be effective, although additional cases should be studied in order to establish effective treatments.

  17. Specificity redirection by CAR with human VEGFR-1 affinity endows T lymphocytes with tumor-killing ability and anti-angiogenic potency.

    PubMed

    Wang, W; Ma, Y; Li, J; Shi, H-S; Wang, L-Q; Guo, F-C; Zhang, J; Li, D; Mo, B-H; Wen, F; Liu, T; Liu, Y-T; Wang, Y-S; Wei, Y-Q

    2013-10-01

    Immunotherapy that is based on adoptive transfer of T lymphocytes, which are genetically modified to express chimeric antigen receptors (CARs) that recognize tumor-associated antigens, has been demonstrated to be an efficient cancer therapy. Vascular endothelial growth factor receptor-1 (VEGFR-1), a vital molecule involved in tumor growth and angiogenesis, has not been targeted by CAR-modified T lymphocytes. In this study, we generated CAR-modified T lymphocytes with human VEGFR-1 specificity (V-1 CAR) by electroporation. V-1 CAR-modified T lymphocytes were demonstrated to elicit lytic cytotoxicity to target cells in a VEGFR-1-dependent manner. The adoptive transfer of V-1 CAR T lymphocytes delayed tumor growth and formation and inhibited pulmonary metastasis in xenograft models and such efficacies were enhanced by cotransfer of T lymphocytes that expressed interleukin-15 (IL-15). Moreover, V-1 CAR-modified T lymphocytes lysed primary endothelial cells and impaired tube formation, in vitro. These data demonstrated the antitumor and anti-angiogenesis ability of V-1 CAR-modified T lymphocytes. Our study provides the rationale for the clinical translation of CAR-modified T lymphocytes with VEGFR-1 specificity.

  18. Decreased membrane potassium permeability and transport in human chronic leukemic and tonsillar lymphocytes

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

    Segel, G.B.; Lichtman, M.A.

    Human blood T-lymphocytes increase their potassium (K/sup +/) permeability and active K/sup +/ transport following lectin or antigen stimulation. We have studied the permeability and active transport of K/sup +/ by lymphocytes in chronic lymphocytic leukemia (CLL) to determine if their membrane K/sup +/ transport was similar to resting or lectin-stimulated normal blood lymphocytes. K/sup +/ transport was assessed both by the rate of isotopic /sup 42/K/sup +/ uptake and by the rate of change in cell K/sup +/ concentration after inhibition of the K/sup +/ transport system with ouabain. CLL lymphocytes had a marked decrease in membrane K/sup +/more » permeability and active transport of K/sup +/ when compared to blood T lymphocytes. K/sup +/ transport in five subjects with CLL (10 mmol . 1 cell water/sup -1/ . h/sup -1/) was half that in normal blood T-lymphocytes (20 mmol . 1 cell water/sup -1/ h/sup -1/). Phytohemagglutinin (PHA) treatment of CLL lymphocytes did not increase significantly their active K/sup +/ transport, whereas K/sup +/ transport by normal T-lymphocytes increased by 100%. Since there were 73% T-lymphocytes in normal blood and 14% in CLL blood, the difference in membrane K/sup +/ turnover could be related either to neoplasia or to the proposed B-lymphocyte origin of CLL. We studied human tonsillar lymphocytes which contained a mean of 34% T-cells. In five studies of tonsils, K/sup +/ transport was 14 mmol . 1 cell water/sup -1/ . h/sup -1/ and treatment with PHA increased K/sup +/ transport only 30%. The intermediate values for basal K/sup +/ transport and K/sup +/ transport in response to PHA in tonsillar lymphocytes were consistent with the proportion of T-lymphocytes present. These data sugges t that B-lymphocytes have reduced membrane permeability and active transport of K/sup +/. Thus the marked decrease in CLL lymphocyte membrane K/sup +/ permeability and transport may be a reflection of its presumed B-cell origin, rather than a membrane

  19. Direct induction of T lymphocyte-specific gene expression by the mammalian Notch signaling pathway

    PubMed Central

    Reizis, Boris; Leder, Philip

    2002-01-01

    The Notch signaling pathway regulates the commitment and early development of T lymphocytes. We studied Notch-mediated induction of the pre-T cell receptor α (pTa) gene, a T-cell-specific transcriptional target of Notch. The pTa enhancer was activated by Notch signaling and contained binding sites for its nuclear effector, CSL. Mutation of the CSL-binding sites abolished enhancer induction by Notch and delayed the up-regulation of pTa transgene expression during T cell lineage commitment. These results show a direct mechanism of stage- and tissue-specific gene induction by the mammalian Notch/CSL signaling pathway. PMID:11825871

  20. Cytotoxic tumour-infiltrating T lymphocytes influence outcome in resected pancreatic ductal adenocarcinoma.

    PubMed

    Lohneis, Philipp; Sinn, Marianne; Bischoff, Sven; Jühling, Anja; Pelzer, Uwe; Wislocka, Lilianna; Bahra, Marcus; Sinn, Bruno V; Denkert, Carsten; Oettle, Helmut; Bläker, Hendrik; Riess, Hanno; Jöhrens, Korinna; Striefler, Jana K

    2017-09-01

    We studied the prognostic effect of CD3-, CD8- and CD103-positive T lymphocytes in a cohort of 165 patients with resected pancreatic ductal adenocarcinomas (PDACs) of the treatment group (adjuvant gemcitabine) and the untreated control group of the CONKO-001 study. Immunohistochemical stainings on tissue microarrays (TMAs) against CD3, CD8 and CD103 were performed according to standard procedures. A high number of CD8-positive lymphocytes were significantly and independently associated with longer disease-free survival (DFS) and overall survival (OS) in the overall study population. Median DFS/OS were 7.4/18.1 months for patients with a low number of CD8-positive intratumoural lymphocytes (≤42 per 1 mm tissue core) and 12.7/25.2 months for patients with high numbers (>42 per 1-mm tissue core; p = 0.008/0.020; HR 0.62/0.65). The ratio of intraepithelial to total CD103-positive lymphocytes, but not total numbers of CD103-positive lymphocytes or CD103-positive intraepithelial lymphocytes, was associated with significantly improved DFS and OS in the overall study population (p = 0.022/0.009). Median DFS/OS was 5.9/15.7 for patients with a ratio of intraepithelial to total CD103-positive intratumoural lymphocytes higher than 0.3 and 11.6/24.7 for patients with a lower ratio. T-lymphocyte subpopulations might be prognostic in resectable PDAC but need standardization and verification by further studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2015-09-15

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

  2. Accumulation of Pol Mutations Selected by HLA-B*52:01-C*12:02 Protective Haplotype-Restricted Cytotoxic T Lymphocytes Causes Low Plasma Viral Load Due to Low Viral Fitness of Mutant Viruses

    PubMed Central

    Murakoshi, Hayato; Koyanagi, Madoka; Chikata, Takayuki; Rahman, Mohammad Arif; Kuse, Nozomi; Sakai, Keiko; Gatanaga, Hiroyuki; Oka, Shinichi

    2016-01-01

    ABSTRACT HLA-B*52:01-C*12:02, which is the most abundant haplotype in Japan, has a protective effect on disease progression in HIV-1-infected Japanese individuals, whereas HLA-B*57 and -B*27 protective alleles are very rare in Japan. A previous study on HLA-associated polymorphisms demonstrated that the number of HLA-B*52:01-associated mutations at four Pol positions was inversely correlated with plasma viral load (pVL) in HLA-B*52:01-negative individuals, suggesting that the transmission of HIV-1 with these mutations could modulate the pVL in the population. However, it remains unknown whether these mutations were selected by HLA-B*52:01-restricted CTLs and also reduced viral fitness. In this study, we identified two HLA-B*52:01-restricted and one HLA-C*12:02-restricted novel cytotoxic T-lymphocyte (CTL) epitopes in Pol. Analysis using CTLs specific for these three epitopes demonstrated that these CTLs failed to recognize mutant epitopes or more weakly recognized cells infected with mutant viruses than wild-type virus, supporting the idea that these mutations were selected by the HLA-B*52:01- or HLA-C*12:02-restricted T cells. We further showed that these mutations reduced viral fitness, although the effect of each mutation was weak. The present study demonstrated that the accumulation of these Pol mutations selected by HLA-B*52:01- or HLA-C*12:02-restricted CTLs impaired viral replication capacity and thus reduced the pVL. The fitness cost imposed by the mutations partially accounted for the effect of the HLA-B*52:01-C*12:02 haplotype on clinical outcome, together with the effect of HLA-B*52:01-restricted CTLs on viral replication, which had been previously demonstrated. IMPORTANCE Numerous population-based studies identified HLA-associated HIV-1 mutations to predict HIV-1 escape mutations from cytotoxic T lymphocytes (CTLs). However, the majority of these HLA-associated mutations have not been identified as CTL escape mutations. Our previous population

  3. Accumulation of Pol Mutations Selected by HLA-B*52:01-C*12:02 Protective Haplotype-Restricted Cytotoxic T Lymphocytes Causes Low Plasma Viral Load Due to Low Viral Fitness of Mutant Viruses.

    PubMed

    Murakoshi, Hayato; Koyanagi, Madoka; Chikata, Takayuki; Rahman, Mohammad Arif; Kuse, Nozomi; Sakai, Keiko; Gatanaga, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

    2017-02-15

    HLA-B*52:01-C*12:02, which is the most abundant haplotype in Japan, has a protective effect on disease progression in HIV-1-infected Japanese individuals, whereas HLA-B*57 and -B*27 protective alleles are very rare in Japan. A previous study on HLA-associated polymorphisms demonstrated that the number of HLA-B*52:01-associated mutations at four Pol positions was inversely correlated with plasma viral load (pVL) in HLA-B*52:01-negative individuals, suggesting that the transmission of HIV-1 with these mutations could modulate the pVL in the population. However, it remains unknown whether these mutations were selected by HLA-B*52:01-restricted CTLs and also reduced viral fitness. In this study, we identified two HLA-B*52:01-restricted and one HLA-C*12:02-restricted novel cytotoxic T-lymphocyte (CTL) epitopes in Pol. Analysis using CTLs specific for these three epitopes demonstrated that these CTLs failed to recognize mutant epitopes or more weakly recognized cells infected with mutant viruses than wild-type virus, supporting the idea that these mutations were selected by the HLA-B*52:01- or HLA-C*12:02-restricted T cells. We further showed that these mutations reduced viral fitness, although the effect of each mutation was weak. The present study demonstrated that the accumulation of these Pol mutations selected by HLA-B*52:01- or HLA-C*12:02-restricted CTLs impaired viral replication capacity and thus reduced the pVL. The fitness cost imposed by the mutations partially accounted for the effect of the HLA-B*52:01-C*12:02 haplotype on clinical outcome, together with the effect of HLA-B*52:01-restricted CTLs on viral replication, which had been previously demonstrated. Numerous population-based studies identified HLA-associated HIV-1 mutations to predict HIV-1 escape mutations from cytotoxic T lymphocytes (CTLs). However, the majority of these HLA-associated mutations have not been identified as CTL escape mutations. Our previous population-based study showed that five

  4. Concanavalin A-induced and spontaneous suppressor cell activities in peripheral blood lymphocytes and spleen cells from gastric cancer patients.

    PubMed

    Toge, T; Hamamoto, S; Itagaki, E; Yajima, K; Tanada, M; Nakane, H; Kohno, H; Nakanishi, K; Hattori, T

    1983-11-01

    In 173 gastric cancer patients, activities of Concanavalin-A-induced suppressor cells (Con-AS) and spontaneous suppressor cells (SpS) in peripheral blood lymphocytes (PBL), splenic vein lymphocytes (SVL), and spleen cells (SCs) were investigated. Suppressions by Con-AS in PBL were significantly effective in patients of Stages III and IV, while suppressions by SpS were effective in patients with recurrent tumors. Thus, in PBLs of cancer patients, suppressor precursors, which are considered to be activated in vitro by Concanavalin-A, seemed to appear with the advances of the disease, and SpS activities, which could be already activated in vivo, seemed to increase in the terminal stage. In SCs, increased activities of Con-AS, but normal activities of SpS, were observed, and these suppressor-cell populations consisted of glass nonadherent cells. Suppressor activities of SCs would be due to suppressor T-cells, not to other types of cells. Furthermore, Con-AS existed in the medium-sized lymphocytes, which were fractionated on the basis of cell size, while SpS in the large-sized lymphocytes. A higher proportion of T-cells, bearing Fc receptors for IgG, was observed in the larger-sized lymphocyte fractions. Cell numbers in the large-sized lymphocyte fraction tended to increase with the advances of tumors. From these results, it is suggested that higher presence of suppressor precursors and the increase of SpS activities may occur in cancer patients, depending on the tumor advancing.

  5. Interleukin-12 in patients with cancer is synthesized by peripheral helper T lymphocytes.

    PubMed

    Michelin, Marcia A; Montes, Leticia; Nomelini, Rosekeila S; Abdalla, Douglas R; Aleixo, Andre A R; Murta, Eddie F C

    2015-09-01

    The production of cytokines by helper T lymphocytes is a critical event in the immune response, as alterations in the regulation of this process may result in an appropriate immune response, persistent infection or the development of autoimmune disease. Previously, this group has used flow cytometry to demonstrate the expression of interleukin-12 (IL-12) in peripheral blood CD4+ T lymphocytes from patients and mice with advanced cancer. The aim of the present study was to investigate whether CD4+ T lymphocytes from the peripheral blood (PB) of patients with cancer produce IL-12, using molecular approaches, flow cytometry and cellular imaging techniques. CD3+ and CD4+ cells, and cells producing IL-12, were isolated from the PB obtained from patients with cancer, using a cell sorting flow cytometry technique. The positivity of cells for CD3, CD4 and IL-12, which were identified by cell sorting, was visualized using immunofluorescent cellular imaging. Total RNA was extracted from the CD3+CD4+IL-12+ cells, obtained by cell sorting, for confirmation of the presence of IL-12 mRNA, using reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR demonstrated the presence of IL-12 mRNA in all patients (n=14), in contrast to the control group, in whom IL-12 expression was not detected. Immunofluorescent analysis of CD4+ T lymphocytes showed positive intracytoplasmatic IL-12 staining. These results demonstrated that CD3+CD4+ T lymphocytes in the PB of patients with cancer have the capacity to synthesize and express IL-12.

  6. Interleukin-12 in patients with cancer is synthesized by peripheral helper T lymphocytes

    PubMed Central

    MICHELIN, MARCIA A.; MONTES, LETICIA; NOMELINI, ROSEKEILA S.; ABDALLA, DOUGLAS R.; ALEIXO, ANDRE A. R.; MURTA, EDDIE F. C.

    2015-01-01

    The production of cytokines by helper T lymphocytes is a critical event in the immune response, as alterations in the regulation of this process may result in an appropriate immune response, persistent infection or the development of autoimmune disease. Previously, this group has used flow cytometry to demonstrate the expression of interleukin-12 (IL-12) in peripheral blood CD4+ T lymphocytes from patients and mice with advanced cancer. The aim of the present study was to investigate whether CD4+ T lymphocytes from the peripheral blood (PB) of patients with cancer produce IL-12, using molecular approaches, flow cytometry and cellular imaging techniques. CD3+ and CD4+ cells, and cells producing IL-12, were isolated from the PB obtained from patients with cancer, using a cell sorting flow cytometry technique. The positivity of cells for CD3, CD4 and IL-12, which were identified by cell sorting, was visualized using immunofluorescent cellular imaging. Total RNA was extracted from the CD3+CD4+IL-12+ cells, obtained by cell sorting, for confirmation of the presence of IL-12 mRNA, using reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR demonstrated the presence of IL-12 mRNA in all patients (n=14), in contrast to the control group, in whom IL-12 expression was not detected. Immunofluorescent analysis of CD4+ T lymphocytes showed positive intracytoplasmatic IL-12 staining. These results demonstrated that CD3+CD4+ T lymphocytes in the PB of patients with cancer have the capacity to synthesize and express IL-12. PMID:26622702

  7. Signal transduction in T lymphocytes in microgravity

    NASA Technical Reports Server (NTRS)

    Cogoli, A.

    1997-01-01

    More than 120 experiments conducted in space in the last 15 years have shown that dramatic changes are occurring in several types of single cells during their exposure to microgravity. One focus of today's research on cells in space is on signal transduction, especially those steps involving the cytoskeleton and cell-cell interactions. Signal transduction is often altered in microgravity as well as in hypergravity. This leads to changes in cell proliferation, genetic expression and differentiation. Interesting examples are leukocytes, HeLa cells, epidermoid cells and osteoblastic cells. Signalling pathways were studied in T lymphocytes in microgravity by several investigators after the discovery that mitogenic activation in vitro is virtually nil at 0g. T cells are a good model to study signal transduction because three extracellular signals (mitogen, IL-1 and IL-2) are required for full activation, and two classical pathways (via proteins G and PKC) are activated within the cell. In addition, low molecular weight GTP-binding proteins (Ras and Rap) are interacting with the cytoskeleton. The data at 0g support the notion that the expression of IL-2 receptor is inhibited at 0g, while mitogen binding and the transmission of IL-1 by accessory cells occur normally. In addition, alterations of the cytoskeleton suggest that the interaction with Rap proteins is disturbed. Data obtained with phorbol esters indicate that the function of PKC is changed in microgravity. Similar conclusions are drawn from the results with epidermoid cells A431.

  8. Cell-mediated immunity to herpes simplex virus: recognition of type-specific and type-common surface antigens by cytotoxic T cell populations.

    PubMed Central

    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

  9. Ferromagnetic nickel silicide nanowires for isolating primary CD4+ T lymphocytes

    NASA Astrophysics Data System (ADS)

    Kim, Dong-Joo; Seol, Jin-Kyeong; Lee, Mi-Ri; Hyung, Jung-Hwan; Kim, Gil-Sung; Ohgai, Takeshi; Lee, Sang-Kwon

    2012-04-01

    Direct CD4+ T lymphocytes were separated from whole mouse splenocytes using 1-dimensional ferromagnetic nickel silicide nanowires (NiSi NWs). NiSi NWs were prepared by silver-assisted wet chemical etching of silicon and subsequent deposition and annealing of Ni. This method exhibits a separation efficiency of ˜93.5%, which is comparable to that of the state-of-the-art superparamagnetic bead-based cell capture (˜96.8%). Furthermore, this research shows potential for separation of other lymphocytes, B, natural killer and natural killer T cells, and even rare tumor cells simply by changing the biotin-conjugated antibodies.

  10. More intensive CMV-infection in chronic heart failure patients contributes to higher T-lymphocyte differentiation degree.

    PubMed

    Moro-García, Marco Antonio; López-Iglesias, Fernando; Marcos-Fernández, Raquel; Bueno-García, Eva; Díaz-Molina, Beatriz; Lambert, José Luis; Suárez-García, Francisco Manuel; Morís de la Tassa, Cesar; Alonso-Arias, Rebeca

    2018-03-30

    Immunosenescence in chronic heart failure (CHF) is characterized by a high frequency of differentiated T-lymphocytes, contributing to an inflammatory status and a deficient ability to generate immunocompetent responses. CMV is the best known inducer of T-lymphocyte differentiation, and is associated with the phenomenon of immunosenescence. In this study, we included 58 elderly chronic heart failure patients (ECHF), 60 healthy elderly controls (HEC), 40 young chronic heart failure patients (YCHF) and 40 healthy young controls (HYC). High differentiation of CD8+ T-lymphocytes was found in CMV-seropositive patients; however, the differentiation of CD4+ T-lymphocytes was increased in CMV-seropositive but also in CHF patients. Anti-CMV antibody titers showed positive correlation with more differentiated CD4+ and CD8+ subsets and inverse correlation with CD4/CD8 ratio. Immunosenescence found in CHF patients is mainly due to the dynamics of CMV-infection, since the differentiation of T-lymphocyte subsets is related not only to CMV-infection, but also to anti-CMV antibody titers. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Elicitation of anti-viral cytotoxic T lymphocytes with purified viral and H-2 antigens.

    PubMed

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

    1980-07-01

    The minimal molecular requirements for elicitation of secondary anti-Sendai virus CTL were investigated. The hemagglutinin-neuraminidase (HN) glycoprotein of Sendai virus and the H-2Kk glycoprotein of YAC tumor cells were purified and incorporated into phospholipid vesicles. These unilamellar liposomes were then tested for the ability to elicit H-2 restricted secondary anti-Sendai virus CTL. The results indicate that these well-defined vesicles were capable of eliciting secondary anti-Sendai virus CTL which lysed only target cells possessing the H-2Kk haplotype and modified with inactivated Sendai virus.

  12. Regulation of expression of the ligand for CD40 on T helper lymphocytes.

    PubMed

    Castle, B E; Kishimoto, K; Stearns, C; Brown, M L; Kehry, M R

    1993-08-15

    Activated Th cells deliver contact-dependent signals to resting B lymphocytes that initiate and drive B cell proliferation. Recently, a ligand for the B lymphocyte membrane protein, CD40, has been identified that delivers contact-dependent Th cell signals to B cells. A dimeric soluble form of CD40 was produced and used to further characterize the regulation of expression of the CD40 ligand. Expression of the CD40 ligand was rapidly induced after Th lymphocyte activation, and its stability depended upon whether Th cells were activated with soluble or plastic-bound stimuli. Th cells activated with soluble stimuli rapidly turned over cell-surface CD40 ligand whereas Th cells activated with plastic-bound stimuli exhibited more stable CD40 ligand expression for up to 48 h. Removal of activated Th cells from the plastic-bound stimulus resulted in a rapid turnover of CD40 ligand, suggesting that continuous stimulation could maintain CD40 ligand expression. Ligation by soluble CD40 could also stabilize expression of CD40 ligand on the Th cell surface. Both CD40 ligand and IL-2 were transiently synthesized from 1 to 12 h after Th cell activation and had similar kinetics of synthesis. In Con A-activated Th cells newly synthesized CD40 ligand exhibited an initial high turnover (1.5 h t1/2) and after 5 h of Th cell activation became more stable (10-h t1/2). In Th cells activated with plastic-bound anti-CD3, CD40 ligand exhibited a similar biphasic turnover except that the rapid turnover phase began significantly later. This delay could allow more time for newly synthesized CD40 ligand to assemble or associate with other molecules and thus become stabilized on the cell surface. Newly synthesized CD40 ligand in Con A-activated Th cells appeared to not be efficient in delivering Th cell-dependent contact signals to resting B cells, implying the need for assembly or accessory proteins. Regulation of CD40 ligand expression was consistent with all the characteristics of Th cell

  13. Receptor type I and type II binding regions and the peptidyl-prolyl isomerase site of cyclophilin B are required for enhancement of T-lymphocyte adhesion to fibronectin.

    PubMed

    Carpentier, Mathieu; Allain, Fabrice; Slomianny, Marie-Christine; Durieux, Sandrine; Vanpouille, Christophe; Haendler, Bernard; Spik, Geneviève

    2002-04-23

    Cyclophilin B (CyPB), a cyclosporin A (CsA) binding protein, interacts with two types of binding sites at the surface of T-lymphocytes. The type I sites correspond to functional receptors involved in endocytosis and the type II sites to sulfated glycosaminoglycans (GAGs). Mutational analysis of CyPB has revealed that W128, which is part of the CsA-binding pocket, is implicated in the binding to the functional type I receptors and that two amino acid clusters located in the N-terminus ensure the binding to GAGs. The peptidyl-prolyl isomerase activity of CyPB is not required for receptor binding. We have recently demonstrated that CyPB enhances adhesion of peripheral blood T-lymphocytes to fibronectin, a component of the extracellular matrix. We intended to identify additional amino acids involved in the binding of CyPB to its functional type I receptor and to determine regions responsible for the stimulation of peripheral blood T-lymphocyte adhesion. We determined that residues R76, G77, K132, D155, and D158 of the calcineurin (CN) interacting region were implicated in the recognition of type I receptor but not of GAGs. We also found that two different changes in the N-terminal extension that abated binding to GAGs prevented adhesion of peripheral blood T-lymphocytes to coated CyPB, whereas abbrogation of the PPIase activity had no effect. On the other hand, the adhesion of peripheral blood T-lymphocytes to coated fibronectin was not stimulated by CyPB mutants devoid of either type I receptor or GAGs binding activity or by mutants of the PPIase site. Altogether, the results demonstrate that different regions of CyPB are involved in peripheral blood T-lymphocyte activation and imply a novel important physiological function for peptidyl-prolyl isomerase activity.

  14. Expression and agonist responsiveness of CXCR3 variants in human T lymphocytes

    PubMed Central

    Korniejewska, Anna; McKnight, Andrew J; Johnson, Zoë; Watson, Malcolm L; Ward, Stephen G

    2011-01-01

    The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 are involved in variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. Two alternatively spliced variants of the human CXCR3-A receptor have been described, termed CXCR3-B and CXCR3-alt. Human CXCR3-B binds CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4. In contrast, CXCR3-alt only binds CXCL11. We report that CXCL4 induces intracellular calcium mobilization as well as Akt and p44/p42 extracellular signal-regulated kinase phosphorylation, in activated human T lymphocytes. These responses have similar concentration dependence and time–courses to those induced by established CXCR3 agonists. Moreover, phosphorylation of Akt and p44/p42 is inhibited by pertussis toxin, suggesting coupling to Gαi protein. Surprisingly, and in contrast with the other CXCR3 agonists, stimulation of T lymphocytes with CXCL4 failed to elicit migratory responses and did not lead to loss of surface CXCR3 expression. Taken together, our findings show that, although CXCL4 is coupled to downstream biochemical machinery, its role in T cells is probably distinct from that of CXCR3-A agonists. PMID:21255008

  15. Evaluation of blood T-lymphocyte subpopulations involved in host cellular immunity in dogs with mammary cancer.

    PubMed

    Karayannopoulou, Maria; Anagnostou, Tilemachos; Margariti, Apostolia; Kostakis, Charalampos; Kritsepi-Konstantinou, Maria; Psalla, Dimitra; Savvas, Ioannis

    2017-04-01

    Cancer-bearing patients are often immunosuppressed. In dogs with mammary or other cancers, various alterations in blood cell populations involved in host cellular immunity have been reported; among these cell populations some T-lymphocyte subsets play an important role against cancer. The purpose of the present study was to investigate any alterations in circulating T-lymphocyte subpopulations involved in cellular immunity in bitches with mammary cancer, in comparison to age-matched healthy intact bitches. Twenty eight dogs with mammary cancer and 14 control dogs were included in this study. Twelve out of the 28 bitches had mammary cancer of clinical stage II and 16/28 of stage III. Histological examination revealed that 23/28 animals had carcinomas, 3/28 sarcomas and 2/28 carcinosarcomas. White blood cell, neutrophil and lymphocyte absolute numbers were measured by complete blood count. Furthermore, blood T-lymphocyte population (CD3 + ) and the subpopulations CD4 + , CD8 + and CD5 low+ were assessed by flow cytometry. White blood cell and neutrophil but not lymphocyte absolute numbers were higher (P=0.003 and P=0.001, respectively) in cancer patients than controls. Flow cytometric analysis revealed that the relative percentage of T-lymphocytes (CD3 + ) and of CD4 + , CD8 + subpopulations was lower (the CD4 + /CD8 + ratio was higher), whereas the percentage of CD5 low+ T-cells was higher, in dogs with cancer compared to controls; however, a statistically significant difference was found only in the case of CD8 + T-cells (P=0.014), whereas in the case of the CD4 + /CD8 + ratio the difference almost reached statistical significance (P=0.059). Based on these findings, it can be suggested that, although the absolute number of blood lymphocytes is unchanged, the relative percentages of T-lymphocyte subpopulations involved in host cell-mediated immunity are altered, but only cytotoxic CD8 + T-cells are significantly suppressed, in dogs with mammary cancer of clinical

  16. Iron differentially modulates the CD4-lck and CD8-lck complexes in resting peripheral blood T-lymphocytes.

    PubMed

    Arosa, F A; de Sousa, M

    1995-03-01

    Clinical and experimental studies performed in situations of iron overload have demonstrated that iron impairs several T-cell functions. We have examined the effect of iron in the form of ferric citrate on the CD4-lck and CD8-lck complexes in view of the key role played by the tyrosine kinase p56lck in regulating T-cell functions. Ferric citrate was seen to differentially modulate the CD4-lck and CD8-lck complexes in resting peripheral blood T-lymphocytes (PBLs) cultured in the presence of this metal salt for periods of 20 to 24 hr. Thus, whereas ferric citrate invariably induced a marked decrease in the in vitro activity of the CD4-associated lck by three- to fourfold at 100 microM (P < 3 x 10(-5)), it did not affect significantly the in vitro activity of the CD8-associated lck, although modest decreases were observed in some experiments. Immunoprecipitation and subsequent lck-immunoblotting revealed that the marked decrease in CD4-lck activity induced by 100 microM of ferric citrate was due to a decrease in the amount of p56lck on CD4 immunoprecipitates. Furthermore, flow cytometry analysis showed a decrease in the surface expression of the CD4 molecule in iron-treated PBLs, as judged by a decrease in the mean fluorescence intensity (MFI), that was accompanied by a decrease in the percentage of CD4+ T-lymphocytes. In marked contrast, whereas the surface expression of the CD8 molecule was slightly decreased, the percentage of CD8+ T-lymphocytes remained constant. This differential effect of ferric citrate on the CD4+ and CD8+ T-cell subsets led to a marked decrease in the CD4/CD8 ratios in iron-treated PBLs after the 20- to 24-hr period (P < 0.001). The present results indicate that iron in the form of ferric citrate can modulate key molecules involved in the process of T-cell activation and therefore influence T-cell-mediated functions.

  17. Action of the photosensitizer QLT0074 upon human T lymphocytes in vitro

    NASA Astrophysics Data System (ADS)

    Hunt, David W. C.; Jiang, Huijun; Salmon, Ruth A.; Granville, David J.; North, John R.; Richter, Anna M.

    2001-04-01

    A new photosensitizer, presently designated QLT0074, may be useful for the treatment of skin conditions, particularly those mediated by T lymphocytes, with photodynamic therapy (PDT). QLT0074 was tested against human peripheral blood T cells and Jurkat T lymphoma cells. Low concentrations of QLT0074 and blue light were sufficient to induce apoptosis in peripheral blood T cells or Jurkat T lymphoma cells as indicated by expression of the apoptosis-associated mitochondrial 7A6 marker, annexin-V labeling or activation of capsase-3 and cleavage of the capsase substrate poly(ADP-ribose)polymerase (PARP). Flow cytometry studies performed following PDT showed that peripheral blood T cells with high expression of the interleukin-2 receptor (CD25) took up greater amounts of QLT0074 and were eliminated to a greater degree than T cells with low CD25 levels. This effect of PDT was also shown by the reduction in the percentage of T cells that expressed other activation-associated markers such as very late activation antigen-4 (CD49d), human leukocyte antigen DR (HLA-DR), intercellular adhesion molecule-1 (CD54) and Fas (CD95). In the case of T cells that remained viable following PDT, CD25 expression was lower while CD54, CD95 and HLA-DR levels were unchanged. Thus, PDT with QLT0074 has selective, dose-dependent effects on T cells in vitro.

  18. A congenital activating mutant of WASp causes altered plasma membrane topography and adhesion under flow in lymphocytes

    PubMed Central

    Burns, Siobhan O.; Killock, David J.; Moulding, Dale A.; Metelo, Joao; Nunes, Joao; Taylor, Ruth R.; Forge, Andrew; Thrasher, Adrian J.

    2010-01-01

    Leukocytes rely on dynamic actin-dependent changes in cell shape to pass through blood vessels, which is fundamental to immune surveillance. Wiskott-Aldrich Syndrome protein (WASp) is a hematopoietic cell–restricted cytoskeletal regulator important for modulating cell shape through Arp2/3-mediated actin polymerization. A recently identified WASpI294T mutation was shown to render WASp constitutively active in vivo, causing increased filamentous (F)–actin polymerization, high podosome turnover in macrophages, and myelodysplasia. The aim of this study was to determine the effect of WASpI294T expression in lymphocytes. Here, we report that lymphocytes isolated from a patient with WASpI294T, and in a cellular model of WASpI294T, displayed abnormal microvillar architecture, associated with an increase in total cellular F-actin. Microvillus function was additionally altered as lymphocytes bearing the WASpI294T mutation failed to roll normally on L-selectin ligand under flow. This was not because of defects in L-selectin expression, shedding, cytoskeletal anchorage, or membranal positioning; however, under static conditions of adhesion, WASpI294T-expressing lymphocytes exhibited altered dynamic interaction with L-selectin ligand, with a significantly reduced rate of adhesion turnover. Together, our results demonstrate that WASpI294T significantly affects lymphocyte membrane topography and L-selectin–dependent adhesion, which may be linked to defective hematopoiesis and leukocyte function in affected patients. PMID:20354175

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

    PubMed

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

    2018-02-23

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

  20. HIV: current opinion in escapology.

    PubMed

    Klenerman, Paul; Wu, Ying; Phillips, Rodney

    2002-08-01

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

  1. Resveratrol Alters Proliferative Responses and Apoptosis in Human Activated B Lymphocytes In Vitro

    USDA-ARS?s Scientific Manuscript database

    We hypothesized that resveratrol, a polyphenol found in grapes, peanuts, and berries would modulate B lymphocyte proliferation, immunoglobulin synthesis, and apoptosis after activation with T-cell dependent pokeweed mitogen. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of ...

  2. Preexisting compensatory amino acids compromise fitness costs of a HIV-1 T cell escape mutation

    DOE PAGES

    Liu, Donglai; Zuo, Tao; Hora, Bhavna; ...

    2014-01-01

    Background: Fitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses. Results: The T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, themore » fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations. Conclusions: Our results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.« less

  3. Herpes simplex virus requires VP11/12 to induce phosphorylation of the activation loop tyrosine (Y394) of the Src family kinase Lck in T lymphocytes.

    PubMed

    Wagner, Melany J; Smiley, James R

    2009-12-01

    Herpes simplex virus (HSV) tegument proteins are released into the cytoplasm during viral entry and hence are among the first viral proteins encountered by an infected cell. Despite the implied importance of these proteins in the evasion of host defenses, the function of some, like virion protein 11/12 (VP11/12), have not been clearly defined. Previously, we reported that VP11/12 is strongly tyrosine phosphorylated during the infection of lymphocytes but not in fibroblasts or an epithelial cell line (G. Zahariadis, M. J. Wagner, R. C. Doepker, J. M. Maciejko, C. M. Crider, K. R. Jerome, and J. R. Smiley, J. Virol. 82:6098-6108, 2008). We also showed that tyrosine phosphorylation depends in part on the activity of the lymphocyte-specific Src family kinase (SFK) Lck in Jurkat T cells. These data suggested that VP11/12 is a substrate of Lck and that Lck is activated during HSV infection. Here, we show that HSV infection markedly increases the fraction of Lck phosphorylated on its activation loop tyrosine (Y394), a feature characteristic of activated Lck. A previous report implicated the immediate-early protein ICP0 and the viral serine/threonine kinases US3 and UL13 in the induction of a similar activated phenotype of SFKs other than Lck in fibroblasts and suggested that ICP0 interacts directly with SFKs through their SH3 domain. However, we were unable to detect an interaction between ICP0 and Lck in T lymphocytes, and we show that ICP0, US3, and UL13 are not strictly required for Lck activation. In contrast, VP11/12 interacted with Lck or Lck signaling complexes and was strictly required for Lck activation during HSV infection. Thus, VP11/12 likely modulates host cell signaling pathways for the benefit of the virus.

  4. Specific ganglioside binding to receptor sites on T lymphocytes that couple to ganglioside-induced decrease of CD4 expression

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

    Morrison, W.J.; Offner, H.; Vandenbark, A.A.

    1989-01-01

    The binding of different gangliosides to rat T-helper lymphocytes was characterized under conditions that decrease CD4 expression on different mammalian T-helper lymphoctyes. Saturation binding by monosialylated ({sub 3}H)-GM{sub 1} to rat T-lymphocytes was time- and temperature-dependent, had a dissociation constant (K{sub D}) of 2.2 {plus minus} 1.4 {mu}M and a binding capacity near 2 fmoles/cell. Competitive inhibition of ({sup 3}H)- GM{sub 1} binding demonstrated a structural-activity related to the number of unconstrained sialic acid moieties on GM{sub 1}-congeneric gangliosides. A comparison between the results of these binding studies and gangliosides-induced decrease of CD4 expression demonstrated that every aspect of ({supmore » 3}H)-GM{sub 1} binding concurs with ganglioside modulation of CD4 expression. It is concluded that the specific decrease of CD4 expression induced by pretreatment with gangliosides involves the initial process of gangliosides binding to specific sites on CD4{sup {double dagger}} T-helper lymphocytes.« less

  5. BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes.

    PubMed

    Kessler, J H; Bres-Vloemans, S A; van Veelen, P A; de Ru, A; Huijbers, I J G; Camps, M; Mulder, A; Offringa, R; Drijfhout, J W; Leeksma, O C; Ossendorp, F; Melief, C J M

    2006-10-01

    For immunotherapy of residual disease in patients with Philadelphia-positive leukemias, the BCR-ABL fusion regions are attractive disease-specific T-cell targets. We analyzed these regions for the prevalence of cytotoxic T lymphocyte (CTL) epitopes by an advanced reverse immunology procedure. Seventeen novel BCR-ABL fusion peptides were identified to bind efficiently to the human lymphocyte antigen (HLA)-A68, HLA-B51, HLA-B61 or HLA-Cw4 HLA class I molecules. Comprehensive enzymatic digestion analysis showed that 10 out of the 28 HLA class I binding fusion peptides were efficiently excised after their C-terminus by the proteasome, which is an essential requirement for efficient cell surface expression. Therefore, these peptides are prime vaccine candidates. The other peptides either completely lacked C-terminal liberation or were only inefficiently excised by the proteasome, rendering them inappropriate or less suitable for inclusion in a vaccine. CTL raised against the properly processed HLA-B61 epitope AEALQRPVA from the BCR-ABL e1a2 fusion region, expressed in acute lymphoblastic leukemia (ALL), specifically recognized ALL tumor cells, proving cell surface presentation of this epitope, its applicability for immunotherapy and underlining the accuracy of our epitope identification strategy. Our study provides a reliable basis for the selection of optimal peptides to be included in immunotherapeutic BCR-ABL vaccines against leukemia.

  6. Cyclophilin B mediates cyclosporin A incorporation in human blood T-lymphocytes through the specific binding of complexed drug to the cell surface.

    PubMed

    Allain, F; Denys, A; Spik, G

    1996-07-15

    Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein located within intracellular vesicles and released in biological fluids. We recently reported the specific binding of this protein to T-cell surface receptor which is internalized even in the presence of CsA. These results suggest that CyPB might target the drug to lymphocytes and consequently modify its activity. To verify this hypothesis, we have first investigated the binding capacity and internalization of the CsA-CyPB complex in human peripheral blood T-lymphocytes and secondly compared the inhibitory effect of both free and CyPB-complexed CsA on the CD3-induced activation and proliferation of T-cells. Here, we present evidence that both the CsA-CyPB complex and free CyPB bind to the T-lymphocyte surface, with similar values of Kd and number of sites. At 37 degrees C, the complex is internalized but, in contrast to the protein, the drug is accumulated within the cell. Moreover, CyPB receptors are internalized together with the ligand and rapidly recycled to the cell surface. Finally, we demonstrate that CyPB-complexed CsA remains as efficient as uncomplexed CsA and that CyPB enhances the immunosuppressive activity of the drug. Taken together, our results support the hypothesis that surface CyPB receptors may be related to the selective and variable action of CsA, through specific binding and targeting of the CyPB-CsA complex to peripheral blood T-lymphocytes.

  7. Higher Numbers of T-Bet+ Tumor-Infiltrating Lymphocytes Associate with Better Survival in Human Epithelial Ovarian Cancer.

    PubMed

    Xu, Yun; Chen, Lujun; Xu, Bin; Xiong, Yuqi; Yang, Min; Rui, Xiaohui; Shi, Liangrong; Wu, Changping; Jiang, Jingting; Lu, Binfeng

    2017-01-01

    T-bet, a member of the T-box family of transcription factors, is a key marker of type I immune response within the tumor microenvironment, and has been previously reported by us to serve as an important prognostic indicator for human gastric cancer patients and a potential biomarker for immunotherapy. In the present study, we aimed to assess the clinical significance and prognostic value of T-bet+ tumor-infiltrating lymphocytes in human epithelial ovarian cancer. The immunohistochemistry was used to analyze the infiltration density of T-bet+ lymphoid cells in human epithelial ovarian cancer tissues, and the flow cytometry analysis was used to further analyze the presence of T-bet+ tumor-infiltrating lymphocytes subgroups in cancer tissues. Our immunohistochemistry analysis showed increased number of T-bet+ lymphoid cells in the human epithelial ovarian cancer tissues, and the flow cytometry analysis further demonstrated the presence of T-bet+ tumor-infiltrating lymphocytes subgroups including CD4+ , CD8+ T cells and NK cells. In addition, we also observed a significant association of T-bet+ tumor-infiltrating lymphocytes density in the tumor nest of cancer with not only serum CA125 levels but also with distant metastasis. However no association was observed with other characteristics like patients' age, pathological type, FIGO stage, tumor site and tumor size. Furthermore, the survival analysis showed that higher density of T-bet+ tumor-infiltrating lymphocytes both in tumor nest and tumor stroma of cancer tissues was significantly associated with better patient survival. In addition, the density of T-bet+ tumor-infiltrating lymphocytes in tumor nest appeared to be an independent risk factor for predicting patients' postoperative prognoses. Our data indicated that the key transcription factor T-bet might play an important role in the type I immune cells mediated antitumor response, and the density of T-bet+ lymphocytes in human epithelial ovarian cancer tissues

  8. Effect of a high and low dose of caffeine on human lymphocyte activation in response to antigen stimulation.

    PubMed

    Dulson, Deborah K; Bishop, Nicolette C

    2016-02-01

    This study investigated the effect of caffeine on antigen-stimulated lymphocyte activation. Six males rested for 3.5 h after ingesting 0 (PLA), 2, or 6 (6CAF) mg·kg(-1) body mass of caffeine. The number of antigen-stimulated NK CD69(+) cells increased in 6CAF at 1 h compared with PLA (P = 0.021). Caffeine did not influence the number of antigen-stimulated CD69(+) T cells or the geometric mean fluorescence intensity expression of CD69 on antigen-stimulated lymphocytes, suggesting caffeine has little effect on antigen-stimulated lymphocyte activation.

  9. Central memory CD8+ T lymphocytes mediate lung allograft acceptance

    PubMed Central

    Krupnick, Alexander Sasha; Lin, Xue; Li, Wenjun; Higashikubo, Ryuiji; Zinselmeyer, Bernd H.; Hartzler, Hollyce; Toth, Kelsey; Ritter, Jon H.; Berezin, Mikhail Y.; Wang, Steven T.; Miller, Mark J.; Gelman, Andrew E.; Kreisel, Daniel

    2014-01-01

    Memory T lymphocytes are commonly viewed as a major barrier for long-term survival of organ allografts and are thought to accelerate rejection responses due to their rapid infiltration into allografts, low threshold for activation, and ability to produce inflammatory mediators. Because memory T cells are usually associated with rejection, preclinical protocols have been developed to target this population in transplant recipients. Here, using a murine model, we found that costimulatory blockade–mediated lung allograft acceptance depended on the rapid infiltration of the graft by central memory CD8+ T cells (CD44hiCD62LhiCCR7+). Chemokine receptor signaling and alloantigen recognition were required for trafficking of these memory T cells to lung allografts. Intravital 2-photon imaging revealed that CCR7 expression on CD8+ T cells was critical for formation of stable synapses with antigen-presenting cells, resulting in IFN-γ production, which induced NO and downregulated alloimmune responses. Thus, we describe a critical role for CD8+ central memory T cells in lung allograft acceptance and highlight the need for tailored approaches for tolerance induction in the lung. PMID:24569377

  10. The Activation and Inactivation of Mature CD4 T cells: A Case for Peripheral Self–Nonself Discrimination

    PubMed Central

    Bretscher, P A

    2014-01-01

    The establishment of central tolerance to most self-antigens results in a repertoire of mature peripheral lymphocytes specific for foreign and peripheral self-antigens. The framework that single, mature lymphocytes are inactivated by antigen, whereas their activation requires lymphocyte cooperation, accounts for diverse observations and incorporates a mechanism of peripheral tolerance. This framework accounts for the generalizations that the sustained activation by antigen of most B cells and CD8 T cells requires CD4 T helper cells; in the absence of CD4 T cells, antigen can inactivate these B cells and CD8 T cells. In this sense, CD4 T cells are the guardians of the fate of most B cells and CD8 T cells when they encounter antigen. I argue here that the single-lymphocyte/multiple-lymphocyte framework for the inactivation/activation of lymphocytes also applies to CD4 T cells. I consider within this framework a model for the activation/inactivation of CD4 T cells that is consistent with the large majority of contemporary observations, including significant clinical observations. I outline the grounds why I feel this model is more plausible than the contemporary and predominant pathogen-associated molecular pattern (PAMP) and Danger Models for CD4 T cell activation. These models are based upon what I consider the radical premise that self–nonself discrimination does not exist at the level of mature CD4 T cells. I explain why I feel this feature renders the PAMP and Danger Models somewhat implausible. The model I propose, in contrast, is conservative in that it embodies such a process of self–nonself discrimination. PMID:24684567

  11. Induction of micronuclei and apoptosis in natural killer cells compared to T lymphocytes after gamma-irradiation.

    PubMed

    Louagie, H; Philippé, J; Vral, A; Cornelissen, M; Thierens, H; De Ridder, L

    1998-02-01

    To investigate the chromosomal damage caused by gamma-irradiation in T lymphocytes and natural killer (NK) cells and compare this with apoptosis induction in both lymphocyte subsets. Apoptosis induction by gamma-irradiation in T lymphocytes and NK cells was quantified using the annexin V flow cytometric assay. The cytokinesis-block micronucleus (MN) assay was used to evaluate the induced cytogenetic damage. For the MN assays on NK cells, gamma-irradiated peripheral blood mononuclear cells were cultured and stimulated with interleukin 15 (IL-15). Afterwards the NK cells (characterized by the CD3-/CD56+ phenotype) were separated with the FACSort flow cytometer and the number of MN in the sorted binuclear cells was scored. Doses of 1 and 2 Gy gamma-irradiation were applied. Higher numbers of MN in NK cells were found compared with the MN yield in T lymphocytes. In contrast, NK cells were less than T lymphocytes prone to apoptosis after gamma-irradiation. The results support the view that cytogenetic damage and apoptosis after gamma-irradiation are not necessarily correlated.

  12. NetMHCstab – predicting stability of peptide–MHC-I complexes; impacts for cytotoxic T lymphocyte epitope discovery

    PubMed Central

    Jørgensen, Kasper W; Rasmussen, Michael; Buus, Søren; Nielsen, Morten

    2014-01-01

    Major histocompatibility complex class I (MHC-I) molecules play an essential role in the cellular immune response, presenting peptides to cytotoxic T lymphocytes (CTLs) allowing the immune system to scrutinize ongoing intracellular production of proteins. In the early 1990s, immunogenicity and stability of the peptide–MHC-I (pMHC-I) complex were shown to be correlated. At that time, measuring stability was cumbersome and time consuming and only small data sets were analysed. Here, we investigate this fairly unexplored area on a large scale compared with earlier studies. A recent small-scale study demonstrated that pMHC-I complex stability was a better correlate of CTL immunogenicity than peptide–MHC-I affinity. We here extended this study and analysed a total of 5509 distinct peptide stability measurements covering 10 different HLA class I molecules. Artificial neural networks were used to construct stability predictors capable of predicting the half-life of the pMHC-I complex. These predictors were shown to predict T-cell epitopes and MHC ligands from SYFPEITHI and IEDB to form significantly more stable MHC-I complexes compared with affinity-matched non-epitopes. Combining the stability predictions with a state-of-the-art affinity predictions NetMHCcons significantly improved the performance for identification of T-cell epitopes and ligands. For the HLA alleles included in the study, we could identify distinct sub-motifs that differentiate between stable and unstable peptide binders and demonstrate that anchor positions in the N-terminal of the binding motif (primarily P2 and P3) play a critical role for the formation of stable pMHC-I complexes. A webserver implementing the method is available at http://www.cbs.dtu.dk/services/NetMHCstab. PMID:23927693

  13. Myeloid Cell COX-2 deletion reduces mammary tumor growth through enhanced cytotoxic T-lymphocyte function

    PubMed Central

    Chen, Edward P.; Markosyan, Nune; Connolly, Emma; Lawson, John A.; Li, Xuanwen; Grant, Gregory R.; Grosser, Tilo; FitzGerald, Garret A.; Smyth, Emer M.

    2014-01-01

    Cyclooxygenase-2 (COX-2) expression is associated with poor prognosis across a range of human cancers, including breast cancer. The contribution of tumor cell-derived COX-2 to tumorigenesis has been examined in numerous studies; however, the role of stromal-derived COX-2 is ill-defined. Here, we examined how COX-2 in myeloid cells, an immune cell subset that includes macrophages, influences mammary tumor progression. In mice engineered to selectively lack myeloid cell COX-2 [myeloid-COX-2 knockout (KO) mice], spontaneous neu oncogene-induced tumor onset was delayed, tumor burden reduced, and tumor growth slowed compared with wild-type (WT). Similarly, growth of neu-transformed mammary tumor cells as orthotopic tumors in immune competent syngeneic myeloid-COX-2 KO host mice was reduced compared with WT. By flow cytometric analysis, orthotopic myeloid-COX-2 KO tumors had lower tumor-associated macrophage (TAM) infiltration consistent with impaired colony stimulating factor-1-dependent chemotaxis by COX-2 deficient macrophages in vitro. Further, in both spontaneous and orthotopic tumors, COX-2-deficient TAM displayed lower immunosuppressive M2 markers and this was coincident with less suppression of CD8+ cytotoxic T lymphocytes (CTLs) in myeloid-COX-2 KO tumors. These studies suggest that reduced tumor growth in myeloid-COX-2 KO mice resulted from disruption of M2-like TAM function, thereby enhancing T-cell survival and immune surveillance. Antibody-mediated depletion of CD8+, but not CD4+ cells, restored tumor growth in myeloid-COX-2 KO to WT levels, indicating that CD8+ CTLs are dominant antitumor effectors in myeloid-COX-2 KO mice. Our studies suggest that inhibition of myeloid cell COX-2 can potentiate CTL-mediated tumor cytotoxicity and may provide a novel therapeutic approach in breast cancer therapy. PMID:24590894

  14. In vitro T lymphocyte adherence capabilities under the influence of lower induction values (0.1 - 0.01 mT) of 50 Hz external magnetic fields

    NASA Astrophysics Data System (ADS)

    Čoček, A.; Jandová, A.; Hahn, A.; Mártonová, J.; Ambruš, M.; Dohnalová, A.; Nedbalová, M.; Pokorný, J.

    2011-12-01

    Our research thus far has concerned the impact of external magnetic fields (50 Hz) and low (0.01-10 mT) induction on adherence capabilities of T lymphocytes obtained from the blood of patients with head and neck tumors. We know that the in vitro adherence capability of T lymphocytes towards surfaces in cancer patients is less than that of control. Previously, we have found that exposure to magnetic fields (50 Hz / 0.01-10 mT) increases the capability of T lymphocytes, in larynx/pharynx cancer patients, to adhere in vitro to surfaces, achieving almost physiological values, in not only pre-treatment patients but also those receiving treatment in the course of follow-up. The capability of T lymphocytes in controls (voluntary blood donors) to adhere to surfaces was also increased (50 Hz / 0.01-0.5 mT). The present study concentrates on the significance of the level of magnetic field induction in order to determine whether low induction values can restore T lymphocytes adherence capabilities. Testing a set of 20 patients showed a statistically significant difference (p < 0.05) in the in vitro adherence capacity of T lymphocytes between both 0.01 and 0.05, and 0.1 mT induction levels. In the control group (patients diagnosed with chronic sensorineural hearing loss) there was even a statistically significant difference between induction values of 0.05 and 0.01 mT. Therefore, we concluded that lower induction values resulted in a more biologically significant response.

  15. AhR activation increases IL-2 production by alloreactive CD4+ T cells initiating the differentiation of mucosal-homing Tim3+ Lag3+ Tr1 cells.

    PubMed

    Ehrlich, Allison K; Pennington, Jamie M; Tilton, Susan; Wang, Xisheng; Marshall, Nikki B; Rohlman, Diana; Funatake, Castle; Punj, Sumit; O'Donnell, Edmond; Yu, Zhen; Kolluri, Siva K; Kerkvliet, Nancy I

    2017-11-01

    Activation of the aryl hydrocarbon receptor (AhR) by immunosuppressive ligands promotes the development of regulatory T (Treg) cells. Although AhR-induced Foxp3 + Treg cells have been well studied, much less is known about the development and fate of AhR-induced Type 1 Treg (AhR-Tr1) cells. In the current study, we identified the unique transcriptional and functional changes in murine CD4 + T cells that accompany the differentiation of AhR-Tr1 cells during the CD4 + T-cell-dependent phase of an allospecific cytotoxic T lymphocyte (allo-CTL) response. AhR activation increased the expression of genes involved in T-cell activation, immune regulation and chemotaxis, as well as a global downregulation of genes involved in cell cycling.  Increased IL-2 production was responsible for the early AhR-Tr1 activation phenotype previously characterized as CD25 + CTLA4 + GITR + on day 2. The AhR-Tr1 phenotype was further defined by the coexpression of the immunoregulatory receptors Lag3 and Tim3 and non-overlapping expression of CCR4 and CCR9. Consistent with the increased expression of CCR9, real-time imaging showed enhanced migration of AhR-Tr1 cells to the lamina propria of the small intestine and colon. The discovery of mucosal imprinting of AhR-Tr1 cells provides an additional mechanism by which therapeutic AhR ligands can control immunopathology. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Use of monoclonal antibodies in a study of the development of T lymphocytes in the human fetus.

    PubMed Central

    Asma, G E; Van den Bergh, R L; Vossen, J M

    1983-01-01

    A panel of monoclonal antibodies (OKT3, 4, 6, 8, 10, 11) was used for the identification of T lymphocyte subpopulations in cell suspensions of human fetal liver, thymus, bone marrow and spleen. In liver suspensions of 8-16 week old fetuses and in bone marrow suspensions (12-20 weeks) less than 5% of lymphocytes reacted with either OKT3, 11, 4, 8 or 6, whereas the OKT10 antibody bound to, respectively, 35 and 86% of lymphocytes in these tissues. In liver suspensions of 17-20 week old fetuses, about 20% of lymphocytes carried either the T3, 11, 4 or 8 antigen and more than 60% of lymphocytes were OKT10+. The maturation stages in fetal thymus (11-20 weeks) are comparable to those in the post-natal thymus, with the exception that a substantial proportion of fetal thymocytes expresses the T3 and T6 antigen simultaneously. In the fetal spleen (12-20 weeks), 40% of lymphocytes reacts with OKT3. These OKT3+ spleen cells may be divided into two subsets expressing either the T4 antigen or the T8 antigen. These OKT3+/OKT4+ and OKT3+/OKT8+ lymphoid cells of the fetal spleen can be further subdivided into a T10+ and T10- subpopulation. These data suggest that T lymphoid precursor cells, reacting with either none of the monoclonal antibodies or only with OKT10, are generated in fetal liver (up till 16 weeks gestational age) and bone marrow. Further maturation takes place in the fetal thymus, but also to a certain extent in peripheral lymphoid organs such as the fetal spleen, as evidenced by the coexistence of a T3+/T10+ and T3+/T10- subpopulation in this organ. PMID:6349881

  17. HTLV-1 Tax-Specific CTL Epitope-Pulsed Dendritic Cell Therapy Reduces Proviral Load in Infected Rats with Immune Tolerance against Tax.

    PubMed

    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.

  18. In vitro stimulation of rabbit T lymphocytes by cells expressing herpes simplex antigens.

    PubMed

    Kapoor, A K; Ling, N R; Nash, A A; Bachan, A; Wildy, P

    1982-04-01

    Lymphocyte stimulation responses to herpes antigens were studied using virus-infected X-irradiated cells. Rabbits were immunized with herpes simplex virus type 1 (strain HFEM) grown in RK 13 cells. For in vitro stimulation assay BHK21 cells were X-irradiated (15 000 rad) and infected with a high m.o.i. of a temperature-sensitive (ts) mutant (N102) of HFEM strain at the non-permissive temperature (38.5 degrees C) of virus. Virus antigens were expressed on the infected cells and there was no leakage of infectious virus into the medium at 38.5 degrees C. T lymphocytes from rabbits immunized with herpes simplex virus were specifically activated by herpesvirus-infected X-irradiated cells; lymph node cells from rabbits immunized with RK13 cells and from non-immune rabbits showed no proliferative response.

  19. Characterization of T-lymphocytes in the anterior uvea of eyes with chronic equine recurrent uveitis.

    PubMed

    Gilger, B C; Malok, E; Cutter, K V; Stewart, T; Horohov, D W; Allen, J B

    1999-10-01

    Equine recurrent uveitis (ERU), a chronic, recurrent inflammation primarily of the anterior uveal tract, is the most common cause of blindness in horses. Recently, T-lymphocytes have been found to be the most numerous cell type to infiltrate the anterior uveal of horses with ERU. In the present study, we characterized the T-lymphocyte population in the anterior uveal tract of eyes of horses with chronic ERU by evaluating the microscopic appearance (histopathologic features), the T-lymphocyte subsets, and the relative levels and amounts of T-lymphocyte cytokine mRNA in the anterior uvea. Seven inflamed eyes (from six horses with chronic ERU) and 5 normal eyes (from five horses with nonocular problems) were studied. After clinical examination, the eyes were removed, ocular fluids were aspirated, and anterior uveal tissues (iris and ciliary body) were processed for histologic and molecular (RNA isolation) analyses. Histologic examination by hematoxylin and eosin (H and E) staining and immunohistochemistry evaluating T-lymphocyte subsets (anti-CD4, CD8, CD5) were performed for each sample. RNA samples were analyzed for levels of messenger (m) RNA specific for interleukin (IL)-2, 4, and interferon-gamma (IFNgamma) by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). Eyes with ERU exhibited characteristic clinical signs, including corneal edema, aqueous flare, posterior synechia, corpora nigra degeneration, and cataract formation. Histologically, infiltration of the uveal tract with lymphocytes, plasma cells, and macrophages was most evident in the ciliary body and base of the iris. Loss of tissue structure (destruction) was most evident in the ciliary processes. Infiltrating lymphocytes were predominantly CD4+ T-cells (e.g. 48% CD4+ and 18% CD8+ in the ciliary body stroma), as determined by immunohistochemistry. Few inflammatory cells were observed in the normal eyes. The QRT-PCR results revealed increased transcription of IL-2 and IFNgamma and low

  20. Role of T-bet, the master regulator of Th1 cells, in the cytotoxicity of murine CD4+ T cells.

    PubMed

    Eshima, Koji; Misawa, Kana; Ohashi, Chihiro; Iwabuchi, Kazuya

    2018-05-01

    Although CD4 + T cells are generally regarded as helper T cells, some activated CD4 + T cells have cytotoxic properties. Given that CD4 + cytotoxic T lymphocytes (CTLs) often secrete IFN-γ, CTL activity among CD4 + T cells may be attributable to Th1 cells, where a T-box family molecule, T-bet serves as the "master regulator". However, although the essential contribution of T-bet to expression of IFN-γ has been well-documented, it remains unclear whether T-bet is involved in CD4 + T cell-mediated cytotoxicity. In this study, to investigate the ability of T-bet to confer cytolytic activity on CD4 + T cells, the T-bet gene (Tbx21) was introduced into non-cytocidal CD4 + T cell lines and their cytolytic function analyzed. Up-regulation of FasL (CD178), which provided the transfectant with cytotoxicity, was observed in Tbx21transfected CD4 + T cells but not in untransfected parental cells. In one cell line, T-bet transduction also induced perforin gene (Prf1) expression and Tbx21 transfectants efficiently killed Fas - target cells. Although T-bet was found to repress up-regulation of CD40L (CD154), which controls FasL-mediated cytolysis, the extent of CD40L up-regulation on in vitro-differentiated Th1 cells was similar to that on Th2 cells, suggesting the existence of a compensatory mechanism. These results collectively indicate that T-bet may be involved in the expression of genes, such as FasL and Prf1, which confer cytotoxicity on Th1 cells. © 2018 The Societies and John Wiley & Sons Australia, Ltd.

  1. Role of the K(Ca)3.1 K+ channel in auricular lymph node CD4+ T-lymphocyte function of the delayed-type hypersensitivity model.

    PubMed

    Ohya, Susumu; Nakamura, Erina; Horiba, Sayuri; Kito, Hiroaki; Matsui, Miki; Yamamura, Hisao; Imaizumi, Yuji

    2013-07-01

    The intermediate-conductance Ca(2+)-activated K(+) channel (K(Ca)3.1) modulates the Ca(2+) response through the control of the membrane potential in the immune system. We investigated the role of K(Ca)3.1 on the pathogenesis of delayed-type hypersensitivity (DTH) in auricular lymph node (ALN) CD4(+) T-lymphocytes of oxazolone (Ox)-induced DTH model mice. The expression patterns of K(Ca)3.1 and its possible transcriptional regulators were compared among ALN T-lymphocytes of three groups [non-sensitized (Ox-/-), Ox-sensitized, but non-challenged (Ox+/-) and Ox-sensitized and -challenged (Ox+/+)] using real-time polymerase chain reaction, Western blotting and flow cytometry. KCa 3.1 activity was measured by whole-cell patch clamp and the voltage-sensitive dye imaging. The effects of K(Ca)3.1 blockade were examined by the administration of selective K(Ca)3.1 blockers. Significant up-regulation of K(Ca)3.1a was observed in CD4(+) T-lymphocytes of Ox+/- and Ox+/+, without any evident changes in the expression of the dominant-negative form, K(Ca)3.1b. Negatively correlated with this, the repressor element-1 silencing transcription factor (REST) was significantly down-regulated. Pharmacological blockade of K(Ca)3.1 resulted in an accumulation of the CD4(+) T-lymphocytes of Ox+/+ at the G0/G1 phase of the cell cycle, and also significantly recovered not only the pathogenesis of DTH, but also the changes in the K(Ca)3.1 expression and activity in the CD4(+) T-lymphocytes of Ox+/- and Ox+/+. The up-regulation of K(Ca)3.1a in conjunction with the down-regulation of REST may be involved in CD4(+) T-lymphocyte proliferation in the ALNs of DTH model mice; and K(Ca)3.1 may be an important target for therapeutic intervention in allergy diseases such as DTH. © 2013 The British Pharmacological Society.

  2. THE LYMPHOCYTE RESPONSE TO PRIMARY MOLONEY SARCOMA VIRUS TUMORS IN BALB/c MICE

    PubMed Central

    Lamon, E. W.; Wigzell, H.; Klein, E.; Andersson, B.; Skurzak, H. M.

    1973-01-01

    Adult BALB/c mice were injected with Moloney sarcoma virus (MSV) after which the animals' lymphocytes were examined for activity against Moloney leukemia virus (MLV) antigen-bearing target cells at 5-day intervals for 30 days. Lymphocytes from these animals and appropriately matched controls were fractionated into B cell-deficient (primarily T cells) and T cell-deficient (primarily B cells) subpopulations. Macrophages were removed using iron powder and magnetism. The unfractionated lymphocytes, T cells, and non-T cells were then tested in microcytotoxicity tests. Antigen-specific activity was found in the unfractionated lymphocytes from animals that had not yet developed palpable tumors and from regressor animals. The T cells were active just before tumor development and just after regression; however, by day 30 after virus infection (8–10 days after regression) the T cell subpopulation was much less active. The non-T cell subpopulation was also active before tumor development and soon after regression. However, this activity continued to rise after regression and was highest at 30 days. At day 15 (peak tumor size) neither subpopulation was active. The activity was demonstrated to be specific for the MLV-determined cell surface antigen by testing on control target cells that were MLV antigen negative and by comparison of the inhibitory effects with lymphocytes immune to a nonpertinent antigen as well as normal lymphocytes. The non-T cells were tested for activity before and after removal of macrophages with iron powder and magnetism. Such cells were significantly more active after removal of the macrophages. These data demonstrate specific T cell and non-T cell activity in microcytotoxicity tests with a tumor-specific system and strongly suggest that the non-T cell activity described herein is a B cell function. PMID:4709269

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

    PubMed

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

    2013-01-01

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

  4. Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development.

    PubMed

    Taniuchi, Ichiro; Osato, Motomi; Egawa, Takeshi; Sunshine, Mary Jean; Bae, Suk Chul; Komori, Toshihisa; Ito, Yoshiaki; Littman, Dan R

    2002-11-27

    T lymphocytes differentiate in discrete stages within the thymus. Immature thymocytes lacking CD4 and CD8 coreceptors differentiate into double-positive cells (CD4(+)CD8(+)), which are selected to become either CD4(+)CD8(-)helper cells or CD4(-)CD8(+) cytotoxic cells. A stage-specific transcriptional silencer regulates expression of CD4 in both immature and CD4(-)CD8(+) thymocytes. We show here that binding sites for Runt domain transcription factors are essential for CD4 silencer function at both stages, and that different Runx family members are required to fulfill unique functions at each stage. Runx1 is required for active repression in CD4(-)CD8(-) thymocytes whereas Runx3 is required for establishing epigenetic silencing in cytotoxic lineage thymocytes. Runx3-deficient cytotoxic T cells, but not helper cells, have defective responses to antigen, suggesting that Runx proteins have critical functions in lineage specification and homeostasis of CD8-lineage T lymphocytes.

  5. Improving therapy of chronic lymphocytic leukemia with chimeric antigen receptor T cells.

    PubMed

    Fraietta, Joseph A; Schwab, Robert D; Maus, Marcela V

    2016-04-01

    Adoptive cell immunotherapy for the treatment of chronic lymphocytic leukemia (CLL) has heralded a new era of synthetic biology. The infusion of genetically engineered, autologous chimeric antigen receptor (CAR) T cells directed against CD19 expressed by normal and malignant B cells represents a novel approach to cancer therapy. The results of recent clinical trials of CAR T cells in relapsed and refractory CLL have demonstrated long-term disease-free remissions, underscoring the power of harnessing and redirecting the immune system against cancer. This review will briefly summarize T-cell therapies in development for CLL disease. We discuss the role of T-cell function and phenotype, T-cell culture optimization, CAR design, and approaches to potentiate the survival and anti-tumor effects of infused lymphocytes. Future efforts will focus on improving the efficacy of CAR T cells for the treatment of CLL and incorporating adoptive cell immunotherapy into standard medical management of CLL. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. The Class III Kinase Vps34 Promotes T Lymphocyte Survival through Regulating IL-7Rα Surface Expression

    PubMed Central

    McLeod, Ian X.; Zhou, Xiang; Li, Qi-Jing; Wang, Fan; He, You-Wen

    2011-01-01

    IL-7Rα mediated signals are essential for naive T lymphocyte survival. Recent studies show that IL-7Rα is internalized and either recycled to cell surface or degraded. However, how the intracellular process of IL-7Rα trafficking is regulated is unclear. Here we show that Vps34, the class III phosphatidylinositol 3-kinase, plays a critical role in proper IL-7Rα intracellular trafficking. Mice lacking Vps34 in T lymphocytes had a severely reduced T lymphocyte compartment. Vps34-deficient T lymphocytes exhibit increased death and reduced IL-7Rα surface expression, though three major forms of autophagy remain intact. Intracellular IL-7Rα in normal T lymphocytes at steady-state is trafficked through either early endosome/multivesicular bodies (MVB) to the late endosome-Golgi for surface expression or to the lysosome for degradation. However, Vps34-deficient T cells have mislocalized intracellular Eea1, HRS, and Vps36 protein levels, the combined consequence of which is the inability to mobilize internalized IL-7Rα into the retromer pathway for surface display. Our studies reveal that Vps34, though dispensible for autophagy induction, is a critical regulator of naïve T cell homeostasis, modulating IL-7Rα trafficking, signaling, and recycling. PMID:22021616

  7. Expression and agonist responsiveness of CXCR3 variants in human T lymphocytes.

    PubMed

    Korniejewska, Anna; McKnight, Andrew J; Johnson, Zoë; Watson, Malcolm L; Ward, Stephen G

    2011-04-01

    The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 are involved in variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. Two alternatively spliced variants of the human CXCR3-A receptor have been described, termed CXCR3-B and CXCR3-alt. Human CXCR3-B binds CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4. In contrast, CXCR3-alt only binds CXCL11. We report that CXCL4 induces intracellular calcium mobilization as well as Akt and p44/p42 extracellular signal-regulated kinase phosphorylation, in activated human T lymphocytes. These responses have similar concentration dependence and time-courses to those induced by established CXCR3 agonists. Moreover, phosphorylation of Akt and p44/p42 is inhibited by pertussis toxin, suggesting coupling to Gα(i) protein. Surprisingly, and in contrast with the other CXCR3 agonists, stimulation of T lymphocytes with CXCL4 failed to elicit migratory responses and did not lead to loss of surface CXCR3 expression. Taken together, our findings show that, although CXCL4 is coupled to downstream biochemical machinery, its role in T cells is probably distinct from that of CXCR3-A agonists. © 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.

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

    PubMed

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

    2015-05-01

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

  9. [A multicenter study of correlation between peripheral lymphocyte counts and CD(+)4T cell counts in HIV/AIDS patients].

    PubMed

    Xie, Jing; Qiu, Zhifeng; Han, Yang; Li, Yanling; Song, Xiaojing; Li, Taisheng

    2015-02-01

    To evaluate the accuracy of lymphocyte count as a surrogate for CD(+)4T cell count in treatment-naїve HIV-infected adults. A total of 2 013 HIV-infected patients were screened at 23 sites in China. CD(+)4T cell counts were measured by flow cytometry. Correlation between CD(+)4T cell count and peripheral lymphocyte count were analyzed by spearman coefficient. AUCROC were used to evaluate the performance of lymphocyte count as a surrogate for CD(+)4T cell count. The lymphocyte count and CD(+)4T cell count of these 2 013 patients were (1 600 ± 670) × 10(6)/L and (244 ± 148) × 10(6)/L respectively. CD(+)4T cell count were positively correlated with lymphocyte count (r = 0.482, P < 0.000 1). AUCROC of lymphocyte count as a surrogate for CD(+)4T cell counts of <100×10(6)/L, <200×10(6)/L and <350×10(6)/L were 0.790 (95%CI 0.761-0.818, P < 0.000 1), 0.733 (95%CI 0.710-0.755, P < 0.000 1) and 0.732 (95%CI 0.706-0.758, P < 0.000 1) respectively. Lymphocyte count could be considerad as a potential surrogate marker for CD(+)4T cell count in HIV/AIDS patients not having access to T cell subset test by flowcytometry.

  10. Herpes Simplex Virus Requires VP11/12 To Induce Phosphorylation of the Activation Loop Tyrosine (Y394) of the Src Family Kinase Lck in T Lymphocytes

    PubMed Central

    Wagner, Melany J.; Smiley, James R.

    2009-01-01

    Herpes simplex virus (HSV) tegument proteins are released into the cytoplasm during viral entry and hence are among the first viral proteins encountered by an infected cell. Despite the implied importance of these proteins in the evasion of host defenses, the function of some, like virion protein 11/12 (VP11/12), have not been clearly defined. Previously, we reported that VP11/12 is strongly tyrosine phosphorylated during the infection of lymphocytes but not in fibroblasts or an epithelial cell line (G. Zahariadis, M. J. Wagner, R. C. Doepker, J. M. Maciejko, C. M. Crider, K. R. Jerome, and J. R. Smiley, J. Virol. 82:6098-6108, 2008). We also showed that tyrosine phosphorylation depends in part on the activity of the lymphocyte-specific Src family kinase (SFK) Lck in Jurkat T cells. These data suggested that VP11/12 is a substrate of Lck and that Lck is activated during HSV infection. Here, we show that HSV infection markedly increases the fraction of Lck phosphorylated on its activation loop tyrosine (Y394), a feature characteristic of activated Lck. A previous report implicated the immediate-early protein ICP0 and the viral serine/threonine kinases US3 and UL13 in the induction of a similar activated phenotype of SFKs other than Lck in fibroblasts and suggested that ICP0 interacts directly with SFKs through their SH3 domain. However, we were unable to detect an interaction between ICP0 and Lck in T lymphocytes, and we show that ICP0, US3, and UL13 are not strictly required for Lck activation. In contrast, VP11/12 interacted with Lck or Lck signaling complexes and was strictly required for Lck activation during HSV infection. Thus, VP11/12 likely modulates host cell signaling pathways for the benefit of the virus. PMID:19776125

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

    PubMed

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

    2001-03-01

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

  12. Proliferation of CD4CD25Foxp3 regulatory T lymphocytes in ex vivo expanded ascitic fluid from primary and recurrent ovarian carcinoma.

    PubMed

    Lee, Shin Wha; Kim, Yong-Man; Lee, Ha-Young; Kim, Dae-Yeon; Kim, Jong-Hyeok; Nam, Joo-Hyun; Kim, Young-Tak

    2010-03-01

    Regulatory T lymphocytes evoke the immune tolerance by suppressing and inactivating cytotoxic T lymphocytes. The objective of this study was to compare the proportion of regulatory T lymphocytes, precisely defined as CD4(+)CD25(high+)Foxp3(+) T lymphocytes, in primary and recurrent ovarian carcinoma before and after ex vivo expansion of ascites with interleukin-2 (IL-2). Ascitic fluid samples were obtained from 26 patients with ovarian carcinoma. Lymphocytes were isolated from ascites and cell markers were analyzed by flow cytometry using anti-CD3/CD4/CD8/CD16/CD56/CD25 and anti-Foxp3 antibodies. Lymphocytes were incubated for 2 to 3 weeks and expanded ex vivo by IL-2 stimulation and their phenotypes were analyzed by flow cytometry. Following ex vivo expansion, ascitic fluid lymphocytes increased by a greater extent in the recurrent group than in the primary group. The proportion of ex vivo-expanded lymphocytes changed as follows; CD4(+) T lymphocytes increased, CD8(+) T lymphocytes decreased, and the proportion of CD3(-)CD16(+)56(+) NK cells was unchanged. The proportion of CD4(+)CD25(high+)Foxp3(+) regulatory T lymphocytes in CD4(+) T lymphocytes increased after ex vivo expansion in both groups, but to a greater degree in the recurrent group. This study showed that regulatory T lymphocytes, neither cytotoxic T lymphocytes nor NK cells, were extensively increased after ex vivo expansion, especially in recurrent ovarian carcinoma. These results may provide information that helps to guide the future development of adoptive immunotherapy against ovarian carcinoma.

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

    PubMed

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

    2013-04-15

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

  14. CD8 down-regulation on cytotoxic T lymphocytes of patients with endometrioid endometrial carcinomas.

    PubMed

    Pascual-García, Mónica; Bértolo, Cristina; Nieto, Juan C; Serrat, Neus; Espinosa, Íñigo; D'Angelo, Emanuela; Muñoz, Raquel; Rovira, Ramón; Vidal, Silvia; Prat, Jaime

    2016-10-01

    Carcinogenesis is a multistep process in which cancer cells and tumor stroma cells play important roles. T lymphocytes are immune constituents of tumor stroma and play a crucial function in anti-tumor response. By immunohistochemistry and flow cytometry, we studied T cytotoxic (CTLs) and T helper lymphocyte distribution and percentage in the tumor microenvironment and peripheral blood from 35 patients with endometrioid endometrial carcinomas (EEC). We also studied 23 healthy donors' blood samples as a control group. Tumor and non-tumoral endometrium samples were obtained. Immunohistochemistry revealed a high number of CTLs and T helper lymphocytes in the tumor stroma of myoinvasive EECs. T lymphocytes were mostly located in the invasive front. By flow cytometry, the percentages of CTLs and T helper lymphocytes were significantly higher in the tumor compared with the non-neoplastic endometrium (P = .0492 and P = .002). The mean fluorescence intensity of CD8 staining was lower in the tumor compared to the non-neoplastic endometrium (P = .001). There was also reduction of the mean fluorescence intensity of CD8 staining on peripheral blood from patients with grade 3 EECs compare to the peripheral blood from healthy donors (P = .0093). No alterations in the expression of granzymes A and B were found in the CTLs from the EEC cases. Finally, in a proteome profiler cytokine array we found that the growth differentiation factor 15 (GDF15) increased in blood in parallel to the tumor grade. EECs are capable of down-regulating CD8 expression of CTLs. Most likely, this effect is mediated by a soluble molecule present in plasma and is not a result of anergy or exhaustion state. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2016-01-01

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

  16. Trans-dissemination of exosomes from HIV-1-infected cells fosters both HIV-1 trans-infection in resting CD4+ T lymphocytes and reactivation of the HIV-1 reservoir.

    PubMed

    Chiozzini, Chiara; Arenaccio, Claudia; Olivetta, Eleonora; Anticoli, Simona; Manfredi, Francesco; Ferrantelli, Flavia; d'Ettorre, Gabriella; Schietroma, Ivan; Andreotti, Mauro; Federico, Maurizio

    2017-09-01

    Intact HIV-1 and exosomes can be internalized by dendritic cells (DCs) through a common pathway leading to their transmission to CD4 + T lymphocytes by means of mechanisms defined as trans-infection and trans-dissemination, respectively. We previously reported that exosomes from HIV-1-infected cells activate both uninfected quiescent CD4 + T lymphocytes, which become permissive to HIV-1, and latently infected cells, with release of HIV-1 particles. However, nothing is known about the effects of trans-dissemination of exosomes produced by HIV-1-infected cells on uninfected or latently HIV-1-infected CD4 + T lymphocytes. Here, we report that trans-dissemination of exosomes from HIV-1-infected cells induces cell activation in resting CD4 + T lymphocytes, which appears stronger with mature than immature DCs. Using purified preparations of both HIV-1 and exosomes, we observed that mDC-mediated trans-dissemination of exosomes from HIV-1-infected cells to resting CD4 + T lymphocytes induces efficient trans-infection and HIV-1 expression in target cells. Most relevant, when both mDCs and CD4 + T lymphocytes were isolated from combination anti-retroviral therapy (ART)-treated HIV-1-infected patients, trans-dissemination of exosomes from HIV-1-infected cells led to HIV-1 reactivation from the viral reservoir. In sum, our data suggest a role of exosome trans-dissemination in both HIV-1 spread in the infected host and reactivation of the HIV-1 reservoir.

  17. T lymphocyte-derived TNF and IFN-γ repress HFE expression in cancer cells.

    PubMed

    Reuben, Alexandre; Godin-Ethier, Jessica; Santos, Manuela M; Lapointe, Réjean

    2015-06-01

    The immune system and tumors are closely intertwined initially upon tumor development. During this period, tumors evolve to promote self-survival through immune escape, including by targeting crucial components involved in the presentation of antigens to the immune system in order to avoid recognition. Accordingly, components involved in MHC I presentation of tumor antigens are often mutated and down-regulated targets in tumors. On the other hand, the immune system has been shown to influence tumors through production of immunosuppressive cytokines, recruitment and polarization of cells favoring or impeding tumor escape or through production of anti-tumor cytokines promoting tumor rejection. We previously discovered that the hemochromatosis protein HFE, a negative regulator of iron absorption, dampens classical MHC I antigen presentation. In this study, we evaluated the impact of activated T lymphocytes purified from peripheral blood mononuclear cells (PBMC) on HFE expression in tumor cell lines. We co-cultured tumor cell lines from melanoma, lung, and kidney cancers with anti-CD3-activated PBMC and established that HFE expression is increased in tumor cell lines compared to healthy tissues, whilst being down-regulated significantly upon exposure to activated PBMC. HFE down-regulation was mediated by both CD4 and CD8 T lymphocytes, through production of soluble mediators, namely TNF and IFN-γ. These results suggest that the immune system may modulate tumor HFE expression in inflammatory conditions in order to regulate MHC I antigen presentation and promote tumor clearance. Copyright © 2015. Published by Elsevier Ltd.

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

    PubMed

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

    2006-08-01

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

  19. Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

    PubMed Central

    Assadian, Farzaneh; Sandström, Karl; Laurell, Göran; Svensson, Catharina; Akusjärvi, Göran; Punga, Tanel

    2015-01-01

    Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term “tonsils” refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses. PMID:26650582

  20. Boosting antitumor responses of T lymphocytes infiltrating human prostate cancers

    PubMed Central

    Bronte, Vincenzo; Kasic, Tihana; Gri, Giorgia; Gallana, Keti; Borsellino, Giovanna; Marigo, Ilaria; Battistini, Luca; Iafrate, Massimo; Prayer-Galetti, Tommaso; Pagano, Francesco; Viola, Antonella

    2005-01-01

    Immunotherapy may provide valid alternative therapy for patients with hormone-refractory metastatic prostate cancer. However, if the tumor environment exerts a suppressive action on antigen-specific tumor-infiltrating lymphocytes (TIL), immunotherapy will achieve little, if any, success. In this study, we analyzed the modulation of TIL responses by the tumor environment using collagen gel matrix–supported organ cultures of human prostate carcinomas. Our results indicate that human prostatic adenocarcinomas are infiltrated by terminally differentiated cytotoxic T lymphocytes that are, however, in an unresponsive status. We demonstrate the presence of high levels of nitrotyrosines in prostatic TIL, suggesting a local production of peroxynitrites. By inhibiting the activity of arginase and nitric oxide synthase, key enzymes of L-arginine metabolism that are highly expressed in malignant but not in normal prostates, reduced tyrosine nitration and restoration of TIL responsiveness to tumor were achieved. The metabolic control exerted by the tumor on TIL function was confirmed in a transgenic mouse prostate model, which exhibits similarities with human prostate cancer. These results identify a novel and dominant mechanism by which cancers induce immunosuppression in situ and suggest novel strategies for tumor immunotherapy. PMID:15824085