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Sample records for cd8 epitope display

  1. Dengue virus-reactive CD8+ T cells display quantitative and qualitative differences in their response to variant epitopes of heterologous viral serotypes.

    PubMed

    Bashyam, Hema S; Green, Sharone; Rothman, Alan L

    2006-03-01

    Reactivation of serotype cross-reactive CD8+ memory T lymphocytes is thought to contribute to the immunopathogenesis of dengue disease during secondary infection by a heterologous serotype. Using cytokine flow cytometry, we have defined four novel HLA-A*02-restricted dengue viral epitopes recognized by up to 1.5% of circulating CD8+ T cells in four donors after primary vaccination. All four donors had the highest cytokine response to the epitope NS4b 2353. We also studied the effect of sequence differences in heterologous dengue serotypes on dengue-reactive CD8+ memory T cell cytokine and proliferative responses. The D3 variant of a different NS4b epitope 2423 and the D2 variant of the NS4a epitope 2148 induced the largest cytokine response, compared with their respective heterologous sequences in all donors regardless of the primary vaccination serotype. Stimulation with variant peptides also altered the relative frequencies of the various subsets of cells that expressed IFN-gamma, TNF-alpha, MIP-1beta, and combinations of these cytokines. These results indicate that the prior infection history of the individual as well as the serotypes of the primary and heterologous secondary viruses influence the nature of the secondary response. These differences in the effector functions of serotype cross-reactive memory T cells induced by heterologous variant epitopes, which are both quantitative and qualitative, may contribute to the clinical outcome of secondary dengue infection.

  2. CD8 epitope escape and reversion in acute HCV infection.

    PubMed

    Timm, Joerg; Lauer, Georg M; Kavanagh, Daniel G; Sheridan, Isabelle; Kim, Arthur Y; Lucas, Michaela; Pillay, Thillagavathie; Ouchi, Kei; Reyor, Laura L; Schulze zur Wiesch, Julian; Gandhi, Rajesh T; Chung, Raymond T; Bhardwaj, Nina; Klenerman, Paul; Walker, Bruce D; Allen, Todd M

    2004-12-20

    In the setting of acute hepatitis C virus (HCV) infection, robust HCV-specific CD8+ cytotoxic T lymphocyte (CTL) responses are associated with initial control of viremia. Despite these responses, 70-80% of individuals develop persistent infection. Although viral escape from CD8 responses has been illustrated in the chimpanzee model of HCV infection, the effect of CD8 selection pressure on viral evolution and containment in acute HCV infection in humans remains unclear. Here, we examined viral evolution in an immunodominant human histocompatibility leukocyte antigen (HLA)-B8-restricted NS3 epitope in subjects with acute HCV infection. Development of mutations within the epitope coincided with loss of strong ex vivo tetramer and interferon gamma enzyme-linked immunospot responses, and endogenous expression of variant NS3 sequences suggested that the selected mutations altered processing and presentation of the variant epitope. Analysis of NS3 sequences from 30 additional chronic HCV-infected subjects revealed a strong association between sequence variation within this region and expression of HLA-B8, supporting reproducible allele-specific selection pressures at the population level. Interestingly, transmission of an HLA-B8-associated escape mutation to an HLA-B8 negative subject resulted in rapid reversion of the mutation. Together, these data indicate that viral escape from CD8+ T cell responses occurs during human HCV infection and that acute immune selection pressure is of sufficient magnitude to influence HCV evolution.

  3. Dengue virus-infected human dendritic cells reveal hierarchies of naturally expressed novel NS3 CD8 T cell epitopes.

    PubMed

    Piazza, P; Campbell, D; Marques, E; Hildebrand, W H; Buchli, R; Mailliard, R; Rinaldo, C R

    2014-09-01

    Detailed knowledge of dengue virus (DENV) cell-mediated immunity is limited. In this study we characterize CD8(+) T lymphocytes recognizing three novel and two known non-structural protein 3 peptide epitopes in DENV-infected dendritic cells. Three epitopes displayed high conservation (75-100%), compared to the others (0-50%). A hierarchy ranking based on magnitude and polyfunctionality of the antigen-specific response showed that dominant epitopes were both highly conserved and cross-reactive against multiple DENV serotypes. These results are relevant to DENV pathogenesis and vaccine design.

  4. Novel CD8(+) cytotoxic T cell epitopes in bovine leukemia virus with cattle.

    PubMed

    Bai, Lanlan; Takeshima, Shin-Nosuke; Isogai, Emiko; Kohara, Junko; Aida, Yoko

    2015-12-16

    Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T cell leukemia virus (HTLV). The cytotoxic T lymphocyte (CTL) plays a key role in suppressing the progression of disease caused by BLV. T and B cell epitopes in BLV have been studied, but CD8(+) CTL epitopes remain poorly understood. We used a library of 115 synthetic peptides covering the entirety of the Env proteins (gp51 and gp30), the Gag proteins (p15, p24, and p12), and the Tax protein of BLV to identify 11 novel CD8(+) T cell epitopes (gp51N5, gp51N11, gp51N12, gp30N5, gp30N6, gp30N8, gp30N16, tax16, tax18, tax19, and tax20) in four calves experimentally infected with BLV. The number of CD8(+) T cell epitopes that could be identified in each calf correlated with the BLV proviral load. Interestingly, among the 11 epitopes identified, only gp51N11 was capable of inducing CD8(+) T cell-mediated cytotoxicity in all four calves, but it is not a suitable vaccine target because it shows a high degree of polymorphism according to the Wu-Kabat variability index. By contrast, no CTL epitopes were identified from the Gag structural protein. In addition, several epitopes were obtained from gp30 and Tax, indicating that cellular immunity against BLV is strongly targeted to these proteins. CD8(+) CTL epitopes from gp30 and Tax were less polymorphic than epitopes from. Indeed, peptides tax16, tax18, tax19, and tax20 include a leucine-rich activation domain that encompasses a transcriptional activation domain, and the gp30N16 peptide contains a proline-rich region that interacts with a protein tyrosine phosphatase SHP1 to regulate B cell activation. Moreover, at least one CD8(+) CTL epitope derived from gp30 was identified in each of the four calves. These results indicate that BLV gp30 may be the best candidate for the development of a BLV vaccine. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. HLA-B*35-Restricted CD8+-T-Cell Epitope in Mycobacterium tuberculosis Rv2903c

    PubMed Central

    Klein, Michèl R.; Hammond, Abdulrahman S.; Smith, Steve M.; Jaye, Assan; Lukey, Pauline T.; McAdam, Keith P. W. J.

    2002-01-01

    Few human CD8+ T-cell epitopes in mycobacterial antigens have been described to date. Here we have identified a novel HLA-B*35-restricted CD8+ T-cell epitope in Mycobacterium tuberculosis Rv2903c based on a reverse immunogenetics approach. Peptide-specific CD8 T cells were able to kill M. tuberculosis-infected macrophages and produce gamma interferon and tumor necrosis factor alpha. PMID:11796635

  6. Chemical Modification of Influenza CD8+ T-Cell Epitopes Enhances Their Immunogenicity Regardless of Immunodominance

    PubMed Central

    van Beek, Josine; Hoppes, Rieuwert; Jacobi, Ronald H. J.; Hendriks, Marion; Kapteijn, Kim; Ouwerkerk, Casper; Rodenko, Boris; Ovaa, Huib; de Jonge, Jørgen

    2016-01-01

    T cells are essential players in the defense against infection. By targeting the MHC class I antigen-presenting pathway with peptide-based vaccines, antigen-specific T cells can be induced. However, low immunogenicity of peptides poses a challenge. Here, we set out to increase immunogenicity of influenza-specific CD8+ T cell epitopes. By substituting amino acids in wild type sequences with non-proteogenic amino acids, affinity for MHC can be increased, which may ultimately enhance cytotoxic CD8+ T cell responses. Since preventive vaccines against viruses should induce a broad immune response, we used this method to optimize influenza-specific epitopes of varying dominance. For this purpose, HLA-A*0201 epitopes GILGFVFTL, FMYSDFHFI and NMLSTVLGV were selected in order of decreasing MHC-affinity and dominance. For all epitopes, we designed chemically enhanced altered peptide ligands (CPLs) that exhibited greater binding affinity than their WT counterparts; even binding scores of the high affinity GILGFVFTL epitope could be improved. When HLA-A*0201 transgenic mice were vaccinated with selected CPLs, at least 2 out of 4 CPLs of each epitope showed an increase in IFN-γ responses of splenocytes. Moreover, modification of the low affinity epitope NMLSTVLGV led to an increase in the number of mice that responded. By optimizing three additional influenza epitopes specific for HLA-A*0301, we show that this strategy can be extended to other alleles. Thus, enhancing binding affinity of peptides provides a valuable tool to improve the immunogenicity and range of preventive T cell-targeted peptide vaccines. PMID:27333291

  7. Chemical Modification of Influenza CD8+ T-Cell Epitopes Enhances Their Immunogenicity Regardless of Immunodominance.

    PubMed

    Rosendahl Huber, Sietske K; Luimstra, Jolien J; van Beek, Josine; Hoppes, Rieuwert; Jacobi, Ronald H J; Hendriks, Marion; Kapteijn, Kim; Ouwerkerk, Casper; Rodenko, Boris; Ovaa, Huib; de Jonge, Jørgen

    2016-01-01

    T cells are essential players in the defense against infection. By targeting the MHC class I antigen-presenting pathway with peptide-based vaccines, antigen-specific T cells can be induced. However, low immunogenicity of peptides poses a challenge. Here, we set out to increase immunogenicity of influenza-specific CD8+ T cell epitopes. By substituting amino acids in wild type sequences with non-proteogenic amino acids, affinity for MHC can be increased, which may ultimately enhance cytotoxic CD8+ T cell responses. Since preventive vaccines against viruses should induce a broad immune response, we used this method to optimize influenza-specific epitopes of varying dominance. For this purpose, HLA-A*0201 epitopes GILGFVFTL, FMYSDFHFI and NMLSTVLGV were selected in order of decreasing MHC-affinity and dominance. For all epitopes, we designed chemically enhanced altered peptide ligands (CPLs) that exhibited greater binding affinity than their WT counterparts; even binding scores of the high affinity GILGFVFTL epitope could be improved. When HLA-A*0201 transgenic mice were vaccinated with selected CPLs, at least 2 out of 4 CPLs of each epitope showed an increase in IFN-γ responses of splenocytes. Moreover, modification of the low affinity epitope NMLSTVLGV led to an increase in the number of mice that responded. By optimizing three additional influenza epitopes specific for HLA-A*0301, we show that this strategy can be extended to other alleles. Thus, enhancing binding affinity of peptides provides a valuable tool to improve the immunogenicity and range of preventive T cell-targeted peptide vaccines.

  8. Dynamics of Haplotype Frequency Change in a CD8+TL Epitope of Simian Immunodeficiency Virus

    PubMed Central

    Hughes, Austin L.; O’Connor, Shelby; Dudley, Dawn M.; Burwitz, Benjamin J.; Bimber, Benjamin N.; O’Connor, David

    2010-01-01

    Deep pyrosequencing of a CD8+TL epitope from the Tat protein of simian immunodeficiency virus (SIV) from four infected rhesus macaques carrying the restricting MHC allele (Mamu-A*01) for that epitope, revealed that natural selection favoring escape mutations led to an increase in the frequency of haplotypes in the epitope region that differed from the inoculum. After 20 weeks of infection, a new sequence haplotype in the epitope region had increased to a frequency greater than 50% in each of the four monkeys (range 57.9%–98.9%); but the predominant haplotype was not the same in all four monkeys. Thus, even under strong selection favoring escape from CD8+TL recognition, the random nature of mutation itself is the primary factor affecting which escape mutation is likely to become predominant within an individual host. The relationship between the frequency of the inoculum haplotype in the epitope region and time post infection approximated a simple hyperbola. On this assumption, the expected ratio of the frequencies at the inoculum at two times t1 and t2, fi (t2)/fi (t1), will be given by t1 / t2. Because standard phylogenetic methods for reconstructing ancestral sequences failed to predict the inoculum sequence correctly, we used this relationship to predict the inoculum sequence with 100% accuracy, given data on haplotype frequencies at different time periods. PMID:20149896

  9. Perturbed CD8(+) T cell immunity across universal influenza epitopes in the elderly.

    PubMed

    Nguyen, Thi H O; Sant, Sneha; Bird, Nicola L; Grant, Emma J; Clemens, E Bridie; Koutsakos, Marios; Valkenburg, Sophie A; Gras, Stephanie; Lappas, Martha; Jaworowski, Anthony; Crowe, Jane; Loh, Liyen; Kedzierska, Katherine

    2017-09-19

    Influenza epidemics lead to severe illness, life-threatening complications, and deaths, especially in the elderly. As CD8(+) T cells are associated with rapid recovery from influenza, we investigated the effects of aging on antigen-specific CD8(+) T cells across the universal influenza epitopes in humans. We show that aging is characterized by altered frequencies in T cell subsets, with naive T cells being partially replaced by activated effector/memory populations. Although we observed no striking differences in TCR signaling capacity, T cells in the elderly had increased expression of transcription factors Eomes and T-bet, and such changes were most apparent in CD8(+) T cells. Strikingly, the numbers of antigen-specific CD8(+) T cells across universal influenza epitopes were reduced in the elderly, although their effector/memory phenotypes remained stable. To understand whether diminished numbers of influenza-specific CD8(+) T cells in the elderly resulted from alteration in TCR clonotypes, we dissected the TCRαβ repertoire specific for the prominent HLA-A*02:01-restricted-M158-66 (A2/M158) influenza epitope. We provide the first ex vivo data on paired antigen-specific TCRαβ clonotypes in the elderly, showing that influenza-specific A2/M158(+) TCRαβ repertoires in the elderly adults varied from those in younger adults, with the main features being a reduction in the frequency of the public TRAV27-TRBV19 TCRαβ clonotype, increased proportion of private TCRαβ signatures, broader use of TRAV and TRBV gene segments, and large clonal expansion of private TCRαβ clonotypes with longer CDR3 loops. Our study supports the development of T cell-targeted influenza vaccines that would boost the T cell compartment during life and maintain the numbers and optimal TCRαβ signatures in the elderly. © Society for Leukocyte Biology.

  10. Parasite stage-specific recognition of endogenous Toxoplasma gondii derived CD8+ T cell epitopes

    PubMed Central

    Frickel, Eva-Maria; Sahoo, Nivedita; Hopp, Johnathan; Gubbels, Marc-Jan; Craver, Mary Patricia J.; Knoll, Laura J.; Ploegh, Hidde L.; Grotenbreg, Gijsbert M.

    2016-01-01

    Background BALB/c mice control infection with the obligate intracellular parasite Toxoplasma gondii and develop a latent chronic infection in the brain, as do immunocompetent humans. IFN-γ producing CD8+ T cells provide essential protection against T. gondii, but the epitopes recognized have so far remained elusive. Methods We employed caged MHC molecules to generate ~ 250 H-2Ld tetramers and distinguish T. gondii-specific CD8+ T cells in BALB/c mice. Results We identify two T. gondii specific H-2Ld-restricted T cell epitopes, one from dense granule protein GRA4 and the other from rhoptry protein ROP7. H-2Ld/GRA4 reactive T cells from multiple organ sources predominate 2 weeks after infection, while the reactivity of the H-2Ld/ROP7 T cells peaks 6–8 weeks after infection. BALB/c animals infected with T. gondii mutants defective in establishing a chronic infection show altered levels of antigen-specific T cells, depending on the T. gondii mutant used. Conclusions Our results shed light on the identity and the parasite stage-specificity of two CD8+ T cell epitopes recognized in the acute and chronic phase of infection with T. gondii. PMID:18922097

  11. Parasite stage-specific recognition of endogenous Toxoplasma gondii-derived CD8+ T cell epitopes.

    PubMed

    Frickel, Eva-Maria; Sahoo, Nivedita; Hopp, Johnathan; Gubbels, Marc-Jan; Craver, Mary Patricia J; Knoll, Laura J; Ploegh, Hidde L; Grotenbreg, Gijsbert M

    2008-12-01

    BALB/c mice control infection with the obligate intracellular parasite Toxoplasma gondii and develop a latent chronic infection in the brain, as do immunocompetent humans. Interferon-gamma-producing CD8+ T cells provide essential protection against T. gondii infection, but the epitopes recognized have so far remained elusive. We employed caged major histocompatibility complex molecules to generate approximately 250 H-2L(d) tetramers and to distinguish T. gondii-specific CD8+ T cells in BALB/c mice. We identified 2 T. gondii-specific H-2L(d)-restricted T cell epitopes, one from dense granule protein GRA4 and the other from rhoptry protein ROP7. H-2L(d)/GRA4 reactive T cells from multiple organ sources predominated 2 weeks after infection, while the reactivity of the H-2L(d)/ROP7 T cells peaked 6-8 weeks after infection. BALB/c animals infected with T. gondii mutants defective in establishing a chronic infection showed altered levels of antigen-specific T cells, depending on the T. gondii mutant used. Our results shed light on the identity and the parasite stage-specificity of 2 CD8+ T cell epitopes recognized in the acute and chronic phase of infection with T. gondii.

  12. Mycobacterium tuberculosis genome-wide screen exposes multiple CD8+ T cell epitopes

    PubMed Central

    Hammond, A S; Klein, M R; Corrah, T; Fox, A; Jaye, A; McAdam, K P; Brookes, R H

    2005-01-01

    Mounting evidence suggests human leucocyte antigen (HLA) class I-restricted CD8+ T cells play a role in protective immunity against tuberculosis yet relatively few epitopes specific for the causative organism, Mycobacterium tuberculosis, are reported. Here a total genome-wide screen of M. tuberculosis was used to identify putative HLA-B*3501 T cell epitopes. Of 479 predicted epitopes, 13 with the highest score were synthesized and used to restimulate lymphocytes from naturally exposed HLA-B*3501 healthy individuals in cultured and ex vivo enzyme-linked immunospot (ELISPOT) assays for interferon (IFN)-γ. All 13 peptides elicited a response that varied considerably between individuals. For three peptides CD8+ T cell lines were expanded and four of the 13 were recognized permissively through the HLA-B7 supertype family. Although further testing is required we show the genome-wide screen to be feasible for the identification of unknown mycobacterial antigens involved in immunity against natural infection. While the mechanisms of protective immunity against M. tuberculosis infection remain unclear, conventional class I-restricted CD8+ T cell responses appear to be widespread throughout the genome. PMID:15762882

  13. TCR contact residue hydrophobicity is a hallmark of immunogenic CD8+ T cell epitopes

    PubMed Central

    Chowell, Diego; Krishna, Sri; Cocita, Clément; Shu, Jack; Tan, Xuefang; Greenberg, Philip D.; Klavinskis, Linda S.; Blattman, Joseph N.; Anderson, Karen S.

    2015-01-01

    Despite the availability of major histocompatibility complex (MHC)-binding peptide prediction algorithms, the development of T-cell vaccines against pathogen and tumor antigens remains challenged by inefficient identification of immunogenic epitopes. CD8+ T cells must distinguish immunogenic epitopes from nonimmunogenic self peptides to respond effectively against an antigen without endangering the viability of the host. Because this discrimination is fundamental to our understanding of immune recognition and critical for rational vaccine design, we interrogated the biochemical properties of 9,888 MHC class I peptides. We identified a strong bias toward hydrophobic amino acids at T-cell receptor contact residues within immunogenic epitopes of MHC allomorphs, which permitted us to develop and train a hydrophobicity-based artificial neural network (ANN-Hydro) to predict immunogenic epitopes. The immunogenicity model was validated in a blinded in vivo overlapping epitope discovery study of 364 peptides from three HIV-1 Gag protein variants. Applying the ANN-Hydro model on existing peptide-MHC algorithms consistently reduced the number of candidate peptides across multiple antigens and may provide a correlate with immunodominance. Hydrophobicity of TCR contact residues is a hallmark of immunogenic epitopes and marks a step toward eliminating the need for empirical epitope testing for vaccine development. PMID:25831525

  14. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

    PubMed

    Shorter, Shayla K; Schnell, Frederick J; McMaster, Sean R; Pinelli, David F; Andargachew, Rakieb; Evavold, Brian D

    2016-01-01

    T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

  15. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses

    PubMed Central

    Shorter, Shayla K.; Schnell, Frederick J.; McMaster, Sean R.; Pinelli, David F.; Andargachew, Rakieb; Evavold, Brian D.

    2016-01-01

    T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant. PMID:26915099

  16. Subdominant CD8 T-cell epitopes account for protection against cytomegalovirus independent of immunodomination.

    PubMed

    Holtappels, Rafaela; Simon, Christian O; Munks, Michael W; Thomas, Doris; Deegen, Petra; Kühnapfel, Birgit; Däubner, Torsten; Emde, Simone F; Podlech, Jürgen; Grzimek, Natascha K A; Oehrlein-Karpi, Silke A; Hill, Ann B; Reddehase, Matthias J

    2008-06-01

    Cytomegalovirus (CMV) infection continues to be a complication in recipients of hematopoietic stem cell transplantation (HSCT). Preexisting donor immunity is recognized as a favorable prognostic factor for the reconstitution of protective antiviral immunity mediated primarily by CD8 T cells. Furthermore, adoptive transfer of CMV-specific memory CD8 T (CD8-T(M)) cells is a therapeutic option for preventing CMV disease in HSCT recipients. Given the different CMV infection histories of donor and recipient, a problem may arise from an antigenic mismatch between the CMV variant that has primed donor immunity and the CMV variant acquired by the recipient. Here, we have used the BALB/c mouse model of CMV infection in the immunocompromised host to evaluate the importance of donor-recipient CMV matching in immundominant epitopes (IDEs). For this, we generated the murine CMV (mCMV) recombinant virus mCMV-DeltaIDE, in which the two memory repertoire IDEs, the IE1-derived peptide 168-YPHFMPTNL-176 presented by the major histocompatibility complex class I (MHC-I) molecule L(d) and the m164-derived peptide 257-AGPPRYSRI-265 presented by the MHC-I molecule D(d), are both functionally deleted. Upon adoptive transfer, polyclonal donor CD8-T(M) cells primed by mCMV-DeltaIDE and the corresponding revertant virus mCMV-revDeltaIDE controlled infection of immunocompromised recipients with comparable efficacy and regardless of whether or not IDEs were presented in the recipients. Importantly, CD8-T(M) cells primed under conditions of immunodomination by IDEs protected recipients in which IDEs were absent. This shows that protection does not depend on compensatory expansion of non-IDE-specific CD8-T(M) cells liberated from immunodomination by the deletion of IDEs. We conclude that protection is, rather, based on the collective antiviral potential of non-IDEs independent of the presence or absence of IDE-mediated immunodomination.

  17. Identification of Zika virus epitopes reveals immunodominant and protective roles for dengue virus cross-reactive CD8(+) T cells.

    PubMed

    Wen, Jinsheng; Tang, William Weihao; Sheets, Nicholas; Ellison, Julia; Sette, Alessandro; Kim, Kenneth; Shresta, Sujan

    2017-03-13

    CD8(+) T cells play an important role in controlling Flavivirus infection, including Zika virus (ZIKV). Here, we have identified 25 HLA-B*0702-restricted epitopes and 1 HLA-A*0101-restricted epitope using interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICS) in ZIKV-infected IFN-α/β receptor-deficient HLA transgenic mice. The cross-reactivity of ZIKV epitopes to dengue virus (DENV) was tested using IFN-γ-ELISPOT and IFN-γ-ICS on CD8(+) T cells from DENV-infected mice, and five cross-reactive HLA-B*0702-binding peptides were identified by both assays. ZIKV/DENV cross-reactive CD8(+) T cells in DENV-immune mice expanded post ZIKV challenge and dominated in the subsequent CD8(+) T cell response. ZIKV challenge following immunization of mice with ZIKV-specific and ZIKV/DENV cross-reactive epitopes elicited CD8(+) T cell responses that reduced infectious ZIKV levels, and CD8(+) T cell depletions confirmed that CD8(+) T cells mediated this protection. These results identify ZIKV-specific and ZIKV/DENV cross-reactive epitopes and demonstrate both an altered immunodominance pattern in the DENV-immune setting relative to naive, as well as a protective role for epitope-specific CD8(+) T cells against ZIKV. These results have important implications for ZIKV vaccine development and provide a mouse model for evaluating anti-ZIKV CD8(+) T cell responses of human relevance.

  18. Apoptotic Epitope-Specific CD8+ T Cells and Interferon Signaling Intersect in Chronic Hepatitis C Virus Infection.

    PubMed

    Martini, Helene; Citro, Alessandra; Martire, Carmela; D'Ettorre, Gabriella; Labbadia, Giancarlo; Accapezzato, Daniele; Piconese, Silvia; De Marzio, Paolo; Cavallari, Eugenio N; Calvo, Ludovica; Rizzo, Fabiana; Severa, Martina; Coccia, Eliana M; Grazi, Gian Luca; Di Filippo, Simona; Sidney, John; Vullo, Vincenzo; Sette, Alessandro; Barnaba, Vincenzo

    2016-02-15

    CD8(+) T cells specific to caspase-cleaved antigens derived from apoptotic T cells represent a principal player in chronic immune activation. Here, we found that both apoptotic epitope-specific and hepatitis C virus (HCV)-specific CD8(+) T cells were mostly confined within the effector memory (EM) or terminally differentiated EM CD45RA(+) cell subsets expressing a dysfunctional T-helper 1-like signature program in chronic HCV infection. However, apoptotic epitope-specific CD8(+) T cells produced tumor necrosis factor α and interleukin 2 at the intrahepatic level significantly more than HCV-specific CD8(+) T cells, despite both populations expressing high levels of programmed death 1 receptor. Contextually, only apoptotic epitope-specific CD8(+) T cells correlated with both interferon-stimulated gene levels in T cells and hepatic fibrosis score. Together, these data suggest that, compared with HCV-specific CD8(+) T cells, apoptotic epitope-specific CD8(+) T cells can better sustain chronic immune activation, owing to their capacity to produce tumor necrosis factor α, and exhibit greater resistance to inhibitory signals during chronic HCV infection.

  19. Subdominant/Cryptic CD8 T Cell Epitopes Contribute to Resistance against Experimental Infection with a Human Protozoan Parasite

    PubMed Central

    Dominguez, Mariana R.; Silveira, Eduardo L. V.; de Vasconcelos, José Ronnie C.; de Alencar, Bruna C. G.; Machado, Alexandre V.; Bruna-Romero, Oscar; Gazzinelli, Ricardo T.; Rodrigues, Mauricio M.

    2011-01-01

    During adaptive immune response, pathogen-specific CD8+ T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8+ T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8+ T cells of H-2a infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8+ T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8+ T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development. PMID:21779365

  20. Simian Immunodeficiency Virus-Specific CD8+ T Cells Recognize Vpr- and Rev-Derived Epitopes Early after Infection ▿

    PubMed Central

    Sacha, Jonah B.; Buechler, Matthew B.; Newman, Laura P.; Reed, Jason; Wallace, Lyle T.; Loffredo, John T.; Wilson, Nancy A.; Watkins, David I.

    2010-01-01

    The kinetics of CD8+ T cell epitope presentation contribute to the antiviral efficacy of these cells yet remain poorly defined. Here, we demonstrate presentation of virion-derived Vpr peptide epitopes early after viral penetration and prior to presentation of Vif-derived epitopes, which required de novo Vif synthesis. Two Rev epitopes exhibited differential presentation kinetics, with one Rev epitope presented within 1 h of infection. We also demonstrate that cytolytic activity mirrors the recognition kinetics of infected cells. These studies show for the first time that Vpr- and Rev-specific CD8+ T cells recognize and kill simian immunodeficiency virus (SIV)-infected CD4+ T cells early after SIV infection. PMID:20686015

  1. Identification and immunogenicity of two new HLA-A*0201-restricted CD8+ T-cell epitopes on dengue NS1 protein.

    PubMed

    Tian, Jiang; Zeng, Gucheng; Pang, Xianwu; Liang, Mifang; Zhou, Junmei; Fang, Danyun; Liu, Yan; Li, Dexin; Jiang, Lifang

    2012-04-01

    Immunopathogenesis of dengue virus (DEN) infection remains poorly studied. Identification and characterization of human CD8(+) T-cell epitopes on DEN are necessary for a better understanding of the immunopathogenesis of dengue infection and would facilitate the development of immunotherapy and vaccines to protect from dengue infection. Here, we identified two new HLA-A*0201-restricted CD8(+) T-cell epitopes, DEN-4 NS1(990)(-998) and DEN-4 NS1(997)(-1005) that are conserved in three or four major DEN serotypes, respectively. Unexpectedly, we found that immunization of HLA-A*0201 transgenic mice with DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005) epitope peptide induced de novo synthesis of tumor necrosis factor (TNF)-α and IFN-γ, two important pro-inflammatory molecules that are hard to be detected directly without in vitro antigenic re-stimulation. Importantly, we demonstrated that CD8(+) T cells specifically activated by DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005) epitope peptide induced de novo synthesis of perforin. Furthermore, we observed that DEN-4 NS1(990)(-998) or DEN-4 NS1(997)(-1005)-specific CD8(+) T cells capable of producing large amounts of perforin, TNF-α and IFN-γ preferentially displayed CD27(+)CD45RA(-), but not CD27(-)CD45RA(+), phenotypes. This study, therefore, suggested the importance of synergistic effects of pro-inflammatory cytokines and cytotoxic molecules which were produced by dengue-specific CD8(+) T cells in immunopathogenesis or anti-dengue immunity during dengue infection.

  2. Identification of a dengue virus-specific HLA-A*0201-restricted CD8+ T cell epitope.

    PubMed

    Wen, Jinsheng; Duan, Zhiliang; Jiang, Lifang

    2010-04-01

    In this study, a combination of epitope-prediction programs and in vitro assays was used to identify dengue virus (DENV)-specific CD8(+) T cell epitopes. Peripheral blood mononuclear cells (PBMCs) isolated from patients who recovered from dengue fever were stimulated with candidate epitope peptides derived from DENV, which were predicted by using SYFPEITHI and RANKpep epitope-prediction programs. The IFN-gamma ELISpot results and the results of intracellular staining of IFN-gamma showed that peptides NS4b_40 (TLYAVATTI), E_256 (QEGAMHTAL), NS3_205 (LPAIVREAI), NS5_210 (SRNSTHEMY), and NS3_207 (AIVREAIKR) could induce the recall response of CD8(+) T cells. Furthermore, the results of the MHC-peptide complex stabilization assay revealed that peptide NS4b_40 (TLYAVATTI) has a high affinity for HLA-A*0201 molecules. The IFN-gamma ELISpot results and staining of intracellular IFN-gamma confirmed that this peptide could induce high-level CD8(+) T cell response in HLA-A*0201 positive PBMCs. Peptide NS4b_40 (TLYAVATTI) was identified as a novel DENV-specific HLA-A*0201-restricted CD8(+) T cell epitope.

  3. Broadly reactive human CD8 T cells that recognize an epitope conserved between VZV, HSV and EBV.

    PubMed

    Chiu, Christopher; McCausland, Megan; Sidney, John; Duh, Fuh-Mei; Rouphael, Nadine; Mehta, Aneesh; Mulligan, Mark; Carrington, Mary; Wieland, Andreas; Sullivan, Nicole L; Weinberg, Adriana; Levin, Myron J; Pulendran, Bali; Peters, Bjoern; Sette, Alessandro; Ahmed, Rafi

    2014-03-01

    Human herpesviruses are important causes of potentially severe chronic infections for which T cells are believed to be necessary for control. In order to examine the role of virus-specific CD8 T cells against Varicella Zoster Virus (VZV), we generated a comprehensive panel of potential epitopes predicted in silico and screened for T cell responses in healthy VZV seropositive donors. We identified a dominant HLA-A*0201-restricted epitope in the VZV ribonucleotide reductase subunit 2 and used a tetramer to analyze the phenotype and function of epitope-specific CD8 T cells. Interestingly, CD8 T cells responding to this VZV epitope also recognized homologous epitopes, not only in the other α-herpesviruses, HSV-1 and HSV-2, but also the γ-herpesvirus, EBV. Responses against these epitopes did not depend on previous infection with the originating virus, thus indicating the cross-reactive nature of this T cell population. Between individuals, the cells demonstrated marked phenotypic heterogeneity. This was associated with differences in functional capacity related to increased inhibitory receptor expression (including PD-1) along with decreased expression of co-stimulatory molecules that potentially reflected their stimulation history. Vaccination with the live attenuated Zostavax vaccine did not efficiently stimulate a proliferative response in this epitope-specific population. Thus, we identified a human CD8 T cell epitope that is conserved in four clinically important herpesviruses but that was poorly boosted by the current adult VZV vaccine. We discuss the concept of a "pan-herpesvirus" vaccine that this discovery raises and the hurdles that may need to be overcome in order to achieve this.

  4. Superimposed epitopes restricted by the same HLA molecule drive distinct HIV-specific CD8+ T cell repertoires.

    PubMed

    Sun, Xiaoming; Fujiwara, Mamoru; Shi, Yi; Kuse, Nozomi; Gatanaga, Hiroyuki; Appay, Victor; Gao, George F; Oka, Shinichi; Takiguchi, Masafumi

    2014-07-01

    Superimposed epitopes, in which a shorter epitope is embedded within a longer one, can be presented by the same HLA class I molecule. CD8(+) CTL responses against such epitopes and the contribution of this phenomenon to immune control are poorly characterized. In this study, we examined HLA-A*24:02-restricted CTLs specific for the superimposed HIV Nef epitopes RYPLTFGWCF (RF10) and RYPLTFGW (RW8). Unexpectedly, RF10-specific and RW8-specific CTLs from HIV-1-infected HLA-A*24:02+ individuals had no overlapping Ag reactivity or clonotypic compositions. Single-cell TCR sequence analyses demonstrated that RF10-specific T cells had a more diverse TCR repertoire than did RW8-specific T cells. Furthermore, RF10-specific CTLs presented a higher Ag sensitivity and HIV suppressive capacity compared with RW8-specific CTLs. Crystallographic analyses revealed important structural differences between RF10- and RW8-HLA-A*24:02 complexes as well, with featured and featureless conformations, respectively, providing an explanation for the induction of distinct T cell responses against these epitopes. The present study shows that a single viral sequence containing superimposed epitopes restricted by the same HLA molecule could elicit distinct CD8+ T cell responses, therefore enhancing the control of HIV replication. This study also showed that a featured epitope (e.g., RF10) could drive the induction of T cells with high TCR diversity and affinity. Copyright © 2014 by The American Association of Immunologists, Inc.

  5. Presentation of an Immunodominant Immediate-Early CD8+ T Cell Epitope Resists Human Cytomegalovirus Immunoevasion

    PubMed Central

    Ameres, Stefanie; Mautner, Josef; Schlott, Fabian; Neuenhahn, Michael; Busch, Dirk H.; Plachter, Bodo; Moosmann, Andreas

    2013-01-01

    Control of human cytomegalovirus (HCMV) depends on CD8+ T cell responses that are shaped by an individual's repertoire of MHC molecules. MHC class I presentation is modulated by a set of HCMV-encoded proteins. Here we show that HCMV immunoevasins differentially impair T cell recognition of epitopes from the same viral antigen, immediate-early 1 (IE-1), that are presented by different MHC class I allotypes. In the presence of immunoevasins, HLA-A- and HLA-B-restricted T cell clones were ineffective, but HLA-C*0702-restricted T cell clones recognized and killed infected cells. Resistance of HLA-C*0702 to viral immunoevasins US2 and US11 was mediated by the alpha3 domain and C-terminal region of the HLA heavy chain. In healthy donors, HLA-C*0702-restricted T cells dominated the T cell response to IE-1. The same HLA-C allotype specifically protected infected cells from attack by NK cells that expressed a corresponding HLA-C-specific KIR. Thus, allotype-specific viral immunoevasion allows HCMV to escape control by NK cells and HLA-A- and HLA-B-restricted T cells, while the virus becomes selectively vulnerable to an immunodominant population of HLA-C-restricted T cells. Our work identifies a T cell population that may be of particular efficiency in HCMV-specific immunotherapy. PMID:23717207

  6. HLA Class I-T Cell Epitopes from trans-Sialidase Proteins Reveal Functionally Distinct Subsets of CD8+ T Cells in Chronic Chagas Disease

    PubMed Central

    Alvarez, María G.; Postan, Miriam; Weatherly, D. Brent; Albareda, María C.; Sidney, John; Sette, Alessandro; Olivera, Carina; Armenti, Alejandro H.

    2008-01-01

    Background Previously, we identified a set of HLA-A020.1-restricted trans-sialidase peptides as targets of CD8+ T cell responses in HLA-A0201+ individuals chronically infected by T. cruzi. Methods and Findings Herein, we report the identification of peptides encoded by the same trans-sialidase gene family that bind alleles representative of the 6 most common class I HLA-supertypes. Based on a combination of bioinformatic predictions and HLA-supertype considerations, a total of 1001 epitopes predicted to bind to HLA A01, A02, A03, A24, B7 and B44 supertypes was selected. Ninety-six supertype-binder epitopes encoded by multiple trans-sialidase genes were tested for the ability to stimulate a recall CD8+ T cell response in the peripheral blood from subjects with chronic T. cruzi infection regardless the HLA haplotype. An overall hierarchy of antigenicity was apparent, with the A02 supertype peptides being the most frequently recognized in the Chagas disease population followed by the A03 and the A24 supertype epitopes. CD8+ T cell responses to promiscuous epitopes revealed that the CD8+ T cell compartment specific for T. cruzi displays a functional profile with T cells secreting interferon-γ alone as the predominant pattern and very low prevalence of single IL-2-secreting or dual IFN-γ/IL-2 secreting T cells denoting a lack of polyfunctional cytokine responses in chronic T. cruzi infection. Conclusions This study identifies a set of T. cruzi peptides that should prove useful for monitoring immune competence and changes in infection and disease status in individuals with chronic Chagas disease. PMID:18846233

  7. Human TCR-αβ+ CD4− CD8− T Cells Can Derive from CD8+ T Cells and Display an Inflammatory Effector Phenotype1

    PubMed Central

    Crispín, José C.; Tsokos, George C.

    2010-01-01

    The origin and function of human double negative (DN) TCR-αβ+ T cells is unknown. They are thought to contribute to the pathogenesis of systemic lupus erythematosus because they expand and accumulate in inflamed organs. In this study, we provide evidence that human TCR-αβ+ CD4− CD8− DN T cells can derive from activated CD8+ T cells. Freshly isolated TCR-αβ+ DN T cells display a distinct gene expression and cytokine production profile. DN cells isolated from peripheral blood as well as DN cells derived in vitro from CD8+ T cells produce a defined array of proinflammatory mediators that includes IL-1β, IL-17, IFN-γ, CXCL3, and CXCL2. These results indicate that, upon activation, CD8+ T cells have the capacity to acquire a distinct phenotype that grants them inflammatory capacity. PMID:19734235

  8. Cross-genotype-reactivity of the immunodominant HCV CD8 T-cell epitope NS3-1073.

    PubMed

    Fytili, P; Dalekos, G N; Schlaphoff, V; Suneetha, P V; Sarrazin, C; Zauner, W; Zachou, K; Berg, T; Manns, M P; Klade, C S; Cornberg, M; Wedemeyer, H

    2008-07-23

    The HCV-specific HLA-A2-restricted NS3(1073) epitope is one of the most frequently recognized epitopes in hepatitis C. NS3(1073)-specific T-cell responses are associated with clearance of acute HCV-infection. Therefore this epitope is an interesting candidate for a HCV-peptide vaccine. However, heterogeneity between genotypes and mutations in the epitope has to be considered as an obstacle. We here identified 34 naturally occurring NS3(1073)-variants, as compared with the wild type genotype-1 variants (CVNGVCWTV/CINGVCWTV) by sequencing sera of 251 Greek and German patients and searching for published HCV-genomes. The frequency of variants among genotype-1 patients was 10%. Importantly, HLA-A2 binding was reduced only in 3 genotype 1 mutants while all non-genotype 1 variants showed strong HLA-A2-binding. By screening 28 variants in ELISPOT assays from T cell lines we could demonstrate that HCV-NS3(1073)-wild-type-specific T-cells displayed cross-genotype-reactivity, in particular against genotypes 4-6 variants. However, single aa changes within the TCR-binding domain completely abolished recognition even in case of conservative aa exchanges within genotype-1. NS3(1073)-specific T-cell lines from recovered, chronically infected, and HCV-negative individuals showed no major difference in the pattern of cross-recognition although the proliferation of NS3(1073)-specific T-cells differed significantly between the groups. Importantly, the recognition pattern against the 28 variants was also identical directly ex vivo in a patient with acute HCV infection and a healthy volunteer vaccinated with the peptide vaccine IC41 containing the NS3(1073)-wild-type peptide. Thus, partial cross-genotype recognition of HCV NS3(1073)-specific CD8 T cells is possible; however, even single aa exchanges can significantly limit the potential efficacy of vaccines containing the NS3(1073)-wild-type peptide.

  9. Transnuclear CD8 T cells specific for the immunodominant epitope Gra6 lower acute-phase Toxoplasma gondii burden.

    PubMed

    Sanecka, Anna; Yoshida, Nagisa; Dougan, Stephanie K; Jackson, John; Shastri, Nilabh; Ploegh, Hidde; Blanchard, Nicolas; Frickel, Eva-Maria

    2016-11-01

    We generated a CD8 T-cell receptor (TCR) transnuclear (TN) mouse specific to the L(d) -restricted immunodominant epitope of GRA6 from Toxoplasma gondii as a source of cells to facilitate further investigation into the CD8 T-cell-mediated response against this pathogen. The TN T cells bound L(d) -Gra6 tetramer and proliferated upon unspecific and peptide-specific stimulation. The TCR beta sequence of the Gra6-specific TN CD8 T cells is identical in its V- and J-region to the TCR-β harboured by a hybridoma line generated in response to Gra6 peptide. Adoptively transferred Gra6 TN CD8 T cells proliferated upon Toxoplasma infection in vivo and exhibited an activated phenotype similar to host CD8 T cells specific to Gra6. The brain of Toxoplasma-infected mice carried Gra6 TN cells already at day 8 post-infection. Both Gra6 TN mice as well as adoptively transferred Gra6 TN cells were able to significantly reduce the parasite burden in the acute phase of Toxoplasma infection. Overall, the Gra6 TN mouse represents a functional tool to study the protective and immunodominant specific CD8 T-cell response to Toxoplasma in both the acute and the chronic phases of infection. © 2016 The Authors. Immunology Published by John Wiley & Sons Ltd.

  10. Translation efficiency of EBNA1 encoded by lymphocryptoviruses influences endogenous presentation of CD8+ T cell epitopes.

    PubMed

    Tellam, Judy; Rist, Michael; Connolly, Geoff; Webb, Natasha; Fazou, Chrysa; Wang, Fred; Khanna, Rajiv

    2007-02-01

    Lymphocryptoviruses (LCV) that infect humans and Old World primates display a significant degree of genetic identity. These viruses use B lymphocytes as primary host cells to establish a long-term latent infection and express highly homologous latent viral proteins. Of particular interest is the expression of the EBV-encoded nuclear antigen-1 (EBNA1), which plays a crucial role in maintaining the viral genome in B cells. Using human and Old World primate homologues of EBNA1, we show that the internal repeat sequences differentially influence their in vitro translation efficiency. Although the glycine-alanine repeat domain of human LCV (EBV) EBNA1 inhibits its self-synthesis, the repeat domains within the simian LCV homologues of EBNA1 do not inhibit self-synthesis. As a consequence, simian LCV EBNA1-expressing cells are more efficiently recognized by virus-specific CTL when compared to human EBV EBNA1, even though both proteins are highly stable in B cells. Interestingly, we also show that similar to human EBNA1, CD8+ T cell epitopes from simian LCV EBNA1 are predominantly derived from newly synthesized protein rather than the long-lived pool of stable protein. These observations provide additional evidence that supports the theory that immune recognition of EBNA1 can occur without compromising the biological maintenance function of this protein.

  11. Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes.

    PubMed

    Hui, Daniel J; Basner-Tschakarjan, Etiena; Chen, Yifeng; Davidson, Robert J; Buchlis, George; Yazicioglu, Mustafa; Pien, Gary C; Finn, Jonathan D; Haurigot, Virginia; Tai, Alex; Scott, David W; Cousens, Leslie P; Zhou, Shangzhen; De Groot, Anne S; Mingozzi, Federico

    2013-09-01

    Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible.

  12. Epitope-specific CD8+ T cell kinetics rather than viral variability determine the timing of immune escape in simian immunodeficiency virus infection.

    PubMed

    Martyushev, Alexey P; Petravic, Janka; Grimm, Andrew J; Alinejad-Rokny, Hamid; Gooneratne, Shayarana L; Reece, Jeanette C; Cromer, Deborah; Kent, Stephen J; Davenport, Miles P

    2015-05-01

    CD8(+) T cells are important for the control of chronic HIV infection. However, the virus rapidly acquires "escape mutations" that reduce CD8(+) T cell recognition and viral control. The timing of when immune escape occurs at a given epitope varies widely among patients and also among different epitopes within a patient. The strength of the CD8(+) T cell response, as well as mutation rates, patterns of particular amino acids undergoing escape, and growth rates of escape mutants, may affect when escape occurs. In this study, we analyze the epitope-specific CD8(+) T cells in 25 SIV-infected pigtail macaques responding to three SIV epitopes. Two epitopes showed a variable escape pattern and one had a highly monomorphic escape pattern. Despite very different patterns, immune escape occurs with a similar delay of on average 18 d after the epitope-specific CD8(+) T cells reach 0.5% of total CD8(+) T cells. We find that the most delayed escape occurs in one of the highly variable epitopes, and that this is associated with a delay in the epitope-specific CD8(+) T cells responding to this epitope. When we analyzed the kinetics of immune escape, we found that multiple escape mutants emerge simultaneously during the escape, implying that a diverse population of potential escape mutants is present during immune selection. Our results suggest that the conservation or variability of an epitope does not appear to affect the timing of immune escape in SIV. Instead, timing of escape is largely determined by the kinetics of epitope-specific CD8(+) T cells.

  13. Experimental myositis inducible with transfer of dendritic cells presenting a skeletal muscle C protein-derived CD8 epitope peptide.

    PubMed

    Okiyama, Naoko; Hasegawa, Hisanori; Oida, Takatoku; Hirata, Shinya; Yokozeki, Hiroo; Fujimoto, Manabu; Miyasaka, Nobuyuki; Kohsaka, Hitoshi

    2015-07-01

    It is suggested that polymyositis, an autoimmune inflammatory myopathy, is mediated by autoaggressive CD8 T cells. Skeletal muscle C protein is a self-antigen that induces C protein-induced myositis, a murine model of polymyositis. To establish a new murine model of myositis inducible with a single CD8 T-cell epitope peptide that derives from the C protein, three internet-based prediction systems were employed to identify 24 candidate peptides of the immunogenic fragment of the C protein and bind theoretically to major histocompatibility complex class I molecules of C57BL/6 (B6) mice. RMA-S cell assay revealed that a HILIYSDV peptide, amino acid position 399-406 of the C protein, had the highest affinity to the H2-K(b) molecules. Transfer of mature bone marrow-derived dendritic cells pulsed with HILIYSDV induced myositis in naive B6 mice. This myositis was suppressed by anti-CD8-depleting antibodies but not by anti-CD4-depleting antibodies. Because this myositis model is mediated by CD8 T cells independently of CD4 T cells, it should be a useful tool to investigate pathology of polymyositis and develop therapies targeting CD8 T cells. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Vaccination with Altered Peptide Ligands of a Plasmodium berghei Circumsporozoite Protein CD8 T-Cell Epitope: A Model to Generate T Cells Resistant to Immune Interference by Polymorphic Epitopes

    PubMed Central

    Minigo, Gabriela; Flanagan, Katie L.; Slattery, Robyn M.; Plebanski, Magdalena

    2017-01-01

    Many pathogens, including the malaria parasite Plasmodium falciparum, display high levels of polymorphism within T-cell epitope regions of proteins associated with protective immunity. The T-cell epitope variants are often non-cross-reactive. Herein, we show in a murine model, which modifies a protective CD8 T-cell epitope from the circumsporozoite protein (CS) of Plasmodium berghei (SYIPSAEKI), that simultaneous or sequential co-stimulation with two of its putative similarly non-cross-reactive altered peptide ligand (APL) epitopes (SYIPSAEDI or SYIPSAEAI) has radically different effects on immunity. Hence, co-immunization or sequential stimulation in vivo of SYIPSAEKI with its APL antagonist SYIPSAEDI decreases immunity to both epitopes. By contrast, co-immunization with SYIPSAEAI has no apparent initial effect, but it renders the immune response to SYIPSAEKI resistant to being turned off by subsequent immunization with SYIPSAEDI. These results suggest a novel strategy for vaccines that target polymorphic epitopes potentially capable of mutual immune interference in the field, by initiating an immune response by co-immunization with the desired index epitope, together with a carefully selected “potentiator” APL peptide. PMID:28261200

  15. DNA vaccines encoding altered peptide ligands for SSX2 enhance epitope-specific CD8+ T-cell immune responses.

    PubMed

    Smith, Heath A; Rekoske, Brian T; McNeel, Douglas G

    2014-03-26

    Plasmid DNA serves as a simple and easily modifiable form of antigen delivery for vaccines. The USDA approval of DNA vaccines for several non-human diseases underscores the potential of this type of antigen delivery method as a cost-effective approach for the treatment or prevention of human diseases, including cancer. However, while DNA vaccines have demonstrated safety and immunological effect in early phase clinical trials, they have not consistently elicited robust anti-tumor responses. Hence many recent efforts have sought to increase the immunological efficacy of DNA vaccines, and we have specifically evaluated several target antigens encoded by DNA vaccine as treatments for human prostate cancer. In particular, we have focused on SSX2 as one potential target antigen, given its frequent expression in metastatic prostate cancer. We have previously identified two peptides, p41-49 and p103-111, as HLA-A2-restricted SSX2-specific epitopes. In the present study we sought to determine whether the efficacy of a DNA vaccine could be enhanced by an altered peptide ligand (APL) strategy wherein modifications were made to anchor residues of these epitopes to enhance or ablate their binding to HLA-A2. A DNA vaccine encoding APL modified to increase epitope binding elicited robust peptide-specific CD8+ T cells producing Th1 cytokines specific for each epitope. Ablation of one epitope in a DNA vaccine did not enhance immune responses to the other epitope. These results demonstrate that APL encoded by a DNA vaccine can be used to elicit increased numbers of antigen-specific T cells specific for multiple epitopes simultaneously, and suggest this could be a general approach to improve the immunogenicity of DNA vaccines encoding tumor antigens.

  16. Susceptibility to chronic hepatitis C virus infection is influenced by sequence differences in immunodominant CD8+ T cell epitopes.

    PubMed

    Ziegler, Susanne; Ruhl, Marianne; Tenckhoff, Hannelore; Wiese, Manfred; Heinemann, Falko M; Horn, Peter A; Spengler, Ulrich; Neumann-Haefelin, Christoph; Nattermann, Jacob; Timm, Jörg

    2013-01-01

    The antiviral immune response against HCV by CD8+ T cells plays a central role in viral containment. In a large HCV genotype 1b outbreak in Ireland, HLA-B(∗)08 was identified as a risk allele for chronic infection and HLA-A(∗)03 and HLA-B(∗)27 were associated with higher clearance rates. Here we took advantage of a similar large common source HCV genotype 1b outbreak (East-German cohort) to determine the role of HLA class I alleles and the sequence of the infection source, in immunodominant CD8+ T cell epitopes for disease outcome. HLA-type and IL28B genotype were determined in 216 patients with chronic and 95 with spontaneously resolved HCV infection. The viral sequence in immunodominant epitopes was determined in the infection source and in patients with chronic infection. In contrast to the Irish cohort, HLA-B(∗)08, HLA-A(∗)03 and HLA-B(∗)27 were neutral for disease outcome even when the cohort was stratified for the IL28B genotype. Sequence analysis of the immunodominant epitopes revealed that pre-existing substitutions in the infection source of both cohorts influenced the impact of the corresponding HLA-allele. The immunodominant epitopes presented by the "protective" alleles HLA-A(∗)03 and -B(∗)27 in the Irish cohort contained substitutions in the source virus of the East-German outbreak. Importantly, the pre-existing substitutions altered subsequent selection pressure and viral evolution in the East-German cohort. This study highlights that subtle sequence differences in the infection source may have profound effects on the ability to clear HCV infection in the presence of particular HLA class I alleles. Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  17. Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals

    PubMed Central

    Comber, Joseph D; Karabudak, Aykan; Huang, Xiaofang; Piazza, Paolo A; Marques, Ernesto T A; Philip, Ramila

    2015-01-01

    Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines. PMID:25668665

  18. Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals.

    PubMed

    Comber, Joseph D; Karabudak, Aykan; Huang, Xiaofang; Piazza, Paolo A; Marques, Ernesto T A; Philip, Ramila

    2014-01-01

    Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines.

  19. Different Vaccine Vectors Delivering the Same Antigen Elicit CD8plus T Cell Responses with Distinct Clonotype and Epitope Specificity

    SciTech Connect

    M Honda; R Wang; W Kong; M Kanekiyo; Q Akahata; L Xu; K Matsuo; K Natarajan; H Robinson; et al.

    2011-12-31

    Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.

  20. Different Vaccine Vectors Delivering the Same Antigen Elicit CD8+ T Cell Responses with Distinct Clonotype and Epitope Specificity

    SciTech Connect

    Honda, M.; Robinson, H.; Wang, R.; Kong, W.-P.; Kanekiyo, M.; Akahata, W.; Xu, L.; Matsuo, K.; Natarajan, K.; Asher, T. E.; Price, D. A.; Douek, D. C.; Margulies, D. H.; Nabel, G. J.

    2009-08-15

    Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.

  1. TCR Affinity Associated with Functional Differences between Dominant and Subdominant SIV Epitope-Specific CD8+ T Cells in Mamu-A*01+ Rhesus Monkeys

    PubMed Central

    Osuna, Christa E.; Gonzalez, Ana Maria; Chang, Hsun-Hsien; Hung, Amy Shi; Ehlinger, Elizabeth; Anasti, Kara; Alam, S. Munir; Letvin, Norman L.

    2014-01-01

    Many of the factors that contribute to CD8+ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8+ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8+ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A*01 during acute and chronic SIV infection. Whole genome expression analyses during acute infection revealed that dominant SIV epitope-specific CD8+ T cells had a gene expression profile consistent with greater maturity and higher cytotoxic potential than subdominant epitope-specific CD8+ T cells. Flow-cytometric measurements of protein expression and anti-viral functionality during chronic infection confirmed these phenotypic and functional differences. Expression analyses of exhaustion-associated genes indicated that LAG-3 and CTLA-4 were more highly expressed in the dominant epitope-specific cells during acute SIV infection. Interestingly, only LAG-3 expression remained high during chronic infection in dominant epitope-specific cells. We also explored the binding interaction between peptide:MHC (pMHC) complexes and their cognate TCRs to determine their role in the establishment of immunodominance hierarchies. We found that epitope dominance was associated with higher TCR:pMHC affinity. These studies demonstrate that significant functional differences exist between dominant and subdominant epitope-specific CD8+ T cells within MHC-restricted immunodominance hierarchies and suggest that TCR:pMHC affinity may play an important role in determining the frequency and functionality of these cell populations. These findings advance our understanding of the regulation of T cell immunodominance and will aid HIV vaccine design. PMID:24743648

  2. TCR affinity associated with functional differences between dominant and subdominant SIV epitope-specific CD8+ T cells in Mamu-A*01+ rhesus monkeys.

    PubMed

    Osuna, Christa E; Gonzalez, Ana Maria; Chang, Hsun-Hsien; Hung, Amy Shi; Ehlinger, Elizabeth; Anasti, Kara; Alam, S Munir; Letvin, Norman L

    2014-04-01

    Many of the factors that contribute to CD8+ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8+ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8+ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A*01 during acute and chronic SIV infection. Whole genome expression analyses during acute infection revealed that dominant SIV epitope-specific CD8+ T cells had a gene expression profile consistent with greater maturity and higher cytotoxic potential than subdominant epitope-specific CD8+ T cells. Flow-cytometric measurements of protein expression and anti-viral functionality during chronic infection confirmed these phenotypic and functional differences. Expression analyses of exhaustion-associated genes indicated that LAG-3 and CTLA-4 were more highly expressed in the dominant epitope-specific cells during acute SIV infection. Interestingly, only LAG-3 expression remained high during chronic infection in dominant epitope-specific cells. We also explored the binding interaction between peptide:MHC (pMHC) complexes and their cognate TCRs to determine their role in the establishment of immunodominance hierarchies. We found that epitope dominance was associated with higher TCR:pMHC affinity. These studies demonstrate that significant functional differences exist between dominant and subdominant epitope-specific CD8+ T cells within MHC-restricted immunodominance hierarchies and suggest that TCR:pMHC affinity may play an important role in determining the frequency and functionality of these cell populations. These findings advance our understanding of the regulation of T cell immunodominance and will aid HIV vaccine design.

  3. A Novel HLA (HLA-A*0201) Transgenic Rabbit Model for Preclinical Evaluation of Human CD8+ T Cell Epitope-Based Vaccines against Ocular Herpes

    PubMed Central

    Chentoufi, Aziz A.; Dasgupta, Gargi; Christensen, Neil D.; Hu, Jiafen; Choudhury, Zareen S.; Azeem, Arfan; Jester, James V.; Nesburn, Anthony B.; Wechsler, Steven L.; BenMohamed, Lbachir

    2013-01-01

    We introduced a novel humanized HLA-A*0201 transgenic (HLA Tg) rabbit model to assess the protective efficacy of a human CD8+ T cell epitope-based vaccine against primary ocular herpes infection and disease. Each of the three immunodominant human CD8+ T cell peptide epitopes from HSV-1 glycoprotein D (gD53–61, gD70–78, and gD278–286) were joined with a promiscuous human CD4+ T cell peptide epitope (gD49–82) to construct three separate pairs of CD4–CD8 peptides. Each CD4–CD8 peptide pair was then covalently linked to an Nε-palmitoyl–lysine residue via a functional base lysine amino group to construct CD4–CD8 lipopeptides. HLA Tg rabbits were immunized s.c. with a mixture of the three CD4–CD8 HSV-1 gD lipopeptides. The HSV-gD–specific T cell responses induced by the mixture of CD4–CD8 lipopeptide vaccine and the protective efficacy against acute virus replication and ocular disease were determined. Immunization induced HSV-gD49–82–specific CD4+ T cells in draining lymph node (DLN); induced HLA-restricted HSV-gD53–61, gD70–78, and gD278–286–specific CD8+ T cells in DLN, conjunctiva, and trigeminal ganglia and reduced HSV-1 replication in tears and corneal eye disease after ocular HSV-1 challenge. In addition, the HSV-1 epitope-specific CD8+ T cells induced in DLNs, conjunctiva, and the trigeminal ganglia were inversely proportional with corneal disease. The humanized HLA Tg rabbits appeared to be a useful preclinical animal model for investigating the immunogenicity and protective efficacy of human CD8+ T cell epitope-based prophylactic vaccines against ocular herpes. The relevance of HLA Tg rabbits for future investigation of human CD4–CD8 epitope-based therapeutic vaccines against recurrent HSV-1 is discussed. PMID:20124097

  4. A novel HLA (HLA-A*0201) transgenic rabbit model for preclinical evaluation of human CD8+ T cell epitope-based vaccines against ocular herpes.

    PubMed

    Chentoufi, Aziz A; Dasgupta, Gargi; Christensen, Neil D; Hu, Jiafen; Choudhury, Zareen S; Azeem, Arfan; Jester, James V; Nesburn, Anthony B; Wechsler, Steven L; BenMohamed, Lbachir

    2010-03-01

    We introduced a novel humanized HLA-A*0201 transgenic (HLA Tg) rabbit model to assess the protective efficacy of a human CD8(+) T cell epitope-based vaccine against primary ocular herpes infection and disease. Each of the three immunodominant human CD8(+) T cell peptide epitopes from HSV-1 glycoprotein D (gD(53-61), gD(70-78), and gD(278-286)) were joined with a promiscuous human CD4(+) T cell peptide epitope (gD(49-82)) to construct three separate pairs of CD4-CD8 peptides. Each CD4-CD8 peptide pair was then covalently linked to an N(epsilon)-palmitoyl-lysine residue via a functional base lysine amino group to construct CD4-CD8 lipopeptides. HLA Tg rabbits were immunized s.c. with a mixture of the three CD4-CD8 HSV-1 gD lipopeptides. The HSV-gD-specific T cell responses induced by the mixture of CD4-CD8 lipopeptide vaccine and the protective efficacy against acute virus replication and ocular disease were determined. Immunization induced HSV-gD(49-82)-specific CD4(+) T cells in draining lymph node (DLN); induced HLA-restricted HSV-gD(53-61), gD(70-78), and gD(278-286)-specific CD8(+) T cells in DLN, conjunctiva, and trigeminal ganglia and reduced HSV-1 replication in tears and corneal eye disease after ocular HSV-1 challenge. In addition, the HSV-1 epitope-specific CD8(+) T cells induced in DLNs, conjunctiva, and the trigeminal ganglia were inversely proportional with corneal disease. The humanized HLA Tg rabbits appeared to be a useful preclinical animal model for investigating the immunogenicity and protective efficacy of human CD8(+) T cell epitope-based prophylactic vaccines against ocular herpes. The relevance of HLA Tg rabbits for future investigation of human CD4-CD8 epitope-based therapeutic vaccines against recurrent HSV-1 is discussed.

  5. Positive Selection in CD8+ T-Cell Epitopes of Influenza Virus Nucleoprotein Revealed by a Comparative Analysis of Human and Swine Viral Lineages.

    PubMed

    Machkovech, Heather M; Bedford, Trevor; Suchard, Marc A; Bloom, Jesse D

    2015-11-01

    Numerous experimental studies have demonstrated that CD8(+) T cells contribute to immunity against influenza by limiting viral replication. It is therefore surprising that rigorous statistical tests have failed to find evidence of positive selection in the epitopes targeted by CD8(+) T cells. Here we use a novel computational approach to test for selection in CD8(+) T-cell epitopes. We define all epitopes in the nucleoprotein (NP) and matrix protein (M1) with experimentally identified human CD8(+) T-cell responses and then compare the evolution of these epitopes in parallel lineages of human and swine influenza viruses that have been diverging since roughly 1918. We find a significant enrichment of substitutions that alter human CD8(+) T-cell epitopes in NP of human versus swine influenza virus, consistent with the idea that these epitopes are under positive selection. Furthermore, we show that epitope-altering substitutions in human influenza virus NP are enriched on the trunk versus the branches of the phylogenetic tree, indicating that viruses that acquire these mutations have a selective advantage. However, even in human influenza virus NP, sites in T-cell epitopes evolve more slowly than do nonepitope sites, presumably because these epitopes are under stronger inherent functional constraint. Overall, our work demonstrates that there is clear selection from CD8(+) T cells in human influenza virus NP and illustrates how comparative analyses of viral lineages from different hosts can identify positive selection that is otherwise obscured by strong functional constraint. There is a strong interest in correlates of anti-influenza immunity that are protective against diverse virus strains. CD8(+) T cells provide such broad immunity, since they target conserved viral proteins. An important question is whether T-cell immunity is sufficiently strong to drive influenza virus evolution. Although many studies have shown that T cells limit viral replication in animal

  6. Positive Selection in CD8+ T-Cell Epitopes of Influenza Virus Nucleoprotein Revealed by a Comparative Analysis of Human and Swine Viral Lineages

    PubMed Central

    Machkovech, Heather M.; Bedford, Trevor; Suchard, Marc A.

    2015-01-01

    ABSTRACT Numerous experimental studies have demonstrated that CD8+ T cells contribute to immunity against influenza by limiting viral replication. It is therefore surprising that rigorous statistical tests have failed to find evidence of positive selection in the epitopes targeted by CD8+ T cells. Here we use a novel computational approach to test for selection in CD8+ T-cell epitopes. We define all epitopes in the nucleoprotein (NP) and matrix protein (M1) with experimentally identified human CD8+ T-cell responses and then compare the evolution of these epitopes in parallel lineages of human and swine influenza viruses that have been diverging since roughly 1918. We find a significant enrichment of substitutions that alter human CD8+ T-cell epitopes in NP of human versus swine influenza virus, consistent with the idea that these epitopes are under positive selection. Furthermore, we show that epitope-altering substitutions in human influenza virus NP are enriched on the trunk versus the branches of the phylogenetic tree, indicating that viruses that acquire these mutations have a selective advantage. However, even in human influenza virus NP, sites in T-cell epitopes evolve more slowly than do nonepitope sites, presumably because these epitopes are under stronger inherent functional constraint. Overall, our work demonstrates that there is clear selection from CD8+ T cells in human influenza virus NP and illustrates how comparative analyses of viral lineages from different hosts can identify positive selection that is otherwise obscured by strong functional constraint. IMPORTANCE There is a strong interest in correlates of anti-influenza immunity that are protective against diverse virus strains. CD8+ T cells provide such broad immunity, since they target conserved viral proteins. An important question is whether T-cell immunity is sufficiently strong to drive influenza virus evolution. Although many studies have shown that T cells limit viral replication in animal

  7. Generation of functional CD8+ T Cells by human dendritic cells expressing glypican-3 epitopes

    PubMed Central

    2010-01-01

    Background Glypican 3 (GPC-3) is an oncofoetal protein that is expressed in most hepatocellular carcinomas (HCC). Since it is a potential target for T cell immunotherapy, we investigated the generation of functional, GPC-3 specific T cells from peripheral blood mononuclear cells (PBMC). Methods Dendritic cells (DC) were derived from adherent PBMC cultured at 37°C for 7 days in X-Vivo, 1% autologous plasma, and 800 u/ml GM-CSF plus 500 u/ml IL-4. Immature DC were transfected with 20 μg of in vitro synthesised GPC-3 mRNA by electroporation using the Easy-ject plus system (Equibio, UK) (300 V, 150 μF and 4 ms pulse time), or pulsed with peptide, and subsequently matured with lipopolysaccharide (LPS). Six predicted GPC-3 peptide epitopes were synthesized using standard f-moc technology and tested for their binding affinity to HLA-A2.1 molecules using the cell line T2. Results DC transfected with GPC-3 mRNA but not control DC demonstrated strong intracellular staining for GPC-3 and in vitro generated interferon-gamma expressing T cells from autologous PBMC harvested from normal subjects. One peptide, GPC-3522-530 FLAELAYDL, fulfilled our criteria as a naturally processed, HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope: i) it showed high affinity binding to HLA-A2, in T2 cell binding assay; ii) it was generated by the MHC class I processing pathway in DC transfected with GPC-3 mRNA, and iii) HLA-A2 positive DC loaded with the peptide stimulated proliferation in autologous T cells and generated CTL that lysed HLA-A2 and GPC-3 positive target cells. Conclusions These findings demonstrate that electroporation of GPC-3 mRNA is an efficient method to load human monocyte-derived DC with antigen because in vitro they generated GPC-3-reactive T cells that were functional, as shown by interferon-gamma production. Furthermore, this study identified a novel naturally processed, HLA-A2-restricted CTL epitope, GPC-3522-530 FLAELAYDL, which can be used to monitor HLA-A2

  8. Novel, in-natural-infection subdominant HIV-1 CD8+ T-cell epitopes revealed in human recipients of conserved-region T-cell vaccines.

    PubMed

    Borthwick, Nicola; Lin, Zhansong; Akahoshi, Tomohiro; Llano, Anuska; Silva-Arrieta, Sandra; Ahmed, Tina; Dorrell, Lucy; Brander, Christian; Murakoshi, Hayato; Takiguchi, Masafumi; Hanke, Tomáš

    2017-01-01

    Fine definition of targeted CD8+ T-cell epitopes and their human leucocyte antigen (HLA) class I restriction informs iterative improvements of HIV-1 T-cell vaccine designs and may predict early vaccine success or failure. Here, lymphocytes from volunteers, who had received candidate HIVconsv vaccines expressing conserved sub-protein regions of HIV-1, were used to define the optimum-length target epitopes and their HLA restriction. In HIV-1-positive patients, CD8+ T-cell responses predominantly recognize immunodominant, but hypervariable and therefore less protective epitopes. The less variable, more protective epitopes in conserved regions are typically subdominant. Therefore, induction of strong responses to conserved regions by vaccination provides an opportunity to discover novel important epitopes. Cryopreserved lymphocytes from vaccine recipients were expanded by stimulation with 15-mer responder peptides for 10 days to establish short term-cell-line (STCL) effector cells. These were subjected to intracellular cytokine staining using serially truncated peptides and peptide-pulsed 721.221 cells expressing individual HLA class I alleles to define minimal epitope length and HLA restriction by stimulation of IFN-γ and TNF-α production and surface expression of CD107a. Using lymphocyte samples of 12 vaccine recipients, we defined 14 previously unreported optimal CD8+ T-cell HIV-1 epitopes and their four-digit HLA allele restriction (6 HLA-A, 7 HLA-B and 1 HLA-C alleles). Further 13 novel targets with incomplete information were revealed. The high rate of discovery of novel CD8+ T-cell effector epitopes warrants further epitope mining in recipients of the conserved-region vaccines in other populations and informs development of HIV-1/AIDS vaccines. ClinicalTrials.gov NCT01151319.

  9. Phenotypic and Functional Characterization of Herpes Simplex Virus Glycoprotein B Epitope-Specific Effector and Memory CD8+ T Cells from Symptomatic and Asymptomatic Individuals with Ocular Herpes

    PubMed Central

    Khan, Arif A.; Srivastava, Ruchi; Spencer, Doran; Garg, Sumit; Fremgen, Daniel; Vahed, Hawa; Lopes, Patricia P.; Pham, Thanh T.; Hewett, Charlie; Kuang, Jasmine; Ong, Nicolas; Huang, Lei; Scarfone, Vanessa M.; Nesburn, Anthony B.

    2015-01-01

    ABSTRACT Herpes simplex virus 1 (HSV-1) glycoprotein B (gB)-specific CD8+ T cells protect mice from herpes infection and disease. However, whether and which HSV-1 gB-specific CD8+ T cells play a key role in the “natural” protection seen in HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we have dissected the phenotypes and the functions of HSV-1 gB-specific CD8+ T cells from HLA-A*02:01 positive, HSV-1 seropositive ASYMP and symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpes disease). We found the following. (i) Healthy ASYMP individuals maintained a significantly higher proportion of differentiated HSV-1 gB-specific effector memory CD8+ T cells (TEM cells) (CD45RAlow CCR7low CD44high CD62Llow). In contrast, SYMP patients had frequent less-differentiated central memory CD8+ T cells (TCM cells) (CD45RAlow CCR7high CD44low CD62Lhigh). (ii) ASYMP individuals had significantly higher proportions of multifunctional effector CD8+ T cells which responded mainly to gB342–350 and gB561–569 “ASYMP” epitopes, and simultaneously produced IFN-γ, CD107a/b, granzyme B, and perforin. In contrast, effector CD8+ T cells from SYMP individuals were mostly monofunctional and were directed mainly against nonoverlapping gB17–25 and gB183–191 “SYMP” epitopes. (iii) Immunization of an HLA-A*02:01 transgenic mouse model of ocular herpes with “ASYMP” CD8+ TEM cell epitopes, but not with “SYMP” CD8+ TCM cell epitopes, induced a strong CD8+ T cell-dependent protective immunity against ocular herpes infection and disease. Our findings provide insights into the role of HSV-specific CD8+ TEM cells in protection against herpes and should be considered in the development of an effective vaccine. IMPORTANCE A significantly higher proportion of differentiated and multifunctional HSV-1 gB-specific effector memory CD8+ T cells (TEM

  10. Phenotypic and functional characterization of herpes simplex virus glycoprotein B epitope-specific effector and memory CD8+ T cells from symptomatic and asymptomatic individuals with ocular herpes.

    PubMed

    Khan, Arif A; Srivastava, Ruchi; Spencer, Doran; Garg, Sumit; Fremgen, Daniel; Vahed, Hawa; Lopes, Patricia P; Pham, Thanh T; Hewett, Charlie; Kuang, Jasmine; Ong, Nicolas; Huang, Lei; Scarfone, Vanessa M; Nesburn, Anthony B; Wechsler, Steven L; BenMohamed, Lbachir

    2015-04-01

    Herpes simplex virus 1 (HSV-1) glycoprotein B (gB)-specific CD8(+) T cells protect mice from herpes infection and disease. However, whether and which HSV-1 gB-specific CD8(+) T cells play a key role in the "natural" protection seen in HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we have dissected the phenotypes and the functions of HSV-1 gB-specific CD8(+) T cells from HLA-A*02:01 positive, HSV-1 seropositive ASYMP and symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpes disease). We found the following. (i) Healthy ASYMP individuals maintained a significantly higher proportion of differentiated HSV-1 gB-specific effector memory CD8(+) T cells (TEM cells) (CD45RA(low) CCR7(low) CD44(high) CD62L(low)). In contrast, SYMP patients had frequent less-differentiated central memory CD8(+) T cells (TCM cells) (CD45RA(low) CCR7(high) CD44(low) CD62L(high)). (ii) ASYMP individuals had significantly higher proportions of multifunctional effector CD8(+) T cells which responded mainly to gB342-350 and gB561-569 "ASYMP" epitopes, and simultaneously produced IFN-γ, CD107(a/b), granzyme B, and perforin. In contrast, effector CD8(+) T cells from SYMP individuals were mostly monofunctional and were directed mainly against nonoverlapping gB17-25 and gB183-191 "SYMP" epitopes. (iii) Immunization of an HLA-A*02:01 transgenic mouse model of ocular herpes with "ASYMP" CD8(+) TEM cell epitopes, but not with "SYMP" CD8(+) TCM cell epitopes, induced a strong CD8(+) T cell-dependent protective immunity against ocular herpes infection and disease. Our findings provide insights into the role of HSV-specific CD8(+) TEM cells in protection against herpes and should be considered in the development of an effective vaccine. A significantly higher proportion of differentiated and multifunctional HSV-1 gB-specific effector memory CD8(+) T cells (TEM cells

  11. Escape from a Dominant HLA-B*15-Restricted CD8+ T Cell Response against Hepatitis C Virus Requires Compensatory Mutations outside the Epitope

    PubMed Central

    Ruhl, Marianne; Chhatwal, Patrick; Strathmann, Heiko; Kuntzen, Thomas; Bankwitz, Dorothea; Skibbe, Kathrin; Walker, Andreas; Heinemann, Falko M.; Horn, Peter A.; Allen, Todd M.; Hoffmann, Daniel; Pietschmann, Thomas

    2012-01-01

    Antiviral CD8+ T cells are a key component of the adaptive immune system against hepatitis C virus (HCV). For the development of immune therapies, it is essential to understand how CD8+ T cells contribute to clearance of infection and why they fail so often. A mechanism for secondary failure is mutational escape of the virus. However, some substitutions in viral epitopes are associated with fitness costs and often require compensatory mutations. We hypothesized that compensatory mutations may point toward epitopes under particularly strong selection pressure that may be beneficial for vaccine design because of a higher genetic barrier to escape. We previously identified two HLA-B*15-restricted CD8+ epitopes in NS5B (LLRHHNMVY2450-2458 and SQRQKKVTF2466-2474), based on sequence analysis of a large HCV genotype 1b outbreak. Both epitopes are targeted in about 70% of HLA-B*15-positive individuals exposed to HCV. Reproducible selection of escape mutations was confirmed in an independent multicenter cohort in the present study. Interestingly, mutations were also selected in the epitope flanking region, suggesting that compensatory evolution may play a role. Covariation analysis of sequences from the database confirmed a significant association between escape mutations inside one of the epitopes (H2454R and M2456L) and substitutions in the epitope flanking region (S2439T and K2440Q). Functional analysis with the subgenomic replicon Con1 confirmed that the primary escape mutations impaired viral replication, while fitness was restored by the additional substitutions in the epitope flanking region. We concluded that selection of escape mutations inside an HLA-B*15 epitope requires secondary substitutions in the epitope flanking region that compensate for fitness costs. PMID:22072759

  12. Epitope Mapping with Random Phage Display Library

    PubMed Central

    Midoro-Horiuti, Terumi; Goldblum, Randall M.

    2017-01-01

    Random phage display library is used to map conformational as well as linear epitopes. These libraries are available in varying lengths and with circularization. We provide here a protocol conveying our experience using a commercially available peptide phage display library, which in our hands provides good results. PMID:24515483

  13. HLA-A*0201 restricted CD8+ T-lymphocyte responses to malaria: identification of new Plasmodium falciparum epitopes by IFN-gamma ELISPOT.

    PubMed

    González, J M; Peter, K; Esposito, F; Nebié, I; Tiercy, J M; Bonelo, A; Arévalo-Herrera, M; Valmori, D; Romero, P; Herrera, S; Corradin, G; López, J A

    2000-10-01

    The role of antigen specific CD8+ T-lymphocytes in mediating protection against sporozoite-induced malaria has been well established in murine models. In humans, indirect evidence has accumulated suggesting a similar protective role for antigen-specific CD8+ T-lymphocytes. Nevertheless, the low frequency of circulating specific cells together with the lack of sensitive methods to quantify them has hampered the direct assessment of their function. Using a combination of short-term cell culture and IFN-gamma ELISPOT, we studied CD8+ T-lymphocyte responses to a panel of HLA-A*0201 binding peptides. In addition to confirming the response to already described epitopes, we also identified five new CD8+ T-lymphocyte epitopes. These epitopes are presented in pre-erythrocytic stages gene products of Plasmodium falciparum 7G8 strain and correspond to the following protein segments: circumsporozoite (CS) 64-72, 104-113, 299-308 and 403-411; liver stage antigen (LSA-1) repeat region; sporozoite surface protein 2 or thrombospondin related anonymous protein (SSP2/TRAP) 78-88 and 504-513. Four of these peptides are conserved amongst all published sequences of P. falciparum strains. We conclude that the modified IFN-gamma ELISPOT assay is a sensitive technique to monitor antigen-specific CD8+ T-lymphocyte responses in human malaria which may help in the improvement and assessment of the efficacy of malaria subunit vaccines.

  14. HLA-B*27 subtype specificity determines targeting and viral evolution of a hepatitis C virus-specific CD8+ T-cell epitope

    PubMed Central

    Nitschke, Katja; Barriga, Alejandro; Schmidt, Julia; Timm, Jörg; Viazov, Sergei; Kuntzen, Thomas; Kim, Arthur Y.; Lauer, Georg M.; Allen, Todd M.; Gaudieri, Silvana; Rauch, Andri; Lange, Christian M.; Sarrazin, Christoph; Eiermann, Thomas; Sidney, John; Sette, Alessandro; Thimme, Robert; López, Daniel; Neumann-Haefelin, Christoph

    2013-01-01

    Background & Aims HLA-B*27 is associated with spontaneous HCV genotype 1 clearance. HLA-B*27-restricted CD8+ T-cells target three NS5B epitopes. Two of these epitopes are dominantly targeted in the majority of HLA-B*27+ patients. In chronic infection, viral escape occurs consistently in these two epitopes. The third epitope (NS5B2820) was dominantly targeted in an acutely infected patient. This was in contrast, however, to the lack of recognition and viral escape in the large majority of HLA-B*27+ patients. Here, we set out to determine the host factors contributing to selective targeting of this epitope. Methods Four-digit HLA class I typing and viral sequence analyses were performed in 78 HLA-B*27+ patients with chronic HCV genotype 1 infection. CD8+ T-cell analyses were performed in a subset of patients. In addition, HLA/peptide affinity was compared for HLA-B*27:02 and 05. Results The NS5B2820 epitope is only restricted by the HLA-B*27 subtype HLA-B*27:02 (that is frequent in Mediterranean populations), but not by the prototype HLA-B*27 subtype B*27:05. Indeed, the epitope is very dominant in HLA-B*27:02+ patients and is associated with viral escape mutations at the anchor position for HLA-binding in 12 out of 13 HLA-B*27:02+ chronically infected patients. Conclusions The NS5B2820 epitope is immunodominant in the context of HLA-B*27:02, but is not restricted by other HLA-B*27 subtypes. This finding suggests an important role of HLA subtypes in the restriction of HCV-specific CD8+ responses. With minor HLA subtypes covering up to 39% of specific populations, these findings may have important implications for the selection of epitopes for global vaccines. PMID:23978718

  15. Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load

    PubMed Central

    Kløverpris, Henrik N.; McGregor, Reuben; McLaren, James E.; Ladell, Kristin; Stryhn, Anette; Koofhethile, Catherine; Brener, Jacqui; Chen, Fabian; Riddell, Lynn; Graziano, Luzzi; Klenerman, Paul; Leslie, Alasdair; Buus, Søren; Price, David A.; Goulder, Philip

    2014-01-01

    Objectives: Although CD8+ T cells play a critical role in the control of HIV-1 infection, their antiviral efficacy can be limited by antigenic variation and immune exhaustion. The latter phenomenon is characterized by the upregulation of multiple inhibitory receptors, such as programmed death-1 (PD-1), CD244 and lymphocyte activation gene-3 (LAG-3), which modulate the functional capabilities of CD8+ T cells. Design and methods: Here, we used an array of different human leukocyte antigen (HLA)-B∗15 : 03 and HLA-B∗42 : 01 tetramers to characterize inhibitory receptor expression as a function of differentiation on HIV-1-specific CD8+ T-cell populations (n = 128) spanning 11 different epitope targets. Results: Expression levels of PD-1, but not CD244 or LAG-3, varied substantially across epitope specificities both within and between individuals. Differential expression of PD-1 on T-cell receptor (TCR) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated by effector memory CD8+ T cells. Conclusion: Collectively, these data suggest that PD-1 expression on HIV-1-specific CD8+ T cells tracks antigen load at the level of epitope specificity and TCR clonotype usage. These findings are important because they provide evidence that PD-1 expression levels are influenced by peptide/HLA class I antigen exposure. PMID:24906112

  16. Defining the mechanism(s) of protection by cytolytic CD8 T cells against a cryptic epitope derived from a retroviral alternative reading frame

    PubMed Central

    Rutkowski, Melanie R.; Ho, On; Green, William R.

    2009-01-01

    The biological significance of protective CD8 T-cell-mediated responses against non-traditional alternative reading frame epitopes remains relatively unknown. Cytolytic CD8 T cells (CTL) specific for a non-traditional cryptic MHC class I epitope, SYNTGRFPPL, are critically involved in the protection of mice during infection with the LP-BM5 murine retrovirus. The goal of this study was to determine the functional properties of the protective SYNTGRFPPL-specific CTL during LP-BM5 infection of susceptible BALB/c CD8−/− mice. Direct infection experiments and adoptive transfer of CD8 T cells derived from perforin (pfp)−/−, IFNγ−/−, FasL−/− and, as a positive control, wild-type BALB/c mice, were utilized to assess the effector mechanisms responsible for protection. Our results indicate that SYNTGRFPPL-specific effector CTL preferentially utilize perforin-mediated cytolysis to provide protection against LP-BM5-induced pathogenesis, whereas CTL production of IFNγ is not required. Our results also suggest a minimal contribution of FasL/Fas-mediated lysis during the effector response. Collectively, these results provide insight into effector mechanisms utilized by protective CTL directed against non-traditional cryptic epitopes during disease protection. PMID:19539970

  17. CD8+ T cell response in HLA-A*0201 transgenic mice is elicited by epitopes from SARS-CoV S protein.

    PubMed

    Zhao, Kai; Yang, Binyan; Xu, Yanquan; Wu, Changyou

    2010-09-24

    Cytotoxic CD8(+) T lymphocytes (CTLs) play an important role in antiviral immunity. Several human HLA-A*0201 restricted CTL epitopes of severe acute respiratory syndrome (SARS) spike (S) protein have been identified in HLA-A*0201 transgenic (Tg) mice, but the mechanisms and properties of immune responses are still not well understood. In this study, HLA-A*0201 Tg mice were primed intramuscularly with SARS S DNA and boosted subcutaneously with HLA-A*0201 restricted peptides. The lymphocytes from draining lymph nodes, spleens and lungs were stimulated with the cognate peptides. Three different methods (ELISA, ELISPOT and FACS) were used to evaluate the immune responses during short and long periods of time after immunization. Results showed that peptide-specific CD8(+) T cells secreted IFN-γ, TNF-α and IL-2 and expressed CD107a/b on cell surface. IFN-γ(+)CD8(+) T cells and CD107a/b(+)CD8(+) T cells distributed throughout the lymphoid and non-lymphoid tissues, but the frequency of peptide-specific CD8(+) T cells was higher in lungs than in spleens and lymph nodes. The phenotype of the CD8(+) T cells was characterized based on the expression of IFN-γ. Most of the HLA-A*0201 restricted peptide-specific CD8(+) T cells represented a memory subset with CD45RB(high) and CD62L(low). Taken together, these data demonstrate that immunization with SARS S DNA and HLA-A*0201 restricted peptides can elicit antigen-specific CD8(+) T cell immune responses which may have a significant implication in the long-term protection. We provide novel information in cellular immune responses of SARS S antigen-specific CD8(+) T cells, which are important in the development of vaccine against SARS-CoV infection. Copyright © 2010 Elsevier Ltd. All rights reserved.

  18. Reconstitution of CD8 T Cells Protective against Cytomegalovirus in a Mouse Model of Hematopoietic Cell Transplantation: Dynamics and Inessentiality of Epitope Immunodominance

    PubMed Central

    Holtappels, Rafaela; Lemmermann, Niels A. W.; Podlech, Jürgen; Ebert, Stefan; Reddehase, Matthias J.

    2016-01-01

    Successful reconstitution of cytomegalovirus (CMV)-specific CD8+ T cells by hematopoietic cell transplantation (HCT) gives a favorable prognosis for the control of CMV reactivation and prevention of CMV disease after hematoablative therapy of hematopoietic malignancies. In the transient immunocompromised state after HCT, pre-emptive cytoimmunotherapy with viral epitope-specific effector or memory CD8+ T cells is a promising option to speed up antiviral control. Despite high-coding capacity of CMVs and a broad CD8+ T-cell response on the population level, which reflects polymorphism in major histocompatibility complex class-I (MHC-I) glycoproteins, the response in terms of quantity of CD8+ T cells in any individual is directed against a limited set of CMV-encoded epitopes selected for presentation by the private repertoire of MHC-I molecules. Such epitopes are known as “immunodominant” epitopes (IDEs). Besides host immunogenetics, genetic variance in CMV strains harbored as latent viruses by an individual HCT recipient can also determine the set of IDEs, which complicates a “personalized immunotherapy.” It is, therefore, an important question if IDE-specific CD8+ T-cell reconstitution after HCT is critical or dispensable for antiviral control. As viruses with targeted mutations of IDEs cannot be experimentally tested in HCT patients, we employed the well-established mouse model of HCT. Notably, control of murine CMV (mCMV) after HCT was comparably efficient for IDE-deletion mutant mCMV-Δ4IDE and the corresponding IDE-expressing revertant virus mCMV-Δ4IDE-rev. Thus, antigenicity-loss mutations in IDEs do not result in loss-of-function of a polyclonal CD8+ T-cell population. Although IDE deletion was not associated with global changes in the response to non-IDE epitopes, the collective of non-IDE-specific CD8+ T-cells infiltrates infected tissue and confines infection within nodular inflammatory foci. We conclude from the model, and predict also for human

  19. Influence of flanking sequences on presentation efficiency of a CD8+ cytotoxic T-cell epitope delivered by parvovirus-like particles.

    PubMed

    Rueda, P; Morón, G; Sarraseca, J; Leclerc, C; Casal, J I

    2004-03-01

    We have previously developed an antigen-delivery system based on hybrid recombinant porcine parvovirus-like particles (PPV-VLPs) formed by the self-assembly of the VP2 protein of PPV carrying a foreign epitope at its N terminus. In this study, different constructs were made containing a CD8(+) T-cell epitope of chicken ovalbumin (OVA) to analyse the influence of the sequence inserted into VP2 on the correct processing of VLPs by antigen-presenting cells. We analysed the presentation of the OVA epitope inserted without flanking sequences or with either different natural flanking sequences or with the natural flanking sequences of a CD8(+) T-cell epitope from the lymphocytic choriomeningitis virus nucleoprotein, and as a dimer with or without linker sequences. All constructs were studied in terms of level of expression, assembly of VLPs and ability to deliver the inserted epitope into the MHC I pathway. The presentation of the OVA epitope was considerably improved by insertion of short natural flanking sequences, which indicated the relevance of the flanking sequences on the processing of PPV-VLPs. Only PPV-VLPs carrying two copies of the OVA epitope linked by two glycines were able to be properly processed, suggesting that the introduction of flexible residues between the two consecutive OVA epitopes may be necessary for the correct presentation of these dimers by PPV-VLPs. These results provide information to improve the insertion of epitopes into PPV-VLPs to facilitate their processing and presentation by MHC class I molecules.

  20. Biological and immunological characterization of recombinant Yellow Fever 17D Viruses expressing a Trypanosoma cruzi Amastigote Surface Protein-2 CD8+ T cell epitope at two distinct regions of the genome

    PubMed Central

    2011-01-01

    Background The attenuated Yellow fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, in which it elicits a polyvalent immune response. Herein, we used the YF 17D backbone to express a Trypanosoma cruzi CD8+ T cell epitope from the Amastigote Surface Protein 2 (ASP-2) to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI CD8+ T cell epitope was cloned and expressed based on two different genomic insertion sites: in the fg loop of the viral Envelope protein and the protease cleavage site between the NS2B and NS3. We investigated whether the site of expression had any influence on immunogenicity of this model epitope. Results Recombinant viruses replicated similarly to vaccine virus YF 17D in cell culture and remained genetically stable after several serial passages in Vero cells. Immunogenicity studies revealed that both recombinant viruses elicited neutralizing antibodies to the YF virus as well as generated an antigen-specific gamma interferon mediated T-cell response in immunized mice. The recombinant viruses displayed a more attenuated phenotype than the YF 17DD vaccine counterpart in mice. Vaccination of a mouse lineage highly susceptible to infection by T. cruzi with a homologous prime-boost regimen of recombinant YF viruses elicited TEWETGQI specific CD8+ T cells which might be correlated with a delay in mouse mortality after a challenge with a lethal dose of T. cruzi. Conclusions We conclude that the YF 17D platform is useful to express T. cruzi (Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model T. cruzi epitope expressed at different regions of the YF 17D genome elicited a similar T cell-based immune response, suggesting that both expression sites are useful. However, the epitope as such is not protective and it remains to be seen whether expression of larger domains of ASP-2

  1. Biological and immunological characterization of recombinant Yellow Fever 17D viruses expressing a Trypanosoma cruzi Amastigote Surface Protein-2 CD8+ T cell epitope at two distinct regions of the genome.

    PubMed

    Nogueira, Raquel T; Nogueira, Alanderson R; Pereira, Mirian C S; Rodrigues, Maurício M; Galler, Ricardo; Bonaldo, Myrna C

    2011-03-18

    The attenuated Yellow fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, in which it elicits a polyvalent immune response. Herein, we used the YF 17D backbone to express a Trypanosoma cruzi CD8+ T cell epitope from the Amastigote Surface Protein 2 (ASP-2) to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI CD8+ T cell epitope was cloned and expressed based on two different genomic insertion sites: in the fg loop of the viral Envelope protein and the protease cleavage site between the NS2B and NS3. We investigated whether the site of expression had any influence on immunogenicity of this model epitope. Recombinant viruses replicated similarly to vaccine virus YF 17D in cell culture and remained genetically stable after several serial passages in Vero cells. Immunogenicity studies revealed that both recombinant viruses elicited neutralizing antibodies to the YF virus as well as generated an antigen-specific gamma interferon mediated T-cell response in immunized mice. The recombinant viruses displayed a more attenuated phenotype than the YF 17DD vaccine counterpart in mice. Vaccination of a mouse lineage highly susceptible to infection by T. cruzi with a homologous prime-boost regimen of recombinant YF viruses elicited TEWETGQI specific CD8+ T cells which might be correlated with a delay in mouse mortality after a challenge with a lethal dose of T. cruzi. We conclude that the YF 17D platform is useful to express T. cruzi (Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model T. cruzi epitope expressed at different regions of the YF 17D genome elicited a similar T cell-based immune response, suggesting that both expression sites are useful. However, the epitope as such is not protective and it remains to be seen whether expression of larger domains of ASP-2, which include the TEWETGQI

  2. Protective Effect of Human Leukocyte Antigen B27 in Hepatitis C Virus Infection Requires the Presence of a Genotype-Specific Immunodominant CD8+ T-Cell Epitope

    PubMed Central

    Kersting, Nadine; Fitzmaurice, Karen; Oniangue-Ndza, Cesar; Kemper, Michael N.; Humphreys, Isla; McKiernan, Susan; Kelleher, Dermot; Lohmann, Volker; Bowness, Paul; Huzly, Daniela; Rosen, Hugo R.; Kim, Arthur Y.; Lauer, Georg M.; Allen, Todd M.; Barnes, Eleanor; Roggendorf, Michael; Blum, Hubert E.; Thimme, Robert

    2015-01-01

    Human leukocyte antigen B27 (HLA-B27) is associated with protection in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. This protective role is linked to single immunodominant HLA-B27-restricted CD8+ T-cell epitopes in both infections. In order to define the relative contribution of a specific HLA-B27-restricted epitope to the natural course of HCV infection, we compared the biological impact of the highly conserved HCV genotype 1 epitope, for which the protective role has been described, with the corresponding region in genotype 3 that differs in its sequence by three amino acid residues. The genotype 3a peptide was not recognized by CD8+ T cells specific for the genotype 1 peptide. Furthermore, patients with acute or chronic infection with HCV genotype 3a did not mount T-cell responses to this epitope region, and their autologous viral sequences showed no evidence of T-cell pressure. Finally, we found a significantly higher frequency of HLA-B27 positivity in patients with chronic HCV genotype 3a infection compared to genotype 1 infection, indicating that there is no protection by HLA-B27 in HCV genotype 3 infection. Conclusion Our data indicate that the protective effect of HLA-B27 is limited to HCV genotype 1 infection and does not expand to other genotypes such as genotype 3a. This can most likely be explained by intergenotype sequence diversity leading to the loss of the immunodominant HLA-B27 epitope in viral strains other than genotype 1. Our results underline the central role of a single HLA-B27-restricted epitope-specific CD8+ T-cell response in mediating protection in HCV genotype 1 infection. PMID:20034048

  3. Polyfunctional CD8(+) T cells are associated with the vaccination-induced control of a novel recombinant influenza virus expressing an HCV epitope.

    PubMed

    Tan, Amabel C L; Eriksson, Emily M Y; Kedzierska, Katherine; Deliyannis, Georgia; Valkenburg, Sophie A; Zeng, Weiguang; Jackson, David C

    2012-05-01

    In hepatitis C virus (HCV) infection, CD8(+) T cell responses have been shown to be important in viral clearance. Examining the efficacy of CD8(+) T cell vaccines against HCV has been limited by the lack of an HCV infectious model in mice and the differences between MHC restriction in humans and mice. Using HLA-A2 transgenic HHD mice, we demonstrate that intranasally delivered Pam2Cys-based lipopeptides containing HLA-A2-restricted HCV epitopes can induce polyfunctional CD8(+) T cell responses in several organs including the liver. To examine the activity of these responses in an infectious context, we developed a recombinant influenza virus that expresses the NS5B(2594-2602) epitope from non-structural protein 5B of hepatitis C virus (PR8-HCV(NS5B)). We showed that mice inoculated with a lipopeptide containing the NS5B epitope had reduced viral loads following challenge with the PR8-HCV(NS5B) virus. This reduction was associated with the induction of NS5B(2594-2602)-specific IFN-γ and TNF-α co-producing CD8(+) T cells. The T cell receptor usage in the NS5B(2594-2602) response was found to exhibit a Vβ8.1/8.2 bias that was characterized by a narrow repertoire and a common CDR3β motif. This work has identified CD8(+) T cell functions induced by lipopeptides that are associated with viral control and demonstrate the potential of lipopeptide-based vaccines as candidates for treatment of HCV infection.

  4. Identification of HLA-A*2402-restricted HCMV immediate early-1 (IE-1) epitopes as targets for CD8+ HCMV-specific cytotoxic T lymphocytes.

    PubMed

    Lim, Jong-Baeck; Kim, Hyun Ok; Jeong, Seok Hoon; Ha, Joo Eun; Jang, Sunphil; Lee, Sang-Guk; Lee, Kyungwon; Stroncek, David

    2009-08-23

    To identify novel HLA-A*2402-restricted human cytomegalovirus (HCMV) immediate early-1 (IE-1) epitopes for adoptive immunotherapy, we explored 120 overlapping 15-amino acid spanning IE-1. These peptides were screened by measuring the frequency of polyclonal CD8+ T cells producing intracellular interferon-gamma (IFN-gamma) using flow cytometry and the epitopes were validated with a HCMV-infected target Cr release cytotoxicity assay. Initial screening was performed with 12 mini-pools of 10 consecutive peptides made from 120 overlapping peptides15-amino acids in length that spanned IE-1. When peripheral blood mononuclear cells (PBMCs) from HLA-A*2402 HCMV-seropositive donors were sensitized with each of the 12 mini-pools, mini-pools 1 and 2 induced the highest frequency of CD8+ cytotoxic T lymphocytes (CTLs) producing IFN-gamma. When PBMCs were stimulated with each of the twenty peptides belonging to mini-pools 1 and 2, peptides IE-11-15MESSAKRKMDPDNPD and IE-15-19AKRKMDPDNPDEGPS induced the greatest quantities of IFN-gamma production and cytotoxicity of HLA-matched HCMV-infected fibroblasts. To determine the exact HLA-A*2402-restricted epitopes within the two IE-1 proteins, we synthesized a total of twenty-one overlapping 9- or 10 amino acid peptides spanning IE-11-15 and IE-15-19. Peptide IE-13-12SSAKRKMDPD induced the greatest quantities of IFN-gamma production and target cell killing by CD8+ CTLs. HCMV IE-13-12SSAKRKMDPD is a HLA-A*2402-restricted HCMV IE-1 epitope that can serve as a common target for CD8+ HCMV-specific CTLs.

  5. A genital tract peptide epitope vaccine targeting TLR-2 efficiently induces local and systemic CD8 + T cells and protects against herpes simplex virus type 2 challenge

    PubMed Central

    Dasgupta, G; Nesburn, AB; Wu, M; Zhu, X; Carpenter, D; Wechsler, SL; You, S; BenMohamed, L

    2015-01-01

    The next generation of needle-free mucosal vaccines is being rationally designed according to rules that govern the way in which the epitopes are recognized by and stimulate the genital mucosal immune system. We hypothesized that synthetic peptide epitopes extended with an agonist of Toll-like receptor 2 (TLR-2), that are abundantly expressed by dendritic and epithelial cells of the vaginal mucosa, would lead to induction of protective immunity against genital herpes. To test this hypothesis, we intravaginally (IVAG) immunized wild-type B6, TLR-2 (TLR2 −/−) or myeloid differentiation factor 88 deficient (MyD88 −/−) mice with a herpes simplex virus type 2 (HSV-2) CD8 + T-cell peptide epitope extended by a palmitic acid moiety (a TLR-2 agonist). IVAG delivery of the lipopeptide generated HSV-2-specific memory CD8 + cytotoxic T cells both locally in the genital tract draining lymph nodes and systemically in the spleen. Moreover, lipopeptide-immunized TLR2 −/− and MyD88 −/− mice developed significantly less HSV-specific CD8 + T-cell response, earlier death, faster disease progression, and higher vaginal HSV-2 titers compared to lipopeptide-immunized wild-type B6 mice. IVAG immunization with self-adjuvanting lipid-tailed peptides appears to be a novel mucosal vaccine approach, which has attractive practical and immunological features. PMID:19129756

  6. CD8(+) T-cell Cytotoxic Capacity Associated with Human Immunodeficiency Virus-1 Control Can Be Mediated through Various Epitopes and Human Leukocyte Antigen Types.

    PubMed

    Migueles, Stephen A; Mendoza, Daniel; Zimmerman, Matthew G; Martins, Kelly M; Toulmin, Sushila A; Kelly, Elizabeth P; Peterson, Bennett A; Johnson, Sarah A; Galson, Eric; Poropatich, Kate O; Patamawenu, Andy; Imamichi, Hiromi; Ober, Alexander; Rehm, Catherine A; Jones, Sara; Hallahan, Claire W; Follmann, Dean A; Connors, Mark

    2015-01-01

    Understanding natural immunologic control over Human Immunodeficiency Virus (HIV)-1 replication, as occurs in rare long-term nonprogressors/elite controllers (LTNP/EC), should inform the design of efficacious HIV vaccines and immunotherapies. Durable control in LTNP/EC is likely mediated by highly functional virus-specific CD8(+) T-cells. Protective Human Leukocyte Antigen (HLA) class I alleles, like B*27 and B*57, are present in most, but not all LTNP/EC, providing an opportunity to investigate features shared by their HIV-specific immune responses. To better understand the contribution of epitope targeting and conservation to immune control, we compared the CD8(+) T-cell specificity and function of B*27/57(neg) LTNP/EC (n = 23), B*27/57(pos) LTNP/EC (n = 23) and B*27/57(neg) progressors (n = 13). Fine mapping revealed 11 previously unreported immunodominant responses. Although B*27/57(neg) LTNP/EC did not target more highly conserved epitopes, their CD8(+) T-cell cytotoxic capacity was significantly higher than progressors. Similar to B*27/57(pos) LTNP/EC, this superior cytotoxicity was mediated by preferential expansion of immunodominant responses and lysis through the predicted HLA. These findings suggest that increased CD8(+) T-cell cytotoxic capacity is a common mechanism of control in most LTNP/EC regardless of HLA type. They also suggest that potent cytotoxicity can be mediated through various epitopes and HLA molecules and could, in theory, be induced in most people.

  7. Control of HIV-1 replication in vitro by vaccine-induced human CD8+ T cells through conserved subdominant Pol epitopes

    PubMed Central

    Ahmed, Tina; Borthwick, Nicola J.; Gilmour, Jill; Hayes, Peter; Dorrell, Lucy; Hanke, Tomáš

    2016-01-01

    Objective The specificity of CD8+ T cells is critical for early control of founder/transmitted and reactivated HIV-1. To tackle HIV-1 variability and escape, we designed vaccine immunogen HIVconsv assembled from 14 highly conserved regions of mainly Gag and Pol proteins. When administered to HIV-1-negative human volunteers in trial HIV-CORE 002, HIVconsv vaccines elicited CD8+ effector T cells which inhibited replication of up to 8 HIV-1 isolates in autologous CD4+ cells. This inhibition correlated with interferon-γ production in response to Gag and Pol peptide pools, but direct evidence of the inhibitory specificity was missing. Here, we aimed to define through recognition of which epitopes these effectors inhibit HIV-1 replication. Design CD8+ T-cells from the 3 broadest HIV-1 inhibitors out of 23 vaccine recipients were expanded in culture by Gag or Pol peptide restimulation and tested in viral inhibition assay (VIA) using HIV-1 clade B and A isolates. Methods Frozen PBMCs were expanded first using peptide pools from Gag or Pol conserved regions and tested on HIV-1-infected cells in VIA or by individual peptides for their effector functions. Single peptide specificities responsible for inhibition of HIV-1 replication were then confirmed by single-peptide expanded effectors tested on HIV-1-infected cells. Results We formally demonstrated that the vaccine-elicited inhibitory human CD8+ T cells recognized conserved epitopes of both Pol and Gag proteins. We defined 7 minimum epitopes, of which 3 were novel, presumably naturally subdominant. The effectors were oligofunctional producing several cytokines and chemokines and killing peptide-pulsed target cells. Conclusions These results implicate the use of functionally conserved regions of Pol in addition to the widely used Gag for T-cell vaccine design. Proportion of volunteers developing these effectors and their frequency in circulating PBMC are separate issues, which can be addressed, if needed, by more efficient

  8. Cutting edge: DNAX accessory molecule 1-deficient CD8+ T cells display immunological synapse defects that impair antitumor immunity.

    PubMed

    Ramsbottom, Kelly M; Hawkins, Edwin D; Shimoni, Raz; McGrath, Mairi; Chan, Christopher J; Russell, Sarah M; Smyth, Mark J; Oliaro, Jane

    2014-01-15

    DNAX accessory molecule 1 (DNAM-1) is expressed on all CD8(+) T cells and promotes their activation and effector function. DNAM-1 interacts with LFA-1, a critical molecule for immunological synapse formation between T cells and APCs, and for cytotoxic killing of target cells. Mice that lack DNAM-1 display abnormal T cell responses and antitumor activity; however, the mechanism involved is unclear. In this article, we show that DNAM-1 deficiency results in reduced proliferation of CD8(+) T cells after Ag presentation and impaired cytotoxic activity. We also demonstrate that DNAM-1-deficient T cells show reduced conjugations with tumor cells and decreased recruitment of both LFA-1 and lipid rafts to the immunological synapse, which correlates with reduced tumor cell killing in vitro. This synapse defect may explain why DNAM-1-deficient mice cannot clear tumors in vivo, and highlights the importance of DNAM-1 and the immunological synapse in T cell-mediated antitumor immunity.

  9. Vaccine Targeting of Subdominant CD8+ T Cell Epitopes Increases the Breadth of the T Cell Response upon Viral Challenge, but May Impair Immediate Virus Control.

    PubMed

    Steffensen, Maria A; Pedersen, Louise H; Jahn, Marie L; Nielsen, Karen N; Christensen, Jan P; Thomsen, Allan R

    2016-03-15

    As a result of the difficulties in making efficient vaccines against genetically unstable viruses such as HIV, it has been suggested that future vaccines should preferentially target subdominant epitopes, the idea being that this should allow a greater breadth of the induced T cell response and, hence, a greater efficiency in controlling escape variants. However, to our knowledge the evidence supporting this concept is limited at best. To improve upon this, we used the murine lymphocytic choriomeningitis virus model and adenoviral vectors to compare a vaccine expressing unmodified Ag to a vaccine expressing the same Ag without its immunodominant epitope. We found that removal of the dominant epitope allowed the induction of CD8(+) T cell responses targeting at least two otherwise subdominant epitopes. Importantly, the overall magnitude of the induced T cell responses was similar, allowing us to directly compare the efficiency of these vaccines. Doing this, we observed that mice vaccinated with the vaccine expressing unmodified Ag more efficiently controlled an acute viral challenge. In the course of a more chronic viral infection, mice vaccinated using the vaccine targeting subdominant epitopes caught up with the conventionally vaccinated mice, and analysis of the breadth of the CD8(+) T cell response revealed that this was notably greater in the former mice. However, under the conditions of our studies, we never saw any functional advantage of this. This may represent a limitation of our model, but clearly our findings underscore the importance of carefully weighing the pros and cons of changes in epitope targeting before any implementation. Copyright © 2016 by The American Association of Immunologists, Inc.

  10. Identification of conserved subdominant HIV Type 1 CD8(+) T Cell epitopes restricted within common HLA Supertypes for therapeutic HIV Type 1 vaccines.

    PubMed

    Karlsson, Ingrid; Kløverpris, Henrik; Jensen, Kristoffer Jarlov; Stryhn, Anette; Buus, Søren; Karlsson, Annika; Vinner, Lasse; Goulder, Philip; Fomsgaard, Anders

    2012-11-01

    The high HIV-1 prevalence, up to 4.6% in Guinea-Bissau, West Africa, makes it a relevant location for testing of therapeutic vaccines. With the aim of performing a clinical study in Guinea-Bissau, after first testing the vaccine for safety in Denmark, Europe, we here describe the design of a universal epitope peptide-based T cell vaccine with relevance for any geographic locations. The two major obstacles when designing such a vaccine are the high diversities of the HIV-1 genome and of the human major histocompatibility complex (MHC) class I. We selected 15 CD8-restricted epitopes predicted from conserved regions of HIV-1 that were subdominant (i.e., infrequently targeted) within natural infections. Moreover, the epitopes were predicted to be restricted to at least one of the five common HLA supertypes (HLA-A01, A02, A03, B07, and B44). Here, we validated the resulting peptide-specific, HLA-restricted T cell specificities using peptide-MHC class I tetramer labeling of CD8(+) T cells from HIV-1-infected individuals. The selected vaccine epitopes are infrequently targeted in HIV-1-infected individuals from both locations. Moreover, we HLA-typed HIV-1-infected individuals and demonstrated that the selected vaccine epitopes, when targeted, are restricted to the five most common HLA supertypes at both locations. Thus, the HLA supertype-directed approach achieved HLA coverage of 95% and 100% of the examined cohorts in Guinea-Bissau and Denmark, respectively. In conclusion, the selected vaccine epitopes match the host populations and HIV-1 strains of these two distant geographic regions, justifying clinical testing in both locations.

  11. Sterile Immunity to Malaria after DNA Prime/Adenovirus Boost Immunization Is Associated with Effector Memory CD8+T Cells Targeting AMA1 Class I Epitopes

    PubMed Central

    Sedegah, Martha; Hollingdale, Michael R.; Farooq, Fouzia; Ganeshan, Harini; Belmonte, Maria; Kim, Yohan; Peters, Bjoern; Sette, Alessandro; Huang, Jun; McGrath, Shannon; Abot, Esteban; Limbach, Keith; Shi, Meng; Soisson, Lorraine; Diggs, Carter; Chuang, Ilin; Tamminga, Cindy; Epstein, Judith E.; Villasante, Eileen; Richie, Thomas L.

    2014-01-01

    Background Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection. Methodology/Principal Findings We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans. Conclusions/Significance We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches

  12. The generation of CD8+ T-cell population specific for vaccinia virus epitope involved in the antiviral protection against ectromelia virus challenge.

    PubMed

    Gierynska, Malgorzata; Szulc-Dabrowska, Lidia; Dzieciatkowski, Tomasz; Golke, Anna; Schollenberger, Ada

    2015-12-01

    Eradication of smallpox has led to cessation of vaccination programs. This has rendered the human population increasingly susceptible not only to variola virus infection but also to infections with other representatives of Poxviridae family that cause zoonotic variola-like diseases. Thus, new approaches for designing improved vaccine against smallpox are required. Discovering that orthopoxviruses, e.g. variola virus, vaccinia virus, ectromelia virus, share common immunodominant antigen, may result in the development of such a vaccine. In our study, the generation of antigen-specific CD8(+) T cells in mice during the acute and memory phase of the immune response was induced using the vaccinia virus immunodominant TSYKFESV epitope and CpG oligodeoxynucleotides as adjuvants. The role of the generated TSYKFESV-specific CD8(+) T cells was evaluated in mice during ectromelia virus infection using systemic and mucosal model. Moreover, the involvement of dendritic cells subsets in the adaptive immune response stimulation was assessed. Our results indicate that the TSYKFESV epitope/TLR9 agonist approach, delivered systemically or mucosally, generated strong CD8(+) T-cell response when measured 10 days after immunization. Furthermore, the TSYKFESV-specific cell population remained functionally active 2 months post-immunization, and gave cross-protection in virally challenged mice, even though the numbers of detectable antigen-specific T cells decreased.

  13. CD8+ T cell recognition of epitopes within the capsid of adeno-associated virus 8-based gene transfer vectors depends on vectors' genome.

    PubMed

    Wu, Te-Lang; Li, Hua; Faust, Susan M; Chi, Emily; Zhou, Shangzhen; Wright, Fraser; High, Katherine A; Ertl, Hildegund C J

    2014-01-01

    Self-complementary adeno-associated viral (AAV) vectors expressing human factor IX (hF.IX) have achieved transient or sustained correction of hemophilia B in human volunteers. High doses of AAV2 or AAV8 vectors delivered to the liver caused in several patients an increase in transaminases accompanied by a rise in AAV capsid-specific T cells and a decrease in circulating hF.IX levels suggesting immune-mediated destruction of vector-transduced cells. Kinetics of these adverse events differed in patients receiving AAV2 or AAV8 vectors causing rise in transaminases at 3 versus 8 weeks after vector injection, respectively. To test if CD8+ T cells to AAV8 vectors, which are similar to AAV2 vectors are fully-gutted vectors and thereby fail to encode structural viral proteins, could cause damage at this late time point, we tested in a series of mouse studies how long major histocompatibility (MHC) class I epitopes within AAV8 capsid can be presented to CD8+ T cells. Our results clearly show that depending on the vectors' genome, CD8+ T cells can detect such epitopes on AAV8's capsid for up to 6 months indicating that the capsid of AAV8 degrades slowly in mice.

  14. Gene Expression Driven by a Strong Viral Promoter in MVA Increases Vaccination Efficiency by Enhancing Antibody Responses and Unmasking CD8⁺ T Cell Epitopes.

    PubMed

    Becker, Pablo D; Nörder, Miriam; Weissmann, Sebastian; Ljapoci, Ronny; Erfle, Volker; Drexler, Ingo; Guzmán, Carlos A

    2014-07-22

    Viral vectors are promising tools for vaccination strategies and immunotherapies. However, CD8⁺ T cell responses against pathogen-derived epitopes are usually limited to dominant epitopes and antibody responses to recombinant encoded antigens (Ags) are mostly weak. We have previously demonstrated that the timing of viral Ag expression in infected professional Ag-presenting cells strongly shapes the epitope immunodominance hierarchy. T cells recognizing determinants derived from late viral proteins have a clear disadvantage to proliferate during secondary responses. In this work we evaluate the effect of overexpressing the recombinant Ag using the modified vaccinia virus early/late promoter H5 (mPH5). Although the Ag-expression from the natural promoter 7.5 (P7.5) and the mPH5 seemed similar, detailed analysis showed that mPH5 not only induces higher expression levels than P7.5 during early phase of infection, but also Ag turnover is enhanced. The strong overexpression during the early phase leads to broader CD8 T cell responses, while preserving the priming efficiency of stable Ags. Moreover, the increase in Ag-secretion favors the induction of strong antibody responses. Our findings provide the rationale to develop new strategies for fine-tuning the responses elicited by recombinant modified vaccinia virus Ankara by using selected promoters to improve the performance of this viral vector.

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

    NASA Astrophysics Data System (ADS)

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

    2017-02-01

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

  16. Human Memory Cytotoxic T-Lymphocyte (CTL) Responses to Hantaan Virus Infection: Identification of Virus-Specific and Cross-Reactive CD8+ CTL Epitopes on Nucleocapsid Protein

    PubMed Central

    Van Epps, Heather L.; Schmaljohn, Connie S.; Ennis, Francis A.

    1999-01-01

    Hantaan virus, the prototypic member of the Hantavirus genus, causes hemorrhagic fever with renal syndrome in humans. We examined the human memory T-lymphocyte responses of three donors who had previous laboratory-acquired infections with Hantaan virus. We demonstrated virus-specific responses in bulk cultures of peripheral blood mononuclear cells (PBMC) from all donors. Bulk T-cell responses were directed against either Hantaan virus nucleocapsid (N) or G1 protein, and these responses varied between donors. We established both CD4+ and CD8+ N-specific cell lines from two donors and CD4+ G1-specific cell lines from a third donor. All CD8+ cytotoxic T-lymphocyte (CTL) lines recognized one of two epitopes on the nucleocapsid protein: one epitope spanning amino acids 12 to 20 and the other spanning amino acids 421 to 429. The CTL lines specific for amino acids 12 to 20 were restricted by HLA B51, and those specific for amino acids 421 to 429 were restricted by HLA A1. The N-specific CTL lines isolated from these two donors included both Hantaan virus-specific CTLs and hantavirus cross-reactive CTLs. Responses to both epitopes are detectable in short-term bulk cultures of PBMC from one donor, and precursor frequency analysis confirms that CTLs specific for these epitopes are present at relatively high precursor frequencies in the peripheral T-cell pool. These data suggest that infection with Hantaan virus results in the generation of CTL to limited epitopes on the nucleocapsid protein and that infection also results in the generation of cross-reactive T-cell responses to distantly related hantaviruses which cause the distinct hantavirus pulmonary syndrome. This is the first demonstration of human T-lymphocyte responses to Hantaan virus. PMID:10364276

  17. Of Mice and not Humans: How Reliable are Animal Models for Evaluation of Herpes CD8+-T cell-Epitopes-Based Immunotherapeutic Vaccine Candidates?

    PubMed Central

    Dasgupta, Gargi; BenMohamed, Lbachir

    2011-01-01

    Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) -specific CD8+ T cells that reside in sensory ganglia, appears to control recurrent herpetic disease by aborting or reducing spontaneous and sporadic reactivations of latent virus. A reliable animal model is the ultimate key factor to test the efficacy of therapeutic vaccines that boost the level and the quality of sensory ganglia-resident CD8+ T cells against spontaneous herpes reactivation from sensory neurons, yet its relevance has been often overlooked. Herpes vaccinologists are hesitant about using mouse as a model in pre-clinical development of therapeutic vaccines because they do not adequately mimic spontaneous viral shedding or recurrent symptomatic diseases, as occurs in human. Alternatives to mouse models are rabbits and guinea pigs in which reactivation arise spontaneously with clinical features relevant to human disease. However, while rabbits and guinea pigs develop spontaneous HSV reactivation and recurrent ocular and genital disease none of them can mount CD8+ T cell responses specific to Human Leukocyte Antigen- (HLA-) restricted epitopes. In this review, we discuss the advantages and limitations of these animal models and describe a novel “humanized” HLA transgenic rabbit, which shows spontaneous HSV-1 reactivation, recurrent ocular disease and mounts CD8+ T cell responses to HLA-restricted epitopes. Adequate investments are needed to develop reliable preclinical animal models, such as HLA class I and class II double transgenic rabbits and guinea pigs to balance the ethical and financial concerns associated with the rising number of unsuccessful clinical trials for therapeutic vaccine formulations tested in unreliable mouse models. PMID:21718746

  18. Localization of CD8 T cell epitope within cardiac myosin heavy chain-α334-352 that induces autoimmune myocarditis in A/J mice.

    PubMed

    Massilamany, Chandirasegaran; Gangaplara, Arunakumar; Basavalingappa, Rakesh H; Rajasekaran, Rajkumar A; Khalilzad-Sharghi, Vahid; Han, Zhongji; Othman, Shadi; Steffen, David; Reddy, Jay

    2016-01-01

    Cardiac myosin heavy chain-α (Myhc), an intracellular protein expressed in the cardiomyocytes, has been identified as a major autoantigen in cardiac autoimmunity. In our studies with Myhc334-352-induced experimental autoimmune myocarditis in A/J mice (H-2a), we discovered that Myhc334-352, supposedly a CD4 T cell epitope, also induced antigen-specific CD8 T cells that transfer disease to naive animals. In our efforts to identify the CD8 T cell determinants, we localized Myhc338-348 within the full length-Myhc334-352, leading to four key findings. (1) By acting as a dual epitope, Myhc338-348 induces both CD4 and CD8 T cell responses. (2) In a major histocompatibility complex (MHC) class I-stabilization assay, Myhc338-348 was found to bind H-2Dd-but not H-2Kk or H-2Ld-alleles. (3) The CD8 T cell response induced by Myhc338-348 was antigen-specific, as evaluated by MHC class I/H-2Dd dextramer staining. The antigen-sensitized T cells predominantly produced interferon-γ, the critical cytokine of effector cytotoxic T lymphocytes. (4) Myhc338-348 was found to induce myocarditis in immunized animals as determined by histology and magnetic resonance microscopy imaging. Our data provide new insights as to how different immune cells can recognize the same antigen and inflict damage through different mechanisms. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  19. A Novel CD8-Independent High-Avidity Cytotoxic T-Lymphocyte Response Directed against an Epitope in the Phosphoprotein of the Paramyxovirus Simian Virus 5

    PubMed Central

    Gray, Peter M.; Parks, Griffith D.; Alexander-Miller, Martha A.

    2001-01-01

    Adoptive transfer studies have shown that cytotoxic T lymphocytes (CTL) of high avidity, capable of recognizing low levels of peptide-MHC I molecules, are more efficient at reducing viral titers than are low-avidity CTL, thus establishing CTL avidity as a critical parameter for the ability of a CTL to clear virus in vivo. It has been well documented that CTL of high avidity are relatively CD8 independent, whereas low-avidity CTL require CD8 engagement in order to become activated. In this study we have analyzed the antiviral CTL response elicited following infection with the paramyxovirus simian virus 5 (SV5). We have identified the immunodominant and subdominant CTL responses and subsequently assessed the avidity of these responses by their CD8 dependence. This is the first study in which the relationship between immunodominance and CTL avidity has been investigated. The immunodominant response was directed against an epitope present in the viral M protein, and subdominant responses were directed against epitopes present in the P, F, and HN proteins. Similarly to other CTL responses we have analyzed, the immunodominant response and the subdominant F and HN responses were comprised of both high- and low-avidity CTL. However, the subdominant response directed against the epitope present in the P protein is novel, as it is exclusively high avidity. This high-avidity response is independent of both the route of infection and expression by recombinant SV5. A further understanding of the inherent properties of P that elicit only high-avidity CTL may allow for the design of more efficacious vaccine vectors that preferentially elicit high-avidity CTL in vivo. PMID:11581375

  20. A Chimera Containing CD4+ and CD8+ T-Cell Epitopes of the Leishmania donovani Nucleoside Hydrolase (NH36) Optimizes Cross-Protection against Leishmania amazonesis Infection

    PubMed Central

    Alves-Silva, Marcus Vinícius; Nico, Dirlei; Morrot, Alexandre; Palatnik, Marcos; Palatnik-de-Sousa, Clarisa B.

    2017-01-01

    The Leishmania donovani nucleoside hydrolase (NH36) and NH A34480 of Leishmania amazonensis share 93% of sequence identity. In mice, the NH36 induced protection against visceral leishmaniasis is mediated by a CD4+ T cell response against its C-terminal domain (F3). Besides this CD4+ Th1 response, prevention and cure of L. amazonensis infection require also additional CD8+ and regulatory T-cell responses to the NH36 N-terminal (F1 domain). We investigated if mice vaccination with F1 and F3 domains cloned in tandem, in a recombinant chimera, with saponin, optimizes the vaccine efficacy against L. amazonensis infection above the levels promoted by the two admixed domains or by each domain independently. The chimera induced the highest IgA, IgG, and IgG2a anti-NH36 antibody, IDR, IFN-γ, and IL-10 responses, while TNF-α was more secreted by mice vaccinated with F3 or all F3-contaning vaccines. Additionally, the chimera and the F1 vaccine also induced the highest proportions of CD4+ and CD8+ T cells secreting IL-2, TNF-α, or IFN-γ alone, TNF-α in combination with IL-2 or IFN-γ, and of CD4+ multifunctional cells secreting IL-2, TNF-α, and IFN-γ. Correlating with the immunological results, the strongest reductions of skin lesions sizes were determined by the admixed domains (80%) and by the chimera (84%), which also promoted the most pronounced and significant reduction of the parasite load (99.8%). Thus, the epitope presentation in a recombinant chimera optimizes immunogenicity and efficacy above the levels induced by the independent or admixed F1 and F3 domains. The multiparameter analysis disclosed that the Th1-CD4+ T helper response induced by the chimera is mainly directed against its FRYPRPKHCHTQVA epitope. Additionally, the YPPEFKTKL epitope of F1 induced the second most important CD4+ T cell response, and, followed by the DVAGIVGVPVAAGCT, FMLQILDFYTKVYE, and ELLAITTVVGNQ sequences, also the most potent CD8+ T cell responses and IL-10 secretion. Remarkably

  1. A Chimera Containing CD4+ and CD8+ T-Cell Epitopes of the Leishmania donovani Nucleoside Hydrolase (NH36) Optimizes Cross-Protection against Leishmania amazonesis Infection.

    PubMed

    Alves-Silva, Marcus Vinícius; Nico, Dirlei; Morrot, Alexandre; Palatnik, Marcos; Palatnik-de-Sousa, Clarisa B

    2017-01-01

    The Leishmania donovani nucleoside hydrolase (NH36) and NH A34480 of Leishmania amazonensis share 93% of sequence identity. In mice, the NH36 induced protection against visceral leishmaniasis is mediated by a CD4+ T cell response against its C-terminal domain (F3). Besides this CD4+ Th1 response, prevention and cure of L. amazonensis infection require also additional CD8+ and regulatory T-cell responses to the NH36 N-terminal (F1 domain). We investigated if mice vaccination with F1 and F3 domains cloned in tandem, in a recombinant chimera, with saponin, optimizes the vaccine efficacy against L. amazonensis infection above the levels promoted by the two admixed domains or by each domain independently. The chimera induced the highest IgA, IgG, and IgG2a anti-NH36 antibody, IDR, IFN-γ, and IL-10 responses, while TNF-α was more secreted by mice vaccinated with F3 or all F3-contaning vaccines. Additionally, the chimera and the F1 vaccine also induced the highest proportions of CD4+ and CD8+ T cells secreting IL-2, TNF-α, or IFN-γ alone, TNF-α in combination with IL-2 or IFN-γ, and of CD4+ multifunctional cells secreting IL-2, TNF-α, and IFN-γ. Correlating with the immunological results, the strongest reductions of skin lesions sizes were determined by the admixed domains (80%) and by the chimera (84%), which also promoted the most pronounced and significant reduction of the parasite load (99.8%). Thus, the epitope presentation in a recombinant chimera optimizes immunogenicity and efficacy above the levels induced by the independent or admixed F1 and F3 domains. The multiparameter analysis disclosed that the Th1-CD4+ T helper response induced by the chimera is mainly directed against its FRYPRPKHCHTQVA epitope. Additionally, the YPPEFKTKL epitope of F1 induced the second most important CD4+ T cell response, and, followed by the DVAGIVGVPVAAGCT, FMLQILDFYTKVYE, and ELLAITTVVGNQ sequences, also the most potent CD8+ T cell responses and IL-10 secretion. Remarkably

  2. Early immunization induces persistent tumor-infiltrating CD8+ T cells against an immunodominant epitope and promotes lifelong control of pancreatic tumor progression in SV40 tumor antigen transgenic mice.

    PubMed

    Otahal, Pavel; Schell, Todd D; Hutchinson, Sandra C; Knowles, Barbara B; Tevethia, Satvir S

    2006-09-01

    The ability to recruit the host's CD8+ T lymphocytes (T(CD8)) against cancer is often limited by the development of peripheral tolerance toward the dominant tumor-associated Ags. Because multiple epitopes derived from a given tumor Ag (T Ag) can be targeted by T(CD8), vaccine approaches should be directed toward those T(CD8) that are more likely to survive under conditions of persistent Ag expression. In this study, we investigated the effect of peripheral tolerance on the endogenous T(CD8) response toward two epitopes, designated epitopes I and IV, from the SV40 large T Ag. Using rat insulin promoter (RIP) 1-Tag4 transgenic mice that express T Ag from the RIP and develop pancreatic insulinomas, we demonstrate that epitope IV- but not epitope I-specific T(CD8) are maintained long term in tumor-bearing RIP1-Tag4 mice. Even large numbers of TCR-transgenic T cells specific for epitope I were rapidly eliminated from RIP1-Tag4 mice after adoptive transfer and recognition of the endogenous T Ag. Importantly, immunization of RIP1-Tag4 mice at 5 wk of age against epitope IV resulted in complete protection from tumor progression over a 2-year period despite continued expression of T Ag in the pancreas. This extensive control of tumor progression was associated with the persistence of functional epitope IV-specific T(CD8) within the pancreas for the lifetime of the mice without the development of diabetes. This study indicates that an equilibrium is reached in which immune surveillance for spontaneous cancer can be achieved for the lifespan of the host while maintaining normal organ function.

  3. Identification of CD8+ cytotoxic T lymphocyte epitopes from porcine reproductive and respiratory syndrome virus matrix protein in BALB/c mice

    PubMed Central

    2011-01-01

    Twenty-seven nanopeptides derived from the matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were screened for their ability to elicit a recall interferon-γ (IFN-γ) response from the splenocytes of BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus rWR-PRRSV-M. We identified two peptides (amino acid residues K93FITSRCRL and F57GYMTFVHF) as CD8+ cytotoxic T lymphocyte (CTL) epitopes. These peptides elicited significant numbers of IFN-γ secreting cells, compared with other M nonapeptides and one irrelevant nonapeptide. Bioinformatics analysis showed that the former is an H-2Kd-restricted CTL epitope, and the latter is an H-2Dd-restricted CTL epitope. Multiple amino acid sequence alignment among different PRRSV M sequences submitted to GenBank indicated that these two CTL epitopes are strongly conserved, and they should therefore be considered for further research on the mechanisms of cellular immune responses to PRRSV. PMID:21619712

  4. Mutational escape of CD8+ T cell epitopes: implications for prevention and therapy of persistent hepatitis virus infections.

    PubMed

    Timm, Joerg; Walker, Christopher M

    2015-02-01

    Over the past two decades, much has been learned about how human viruses evade T cell immunity to establish persistent infection. The lessons are particularly relevant to two hepatotropic viruses, HBV and HCV, that are very significant global public health problems. Although HCV and HBV are very different, the natural history of persistent infections with these viruses in humans shares some common features including failure of T cell immunity. During recent years, large sequence studies of HCV have characterized intra-host evolution as well as sequence diversity between hosts in great detail. Combined with studies of CD8+ T cell phenotype and function, it is now apparent that the T cell response shapes viral evolution. In turn, HCV sequence diversity influences the quality of the CD8+ T cell response and thus infection outcome. Here, we review published studies of CD8+ T cell selection pressure and mutational escape of the virus. Potential consequences for therapeutic strategies to restore T cell immunity against persistent human viruses, most notably HBV, are discussed.

  5. Definition of an epitope on Japanese encephalitis virus (JEV) envelope protein recognized by JEV-specific murine CD8+ cytotoxic T lymphocytes.

    PubMed

    Takada, K; Masaki, H; Konishi, E; Takahashi, M; Kurane, I

    2000-01-01

    We defined an epitope on the Japanese encephalitis virus (JEV) envelope (E) protein recognized by CD8+ cytotoxic T lymphocytes (CTLs). CTLs induced in JEV-infected BALB/c (H-2d) mice recognized E and/or premembrane (PrM) proteins, while CTLs in C57BL/6J (H-2b) and C3H/HeJ (H-2k) mice did not. JEV-specific CTLs had a phenotype of CD3+ CD4- CD8+. Twenty-four 9-amino acid (a.a.) peptides, which had binding motifs for H-2Kd, H-2Ld or H-2Dd, were synthesized according to the amino acid sequences of PrM and E proteins. CTLs induced by JEV infection recognized only the peptide K-3. Immunization of BALB/c mice with only a group of peptides including K-3 induced CTLs which recognized the homologous K-3 peptide, while immunization with other peptides did not. The peptide K-3 had a binding motif for H-2Kd. This is consistent with the finding that JEV-specific CTLs in BALB/c mice was H-2Kd-restricted. These results indicate that the epitope recognized by CTLs in BALB/c mice is located between a.a. 60 and 68 on the E protein, corresponding to an a.a. sequence of CYHASVTDI.

  6. Control of HIV-1 replication in vitro by vaccine-induced human CD8(+) T cells through conserved subdominant Pol epitopes.

    PubMed

    Ahmed, Tina; Borthwick, Nicola J; Gilmour, Jill; Hayes, Peter; Dorrell, Lucy; Hanke, Tomáš

    2016-02-24

    The specificity of CD8(+) T cells is critical for early control of founder/transmitted and reactivated HIV-1. To tackle HIV-1 variability and escape, we designed vaccine immunogen HIVconsv assembled from 14 highly conserved regions of mainly Gag and Pol proteins. When administered to HIV-1-negative human volunteers in trial HIV-CORE 002, HIVconsv vaccines elicited CD8(+) effector T cells which inhibited replication of up to 8 HIV-1 isolates in autologous CD4(+) cells. This inhibition correlated with interferon-γ production in response to Gag and Pol peptide pools, but direct evidence of the inhibitory specificity was missing. Here, we aimed to define through recognition of which epitopes these effectors inhibit HIV-1 replication. CD8(+) T-cells from the 3 broadest HIV-1 inhibitors out of 23 vaccine recipients were expanded in culture by Gag or Pol peptide restimulation and tested in viral inhibition assay (VIA) using HIV-1 clade B and A isolates. Frozen PBMCs were expanded first using peptide pools from Gag or Pol conserved regions and tested on HIV-1-infected cells in VIA or by individual peptides for their effector functions. Single peptide specificities responsible for inhibition of HIV-1 replication were then confirmed by single-peptide expanded effectors tested on HIV-1-infected cells. We formally demonstrated that the vaccine-elicited inhibitory human CD8(+) T cells recognized conserved epitopes of both Pol and Gag proteins. We defined 7 minimum epitopes, of which 3 were novel, presumably naturally subdominant. The effectors were oligofunctional producing several cytokines and chemokines and killing peptide-pulsed target cells. These results implicate the use of functionally conserved regions of Pol in addition to the widely used Gag for T-cell vaccine design. Proportion of volunteers developing these effectors and their frequency in circulating PBMC are separate issues, which can be addressed, if needed, by more efficient vector and regimen delivery of

  7. The role of a glycoprotein K (gK) CD8+ T-cell epitope of herpes simplex virus on virus replication and pathogenicity.

    PubMed

    Mott, Kevin R; Chentoufi, Aziz A; Carpenter, Dale; BenMohamed, Lbachir; Wechsler, Steven L; Ghiasi, Homayon

    2009-06-01

    The authors recently reported that a recombinant HSV-1 expressing two extra copies of glycoprotein K (gK) exacerbated corneal scarring (CS) in mice. The authors also identified a peptide, STVVLITAYGLVLVW, within the signal sequence of gK as an immunodominant gK T-cell-stimulatory region both in vitro and in vivo and identified a highly conserved potential CD8(+) T-cell epitope (ITAYGLVL) within the peptide. In this study, the effect of giving this octamer (8mer) as an eye drop 1 hour before ocular infection with HSV-1 was investigated. Naive mice and rabbits received the gK 8mer or control peptides as eye drops and were then ocularly infected with HSV-1. Virus replication in the eye and trigeminal ganglia (TG), survival, CS, and relative amounts of gB, gK, CD4, CD8, IFN-gamma, and granzyme A/B transcripts were determined in the cornea and TG of infected animals at various times after infection. The effect of the gK 8mer was also analyzed in immunized HLA transgenic mice. The gK 8mer resulted in a short-term significant increase in virus replication in the eyes of BALB/c mice, C57BL/6 mice, and NZW rabbits. gK 8mer treatment also increased viral neurovirulence and viral induced CS in ocularly infected mice. Moreover, in HSV-infected humanized HLA-A*0201 transgenic mice, the gK 8mer epitope induced strong IFN-gamma-producing cytotoxic CD8(+) T-cell responses, as assessed by CD107a/b expression and IFN-gamma ELISAs. gK 8mer induced CD8(+) T-cell responses were unlikely to occur soon enough to account for increased virus replication on day 1 after infection. In contrast, the data are consistent with CD8(+) T cells being involved in the appearance of CS at late times after infection. In addition, the gK peptide may affect viral replication and innate immune responses through other undefined mechanisms.

  8. Selection Pressure in CD8+ T-cell Epitopes in the pol Gene of HIV-1 Infected Individuals in Colombia. A Bioinformatic Approach

    PubMed Central

    Acevedo-Sáenz, Liliana; Ochoa, Rodrigo; Rugeles, Maria Teresa; Olaya-García, Patricia; Velilla-Hernández, Paula Andrea; Diaz, Francisco J.

    2015-01-01

    One of the main characteristics of the human immunodeficiency virus is its genetic variability and rapid adaptation to changing environmental conditions. This variability, resulting from the lack of proofreading activity of the viral reverse transcriptase, generates mutations that could be fixed either by random genetic drift or by positive selection. Among the forces driving positive selection are antiretroviral therapy and CD8+ T-cells, the most important immune mechanism involved in viral control. Here, we describe mutations induced by these selective forces acting on the pol gene of HIV in a group of infected individuals. We used Maximum Likelihood analyses of the ratio of non-synonymous to synonymous mutations per site (dN/dS) to study the extent of positive selection in the protease and the reverse transcriptase, using 614 viral sequences from Colombian patients. We also performed computational approaches, docking and algorithmic analyses, to assess whether the positively selected mutations affected binding to the HLA molecules. We found 19 positively-selected codons in drug resistance-associated sites and 22 located within CD8+ T-cell epitopes. A high percentage of mutations in these epitopes has not been previously reported. According to the docking analyses only one of those mutations affected HLA binding. However, algorithmic methods predicted a decrease in the affinity for the HLA molecule in seven mutated peptides. The bioinformatics strategies described here are useful to identify putative positively selected mutations associated with immune escape but should be complemented with an experimental approach to define the impact of these mutations on the functional profile of the CD8+ T-cells. PMID:25803098

  9. SIV Genome-Wide Pyrosequencing Provides a Comprehensive and Unbiased View of Variation within and outside CD8 T Lymphocyte Epitopes

    PubMed Central

    Hughes, Austin L.; Becker, Ericka A.; Lauck, Michael; Karl, Julie A.; Braasch, Andrew T.; O’Connor, David H.; O’Connor, Shelby L.

    2012-01-01

    Deep sequencing technology is revolutionizing our understanding of HIV/SIV evolution. It is known that acute SIV sequence variation within CD8 T lymphocyte (CD8-TL) epitopes is similar among MHC-identical animals, but we do not know whether this persists into the chronic phase. We now determine whether chronic viral variation in MHC-identical animals infected with clonal SIV is similar throughout the entire coding sequence when using a sensitive deep sequencing approach. We pyrosequenced the entire coding sequence of the SIV genome isolated from a unique cohort of four SIVmac239-infected, MHC-identical Mauritian cynomolgus macaques (MCM) 48 weeks after infection; one MCM in the cohort became an elite controller. Among the three non-controllers, we found that genome-wide sequences were similar between animals and we detected increased sequence complexity within 64% of CD8-TL epitopes when compared to Sanger sequencing methods. When we compared sequences between the MHC-matched controller and the three non-controllers, we found the viral population in the controller was less diverse and accumulated different variants than the viral populations in the non-controllers. Importantly, we found that initial PCR amplification of viral cDNA did not significantly affect the sequences detected, suggesting that data obtained by pyrosequencing PCR-amplified viral cDNA accurately represents the diversity of sequences replicating within an animal. This demonstrates that chronic sequence diversity across the entire SIV coding sequence is similar among MHC-identical animals with comparable viral loads when infected with the same clonal virus stock. Additionally, our approach to genome-wide SIV sequencing accurately reflects the diversity of sequences present in the replicating viral population. In sum, our study suggests that genome-wide pyrosequencing of immunodeficiency viruses captures a thorough and unbiased picture of sequence diversity, and may be a useful approach to employ

  10. Selection pressure in CD8⁺ T-cell epitopes in the pol gene of HIV-1 infected individuals in Colombia. A bioinformatic approach.

    PubMed

    Acevedo-Sáenz, Liliana; Ochoa, Rodrigo; Rugeles, Maria Teresa; Olaya-García, Patricia; Velilla-Hernández, Paula Andrea; Diaz, Francisco J

    2015-03-20

    One of the main characteristics of the human immunodeficiency virus is its genetic variability and rapid adaptation to changing environmental conditions. This variability, resulting from the lack of proofreading activity of the viral reverse transcriptase, generates mutations that could be fixed either by random genetic drift or by positive selection. Among the forces driving positive selection are antiretroviral therapy and CD8+ T-cells, the most important immune mechanism involved in viral control. Here, we describe mutations induced by these selective forces acting on the pol gene of HIV in a group of infected individuals. We used Maximum Likelihood analyses of the ratio of non-synonymous to synonymous mutations per site (dN/dS) to study the extent of positive selection in the protease and the reverse transcriptase, using 614 viral sequences from Colombian patients. We also performed computational approaches, docking and algorithmic analyses, to assess whether the positively selected mutations affected binding to the HLA molecules. We found 19 positively-selected codons in drug resistance-associated sites and 22 located within CD8+ T-cell epitopes. A high percentage of mutations in these epitopes has not been previously reported. According to the docking analyses only one of those mutations affected HLA binding. However, algorithmic methods predicted a decrease in the affinity for the HLA molecule in seven mutated peptides. The bioinformatics strategies described here are useful to identify putative positively selected mutations associated with immune escape but should be complemented with an experimental approach to define the impact of these mutations on the functional profile of the CD8+ T-cells.

  11. Adoptive transfer of Mammaglobin-A epitope specific CD8 T cells combined with a single low dose of total body irradiation eradicates breast tumors.

    PubMed

    Lerret, Nadine M; Rogozinska, Magdalena; Jaramillo, Andrés; Marzo, Amanda L

    2012-01-01

    Adoptive T cell therapy has proven to be beneficial in a number of tumor systems by targeting the relevant tumor antigen. The tumor antigen targeted in our model is Mammaglobin-A, expressed by approximately 80% of human breast tumors. Here we evaluated the use of adoptively transferred Mammaglobin-A specific CD8 T cells in combination with low dose irradiation to induce breast tumor rejection and prevent relapse. We show Mammaglobin-A specific CD8 T cells generated by DNA vaccination with all epitopes (Mammaglobin-A2.1, A2.2, A2.4 and A2.6) and full-length DNA in vivo resulted in heterogeneous T cell populations consisting of both effector and central memory CD8 T cell subsets. Adoptive transfer of spleen cells from all Mammaglobin-A2 immunized mice into tumor-bearing SCID/beige mice induced tumor regression but this anti-tumor response was not sustained long-term. Additionally, we demonstrate that only the adoptive transfer of Mammaglobin-A2 specific CD8 T cells in combination with a single low dose of irradiation prevents tumors from recurring. More importantly we show that this single dose of irradiation results in the down regulation of the macrophage scavenger receptor 1 on dendritic cells within the tumor and reduces lipid uptake by tumor resident dendritic cells potentially enabling the dendritic cells to present tumor antigen more efficiently and aid in tumor clearance. These data reveal the potential for adoptive transfer combined with a single low dose of total body irradiation as a suitable therapy for the treatment of established breast tumors and the prevention of tumor recurrence.

  12. CD8+ T-Lymphocyte Response to Major Immunodominant Epitopes after Vaginal Exposure to Simian Immunodeficiency Virus: Too Late and Too Little

    PubMed Central

    Reynolds, Matthew R.; Rakasz, Eva; Skinner, Pamela J.; White, Cara; Abel, Kristina; Ma, Zhong-Min; Compton, Lara; Napoé, Gnankang; Wilson, Nancy; Miller, Christopher J.; Haase, Ashley; Watkins, David I.

    2005-01-01

    In the acute stage of infection following sexual transmission of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), virus-specific CD8+ T-lymphocyte responses partially control but do not eradicate infection from the lymphatic tissues (LTs) or prevent the particularly massive depletion of CD4+ T lymphocytes in gut-associated lymphatic tissue (GALT). We explored hypothetical explanations for this failure to clear infection and prevent CD4+ T-lymphocyte loss in the SIV/rhesus macaque model of intravaginal transmission. We examined the relationship between the timing and magnitude of the CD8+ T-lymphocyte response to immunodominant SIV epitopes and viral replication, and we show first that the failure to contain infection is not because the female reproductive tract is a poor inductive site. We documented robust responses in cervicovaginal tissues and uterus, but only several days after the peak of virus production. Second, while we also documented a modest response in the draining genital and peripheral lymph nodes, the response at these sites also lagged behind peak virus production in these LT compartments. Third, we found that the response in GALT was surprisingly low or undetectable, possibly contributing to the severe and sustained depletion of CD4+ T lymphocytes in the GALT. Thus, the virus-specific CD8+ T-lymphocyte response is “too late and too little” to clear infection and prevent CD4+ T-lymphocyte loss. However, the robust response in female reproductive tissues may be an encouraging sign that vaccines that rapidly induce high-frequency CD8+ T-lymphocyte responses might be able to prevent acquisition of HIV-1 infection by the most common route of transmission. PMID:15994817

  13. Analysis of the T Cell Response to Zika Virus and Identification of a Novel CD8+ T Cell Epitope in Immunocompetent Mice

    PubMed Central

    Pardy, Ryan D.; Rajah, Maaran M.; Taylor, Nathan G.

    2017-01-01

    Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family. Although ZIKV infection is typically mild and self-limiting in healthy adults, infection has been associated with neurological symptoms such as Guillain-Barré syndrome, and a causal link has been established between fetal microcephaly and ZIKV infection during pregnancy. These risks, and the magnitude of the ongoing ZIKV pandemic, have created an urgent need for the development of animal models to study the immune response to ZIKV infection. Previous animal models have primarily focused on pathogenesis in immunocompromised mice. In this study, we provide a model of ZIKV infection in wild-type immunocompetent C57BL/6 mice, and have provided an analysis of the immune response to infection. We evaluated the activation of several innate immune cell types, and studied the kinetics, phenotype, and functionality of T cell responses to ZIKV infection. Our results demonstrate that ZIKV infection is mild in wild-type immunocompetent C57BL/6 mice, resulting in minimal morbidity. Our data establish that at the peak of the adaptive response, antigen-experienced CD4+ T cells polarize to a Th1 phenotype, and antigen-experienced CD8+ T cells exhibit an activated effector phenotype, producing both effector cytokines and cytolytic molecules. Furthermore, we have identified a novel ZIKV CD8+ T cell epitope in the envelope protein that is recognized by the majority of responding cells. Our model provides an important reference point that will help dissect the impact of polymorphisms in the circulating ZIKV strains on the immune response and ZIKV pathogenesis. In addition, the identification of a ZIKV epitope will allow for the design of tetramers to study epitope-specific T cell responses, and will have important implications for the design and development of ZIKV vaccine strategies. PMID:28231312

  14. Analysis of the T Cell Response to Zika Virus and Identification of a Novel CD8+ T Cell Epitope in Immunocompetent Mice.

    PubMed

    Pardy, Ryan D; Rajah, Maaran M; Condotta, Stephanie A; Taylor, Nathan G; Sagan, Selena M; Richer, Martin J

    2017-02-01

    Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family. Although ZIKV infection is typically mild and self-limiting in healthy adults, infection has been associated with neurological symptoms such as Guillain-Barré syndrome, and a causal link has been established between fetal microcephaly and ZIKV infection during pregnancy. These risks, and the magnitude of the ongoing ZIKV pandemic, have created an urgent need for the development of animal models to study the immune response to ZIKV infection. Previous animal models have primarily focused on pathogenesis in immunocompromised mice. In this study, we provide a model of ZIKV infection in wild-type immunocompetent C57BL/6 mice, and have provided an analysis of the immune response to infection. We evaluated the activation of several innate immune cell types, and studied the kinetics, phenotype, and functionality of T cell responses to ZIKV infection. Our results demonstrate that ZIKV infection is mild in wild-type immunocompetent C57BL/6 mice, resulting in minimal morbidity. Our data establish that at the peak of the adaptive response, antigen-experienced CD4+ T cells polarize to a Th1 phenotype, and antigen-experienced CD8+ T cells exhibit an activated effector phenotype, producing both effector cytokines and cytolytic molecules. Furthermore, we have identified a novel ZIKV CD8+ T cell epitope in the envelope protein that is recognized by the majority of responding cells. Our model provides an important reference point that will help dissect the impact of polymorphisms in the circulating ZIKV strains on the immune response and ZIKV pathogenesis. In addition, the identification of a ZIKV epitope will allow for the design of tetramers to study epitope-specific T cell responses, and will have important implications for the design and development of ZIKV vaccine strategies.

  15. A Novel HLA-B18 Restricted CD8+ T Cell Epitope Is Efficiently Cross-Presented by Dendritic Cells from Soluble Tumor Antigen

    PubMed Central

    Chan, Kok-Fei; Oveissi, Sara; Jackson, Heather M.; Dimopoulos, Nektaria; Guillaume, Philippe; Knights, Ashley J.; Lowen, Tamara; Robson, Neil C.; Russell, Sarah E.; Scotet, Emmanuel; Davis, Ian D.; Maraskovsky, Eugene; Cebon, Jonathan; Luescher, Immanuel F.; Chen, Weisan

    2012-01-01

    NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8+ T cell epitope, NY-ESO-188–96 (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1157–165 epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-188–96 is much more efficiently cross-presented from the soluble form, than NY-ESO-1157–165. On the other hand, NY-ESO-1157–165 is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A26–35; whereas NY-ESO-188–96 was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-188–96 is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18+ melanoma patients. Surprisingly, all the detectable responses to NY-ESO-188–96 from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8+ T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed. PMID:22970293

  16. Flavivirus-cross-reactive, HLA-DR15-restricted epitope on NS3 recognized by human CD4+ CD8- cytotoxic T lymphocyte clones.

    PubMed

    Kurane, I; Okamoto, Y; Dai, L C; Zeng, L L; Brinton, M A; Ennis, F A

    1995-09-01

    The role of flavivirus-cross-reactive T lymphocytes in recovery from and pathogenesis of flavivirus infections is not known. In the present paper, we have defined a flavivirus-cross-reactive epitope recognized by two CD4+ CD8- cytotoxic T lymphocyte (CTL) clones, JK4 and JK43. The T cell clones were established from the peripheral blood T lymphocytes of a dengue-4-immune donor, using a limiting-dilution method with dengue-4 antigen. These two T cell clones were cross-reactive for dengue virus types 1, 2, 3 and 4, yellow fever virus and West Nile virus, and recognized NS3 protein. The smallest synthetic peptide recognized by these T cell clones was an identical 9 amino acid peptide which contains amino acids 146 to 154 (VIGLYGNGV) of dengue-4 NS3. HLA-DR15 was the restriction allele for recognition of this epitope by JK4 and JK43. JK4 and JK43 both used T cell receptor V alpha 8, but JK4 used V beta 8 and JK43 used V beta 2. This result indicates that this epitope is recognized by two flavivirus-cross-reactive CD4+ T cell clones which originated from different T cells in vivo.

  17. Cross-Protective Immunity to Leishmania amazonensis is Mediated by CD4+ and CD8+ Epitopes of Leishmania donovani Nucleoside Hydrolase Terminal Domains

    PubMed Central

    Nico, Dirlei; Gomes, Daniele Crespo; Alves-Silva, Marcus Vinícius; Freitas, Elisangela Oliveira; Morrot, Alexandre; Bahia, Diana; Palatnik, Marcos; Rodrigues, Mauricio M.; Palatnik-de-Sousa, Clarisa B.

    2014-01-01

    The nucleoside hydrolase (NH) of Leishmania donovani (NH36) is a phylogenetic marker of high homology among Leishmania parasites. In mice and dog vaccination, NH36 induces a CD4+ T cell-driven protective response against Leishmania chagasi infection directed against its C-terminal domain (F3). The C-terminal and N-terminal domain vaccines also decreased the footpad lesion caused by Leishmania amazonensis. We studied the basis of the crossed immune response using recombinant generated peptides covering the whole NH36 sequence and saponin for mice prophylaxis against L. amazonensis. The F1 (amino acids 1–103) and F3 peptide (amino acids 199–314) vaccines enhanced the IgG and IgG2a anti-NH36 antibodies to similar levels. The F3 vaccine induced the strongest DTH response, the highest proportions of NH36-specific CD4+ and CD8+ T cells after challenge and the highest expression of IFN-γ and TNF-α. The F1 vaccine, on the other hand, induced a weaker but significant DTH response and a mild enhancement of IFN-γ and TNF-α levels. The in vivo depletion with anti-CD4 or CD8 monoclonal antibodies disclosed that cross-protection against L. amazonensis infection was mediated by a CD4+ T cell response directed against the C-terminal domain (75% of reduction of the size of footpad lesion) followed by a CD8+ T cell response against the N-terminal domain of NH36 (57% of reduction of footpad lesions). Both vaccines were capable of inducing long-term cross-immunity. The amino acid sequence of NH36 showed 93% identity to the sequence of the NH A34480 of L. amazonensis, which also showed the presence of completely conserved predicted epitopes for CD4+ and CD8+ T cells in F1 domain, and of CD4+ epitopes differing by a single amino acid, in F1 and F3 domains. The identification of the C-terminal and N-terminal domains as the targets of the immune response to NH36 in the model of L. amazonensis infection represents a basis for the rationale development of a bivalent vaccine against

  18. Cross-Protective Immunity to Leishmania amazonensis is Mediated by CD4+ and CD8+ Epitopes of Leishmania donovani Nucleoside Hydrolase Terminal Domains.

    PubMed

    Nico, Dirlei; Gomes, Daniele Crespo; Alves-Silva, Marcus Vinícius; Freitas, Elisangela Oliveira; Morrot, Alexandre; Bahia, Diana; Palatnik, Marcos; Rodrigues, Mauricio M; Palatnik-de-Sousa, Clarisa B

    2014-01-01

    The nucleoside hydrolase (NH) of Leishmania donovani (NH36) is a phylogenetic marker of high homology among Leishmania parasites. In mice and dog vaccination, NH36 induces a CD4+ T cell-driven protective response against Leishmania chagasi infection directed against its C-terminal domain (F3). The C-terminal and N-terminal domain vaccines also decreased the footpad lesion caused by Leishmania amazonensis. We studied the basis of the crossed immune response using recombinant generated peptides covering the whole NH36 sequence and saponin for mice prophylaxis against L. amazonensis. The F1 (amino acids 1-103) and F3 peptide (amino acids 199-314) vaccines enhanced the IgG and IgG2a anti-NH36 antibodies to similar levels. The F3 vaccine induced the strongest DTH response, the highest proportions of NH36-specific CD4+ and CD8+ T cells after challenge and the highest expression of IFN-γ and TNF-α. The F1 vaccine, on the other hand, induced a weaker but significant DTH response and a mild enhancement of IFN-γ and TNF-α levels. The in vivo depletion with anti-CD4 or CD8 monoclonal antibodies disclosed that cross-protection against L. amazonensis infection was mediated by a CD4+ T cell response directed against the C-terminal domain (75% of reduction of the size of footpad lesion) followed by a CD8+ T cell response against the N-terminal domain of NH36 (57% of reduction of footpad lesions). Both vaccines were capable of inducing long-term cross-immunity. The amino acid sequence of NH36 showed 93% identity to the sequence of the NH A34480 of L. amazonensis, which also showed the presence of completely conserved predicted epitopes for CD4+ and CD8+ T cells in F1 domain, and of CD4+ epitopes differing by a single amino acid, in F1 and F3 domains. The identification of the C-terminal and N-terminal domains as the targets of the immune response to NH36 in the model of L. amazonensis infection represents a basis for the rationale development of a bivalent vaccine against

  19. In Silico Analysis of Six Known Leishmania major Antigens and In Vitro Evaluation of Specific Epitopes Eliciting HLA-A2 Restricted CD8 T Cell Response

    PubMed Central

    Seyed, Negar; Zahedifard, Farnaz; Safaiyan, Shima; Gholami, Elham; Doustdari, Fatemeh; Azadmanesh, Kayhan; Mirzaei, Maryam; Saeedi Eslami, Nasir; Khadem Sadegh, Akbar; Eslami far, Ali; Sharifi, Iraj; Rafati, Sima

    2011-01-01

    Background As a potent CD8+ T cell activator, peptide vaccine has found its way in vaccine development against intracellular infections and cancer, but not against leishmaniasis. The first step toward a peptide vaccine is epitope mapping of different proteins according to the most frequent HLA types in a population. Methods and Findings Six Leishmania (L.) major-related candidate antigens (CPB,CPC,LmsTI-1,TSA,LeIF and LPG-3) were screened for potential CD8+ T cell activating 9-mer epitopes presented by HLA-A*0201 (the most frequent HLA-A allele). Online software including SYFPEITHI, BIMAS, EpiJen, Rankpep, nHLApred, NetCTL and Multipred were used. Peptides were selected only if predicted by almost all programs, according to their predictive scores. Pan-A2 presentation of selected peptides was confirmed by NetMHCPan1.1. Selected peptides were pooled in four peptide groups and the immunogenicity was evaluated by in vitro stimulation and intracellular cytokine assay of PBMCs from HLA-A2+ individuals recovered from L. major. HLA-A2− individuals recovered from L. major and HLA-A2+ healthy donors were included as control groups. Individual response of HLA-A2+ recovered volunteers as percent of CD8+/IFN-γ+ T cells after in vitro stimulation against peptide pools II and IV was notably higher than that of HLA-A2− recovered individuals. Based on cutoff scores calculated from the response of HLA-A2− recovered individuals, 31.6% and 13.3% of HLA-A2+ recovered persons responded above cutoff in pools II and IV, respectively. ELISpot and ELISA results confirmed flow cytometry analysis. The response of HLA-A2− recovered individuals against peptide pools I and III was detected similar and even higher than HLA-A2+ recovered individuals. Conclusion Using in silico prediction we demonstrated specific response to LmsTI-1 (pool II) and LPG-3- (pool IV) related peptides specifically presented in HLA-A*0201 context. This is among the very few reports mapping L. major epitopes for

  20. Immunization with a peptide containing MHC class I and II epitopes derived from the tumor antigen SIM2 induces an effective CD4 and CD8 T-cell response.

    PubMed

    Kissick, Haydn T; Sanda, Martin G; Dunn, Laura K; Arredouani, Mohamed S

    2014-01-01

    Here, we sought to determine whether peptide vaccines designed harbor both class I as well as class II restricted antigenic motifs could concurrently induce CD4 and CD8 T cell activation against autologous tumor antigens. Based on our prior genome-wide interrogation of human prostate cancer tissues to identify genes over-expressed in cancer and absent in the periphery, we targeted SIM2 as a prototype autologous tumor antigen for these studies. Using humanized transgenic mice we found that the 9aa HLA-A*0201 epitope, SIM2(237-245), was effective at inducing an antigen specific response against SIM2-expressing prostate cancer cell line, PC3. Immunization with a multi-epitope peptide harboring both MHC-I and MHC-II restricted epitopes induced an IFN-γ response in CD8 T cells to the HLA-A*0201-restricted SIM2(237-245) epitope, and an IL-2 response by CD4 T cells to the SIM2(240-254) epitope. This peptide was also effective at inducing CD8+ T-cells that responded specifically to SIM2-expressing tumor cells. Collectively, the data presented in this study suggest that a single peptide containing multiple SIM2 epitopes can be used to induce both a CD4 and CD8 T cell response, providing a peptide-based vaccine formulation for potential use in immunotherapy of various cancers.

  1. Immunization of mice with vaccinia virus-M2 recombinant induces epitope-specific and cross-reactive Kd-restricted CD8+ cytotoxic T cells.

    PubMed Central

    Kulkarni, A B; Morse, H C; Bennink, J R; Yewdell, J W; Murphy, B R

    1993-01-01

    The M2 protein of respiratory syncytial virus (RSV) is a protective antigen in H-2d, but not H-2b or H-2k mice. None of the other RSV proteins, excluding the surface glycoproteins that induce neutralizing antibodies, is protective in mice bearing these haplotypes. Thus, the M2 protein stands alone as a nonglycoprotein-protective antigen of RSV. The M2 protein is a target for murine Kd-restricted cytotoxic T lymphocytes (CTLs), and the resistance induced by infection with a vaccinia virus-RSV M2 (vac-M2) recombinant is mediated by CD8+ CTLs. Since the nonameric consensus sequence for H-2 Kd-restricted T-cell epitopes and the amino acid sequence of the M2 protein of subgroup A and B strains of RSV are known, the present study sought to identify the specific epitope(s) on the M2 protein recognized by CD8+ CTLs. This was done by examining the ability of four predicted Kd-specific motif peptides present in the M2 amino acid sequence of an RSV subgroup A strain to sensitize target cells for lysis by pulmonary or splenic CTLs obtained from mice infected with RSV or vac-M2. The following observations were made. First, two of the four peptides sensitized target cells for lysis by pulmonary or splenic CTLs induced by infection with either vac-M2 or RSV. Second, one of the two peptides, namely the 82-90 (M2) peptide, sensitized targets at a very low peptide concentration (10(-10) to 10(-12) M). Third, cold-target competition experiments revealed that the predominant CTL population induced by infection with vac-M2 or RSV recognized the 82-90 (M2) peptide, and this CTL population appeared to recognize the 71-79 (M2) peptide in a cross-reactive manner. Fourth, CTL recognition of targets sensitized with either the 71-79 (M2) or the 82-90 (M2) peptide was Kd restricted. Fifth, CTLs induced by infection with RSV subgroup A or B strains recognized the two M2 peptides. The findings suggest that the M2 protein of RSV contains an immunodominant Kd-restricted CTL epitope consisting of

  2. Generation of robust CD8+ T cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes

    PubMed Central

    Schaubert, Keri L.; Price, David A.; Salkowitz, Janelle R.; Sewell, Andrew K.; Sidney, John; Asher, Tedi E.; Blondelle, Sylvie E.; Adams, Sharon; Marincola, Francesco M.; Joseph, Aviva; Sette, Alessandro; Douek, Daniel C.; Ayyavoo, Velpandi; Storkus, Walter; Leung, Ming-Ying; Ng, Hwee L.; Yang, Otto O.; Goldstein, Harris; Wilson, Darcy B.; Kan-Mitchell, June

    2011-01-01

    Summary HLA-A*0201-restricted virus-specific CD8+ cytotoxic T lymphocytes (CTLs) do not appear to control HIV effectively in vivo. To enhance the immunogenicity of a highly conserved subdominant epitope, TV9 (TLNAWVKVV, p24 Gag19–27), mimotopes were designed by screening a large combinatorial nonapeptide library with TV9-specific CTLs primed in vitro from healthy donors. A mimic peptide with a low binding affinity to HLA-A*0201, TV9p6 (KINAWIKVV), was studied further. Parallel cultures of in vitro-primed CTLs showed that TV9p6 consistently activated crossreactive and equally functional CTLs as measured by cytotoxicity, cytokine production and suppression of HIV replication in vitro. Comparison of TCRB gene usage between CTLs primed from the same donors with TV9 or TV9p6 revealed a degree of clonal overlap in some cases and an example of a conserved TCRB sequence encoded distinctly at the nucleotide level between individuals (a “public” TCR); however, in the main, distinct clonotypes were recruited by each peptide antigen. These findings indicate that mimotopes can mobilize functional crossreactive clonotypes that are less readily recruited from the naïve T cell pool by the corresponding wildtype epitope. Mimotope-induced repertoire diversification could potentially override subdominance under certain circumstances and enhance vaccine-induced responses to conserved but poorly immunogenic determinants within the HIV proteome. PMID:20432235

  3. The Human CD8+ T Cell Responses Induced by a Live Attenuated Tetravalent Dengue Vaccine Are Directed against Highly Conserved Epitopes

    PubMed Central

    Angelo, Michael A.; Bangs, Derek J.; Sidney, John; Paul, Sinu; Peters, Bjoern; de Silva, Aruna D.; Lindow, Janet C.; Diehl, Sean A.; Whitehead, Stephen; Durbin, Anna; Kirkpatrick, Beth; Sette, Alessandro

    2014-01-01

    ABSTRACT The incidence of infection with any of the four dengue virus serotypes (DENV1 to -4) has increased dramatically in the last few decades, and the lack of a treatment or vaccine has contributed to significant morbidity and mortality worldwide. A recent comprehensive analysis of the human T cell response against wild-type DENV suggested an human lymphocyte antigen (HLA)-linked protective role for CD8+ T cells. We have collected one-unit blood donations from study participants receiving the monovalent or tetravalent live attenuated DENV vaccine (DLAV), developed by the U.S. National Institutes of Health. Peripheral blood mononuclear cells from these donors were screened in gamma interferon enzyme-linked immunosorbent spot assays with pools of predicted, HLA-matched, class I binding peptides covering the entire DENV proteome. Here, we characterize for the first time CD8+ T cell responses after live attenuated dengue vaccination and show that CD8+ T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Interestingly, whereas broad responses to structural and nonstructural (NS) proteins were observed after monovalent vaccination, T cell responses following tetravalent vaccination were, dramatically, focused toward the highly conserved NS proteins. Epitopes were highly conserved in a vast variety of field isolates and able to elicit multifunctional T cell responses. Detailed knowledge of the T cell response will contribute to the identification of robust correlates of protection in natural immunity and following vaccination against DENV. IMPORTANCE The development of effective vaccination strategies against dengue virus (DENV) infection and clinically significant disease is a task of high global public health value and significance, while also being a challenge of significant complexity. A recent efficacy trial of the most advanced dengue vaccine candidate, demonstrated only partial protection against all four DENV

  4. A cytomegalovirus-based vaccine expressing a single tumor-specific CD8+ T-cell epitope delays tumor growth in a murine model of prostate cancer.

    PubMed

    Klyushnenkova, Elena N; Kouiavskaia, Diana V; Parkins, Christopher J; Caposio, Patrizia; Botto, Sara; Alexander, Richard B; Jarvis, Michael A

    2012-06-01

    Cytomegalovirus (CMV) is a highly immunogenic virus that results in a persistent, life-long infection in the host typically with no ill effects. Certain unique features of CMV, including its capacity to actively replicate in the presence of strong host CMV-specific immunity, may give CMV an advantage compared with other virus-based vaccine delivery platforms. In the present study, we tested the utility of mouse CMV (mCMV)-based vaccines expressing human prostate-specific antigen (PSA) for prostate cancer immunotherapy in double-transgenic mice expressing PSA and HLA-DRB1*1501 (DR2bxPSA F1 mice). We assessed the capacity of 2 mCMV-based vectors to induce PSA-specific CD8 T-cell responses and affect the growth of PSA-expressing Transgenic Adenocarcinoma of the Mouse Prostate tumors (TRAMP-PSA). In the absence of tumor challenge, immunization with mCMV vectors expressing either a H2-D(b)-restricted epitope PSA(65-73) (mCMV/PSA(65-73)) or the full-length gene for PSA (mCMV/PSA(FL)) induced comparable levels of CD8 T-cell responses that increased (inflated) with time. Upon challenge with TRAMP-PSA tumor cells, animals immunized with mCMV/PSA(65-73) had delay of tumor growth and increased PSA-specific CD8 T-cell responses, whereas animals immunized with mCMV/PSA(FL) showed progressive tumor growth and no increase in number of splenic PSA(65-73)-specific T cells. The data show that a prototype CMV-based prostate cancer vaccine can induce an effective antitumor immune response in a "humanized" double-transgenic mouse model. The observation that mCMV/PSA(FL) is not effective against TRAMP-PSA is consistent with our previous findings that HLA-DRB1*1501-restricted immune responses to PSA are associated with suppression of effective CD8 T-cell responses to TRAMP-PSA tumors.

  5. Persistence, immune specificity, and functional ability of murine mutant ras epitope-specific CD4(+) and CD8(+) T lymphocytes following in vivo adoptive transfer.

    PubMed

    Bristol, J A; Schlom, J; Abrams, S I

    1999-05-25

    Adoptive T-cell transfer has been shown to be a potentially effective strategy for cellular immunotherapy in some murine models of disease. However, several issues remain unresolved regarding some of the basic features involved in effective adoptive transfer, such as the influence of specific peptide antigen (Ag) boost after T-cell transfer, the addition of IL-2 post-T-cell transfer, the trafficking of transferred T cells to lymphoid and nonlymphoid tissues, and the functional stability of recoverable CD4(+) and CD8(+) T cells. We investigated several of these parameters, particularly as they relate to the persistence and maintenance of effector functions of murine CD4(+) and/or CD8(+) T lymphocytes after adoptive cellular transfer into partially gamma-irradiated syngeneic hosts. Our laboratory previously identified murine (H-2(d)) immunogenic CD4(+) and CD8(+) T-cell peptide epitopes reflecting codon 12 ras mutations as tumor-specific Ag. Therefore, the model system chosen here employed epitope-specific MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells produced from previously immunized BALB/c mice. Between 2 and 7 days after T-cell transfer, recipient mice received various combinations of peptide boosts and/or IL-2 treatments. At different times after the T-cell transfer, spleen and lung tissues were analyzed phenotypically to monitor the persistence of the immune T cells and functionally (via proliferation or cytotoxicity assays) to assess the maintenance of peptide specificity. The results showed that immune donor T lymphocytes (uncultured immune T cells or cloned T cells) were recoverable from the spleens and lungs of recipient mice after transfer. The recovery of Ag-specific T-cell responses was greatest from recipient mice that received peptide boosts and IL-2 treatment. However, mice that received a peptide boost without IL-2 treatment responded nearly as well, which suggested that including a peptide boost after T

  6. Inferring Protective CD8+ T-Cell Epitopes for NS5 Protein of Four Serotypes of Dengue Virus Chinese Isolates Based on HLA-A, -B and -C Allelic Distribution: Implications for Epitope-Based Universal Vaccine Design.

    PubMed

    Shi, Jiandong; Sun, Jing; Wu, Meini; Hu, Ningzhu; Li, Jianfan; Li, Yanhan; Wang, Haixuan; Hu, Yunzhang

    2015-01-01

    Dengue is one of the most globally serious vector-borne infectious diseases in tropical and subtropical areas for which there are currently no effective vaccines. The most highly conserved flavivirus protein, NS5, is an indispensable target of CD8+ T-cells, making it an ideal vaccine design target. Using the Immune Epitope Database (IEDB), CD8+ T-cell epitopes of the dengue virus (DENV) NS5 protein were predicted by genotypic frequency of the HLA-A,-B, and-C alleles in Chinese population. Antigenicity scores of all predicted epitopes were analyzed using VaxiJen v2.0. The IEDB analysis revealed that 116 antigenic epitopes for HLA-A (21),-B (53), and-C (42) had high affinity for HLA molecules. Of them, 14 had 90.97-99.35% conversancy among the four serotypes. Moreover, five candidate epitopes, including 200NS5210 (94.84%, A*11:01), 515NS5525 (98.71%, A*24:02), 225NS5232 (99.35%, A*33:03), 516NS5523 (98.71%, A*33:03), and 284NS5291 (98.06%, A*33:03), were presented by HLA-A. Four candidate epitopes, including 234NS5241 (96.77%, B*13:01), 92NS599 (98.06%, B*15:01, B*15:02, and B*46:01), 262NS5269 (92.90%, B*38:02), and 538NS5547 (90.97%, B*51:01), were presented by HLA-B. Another 9 candidate epitopes, including 514NS5522 (98.71%, C*01:02), 514NS5524 (98.71%, C*01:02 and C*14:02), 92NS599 (98.06%, C*03:02 and C*15:02), 362NS5369 (44.84%, C*03:04 and C*08:01), 225NS5232 (99.35%, C*04:01), 234NS5241(96.77%, C*04:01), 361NS5369 (94.84%, C*04:01), 515NS5522 (98.71%, C*14:02), 515NS5524 (98.71%, C*14:02), were presented by HLA-C. Further data showed that the four-epitope combination of 92NS599 (B*15:01, B*15:02, B*46:01, C*03:02 and C*15:02), 200NS5210 (A*11:01), 362NS5369 (C*03:04, C*08:01), and 514NS5524 (C*01:02, C*14:02) could vaccinate >90% of individuals in China. Further in vivo study of our inferred novel epitopes will be needed for a T-cell epitope-based universal vaccine development that may prevent all four China-endemic DENV serotypes.

  7. A recombinant chimeric protein composed of human and mice-specific CD4(+) and CD8(+) T-cell epitopes protects against visceral leishmaniasis.

    PubMed

    Martins, V T; Duarte, M C; Lage, D P; Costa, L E; Carvalho, A M R S; Mendes, T A O; Roatt, B M; Menezes-Souza, D; Soto, M; Coelho, E A F

    2017-01-01

    In this study, a recombinant chimeric protein (RCP), which was composed of specific CD4(+) and CD8(+) T-cell epitopes to murine and human haplotypes, was evaluated as an immunogen against Leishmania infantum infection in a murine model. BALB/c mice received saline were immunized with saponin or with RCP with or without an adjuvant. The results showed that RCP/saponin-vaccinated mice presented significantly higher levels of antileishmanial IFN-γ, IL-12 and GM-CSF before and after challenge, which were associated with the reduction of IL-4 and IL-10 mediated responses. These animals showed significant reductions in the parasite burden in all evaluated organs, when both limiting dilution and quantitative real-time PCR techniques were used. In addition, the protected animals presented higher levels of parasite-specific nitrite, as well as the presence of anti-Leishmania IgG2a isotype antibodies. In conclusion, the RCP/saponin vaccine could be considered as a prophylactic alternative to prevent against VL. © 2016 John Wiley & Sons Ltd.

  8. Actively induced antigen-specific CD8+ T cells by epitope-bearing parasite pre-infection but not prime/boost virus vector vaccination could ameliorate the course of Plasmodium yoelii blood-stage infection.

    PubMed

    Ono, Takeshi; Yamaguchi, Yoko; Oguma, Takemi; Takayama, Eiji; Takashima, Yasuhiro; Tadakuma, Takushi; Miyahira, Yasushi

    2012-09-28

    The lack of MHC molecules on red blood cells (RBCs) has led to questions regarding the immunological function of CD8(+) T cells against malarial blood-stage (MBS). However, several recent reports contradicting with this concept have suggested that they play an important role in the course of MBS infection. The present study generated genetically engineered murine malaria, Plasmodium yoelii, which expresses a well-defined Trypanosoma cruzi-derived, H-2K(b)-restricted CD8(+) T cell epitope, ANYNFTLV. Prime/boost vaccination by the use of recombinant adenovirus and recombinant modified vaccinia virus Ankara (MVA), which induced an enhanced number of ANYNFTLV-specific CD8(+) T cells, failed to prevent a pathological outcome to occur upon ANYNFTLV-expressing murine MBS infection. This outcome did not change even with the combination of passive transfer of an appreciable number of in vitro-expanded ANYNFTLV-specific CD8(+) T cells. In contrast, the pre-infection of mice with T. cruzi, which intrinsically bears the same CD8(+) T cell epitope significantly improved the survival of ANYNFTLV-expressing malaria-infected mice but not that of control malaria-infected ones. This protective effect was abrogated by the use of a CD8(+) T cell-depleting monoclonal antibody. Although the protective effect was observed only in certain situations, the actively induced antigen-specific CD8(+) T cells could ameliorate the pathologies caused by the MBS. This is the first study to implicate that the active induction of antigen-specific CD8(+) T cells should be included in the development of a vaccine against MBS. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. CD8 and CD4 epitope predictions in RV144: no strong evidence of a T-cell driven sieve effect in HIV-1 breakthrough sequences from trial participants.

    PubMed

    Dommaraju, Kalpana; Kijak, Gustavo; Carlson, Jonathan M; Larsen, Brendan B; Tovanabutra, Sodsai; Geraghty, Dan E; Deng, Wenjie; Maust, Brandon S; Edlefsen, Paul T; Sanders-Buell, Eric; Ratto-Kim, Silvia; deSouza, Mark S; Rerks-Ngarm, Supachai; Nitayaphan, Sorachai; Pitisuttihum, Punnee; Kaewkungwal, Jaranit; O'Connell, Robert J; Robb, Merlin L; Michael, Nelson L; Mullins, James I; Kim, Jerome H; Rolland, Morgane

    2014-01-01

    The modest protection afforded by the RV144 vaccine offers an opportunity to evaluate its mechanisms of protection. Differences between HIV-1 breakthrough viruses from vaccine and placebo recipients can be attributed to the RV144 vaccine as this was a randomized and double-blinded trial. CD8 and CD4 T cell epitope repertoires were predicted in HIV-1 proteomes from 110 RV144 participants. Predicted Gag epitope repertoires were smaller in vaccine than in placebo recipients (p = 0.019). After comparing participant-derived epitopes to corresponding epitopes in the RV144 vaccine, the proportion of epitopes that could be matched differed depending on the protein conservation (only 36% of epitopes in Env vs 84-91% in Gag/Pol/Nef for CD8 predicted epitopes) or on vaccine insert subtype (55% against CRF01_AE vs 7% against subtype B). To compare predicted epitopes to the vaccine, we analyzed predicted binding affinity and evolutionary distance measurements. Comparisons between the vaccine and placebo arm did not reveal robust evidence for a T cell driven sieve effect, although some differences were noted in Env-V2 (0.022≤p-value≤0.231). The paucity of CD8 T cell responses identified following RV144 vaccination, with no evidence for V2 specificity, considered together both with the association of decreased infection risk in RV 144 participants with V-specific antibody responses and a V2 sieve effect, lead us to hypothesize that this sieve effect was not T cell specific. Overall, our results did not reveal a strong differential impact of vaccine-induced T cell responses among breakthrough infections in RV144 participants.

  10. CD8 and CD4 Epitope Predictions in RV144: No Strong Evidence of a T-Cell Driven Sieve Effect in HIV-1 Breakthrough Sequences from Trial Participants

    PubMed Central

    Dommaraju, Kalpana; Kijak, Gustavo; Carlson, Jonathan M.; Larsen, Brendan B.; Tovanabutra, Sodsai; Geraghty, Dan E.; Deng, Wenjie; Maust, Brandon S.; Edlefsen, Paul T.; Sanders-Buell, Eric; Ratto-Kim, Silvia; deSouza, Mark S.; Rerks-Ngarm, Supachai; Nitayaphan, Sorachai; Pitisuttihum, Punnee; Kaewkungwal, Jaranit; O'Connell, Robert J.; Robb, Merlin L.; Michael, Nelson L.; Mullins, James I.; Kim, Jerome H.; Rolland, Morgane

    2014-01-01

    The modest protection afforded by the RV144 vaccine offers an opportunity to evaluate its mechanisms of protection. Differences between HIV-1 breakthrough viruses from vaccine and placebo recipients can be attributed to the RV144 vaccine as this was a randomized and double-blinded trial. CD8 and CD4 T cell epitope repertoires were predicted in HIV-1 proteomes from 110 RV144 participants. Predicted Gag epitope repertoires were smaller in vaccine than in placebo recipients (p = 0.019). After comparing participant-derived epitopes to corresponding epitopes in the RV144 vaccine, the proportion of epitopes that could be matched differed depending on the protein conservation (only 36% of epitopes in Env vs 84–91% in Gag/Pol/Nef for CD8 predicted epitopes) or on vaccine insert subtype (55% against CRF01_AE vs 7% against subtype B). To compare predicted epitopes to the vaccine, we analyzed predicted binding affinity and evolutionary distance measurements. Comparisons between the vaccine and placebo arm did not reveal robust evidence for a T cell driven sieve effect, although some differences were noted in Env-V2 (0.022≤p-value≤0.231). The paucity of CD8 T cell responses identified following RV144 vaccination, with no evidence for V2 specificity, considered together both with the association of decreased infection risk in RV 144 participants with V-specific antibody responses and a V2 sieve effect, lead us to hypothesize that this sieve effect was not T cell specific. Overall, our results did not reveal a strong differential impact of vaccine-induced T cell responses among breakthrough infections in RV144 participants. PMID:25350851

  11. CCR2+ Inflammatory Dendritic Cells and Translocation of Antigen by Type III Secretion Are Required for the Exceptionally Large CD8+ T Cell Response to the Protective YopE69-77 Epitope during Yersinia Infection

    PubMed Central

    Zhang, Yue; Tam, Jason W.; Mena, Patricio; van der Velden, Adrianus W. M.; Bliska, James B.

    2015-01-01

    During Yersinia pseudotuberculosis infection of C57BL/6 mice, an exceptionally large CD8+ T cell response to a protective epitope in the type III secretion system effector YopE is produced. At the peak of the response, up to 50% of splenic CD8+ T cells recognize the epitope YopE69-77. The features of the interaction between pathogen and host that result in this large CD8+ T cell response are unknown. Here, we used Y. pseudotuberculosis strains defective for production, secretion and/or translocation of YopE to infect wild-type or mutant mice deficient in specific dendritic cells (DCs). Bacterial colonization of organs and translocation of YopE into spleen cells was measured, and flow cytometry and tetramer staining were used to characterize the cellular immune response. We show that the splenic YopE69-77-specific CD8+ T cells generated during the large response are polyclonal and are produced by a “translocation-dependent” pathway that requires injection of YopE into host cell cytosol. Additionally, a smaller YopE69-77-specific CD8+ T cell response (~10% of the large expansion) can be generated in a “translocation-independent” pathway in which CD8α+ DCs cross present secreted YopE. CCR2-expressing inflammatory DCs were required for the large YopE69-77-specific CD8+ T cell expansion because this response was significantly reduced in Ccr2-/- mice, YopE was translocated into inflammatory DCs in vivo, inflammatory DCs purified from infected spleens activated YopE69-77-specific CD8+ T cells ex vivo and promoted the expansion of YopE69-77-specific CD8+ T cells in infected Ccr2-/- mice after adoptive transfer. A requirement for inflammatory DCs in producing a protective CD8+ T cell response to a bacterial antigen has not previously been demonstrated. Therefore, the production of YopE69-77-specific CD8+ T cells by inflammatory DCs that are injected with YopE during Y. pseudotuberculosis infection represents a novel mechanism for generating a massive and protective

  12. Therapeutic Immunization with a Mixture of Herpes Simplex Virus 1 Glycoprotein D-Derived “Asymptomatic” Human CD8+ T-Cell Epitopes Decreases Spontaneous Ocular Shedding in Latently Infected HLA Transgenic Rabbits: Association with Low Frequency of Local PD-1+ TIM-3+ CD8+ Exhausted T Cells

    PubMed Central

    Khan, Arif A.; Srivastava, Ruchi; Chentoufi, Aziz A.; Geertsema, Roger; Thai, Nhi Thi Uyen; Dasgupta, Gargi; Osorio, Nelson; Kalantari, Mina; Nesburn, Anthony B.; Wechsler, Steven L.

    2015-01-01

    ABSTRACT Most blinding ocular herpetic disease is due to reactivation of herpes simplex virus 1 (HSV-1) from latency rather than to primary acute infection. No herpes simplex vaccine is currently available for use in humans. In this study, we used the HLA-A*02:01 transgenic (HLA Tg) rabbit model of ocular herpes to assess the efficacy of a therapeutic vaccine based on HSV-1 gD epitopes that are recognized mainly by CD8+ T cells from “naturally” protected HLA-A*02:01-positive, HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease). Three ASYMP CD8+ T-cell epitopes (gD53–61, gD70–78, and gD278–286) were linked with a promiscuous CD4+ T-cell epitope (gD287–317) to create 3 separate pairs of CD4-CD8 peptides, which were then each covalently coupled to an Nε-palmitoyl-lysine moiety, a Toll-like receptor 2 (TLR-2) ligand. This resulted in the construction of 3 CD4-CD8 lipopeptide vaccines. Latently infected HLA Tg rabbits were immunized with a mixture of these 3 ASYMP lipopeptide vaccines, delivered as eye drops in sterile phosphate-buffered saline (PBS). The ASYMP therapeutic vaccination (i) induced HSV-specific CD8+ T cells that prevent HSV-1 reactivation ex vivo from latently infected explanted trigeminal ganglia (TG), (ii) significantly reduced HSV-1 shedding detected in tears, (iii) boosted the number and function of HSV-1 gD epitope-specific CD8+ T cells in draining lymph nodes (DLN), conjunctiva, and TG, and (iv) was associated with fewer exhausted HSV-1 gD-specific PD-1+ TIM-3+ CD8+ T cells. The results underscore the potential of an ASYMP CD8+ T-cell epitope-based therapeutic vaccine strategy against recurrent ocular herpes. IMPORTANCE Seventy percent to 90% of adults harbor herpes simplex virus 1 (HSV-1), which establishes lifelong latency in sensory neurons of the trigeminal ganglia. This latent state sporadically switches to spontaneous reactivation, resulting in viral shedding in tears. Most

  13. Therapeutic immunization with a mixture of herpes simplex virus 1 glycoprotein D-derived “asymptomatic” human CD8+ T-cell epitopes decreases spontaneous ocular shedding in latently infected HLA transgenic rabbits: association with low frequency of local PD-1+ TIM-3+ CD8+ exhausted T cells.

    PubMed

    Khan, Arif A; Srivastava, Ruchi; Chentoufi, Aziz A; Geertsema, Roger; Thai, Nhi Thi Uyen; Dasgupta, Gargi; Osorio, Nelson; Kalantari, Mina; Nesburn, Anthony B; Wechsler, Steven L; BenMohamed, Lbachir

    2015-07-01

    Most blinding ocular herpetic disease is due to reactivation of herpes simplex virus 1 (HSV-1) from latency rather than to primary acute infection. No herpes simplex vaccine is currently available for use in humans. In this study, we used the HLA-A*02:01 transgenic (HLA Tg) rabbit model of ocular herpes to assess the efficacy of a therapeutic vaccine based on HSV-1 gD epitopes that are recognized mainly by CD8(+) T cells from "naturally" protected HLA-A*02:01-positive, HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease). Three ASYMP CD8(+) T-cell epitopes (gD(53-61), gD(70-78), and gD(278-286)) were linked with a promiscuous CD4(+) T-cell epitope (gD(287-317)) to create 3 separate pairs of CD4-CD8 peptides, which were then each covalently coupled to an Nε-palmitoyl-lysine moiety, a Toll-like receptor 2 (TLR-2) ligand. This resulted in the construction of 3 CD4-CD8 lipopeptide vaccines. Latently infected HLA Tg rabbits were immunized with a mixture of these 3 ASYMP lipopeptide vaccines, delivered as eye drops in sterile phosphate-buffered saline (PBS). The ASYMP therapeutic vaccination (i) induced HSV-specific CD8(+) T cells that prevent HSV-1 reactivation ex vivo from latently infected explanted trigeminal ganglia (TG), (ii) significantly reduced HSV-1 shedding detected in tears, (iii) boosted the number and function of HSV-1 gD epitope-specific CD8(+) T cells in draining lymph nodes (DLN), conjunctiva, and TG, and (iv) was associated with fewer exhausted HSV-1 gD-specific PD-1(+) TIM-3+ CD8(+) T cells. The results underscore the potential of an ASYMP CD8(+) T-cell epitope-based therapeutic vaccine strategy against recurrent ocular herpes. Seventy percent to 90% of adults harbor herpes simplex virus 1 (HSV-1), which establishes lifelong latency in sensory neurons of the trigeminal ganglia. This latent state sporadically switches to spontaneous reactivation, resulting in viral shedding in tears. Most blinding

  14. Nanoparticle orientationally displayed antigen epitopes improve neutralizing antibody level in a model of porcine circovirus type 2

    PubMed Central

    Ding, Peiyang; Zhang, Teng; Li, Yafei; Teng, Man; Sun, Yaning; Liu, Xiao; Chai, Shujun; Zhou, Enmin; Jin, Qianyue; Zhang, Gaiping

    2017-01-01

    Recent advancements in biotechnology have enabled the rapid identification and subsequent expression of pathogenic microbial major antigens that induce protective immune responses. However, subunit vaccines have not been successfully commercialized mainly due to the lack of sufficient levels of neutralizing antibodies (NAs). High levels of NA rely on the efficient recognition and cross-linking of multiple neutralizing epitopes with B-cell receptors (BCRs). Nanoparticles are able to display coupled antigenic arrays at high density and provide multiple binding molecular scenarios with BCRs. The high-resolution antigenic structure makes it possible to accurately display stable neutralizing epitopes. Therefore, the development of a nanovaccine that orientationally displays neutralizing epitopes is a feasible strategy. To address this hypothesis, the capsid (Cap) protein of porcine circovirus type 2 as model antigen was conjugated to gold nanoparticles (AuNPs) through direct reaction of the mercapto group of the unique cysteines with AuNPs, rendering Cap-AuNPs to have neutralizing epitopes on outer surface and an immunodominant epitope buried within the inner surface. In vitro studies showed that AuNPs promoted the phagocytosis of Cap protein and NA levels were significantly improved, meanwhile antibody levels against the immunodominant epitope was significantly reduced. In mouse studies, Cap-AuNP-immunized mice displayed a high production of interleukin (IL)-4, IL-10, and interferon-γ, suggesting that Cap-AuNPs can effectively activate CD4+ and CD8+ T cells and balance Th1 and Th2 cellular responses. This study presents a new vaccine design strategy based on antigen structure, where nanoparticles are coupled to antigens in well-ordered arrays and orientationally display neutralizing epitopes to enhance NA levels. PMID:28769561

  15. Beyond HLA-A*0201: new HLA-transgenic nonobese diabetic mouse models of type 1 diabetes identify the insulin C-peptide as a rich source of CD8+ T cell epitopes.

    PubMed

    Antal, Zoltan; Baker, Jason C; Smith, Carla; Jarchum, Irene; Babad, Jeffrey; Mukherjee, Gayatri; Yang, Yang; Sidney, John; Sette, Alessandro; Santamaria, Pere; DiLorenzo, Teresa P

    2012-06-01

    Type 1 diabetes is an autoimmune disease characterized by T cell responses to β cell Ags, including insulin. Investigations employing the NOD mouse model of the disease have revealed an essential role for β cell-specific CD8(+) T cells in the pathogenic process. As CD8(+) T cells specific for β cell Ags are also present in patients, these reactivities have the potential to serve as therapeutic targets or markers for autoimmune activity. NOD mice transgenic for human class I MHC molecules have previously been employed to identify T cell epitopes having important relevance to the human disease. However, most studies have focused exclusively on HLA-A*0201. To broaden the reach of epitope-based monitoring and therapeutic strategies, we have looked beyond this allele and developed NOD mice expressing human β(2)-microglobulin and HLA-A*1101 or HLA-B*0702, which are representative members of the A3 and B7 HLA supertypes, respectively. We have used islet-infiltrating T cells spontaneously arising in these strains to identify β cell peptides recognized in the context of the transgenic HLA molecules. This work has identified the insulin C-peptide as an abundant source of CD8(+) T cell epitopes. Responses to these epitopes should be of considerable utility for immune monitoring, as they cannot reflect an immune reaction to exogenously administered insulin, which lacks the C-peptide. Because the peptides bound by one supertype member were found to bind certain other members also, the epitopes identified in this study have the potential to result in therapeutic and monitoring tools applicable to large numbers of patients and at-risk individuals.

  16. Beyond HLA-A*0201: New HLA-transgenic NOD Mouse Models of Type 1 Diabetes Identify the Insulin C-peptide As a Rich Source of CD8+ T Cell Epitopes1

    PubMed Central

    Antal, Zoltan; Baker, Jason C.; Smith, Carla; Jarchum, Irene; Babad, Jeffrey; Mukherjee, Gayatri; Yang, Yang; Sidney, John; Sette, Alessandro; Santamaria, Pere; DiLorenzo, Teresa P.

    2012-01-01

    Type 1 diabetes is an autoimmune disease characterized by T cell responses to beta cell antigens, including insulin. Investigations employing the NOD mouse model of the disease have revealed an essential role for beta cell-specific CD8+ T cells in the pathogenic process. As CD8+ T cells specific for beta cell antigens are also present in patients, these reactivities have the potential to serve as therapeutic targets or markers for autoimmune activity. NOD mice transgenic for human class I MHC molecules have previously been employed to identify T cell epitopes having important relevance to the human disease. However, most studies have focused exclusively on HLA-A*0201. To broaden the reach of epitope-based monitoring and therapeutic strategies, we have looked beyond this allele and developed NOD mice expressing human β2-microglobulin and HLA-A*1101 or HLA-B*0702, which are representative members of the A3 and B7 HLA supertypes, respectively. We have used islet-infiltrating T cells spontaneously arising in these strains to identify beta cell peptides recognized in the context of the transgenic HLA molecules. This work has identified the insulin C-peptide as an abundant source of CD8+ T cell epitopes. Responses to these epitopes should be of considerable utility for immune monitoring, as they cannot reflect an immune reaction to exogenously administered insulin, which lacks the C-peptide. Because the peptides bound by one supertype member were found to bind certain other members also, the epitopes identified here have the potential to result in therapeutic and monitoring tools applicable to large numbers of patients and at-risk individuals. PMID:22539795

  17. Eliciting Epitope-Specific CD8+ T Cell Response by Immunization with Microbial Protein Antigens Formulated with α-Galactosylceramide: Theory, Practice, and Protocols.

    PubMed

    Gilchuk, Pavlo; Knight, Frances C; Wilson, John T; Joyce, Sebastian

    2017-01-01

    CD8+ cytotoxic T lymphocytes confer protection against infectious diseases caused by viruses, bacteria, and parasites. Hence, significant efforts have been invested into devising ways to generate CD8+ T cell-targeted vaccines. Generation of microbe-free protein subunit vaccines requires a thorough knowledge of protective target antigens. Such antigens are proteolytically processed peptides presented by MHC class I molecules. To induce a robust antigen-specific CD8+ T cell response through vaccination, it is essential to formulate the antigen with an effective adjuvant. Here, we describe a versatile method for generating high-frequency antigen-specific CD8+ T cells through immunization of mice using the invariant natural killer T cell agonist α-galactosylceramide as the adjuvant.

  18. A Herpes Simplex Virus Type 1 Human Asymptomatic CD8+ T-Cell Epitopes-Based Vaccine Protects Against Ocular Herpes in a “Humanized” HLA Transgenic Rabbit Model

    PubMed Central

    Srivastava, Ruchi; Khan, Arif A.; Huang, Jiawei; Nesburn, Anthony B.; Wechsler, Steven L.; BenMohamed, Lbachir

    2015-01-01

    Purpose. A clinical vaccine that protects from ocular herpes simplex virus type 1 (HSV-1) infection and disease still is lacking. In the present study, preclinical vaccine trials of nine asymptomatic (ASYMP) peptides, selected from HSV-1 glycoproteins B (gB), and tegument proteins VP11/12 and VP13/14, were performed in the “humanized” HLA–transgenic rabbit (HLA-Tg rabbit) model of ocular herpes. We recently reported that these peptides are highly recognized by CD8+ T cells from “naturally” protected HSV-1–seropositive healthy ASYMP individuals (who have never had clinical herpes disease). Methods. Mixtures of three ASYMP CD8+ T-cell peptides derived from either HSV-1 gB, VP11/12, or VP13/14 were delivered subcutaneously to different groups of HLA-Tg rabbits (n = 10) in incomplete Freund's adjuvant, twice at 15-day intervals. The frequency and function of HSV-1 epitope-specific CD8+ T cells induced by these peptides and their protective efficacy, in terms of survival, virus replication in the eye, and ocular herpetic disease were assessed after an ocular challenge with HSV-1 (strain McKrae). Results. All mixtures elicited strong and polyfunctional IFN-γ– and TNF-α–producing CD107+CD8+ cytotoxic T cells, associated with a significant reduction in death, ocular herpes infection, and disease (P < 0.015). Conclusions. The results of this preclinical trial support the screening strategy used to select the HSV-1 ASYMP CD8+ T-cell epitopes, emphasize their valuable immunogenic and protective efficacy against ocular herpes, and provide a prototype vaccine formulation that may be highly efficacious for preventing ocular herpes in humans. PMID:26098469

  19. Signal peptides and trans-membrane regions are broadly immunogenic and have high CD8+ T cell epitope densities: Implications for vaccine development.

    PubMed

    Kovjazin, Riva; Volovitz, Ilan; Daon, Yair; Vider-Shalit, Tal; Azran, Roy; Tsaban, Lea; Carmon, Lior; Louzoun, Yoram

    2011-04-01

    Cell mediated immune response has a major role in controlling the elimination of infectious agents. The rational design of sub-unit peptide vaccines against intracellular pathogens or cancer requires the use of antigenic sequence/s that can induce highly potent, long lasting and antigen-specific responses in the majority of the population. A promising peptide selection strategy is the detection of multi-epitope peptide sequences with an ability to bind multiple MHC alleles. While past research sought the best epitopes based on their specific antigenicity, we ask whether specific defined domains have high epitope densities. Signal peptides and trans-membrane domains were found to have exceptionally high epitope densities. The improved MHC binding of these domains relies on their hydrophobic nature and, in signal peptides, also on their specific sequence. The high epitope density of SP was computed using in-silico methods and corroborated by the high percentage of identified SP epitope in the IEDB (immune epitope database). The enhanced immunogenicity of SP was then experimentally confirmed using a panel of nine peptides derived from Mycobacterium tuberculosis (MTb) proteins used in human PBMC proliferation assays and T cell lines functional assays. Our results show the exceptionally high antigen specific response rates and population coverage to SP sequences compared with non-SP peptide antigens derived from the same proteins. The results suggest a novel scheme for the rational design of T cell vaccines using a domain based rather than an epitope based approach.

  20. Combination of In Silico Methods in the Search for Potential CD4+ and CD8+ T Cell Epitopes in the Proteome of Leishmania braziliensis

    PubMed Central

    e Silva, Rafael de Freitas; Ferreira, Luiz Felipe Gomes Rebello; Hernandes, Marcelo Zaldini; de Brito, Maria Edileuza Felinto; de Oliveira, Beatriz Coutinho; da Silva, Ailton Alvaro; de-Melo-Neto, Osvaldo Pompílio; Rezende, Antônio Mauro; Pereira, Valéria Rêgo Alves

    2016-01-01

    The leishmaniases are neglected tropical diseases widespread throughout the globe, which are caused by protozoans from the genus Leishmania and are transmitted by infected phlebotomine flies. The development of a safe and effective vaccine against these diseases has been seen as the best alternative to control and reduce the number of cases. To support vaccine development, this work has applied an in silico approach to search for high potential peptide epitopes able to bind to different major histocompatibility complex Class I and Class II (MHC I and MHC II) molecules from different human populations. First, the predicted proteome of Leishmania braziliensis was compared and analyzed by modern linear programs to find epitopes with the capacity to trigger an immune response. This approach resulted in thousands of epitopes derived from 8,000 proteins conserved among different Leishmania species. Epitopes from proteins similar to those found in host species were excluded, and epitopes from proteins conserved between different Leishmania species and belonging to surface proteins were preferentially selected. The resulting epitopes were then clustered, to avoid redundancies, resulting in a total of 230 individual epitopes for MHC I and 2,319 for MHC II. These were used for molecular modeling and docking with MHC structures retrieved from the Protein Data Bank. Molecular docking then ranked epitopes based on their predicted binding affinity to both MHC I and II. Peptides corresponding to the top 10 ranked epitopes were synthesized and evaluated in vitro for their capacity to stimulate peripheral blood mononuclear cells (PBMC) from post-treated cutaneous leishmaniasis patients, with PBMC from healthy donors used as control. From the 10 peptides tested, 50% showed to be immunogenic and capable to stimulate the proliferation of lymphocytes from recovered individuals. PMID:27621732

  1. Diversified phenotype of antigen specific CD8+ T cells responding to the immunodominant epitopes of IE and pp65 antigens of human cytomegalovirus.

    PubMed

    Xu, Jian; Wu, Rong; Xiang, Fenfen; Kong, Qianqian; Hong, Jian; Kang, Xiangdong

    2015-06-01

    To study the cytomegalovirus (CMV)-specific CD8+ T cells in individuals with HLA A*1101, A*0201 and A*2402, our findings showed that peptide SK-10-2, KI-10 and KV-10 of CMV IE and pp65 antigens were immunodominant in 198 individuals with HLA A*1101, A*0201 and A*2402, the most frequent genotypes in Chinese. Interestingly, SK-10-2 induced the strongest T cell response to produce IFN-γ whereas the others did not induce prominent IFN-γ production despite they all induced remarkable T cell proliferation. The peptides induced different phenotypes including IFN-γ(high)TNF-α(low) and TNF-α(low)Foxp3(low). It suggests that only some of CMV-reactive CD8+ T cells are real protective IFN-γ(high) cytotoxic T cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Naive CD8⁺ T-cell precursors display structured TCR repertoires and composite antigen-driven selection dynamics.

    PubMed

    Neller, Michelle A; Ladell, Kristin; McLaren, James E; Matthews, Katherine K; Gostick, Emma; Pentier, Johanne M; Dolton, Garry; Schauenburg, Andrea J A; Koning, Dan; Fontaine Costa, Ana Isabel C A; Watkins, Thomas S; Venturi, Vanessa; Smith, Corey; Khanna, Rajiv; Miners, Kelly; Clement, Mathew; Wooldridge, Linda; Cole, David K; van Baarle, Debbie; Sewell, Andrew K; Burrows, Scott R; Price, David A; Miles, John J

    2015-08-01

    Basic parameters of the naive antigen (Ag)-specific T-cell repertoire in humans remain poorly defined. Systematic characterization of this 'ground state' immunity in comparison with memory will allow a better understanding of clonal selection during immune challenge. Here, we used high-definition cell isolation from umbilical cord blood samples to establish the baseline frequency, phenotype and T-cell antigen receptor (TCR) repertoire of CD8(+) T-cell precursor populations specific for a range of viral and self-derived Ags. Across the board, these precursor populations were phenotypically naive and occurred with hierarchical frequencies clustered by Ag specificity. The corresponding patterns of TCR architecture were highly ordered and displayed partial overlap with adult memory, indicating biased structuring of the T-cell repertoire during Ag-driven selection. Collectively, these results provide new insights into the complex nature and dynamics of the naive T-cell compartment.

  3. Upregulation of interleukin 7 receptor alpha and programmed death 1 marks an epitope-specific CD8+ T-cell response that disappears following primary Epstein-Barr virus infection.

    PubMed

    Sauce, Delphine; Larsen, Martin; Abbott, Rachel J M; Hislop, Andrew D; Leese, Alison M; Khan, Naeem; Papagno, Laura; Freeman, Gordon J; Rickinson, Alan B

    2009-09-01

    In immunocompetent individuals, the stability of the herpesvirus-host balance limits opportunities to study the disappearance of a virus-specific CD8(+) T-cell response. However, we noticed that in HLA-A 0201-positive infectious mononucleosis (IM) patients undergoing primary Epstein-Barr virus (EBV) infection, the initial CD8 response targets three EBV lytic antigen-derived epitopes, YVLDHLIVV (YVL), GLCTLVAML (GLC), and TLDYKPLSV (TLD), but only the YVL and GLC reactivities persist long-term; the TLD response disappears within 10 to 27 months. While present, TLD-specific cells remained largely indistinguishable from YVL and GLC reactivities in many phenotypic and functional respects but showed unique temporal changes in two markers of T-cell fate, interleukin 7 receptor alpha (IL-7Ralpha; CD127) and programmed death 1 (PD-1). Thus, following the antigen-driven downregulation of IL-7Ralpha seen on all populations in acute IM, in every case, the TLD-specific population recovered expression unusually quickly post-IM. As well, in four of six patients studied, TLD-specific cells showed very strong PD-1 upregulation in the last blood sample obtained before the cells' disappearance. Our data suggest that the disappearance of this individual epitope reactivity from an otherwise stable EBV-specific response (i) reflects a selective loss of cognate antigen restimulation (rather than of IL-7-dependent signals) and (ii) is immediately preceded, and perhaps mediated, by PD-1 upregulation to unprecedented levels.

  4. Toward the development of multi-epitope p53 cancer vaccines: an in vitro assessment of CD8(+) T cell responses to HLA class I-restricted wild-type sequence p53 peptides.

    PubMed

    Sakakura, Koichi; Chikamatsu, Kazuaki; Furuya, Nobuhiko; Appella, Ettore; Whiteside, Theresa L; Deleo, Albert B

    2007-10-01

    Wild-type sequence (wt) p53 peptides are attractive candidates for broadly applicable cancer vaccines. Six HLA-A2 or HLA-A24-restricted wt p53 peptides were evaluated for their ex vivo immunogenicity and their potential for use in cancer vaccines. Peripheral blood mononuclear cells (PBMC) obtained from HLA-A*0201(+) and/or HLA-A*2402(+) normal donors and subjects with squamous cell carcinoma of the head and neck (SCCHN) were analyzed for p53 peptide-specific reactivity in ELISPOT IFN-gamma assays. CD8(+) T cells in 7/10 normal donors (HD) and 11/23 subjects with SCCHN responded to at least one of the wt p53 peptides. CD8(+) T cell precursors responsive to wt p53 epitopes were detected in the circulation of most subjects with early disease, and an elevated blood Tc(1)/Tc(2) ratio distinguished wt p53 peptide responders from non-responders. The identification of multiple wt p53 peptides able to induce cytolytic T lymphocytes in most subjects with cancer promotes the development of multi-epitope p53 vaccines.

  5. Characterization of cross-reactive CD8+ T-cell recognition of HLA-A2-restricted HIV-Gag (SLYNTVATL) and HCV-NS5b (ALYDVVSKL) epitopes in individuals infected with human immunodeficiency and hepatitis C viruses.

    PubMed

    Vali, Bahareh; Tohn, Robert; Cohen, Michael J; Sakhdari, Ali; Sheth, Prameet M; Yue, Feng Yun; Wong, David; Kovacs, Colin; Kaul, Rupert; Ostrowski, Mario A

    2011-01-01

    The immunologic mechanisms underlying the faster progression of hepatitis C virus (HCV) disease in the presence of human immunodeficiency virus (HIV) coinfection are not clearly understood. T-cell cross-reactivity between HCV and influenza virus-specific epitopes has been associated with rapid progression of HCV disease (S. Urbani, B. Amadei, P. Fisicaro, M. Pilli, G. Missale, A. Bertoletti, and C. Ferrari, J. Exp. Med. 201:675-680, 2005). We asked whether T-cell cross-reactivity between HCV and HIV could exist during HCV/HIV coinfection and affect pathogenesis. Our search for amino acid sequence homology between the HCV and HIV proteomes revealed two similar HLA-A2-restricted epitopes, HIV-Gag (SLYNTVATL [HIV-SL9]) and HCV-NS5b (ALYDVVSKL [HCV-AL9]). We found that 4 out of 20 HLA-A2-positive (HLA-A2(+)) HIV-infected individuals had CD8(+) T cells that recognized both the HIV-SL9 and HCV-AL9 epitopes. However, the AL9 epitope was generally shown to be a weak agonist. Although HCV-monoinfected individuals in our study did not show AL9-specific responses, we found that about half of HCV/HIV-coinfected individuals had dual responses to both epitopes. High dual T-cell recognition among coinfected subjects was usually due to separate T-cell populations targeting each epitope, as determined by pentamer staining. The one individual demonstrating cross-reactive T cells to both epitopes showed the most advanced degree of liver disease. In coinfected individuals, we observed a positive correlation between the magnitudes of T-cell responses to both the SL9 and the AL9 epitopes, which was also positively associated with the clinical parameter of liver damage. Thus, we find that HIV infection induces T cells that can cross-react to heterologous viruses or prime for T cells that are closely related in sequence. However, the induction of cross-reactive T cells may not be associated with control of disease caused by the heterologous virus. This demonstrates that degeneracy of HIV

  6. Lymphocryptovirus Infection of Nonhuman Primate B Cells Converts Destructive into Productive Processing of the Pathogenic CD8 T Cell Epitope in Myelin Oligodendrocyte Glycoprotein

    PubMed Central

    Jagessar, S. Anwar; Holtman, Inge R.; Hofman, Sam; Morandi, Elena; Heijmans, Nicole; Laman, Jon D.; Gran, Bruno; Faber, Bart W.; van Kasteren, Sander I.; Eggen, Bart J. L.

    2016-01-01

    EBV is the major infectious environmental risk factor for multiple sclerosis (MS), but the underlying mechanisms remain obscure. Patient studies do not allow manipulation in vivo. We used the experimental autoimmune encephalomyelitis (EAE) models in the common marmoset and rhesus monkey to model the association of EBV and MS. We report that B cells infected with EBV-related lymphocryptovirus (LCV) are requisite APCs for MHC-E–restricted autoaggressive effector memory CTLs specific for the immunodominant epitope 40-48 of myelin oligodendrocyte glycoprotein (MOG). These T cells drive the EAE pathogenesis to irreversible neurologic deficit. The aim of this study was to determine why LCV infection is important for this pathogenic role of B cells. Transcriptome comparison of LCV-infected B cells and CD20+ spleen cells from rhesus monkeys shows increased expression of genes encoding elements of the Ag cross-presentation machinery (i.e., of proteasome maturation protein and immunoproteasome subunits) and enhanced expression of MHC-E and of costimulatory molecules (CD70 and CD80, but not CD86). It was also shown that altered expression of endolysosomal proteases (cathepsins) mitigates the fast endolysosomal degradation of the MOG40–48 core epitope. Finally, LCV infection also induced expression of LC3-II+ cytosolic structures resembling autophagosomes, which seem to form an intracellular compartment where the MOG40–48 epitope is protected against proteolytic degradation by the endolysosomal serine protease cathepsin G. In conclusion, LCV infection induces a variety of changes in B cells that underlies the conversion of destructive processing of the immunodominant MOG40–48 epitope into productive processing and cross-presentation to strongly autoaggressive CTLs. PMID:27412414

  7. CD8 Cells of Patients with Diffuse Cutaneous Leishmaniasis Display Functional Exhaustion: The Latter Is Reversed, In Vitro, by TLR2 Agonists

    PubMed Central

    Hernández-Ruiz, Joselín; Salaiza-Suazo, Norma; Carrada, Georgina; Escoto, Sofía; Ruiz-Remigio, Adriana; Rosenstein, Yvonne; Zentella, Alejandro; Becker, Ingeborg

    2010-01-01

    Leishmania mexicana (Lm) causes localized (LCL) and diffuse (DCL) cutaneous leishmaniasis. DCL patients have a poor cellular immune response leading to chronicity. It has been proposed that CD8 T lymphocytes (CD8) play a crucial role in infection clearance, although the role of CD8 cytotoxicity in disease control has not been elucidated. Lesions of DCL patients have been shown to harbor low numbers of CD8, as compared to patients with LCL, and leishmanicidal treatment restores CD8 numbers. The marked response of CD8 towards Leishmania parasites led us to analyze possible functional differences between CD8 from patients with LCL and DCL. We compared IFNγ production, antigen-specific proliferation, and cytotoxicity of CD8 purified from PBMC against autologous macrophages (MO) infected with Leishmania mexicana (MOi). Additionally, we analyzed tissue biopsies from both groups of patients for evidence of cytotoxicity associated with apoptotic cells in the lesions. We found that CD8 cell of DCL patients exhibited low cytotoxicity, low antigen-specific proliferation and low IFNγ production when stimulated with MOi, as compared to LCL patients. Additionally, DCL patients had significantly less TUNEL+ cells in their lesions. These characteristics are similar to cellular “exhaustion” described in chronic infections. We intended to restore the functional capacity of CD8 cells of DCL patients by preincubating them with TLR2 agonists: Lm lipophosphoglycan (LPG) or Pam3Cys. Cytotoxicity against MOi, antigen-specific proliferation and IFNγ production were restored with both stimuli, whereas PD-1 (a molecule associated with cellular exhaustion) expression, was reduced. Our work suggests that CD8 response is associated with control of Lm infection in LCL patients and that chronic infection in DCL patients leads to a state of CD8 functional exhaustion, which could facilitate disease spread. This is the first report that shows the presence of functionally exhausted CD8 T

  8. Efficient epitope mapping by bacteriophage {lambda} surface display

    SciTech Connect

    Kuwabara, I.; Maruyama, H.; Zuberi, R.I.

    1997-01-01

    A bacteriophage {lambda} surface expression system, {lambda}foo, was used for epitope mapping of human galectin-3. We constructed random epitope and peptide libraries and compared their efficiencies in the mapping. The galectin-3 cDNA was randomly digested by DNase I to make random epitope libraries. The libraries were screened by affinity selection using a microtiter plate coated with monoclonal antibodies. Direct DNA sequencing of the selected clones defined two distinct epitope sites consisting of nine and 11 amino-acid residues. Affinity selection of random peptide libraries recovered a number of sequences that were similar to each other but distinct from the galectin-3 sequence. These results demonstrate that a single affinity selection of epitope libraries with antibodies is able to define an epitope determinant as small as nine residues long and is more efficient in epitope mapping than random peptide libraries. 25 refs., 4 figs., 1 tab.

  9. Strategies to Query and Display Allergy-Derived Epitope Data from the Immune Epitope Database

    PubMed Central

    Vaughan, Kerrie; Peters, Bjoern; Larche, Mark; Pomes, Anna; Broide, David; Sette, Alessandro

    2013-01-01

    The recognition of specific epitopes on allergens by antibodies and T cells is a key element in allergic processes. Analysis of epitope data may be of interest for basic immunopathology or for potential application in diagnostics or immunotherapy. The Immune Epitope Database (IEDB) is a freely available repository of epitope data from infectious disease agents, as well as epitopes defined for allergy, autoimmunity, and transplantation. The IEDB curates the experiments associated with each epitope and thus provides a variety of different ways to search the data. This review aims to demonstrate the utility of the IEDB and its query strategies, including searching by epitope structure (peptidic/nonpeptidic), by assay methodology, by host, by the allergen itself, or by the organism from which the allergen was derived. Links to tools for visualization of 3-D structures, epitope prediction, and analyses of B and T cell reactivity by host response frequency score are also highlighted. PMID:23172234

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

    PubMed

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

    2016-05-04

    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.

  11. 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

  12. IL-2 production by virus- and tumor-specific human CD8 T cells is determined by their fine specificity.

    PubMed

    Mallard, Eric; Vernel-Pauillac, Frédérique; Velu, Thierry; Lehmann, Frédéric; Abastado, Jean-Pierre; Salcedo, Margarita; Bercovici, Nadège

    2004-03-15

    Memory CD8 T cells mediate rapid and effective immune responses against previously encountered Ags. However, these cells display considerable phenotypic and functional heterogeneity. In an effort to identify parameters that correlate with immune protection, we compared cell surface markers, proliferation, and cytokine production of distinct virus- and tumor-specific human CD8 populations. Phenotypic analysis of epitope-specific CD8 T cells showed that Ag specificity is associated with distinct CCR7/CD45RA expression profiles, suggesting that Ag recognition drives the expression of these molecules on effector/memory T cells. Moreover, the majority of central memory T cells (CD45RAlowCCR7dull) secreting cytokines in response to an EBV epitope produces both IL-2 and IFN-gamma, whereas effector memory CD8 cells (CD45RAdullCCR7-) found in EBV, CMV, or Melan-A memory pools are mostly composed of cells secreting exclusively IFN-gamma. However, these various subsets, including Melan-A-specific effector memory cells differentiated in cancer patients, display similar Ag-driven proliferation in vitro. Our findings show for the first time that human epitope-specific CD8 memory pools differ in IL-2 production after antigenic stimulation, although they display similar intrinsic proliferation capacity. These results provide new insights in the characterization of human virus- and tumor-specific CD8 lymphocytes.

  13. Human Asymptomatic Epitopes Identified from the Herpes Simplex Virus Tegument Protein VP13/14 (UL47) Preferentially Recall Polyfunctional Effector Memory CD44high CD62Llow CD8+ TEM Cells and Protect Humanized HLA-A*02:01 Transgenic Mice against Ocular Herpesvirus Infection

    PubMed Central

    Srivastava, Ruchi; Khan, Arif A.; Garg, Sumit; Syed, Sabrina A.; Furness, Julie N.; Vahed, Hawa; Pham, Tiffany; Yu, Howard T.; Nesburn, Anthony B.

    2016-01-01

    ABSTRACT Herpes simplex virus 1 (HSV-1) infection is widespread among humans. The HSV-1 virion protein 13/14 (VP13/14), also known as UL47, is a tegument antigen targeted by CD8+ T cells from HSV-seropositive individuals. However, whether VP13/14-specific CD8+ T cells play a role in the natural protection seen in asymptomatic (ASYMP) individuals (individuals who have never had a clinical herpetic disease) has not been elucidated. Using predictive computer-assisted algorithms, we identified 10 potential HLA-A*02:01-restricted CD8+ T-cell epitopes from the 693-amino-acid sequence of the VP13/14 protein. Three out of 10 epitopes exhibited a high to moderate affinity of binding to soluble HLA-A*02:01 molecules. The phenotype and function of CD8+ T cells specific for each epitope were compared in HLA-A*02:01-positive ASYMP individuals and symptomatic (SYMP) individuals (individuals who have frequent clinical herpetic diseases) using determination of a combination of tetramer frequency and the levels of granzyme B, granzyme K, perforin, gamma interferon, tumor necrosis factor alpha, and interleukin-2 production and CD107a/b cytotoxic degranulation. High frequencies of multifunctional CD8+ T cells directed against three epitopes, VP13/14 from amino acids 286 to 294 (VP13/14286–294), VP13/14 from amino acids 504 to 512 (VP13/14504–512), and VP13/14 from amino acids 544 to 552 (VP13/14544–552), were detected in ASYMP individuals, while only low frequencies were detected in SYMP individuals. The three epitopes also predominantly recalled more CD45RAlow CD44high CCR7low CD62Llow CD8+ effector memory T cells (TEM cells) in ASYMP individuals than SYMP individuals. Moreover, immunization of HLA-A*02:01 transgenic mice with the three CD8+ TEM-cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8+ TEM cells associated with strong protective immunity against ocular herpesvirus infection and disease. Our findings outline the phenotypic and

  14. HLA-A02:01-restricted epitopes identified from the herpes simplex virus tegument protein VP11/12 preferentially recall polyfunctional effector memory CD8+ T cells from seropositive asymptomatic individuals and protect humanized HLA-A*02:01 transgenic mice against ocular herpes.

    PubMed

    Srivastava, Ruchi; Khan, Arif A; Spencer, Doran; Vahed, Hawa; Lopes, Patricia P; Thai, Nhi Thi Uyen; Wang, Christine; Pham, Thanh T; Huang, Jiawei; Scarfone, Vanessa M; Nesburn, Anthony B; Wechsler, Steven L; BenMohamed, Lbachir

    2015-03-01

    The HSV type 1 tegument virion phosphoprotein (VP) 11/12 (VP11/12) is a major Ag targeted by CD8(+) T cells from HSV-seropositive individuals. However, whether and which VP11/12 epitope-specific CD8(+) T cells play a role in the "natural" protection seen in seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we used multiple prediction computer-assisted algorithms to identify 10 potential HLA-A*02:01-restricted CD8(+) T cell epitopes from the 718-aa sequence of VP11/12. Three of 10 epitopes exhibited high-to-moderate binding affinity to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01-positive and HSV-1-seropositive ASYMP individuals, the most frequent, robust, and polyfunctional effector CD8(+) T cell responses, as assessed by a combination of tetramer frequency, granzyme B, granzyme K, perforin, CD107(a/b) cytotoxic degranulation, IFN-γ, and multiplex cytokines assays, were predominantly directed against three epitopes: VP11/1266-74, VP11/12220-228, and VP11/12702-710. Interestingly, ASYMP individuals had a significantly higher proportion of CD45RA(low)CCR7(low)CD44(high)CD62L(low)CD27(low)CD28(low)CD8(+) effector memory CD8(+) T cells (TEMs) specific to the three epitopes, compared with symptomatic individuals (with a history of numerous episodes of recurrent ocular herpetic disease). Moreover, immunization of HLA-A*02:01 transgenic mice with the three ASYMP CD8(+) TEM cell epitopes induced robust and polyfunctional epitope-specific CD8(+) TEM cells that were associated with a strong protective immunity against ocular herpes infection and disease. Our findings outline phenotypic and functional features of protective HSV-specific CD8(+) T cells that should guide the development of an effective T cell-based herpes vaccine. Copyright © 2015 by The American Association of Immunologists, Inc.

  15. Human Asymptomatic Epitopes Identified from the Herpes Simplex Virus Tegument Protein VP13/14 (UL47) Preferentially Recall Polyfunctional Effector Memory CD44high CD62Llow CD8+ TEM Cells and Protect Humanized HLA-A*02:01 Transgenic Mice against Ocular Herpesvirus Infection.

    PubMed

    Srivastava, Ruchi; Khan, Arif A; Garg, Sumit; Syed, Sabrina A; Furness, Julie N; Vahed, Hawa; Pham, Tiffany; Yu, Howard T; Nesburn, Anthony B; BenMohamed, Lbachir

    2017-01-15

    Herpes simplex virus 1 (HSV-1) infection is widespread among humans. The HSV-1 virion protein 13/14 (VP13/14), also known as UL47, is a tegument antigen targeted by CD8(+) T cells from HSV-seropositive individuals. However, whether VP13/14-specific CD8(+) T cells play a role in the natural protection seen in asymptomatic (ASYMP) individuals (individuals who have never had a clinical herpetic disease) has not been elucidated. Using predictive computer-assisted algorithms, we identified 10 potential HLA-A*02:01-restricted CD8(+) T-cell epitopes from the 693-amino-acid sequence of the VP13/14 protein. Three out of 10 epitopes exhibited a high to moderate affinity of binding to soluble HLA-A*02:01 molecules. The phenotype and function of CD8(+) T cells specific for each epitope were compared in HLA-A*02:01-positive ASYMP individuals and symptomatic (SYMP) individuals (individuals who have frequent clinical herpetic diseases) using determination of a combination of tetramer frequency and the levels of granzyme B, granzyme K, perforin, gamma interferon, tumor necrosis factor alpha, and interleukin-2 production and CD107(a/b) cytotoxic degranulation. High frequencies of multifunctional CD8(+) T cells directed against three epitopes, VP13/14 from amino acids 286 to 294 (VP13/14286-294), VP13/14 from amino acids 504 to 512 (VP13/14504-512), and VP13/14 from amino acids 544 to 552 (VP13/14544-552), were detected in ASYMP individuals, while only low frequencies were detected in SYMP individuals. The three epitopes also predominantly recalled more CD45RA(low) CD44(high) CCR7(low) CD62L(low) CD8(+) effector memory T cells (TEM cells) in ASYMP individuals than SYMP individuals. Moreover, immunization of HLA-A*02:01 transgenic mice with the three CD8(+) TEM-cell epitopes from ASYMP individuals induced robust and polyfunctional HSV-specific CD8(+) TEM cells associated with strong protective immunity against ocular herpesvirus infection and disease. Our findings outline the

  16. HLA-A02:01-Restricted Epitopes Identified from the Herpes Simplex Virus Tegument Protein VP11/12 Preferentially Recall Polyfunctional Effector Memory CD8+ T Cells from Seropositive Asymptomatic Individuals and Protect “Humanized” HLA-A*02:01 Transgenic Mice Against Ocular Herpes

    PubMed Central

    Srivastava, Ruchi; Khan, Arif A.; Spencer, Doran; Vahed, Hawa; Lopes, Patricia P.; Thai, Nhi Thi Uyen; Wang, Christine; Pham, Thanh T.; Huang, Jiawei; Scarfone, Vanessa M.; Nesburn, Anthony B.; Wechsler, Steven L.; BenMohamed, Lbachir

    2014-01-01

    The Herpes Simplex Virus type 1 virion tegument phosphoprotein 11/12 (HSV-1 VP11/12) is a major antigen targeted by CD8+ T cells from HSV-seropositive individuals. However, whether and which VP11/12-epitope-specific CD8+ T cells play a role in the “natural” protection seen in seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we used multiple prediction computer-assisted algorithms to identify 10 potential HLA-A*02:01-restricted CD8+ T cell epitopes from the 716 amino acids sequence of VP11/12. Three out of ten epitopes exhibited high to moderate binding affinity to HLA-A*02:01 molecules. In ten sequentially studied HLA-A*02:01 positive and HSV-1-seropositive ASYMP individuals, the most frequent, robust and polyfunctional effector CD8+ T-cell responses, as assessed by a combination of tetramer frequency, granzyme B, granzyme K, perforin, CD107a/b cytotoxic degranulation, IFN-γ and multiplex cytokines assays, were predominantly directed against three epitopes: VP11/1266–74, VP11/12220–228 and VP11/12702–710. Interestingly, ASYMP individuals had significantly higher proportion of CD45RAlowCCR7lowCD44highCD62LlowCD27lowCD28lowCD8+ effector memory T cells (TEM) specific to the three epitopes, compared to symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpetic disease). Moreover, immunization of HLA-A*02:01 transgenic mice with the three ASYMP CD8+ TEM cell epitopes induced robust and polyfunctional epitope-specific CD8+ TEM cells that were associated with a strong protective immunity against ocular herpes infection and disease. Our findings outline phenotypic and functional features of protective HSV-specific CD8+ T cells that should guide the development of an effective T-cell-based herpes vaccine. PMID:25617474

  17. Characterization of cloned class I MHC-restricted, CD8+ anti-Meth A cytotoxic T-lymphocytes: recognition of an epitope derived from the Meth A gp110 tumor rejection antigen.

    PubMed

    Fassanito, M A; Loftus, D; De Leo, R M; Law, L W; Appella, E; De Leo, A B

    1994-08-15

    Meth A gp110 has been tentatively identified as a tumor rejection antigen. Following isolation of a class I major histocompatibility complex (MHC)-restricted, CD8+ anti-Meth A cytotoxic T-lymphocyte (CTL), we sought to determine whether the determinant recognized by this CTL was: (a) functional in tumor rejection of Meth A sarcoma; and (b) derived from Meth A gp110. Initially, we isolated an anti-Meth A CTL-resistant variant of Meth A sarcoma, Meth A4R, by immunoselection. The results of the subsequent analysis of Meth A4R cells showed the CTL-defined determinant as having a functional role in transplantation rejection of Meth A sarcoma. Walker et al. (Proc. Natl. Acad. Sci. USA, 89: 7915-7918, 1993) showed that the cationic lipid, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium-methyl sulfate, mediated delivery of a recombinant glycoprotein into the cytosol of target cells, making it available for processing and presentation by class I MHC molecules. As a result, the cells were sensitized for cytolysis by a class I MHC-restricted CD8+ CTL, which recognized an epitope expressed by the glycoprotein. In a similar manner, we treated the SV40-transformed BALB/c cell line, SVBalb, which is relatively insensitive to cytolysis by the anti-Meth A CTL, with Meth A gp110 and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate. The sensitivities of the treated cells and control cell lines to the anti-Meth A CTL were then examined. The results of these experiments permit us to conclude that the determinant recognized by the anti-Meth A CTL line is derived from Meth A gp110.

  18. Intravaginal immunization with HPV vectors induces tissue-resident CD8+ T cell responses

    PubMed Central

    Çuburu, Nicolas; Graham, Barney S.; Buck, Christopher B.; Kines, Rhonda C.; Pang, Yuk-Ying S.; Day, Patricia M.; Lowy, Douglas R.; Schiller, John T.

    2012-01-01

    The induction of persistent intraepithelial CD8+ T cell responses may be key to the development of vaccines against mucosally transmitted pathogens, particularly for sexually transmitted diseases. Here we investigated CD8+ T cell responses in the female mouse cervicovaginal mucosa after intravaginal immunization with human papillomavirus vectors (HPV pseudoviruses) that transiently expressed a model antigen, respiratory syncytial virus (RSV) M/M2, in cervicovaginal keratinocytes. An HPV intravaginal prime/boost with different HPV serotypes induced 10-fold more cervicovaginal antigen-specific CD8+ T cells than priming alone. Antigen-specific T cell numbers decreased only 2-fold after 6 months. Most genital antigen-specific CD8+ T cells were intra- or subepithelial, expressed αE-integrin CD103, produced IFN-γ and TNF-α, and displayed in vivo cytotoxicity. Using a sphingosine-1-phosphate analog (FTY720), we found that the primed CD8+ T cells proliferated in the cervicovaginal mucosa upon HPV intravaginal boost. Intravaginal HPV prime/boost reduced cervicovaginal viral titers 1,000-fold after intravaginal challenge with vaccinia virus expressing the CD8 epitope M2. In contrast, intramuscular prime/boost with an adenovirus type 5 vector induced a higher level of systemic CD8+ T cells but failed to induce intraepithelial CD103+CD8+ T cells or protect against recombinant vaccinia vaginal challenge. Thus, HPV vectors are attractive gene-delivery platforms for inducing durable intraepithelial cervicovaginal CD8+ T cell responses by promoting local proliferation and retention of primed antigen-specific CD8+ T cells. PMID:23143305

  19. PD-1+Tim-3+ CD8+ T Lymphocytes Display Varied Degrees of Functional Exhaustion in Patients with Regionally Metastatic Differentiated Thyroid Cancer

    PubMed Central

    Severson, Jill J.; Serracino, Hilary S.; Mateescu, Valerica; Raeburn, Christopher D.; C.McIntyre, Robert; Sams, Sharon B.; Haugen, Bryan R.; French, Jena D.

    2015-01-01

    Regional metastatic differentiated thyroid cancer (mDTC) provides a unique model in which to study the tumor-immune interface. These lymph node (LN) metastases persist for years, generally without progression to distant metastases. While the immune system likely impedes disease progression, it is unsuccessful in eliminating disease. Our previous studies revealed that programmed death-1 (PD-1)+ T cells were enriched in tumor-involved lymph nodes (TILN). Tumor-associated leukocytes and tumor cells were collected from grossly involved LNs from 12 patients to further characterize the phenotype and functional potential of mDTC-associated PD-1+ T cells. PD-1+CD4+ and PD-1+CD8+ T cells were enriched in 8/12 TILN samples. PD-1+ T cells co-expressed Tim-3 and CD69 and failed to down-regulate CD27. CD8+ T cells, but not CD4+ T cells, from these samples were variably deficient in their ability to produce effector cytokines when compared to control TILNs that lacked resident PD-1+ T cells. PD-1+CD8+ T cells were capable of exocytosis but lacked intracellular perforin. Surprisingly, T-cell proliferative capacity was largely maintained in all samples. Thus, while PD-1 expression by mDTC-associated CD8+ T cells was associated with dysfunction, exhaustion was not complete. Notably, molecular markers of exhaustion did not translate to dysfunction in all samples or in CD4+ T cells. Regulatory T (Treg) cells, PD-L1, and galectin-9 were commonly found in mDTC and likely contributed to the initiation of T-cell exhaustion and disease progression. Therapies that release the effects of PD-1 and Tim-3 and reduce the suppressive effects of Tregs may encourage tumor elimination in patients with mDTC. PMID:25701326

  20. Application of phage peptide display technology for the study of food allergen epitopes.

    PubMed

    Chen, Xueni; Dreskin, Stephen C

    2017-06-01

    Phage peptide display technology has been used to identify IgE-binding mimotopes (mimics of natural epitopes) that mimic conformational epitopes. This approach is effective in the characterization of those epitopes that are important for eliciting IgE-mediated allergic responses by food allergens and those that are responsible for cross-reactivity among allergenic food proteins. Application of this technology will increase our understanding of the mechanisms whereby food allergens elicit allergic reactions, will facilitate the discovery of diagnostic reagents and may lead to mimotope-based immunotherapy. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Phage-Displayed T-Cell Epitope Grafted into Immunoglobulin Heavy-Chain Complementarity-Determining Regions: an Effective Vaccine Design Tested in Murine Cysticercosis

    PubMed Central

    Manoutcharian, Karen; Terrazas, Luis Ignacio; Gevorkian, Goar; Acero, Gonzalo; Petrossian, Pavel; Rodriguez, Miriam; Govezensky, Tzipe

    1999-01-01

    A new type of immunogenic molecule was engineered by replacing all three complementarity-determining-region (CDR) loops of the human immunoglobulin (Ig) heavy-chain variable (VH) domain with the Taenia crassiceps epitope PT1 (PPPVDYLYQT) and by displaying this construct on the surfaces of M13 bacteriophage. When BALB/c mice were immunized with such phage particles (PIgphage), a strong protection against challenge infection in very susceptible female hosts was obtained. When specifically stimulated, the in vivo-primed CD4+ and CD8+ T cells isolated from mice immunized with PT1, both as a free peptide and as the PIgphage construct, proliferated in vitro, indicating efficient epitope presentation by both major histocompatibility complex class II and class I molecules in the specifically antigen-pulsed macrophages used as antigen-presenting cells. These data demonstrate the immunogenic potential of recombinant phage particles displaying CDR epitope-grafted Ig VH domains and establish an alternative approach to the design of an effective subunit vaccine for prevention of cysticercosis. The key advantage of this type of immunogen is that no adjuvant is required for its application. The proposed strategy for immunogen construction is potentially suitable for use in any host-pathogen interaction. PMID:10456929

  2. CD8(+) T cells of Listeria monocytogenes-infected mice recognize both linear and spliced proteasome products.

    PubMed

    Platteel, Anouk C M; Mishto, Michele; Textoris-Taube, Kathrin; Keller, Christin; Liepe, Juliane; Busch, Dirk H; Kloetzel, Peter M; Sijts, Alice J A M

    2016-05-01

    CD8(+) T cells responding to infection recognize pathogen-derived epitopes presented by MHC class-I molecules. While most of such epitopes are generated by proteasome-mediated antigen cleavage, analysis of tumor antigen processing has revealed that epitopes may also derive from proteasome-catalyzed peptide splicing (PCPS). To determine whether PCPS contributes to epitope processing during infection, we analyzed the fragments produced by purified proteasomes from a Listeria monocytogenes polypeptide. Mass spectrometry identified a known H-2K(b) -presented linear epitope (LLO296-304 ) in the digests, as well as four spliced peptides that were trimmed by ERAP into peptides with in silico predicted H-2K(b) binding affinity. These spliced peptides, which displayed sequence similarity with LLO296-304 , bound to H-2K(b) molecules in cellular assays and one of the peptides was recognized by CD8(+) T cells of infected mice. This spliced epitope differed by one amino acid from LLO296-304 and double staining with LLO296-304 - and spliced peptide-folded MHC multimers showed that LLO296-304 and its spliced variant were recognized by the same CD8(+) T cells. Thus, PCPS multiplies the variety of peptides that is processed from an antigen and leads to the production of epitope variants that can be recognized by cross-reacting pathogen-specific CD8(+) T cells. Such mechanism may reduce the chances for pathogen immune evasion. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Protective Antibody and CD8+ T-Cell Responses to the Plasmodium falciparum Circumsporozoite Protein Induced by a Nanoparticle Vaccine

    PubMed Central

    Kaba, Stephen A.; McCoy, Margaret E.; Doll, Tais A. P. F.; Brando, Clara; Guo, Qin; Dasgupta, Debleena; Yang, Yongkun; Mittelholzer, Christian; Spaccapelo, Roberta; Crisanti, Andrea; Burkhard, Peter; Lanar, David E.

    2012-01-01

    Background The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8+ and CD4+ T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN) vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects. Methodology/Principal Findings To establish the basis for a SAPN-based vaccine, B- and CD8+ T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP) and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (∼125 amino acids) that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP). We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year) protective antibody and poly-functional (IFNγ+, IL-2+) long-lived central memory CD8+ T-cells. Furthermore, we demonstrated that these Ab or CD8+ T–cells can independently provide sterile protection against a lethal challenge of the transgenic parasites. Conclusion The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP) and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP. PMID:23144750

  4. Identification of Theileria lestoquardi Antigens Recognized by CD8+ T Cells

    PubMed Central

    Ngugi, Daniel; Lizundia, Regina; Hostettler, Isabel; Woods, Kerry; Ballingall, Keith; MacHugh, Niall D.; Morrison, W. Ivan; Weir, Willie; Shiels, Brian; Werling, Dirk

    2016-01-01

    As part of an international effort to develop vaccines for Theileria lestoquardi, we undertook a limited screen to test T. lestoquardi orthologues of antigens recognised by CD8+ T lymphocyte responses against T. annulata and T. parva in cattle. Five MHC defined sheep were immunized by live T. lestoquardi infection and their CD8+ T lymphocyte responses determined. Thirteen T. lestoquardi orthologues of T. parva and T. annulata genes, previously shown to be targets of CD8+ T lymphocyte responses of immune cattle, were expressed in autologous fibroblasts and screened for T cell recognition using an IFNγ assay. Genes encoding T. lestoquardi antigens Tl8 (putative cysteine proteinase, 349 aa) or Tl9 (hypothetical secreted protein, 293 aa) were recognise by T cells from one animal that displayed a unique MHC class I genotype. Antigenic 9-mer peptide epitopes of Tl8 and Tl9 were identified through peptide scans using CD8+ T cells from the responding animal. These experiments identify the first T. lestoquardi antigens recognised by CD8+ T cell responses linked to specific MHC class I alleles. PMID:27611868

  5. Surface Display of Foreign Epitopes on the Lactobacillus brevis S-Layer

    PubMed Central

    Åvall-Jääskeläinen, Silja; Kylä-Nikkilä, Kari; Kahala, Minna; Miikkulainen-Lahti, Terhi; Palva, Airi

    2002-01-01

    So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure. PMID:12450814

  6. Surface display of foreign epitopes on the Lactobacillus brevis S-layer.

    PubMed

    Avall-Jääskeläinen, Silja; Kylä-Nikkilä, Kari; Kahala, Minna; Miikkulainen-Lahti, Terhi; Palva, Airi

    2002-12-01

    So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure.

  7. Epitope mapping of metuximab on CD147 using phage display and molecular docking.

    PubMed

    He, Bifang; Mao, Canquan; Ru, Beibei; Han, Hesong; Zhou, Peng; Huang, Jian

    2013-01-01

    Metuximab is the generic name of Licartin, a new drug for radioimmunotherapy of hepatocellular carcinoma. Although it is known to be a mouse monoclonal antibody against CD147, the complete epitope mediating the binding of metuximab to CD147 remains unknown. We panned the Ph.D.-12 phage display peptide library against metuximab and got six mimotopes. The following bioinformatics analysis based on mimotopes suggested that metuximab recognizes a conformational epitope composed of more than 20 residues. The residues of its epitope may include T28, V30, K36, L38, K57, F74, D77, S78, D79, D80, Q81, G83, S86, N98, Q100, L101, H102, G103, P104, V131, P132, and K191. The homology modeling of metuximab and the docking of CD147 to metuximab were also performed. Based on the top one docking model, the epitope was predicted to contain 28 residues: AGTVFTTV (23-30), I37, D45, E84, V88, EPMGTANIQLH (92-102), VPP (131-133), Q164, and K191. Almost half of the residues predicted on the basis of mimotope analysis also appear in the docking result, indicating that both results are reliable. As the predicted epitopes of metuximab largely overlap with interfaces of CD147-CD147 interactions, a structural mechanism of metuximab is proposed as blocking the formation of CD147 dimer.

  8. Tumor-reactive CD8+ T cells in metastatic gastrointestinal cancer refractory to chemotherapy.

    PubMed

    Turcotte, Simon; Gros, Alena; Tran, Eric; Lee, Chyi-Chia R; Wunderlich, John R; Robbins, Paul F; Rosenberg, Steven A

    2014-01-15

    To evaluate whether patients with metastatic gastrointestinal adenocarcinomas refractory to chemotherapy harbor tumor-reactive cytotoxic T cells. Expansion of CD8(+) tumor-infiltrating lymphocytes (TIL) and cancer cell lines was attempted from gastrointestinal cancer metastases in 16 consecutive patients for the study of antitumor immune recognition. Retroviral transduction of genes encoding T-cell receptors (TCR) was used to define HLA-restriction elements and specific reactivity. TIL were expanded from metastases in all patients, and new tumor cell lines were generated in 5 patients. Autologous tumor recognition without cross-reactivity against allogeneic HLA-matched gastrointestinal tumors was found in CD8(+) TIL from 3 of these 5 patients. In a patient with gastric cancer liver metastases, the repertoire of CD8(+) TIL was dominated by cytolytic sister clones reactive to 2 out of 4 autologous cancer cell lines restricted by HLA-C*0701. From the same patient, a rare CD8(+) TIL clone with a distinct TCR recognized all four cancer cell lines restricted by HLA-B*4901. In a patient with bile duct cancer, two distinct antitumor cytolytic clones were isolated from a highly polyclonal CD8(+) TIL repertoire. TCRs isolated from these clones recognized epitopes restricted by HLA-A*0201. In a third patient, CD8(+) TIL reactivity was progressively lost against an autologous colon cancer cell line that displayed loss of HLA haplotype. This study provides a basis for the development of immunotherapy for patients with advanced gastrointestinal malignancies by first establishing the presence of naturally occurring tumor-reactive CD8(+) TIL at the molecular level. ©2013 AACR.

  9. Tumor-reactive CD8+ T cells in metastatic gastrointestinal cancer refractory to chemotherapy

    PubMed Central

    Turcotte, S.; Gros, A.; Tran, E.; Lee, C.-C. R.; Wunderlich, J.R.; Robbins, P.F.; Rosenberg, S.A.

    2014-01-01

    Purpose To evaluate whether patients with metastatic gastrointestinal (GI) adenocarcinomas refractory to chemotherapy harbor tumor-reactive cytotoxic T cells. Experimental design Expansion of CD8+ tumor-infiltrating lymphocytes (TIL) and cancer cell lines was attempted from GI cancer metastases in 16 consecutive patients for the study of anti-tumor immune recognition. Retroviral transduction of genes encoding T-cell receptors (TCR) was used to define HLA-restriction elements and specific reactivity. Results TIL were expanded from metastases in all patients, and new tumor cell lines generated in five patients. Autologous tumor-recognition without cross-reactivity against allogeneic HLA-matched GI tumors was found in CD8+ TIL from three of these five patients. In a patient with gastric cancer liver metastases, the repertoire of CD8+ TIL was dominated by cytolytic sister clones reactive to 2 out of 4 autologous cancer cell lines restricted by HLA-C*0701. From the same patient, a rare CD8+ TIL clone with a distinct TCR recognized all four cancer cell lines restricted by HLA-B*4901. In a patient with bile duct cancer, two distinct anti-tumor cytolytic clones were isolated from a highly polyclonal CD8+ TIL repertoire. TCRs isolated from these clones recognized epitopes restricted by HLA-A*0201. In a third patient, CD8+ TIL reactivity was progressively lost against an autologous colon cancer cell line that displayed loss of HLA haplotype. Conclusions This study provides a basis for the development of immunotherapy for patients with advanced GI malignancies by first establishing the presence of naturally occurring tumor-reactive CD8+ TIL at the molecular level. PMID:24218514

  10. CD4⁺ and CD8⁺ regulatory T cells (Tregs) are elevated and display an active phenotype in patients with chronic HCV mono-infection and HIV/HCV co-infection.

    PubMed

    Hartling, H J; Gaardbo, J C; Ronit, A; Knudsen, L S; Ullum, H; Vainer, B; Clausen, M R; Skogstrand, K; Gerstoft, J; Nielsen, S D

    2012-09-01

    The aim of this study was to examine regulatory T cells (Tregs) in peripheral blood and liver tissue in patients with chronic hepatitis C virus (HCV) mono-infection and in patients with HIV/HCV co-infection. In a cross-sectional study were included 51 patients with chronic HCV infection, 24 patients with HIV/HCV co-infection and 24 healthy individuals. CD4⁺ and CD8⁺ Tregs were determined using flow cytometry. Fibrosis was examined by transient elastography. Inflammation, fibrosis and Tregs were determined in liver biopsies from 12 patients. Increased frequency of CD4⁺ and CD8⁺ Tregs was found in HIV/HCV co-infected patients [median: 6.4% (IQR: 5.7-6.9) and 1.0% (0.7-1.2), respectively] compared to HCV mono-infected patients [5.6% (4.2-6.3), P = 0.01 and 0.5% (0.3-0.7), P < 0.001, respectively]. Furthermore, HCV mono-infected patients had increased frequencies of Tregs compared with healthy controls (P < 0.05). However, no associations between the frequency of Tregs and fibrosis were found. Furthermore, characterization of CD4⁺ Tregs using CD45RA demonstrated a higher frequency of activated Tregs in both HCV mono-infected and HIV/HCV co-infected patients compared with healthy controls. Finally, number of intrahepatic Tregs was associated with both peripheral CD8⁺ Tregs and intrahepatic inflammation. In conclusion, HCV mono-infected patients and particularly HIV/HCV co-infected patients have increased the frequency of CD4⁺ and CD8⁺ Tregs compared with healthy controls. Furthermore, CD4⁺ Tregs in infected patients displayed an active phenotype. Tregs were not associated with fibrosis, but a positive correlation between intrahepatic Tregs and inflammation was found. Taken together, these results suggest a role for Tregs in the pathogenesis of chronic HCV infection.

  11. Infection with Trypanosoma cruzi restricts the repertoire of parasite-specific CD8+ T cells leading to immunodominance.

    PubMed

    Tzelepis, Fanny; de Alencar, Bruna C G; Penido, Marcus L O; Claser, Carla; Machado, Alexandre V; Bruna-Romero, Oscar; Gazzinelli, Ricardo T; Rodrigues, Mauricio M

    2008-02-01

    Interference or competition between CD8(+) T cells restricted by distinct MHC-I molecules can be a powerful means to establish an immunodominant response. However, its importance during infections is still questionable. In this study, we describe that following infection of mice with the human pathogen Trypanosoma cruzi, an immunodominant CD8(+) T cell immune response is developed directed to an H-2K(b)-restricted epitope expressed by members of the trans-sialidase family of surface proteins. To determine whether this immunodominance was exerted over other non-H-2K(b)-restricted epitopes, we measured during infection of heterozygote mice, immune responses to three distinct epitopes, all expressed by members of the trans-sialidase family, recognized by H-2K(b)-, H-2K(k)-, or H-2K(d)-restricted CD8(+) T cells. Infected heterozygote or homozygote mice displayed comparably strong immune responses to the H-2K(b)-restricted immunodominant epitope. In contrast, H-2K(k)- or H-2K(d)-restricted immune responses were significantly impaired in heterozygote infected mice when compared with homozygote ones. This interference was not dependent on the dose of parasite or the timing of infection. Also, it was not seen in heterozygote mice immunized with recombinant adenoviruses expressing T. cruzi Ags. Finally, we observed that the immunodominance was circumvented by concomitant infection with two T. cruzi strains containing distinct immunodominant epitopes, suggesting that the operating mechanism most likely involves competition of T cells for limiting APCs. This type of interference never described during infection with a human parasite may represent a sophisticated strategy to restrict priming of CD8(+) T cells of distinct specificities, avoiding complete pathogen elimination by host effector cells, and thus favoring host parasitism.

  12. Vaccination of stage III/IV melanoma patients with long NY-ESO-1 peptide and CpG-B elicits robust CD8+ and CD4+ T-cell responses with multiple specificities including a novel DR7-restricted epitope

    PubMed Central

    Baumgaertner, P.; Costa Nunes, C.; Cachot, A.; Maby-El Hajjami, H.; Cagnon, L.; Braun, M.; Derré, L.; Rivals, J.-P.; Rimoldi, D.; Gnjatic, S.; Abed Maillard, S.; Marcos Mondéjar, P.; Protti, M. P.; Romano, E.; Michielin, O.; Romero, P.; Speiser, D. E.; Jandus, C.

    2016-01-01

    ABSTRACT Long synthetic peptides and CpG-containing oligodeoxynucleotides are promising components for cancer vaccines. In this phase I trial, 19 patients received a mean of 8 (range 1–12) monthly vaccines s.c. composed of the long synthetic NY-ESO-179–108 peptide and CpG-B (PF-3512676), emulsified in Montanide ISA-51. In 18/18 evaluable patients, vaccination induced antigen-specific CD8+ and CD4+ T-cell and antibody responses, starting early after initiation of immunotherapy and lasting at least one year. The T-cells responded antigen-specifically, with strong secretion of IFNγ and TNFα, irrespective of patients' HLAs. The most immunogenic regions of the vaccine peptide were NY-ESO-189–102 for CD8+ and NY-ESO-183–99 for CD4+ T-cells. We discovered a novel and highly immunogenic epitope (HLA-DR7/NY-ESO-187–99); 7/7 HLA-DR7+ patients generated strong CD4+ T-cell responses, as detected directly ex vivo with fluorescent multimers. Thus, vaccination with the long synthetic NY-ESO-179–108 peptide combined with the strong immune adjuvant CpG-B induced integrated, robust and functional CD8+ and CD4+ T-cell responses in melanoma patients, supporting the further development of this immunotherapeutic approach. PMID:27853637

  13. Vaccination of stage III/IV melanoma patients with long NY-ESO-1 peptide and CpG-B elicits robust CD8(+) and CD4(+) T-cell responses with multiple specificities including a novel DR7-restricted epitope.

    PubMed

    Baumgaertner, P; Costa Nunes, C; Cachot, A; Maby-El Hajjami, H; Cagnon, L; Braun, M; Derré, L; Rivals, J-P; Rimoldi, D; Gnjatic, S; Abed Maillard, S; Marcos Mondéjar, P; Protti, M P; Romano, E; Michielin, O; Romero, P; Speiser, D E; Jandus, C

    2016-01-01

    Long synthetic peptides and CpG-containing oligodeoxynucleotides are promising components for cancer vaccines. In this phase I trial, 19 patients received a mean of 8 (range 1-12) monthly vaccines s.c. composed of the long synthetic NY-ESO-179-108 peptide and CpG-B (PF-3512676), emulsified in Montanide ISA-51. In 18/18 evaluable patients, vaccination induced antigen-specific CD8(+) and CD4(+) T-cell and antibody responses, starting early after initiation of immunotherapy and lasting at least one year. The T-cells responded antigen-specifically, with strong secretion of IFNγ and TNFα, irrespective of patients' HLAs. The most immunogenic regions of the vaccine peptide were NY-ESO-189-102 for CD8(+) and NY-ESO-183-99 for CD4(+) T-cells. We discovered a novel and highly immunogenic epitope (HLA-DR7/NY-ESO-187-99); 7/7 HLA-DR7(+) patients generated strong CD4(+) T-cell responses, as detected directly ex vivo with fluorescent multimers. Thus, vaccination with the long synthetic NY-ESO-179-108 peptide combined with the strong immune adjuvant CpG-B induced integrated, robust and functional CD8(+) and CD4(+) T-cell responses in melanoma patients, supporting the further development of this immunotherapeutic approach.

  14. Identification of Relevant Conformational Epitopes on the HER2 Oncoprotein by Using Large Fragment Phage Display (LFPD)

    PubMed Central

    Gabrielli, Federico; Salvi, Roberto; Garulli, Chiara; Kalogris, Cristina; Arima, Serena; Tardella, Luca; Monaci, Paolo; Pupa, Serenella M.; Tagliabue, Elda; Montani, Maura; Quaglino, Elena; Stramucci, Lorenzo; Curcio, Claudia

    2013-01-01

    We developed a new phage-display based approach, the Large Fragment Phage Display (LFPD), that can be used for mapping conformational epitopes on target molecules of immunological interest. LFPD uses a simplified and more effective phage-display approach in which only a limited set of larger fragments (about 100 aa in length) are expressed on the phage surface. Using the human HER2 oncoprotein as a target, we identified novel B-cell conformational epitopes. The same homologous epitopes were also detected in rat HER2 and all corresponded to the epitopes predicted by computational analysis (PEPITO software), showing that LFPD gives reproducible and accurate results. Interestingly, these newly identified HER2 epitopes seem to be crucial for an effective immune response against HER2-overexpressing breast cancers and might help discriminating between metastatic breast cancer and early breast cancer patients. Overall, the results obtained in this study demonstrated the utility of LFPD and its potential application to the detection of conformational epitopes on many other molecules of interest, as well as, the development of new and potentially more effective B-cell conformational epitopes based vaccines. PMID:23555577

  15. Promiscuous Recognition of a Trypanosoma cruzi CD8+ T Cell Epitope among HLA-A2, HLA-A24 and HLA-A1 Supertypes in Chagasic Patients

    PubMed Central

    Guzmán, Fanny; Rosas, Fernando; Thomas, M. Carmen; López, Manuel Carlos; González, John Mario; Cuéllar, Adriana; Puerta, Concepción J.

    2016-01-01

    Background TcTLE is a nonamer peptide from Trypanosoma cruzi KMP-11 protein that is conserved among different parasite strains and that is presented by different HLA-A molecules from the A2 supertype. Because peptides presented by several major histocompatibility complex (MHC) supertypes are potential targets for immunotherapy, the aim of this study was to determine whether MHC molecules other than the A2 supertype present the TcTLE peptide. Methodology/Principal Findings From 36 HLA-A2-negative chagasic patients, the HLA-A genotypes of twenty-eight patients with CD8+ T cells that recognized the TcTLE peptide using tetramer (twenty) or functional (eight) assays, were determined. SSP-PCR was used to identify the A locus and the allelic variants. Flow cytometry was used to analyze the frequency of TcTLE-specific CD8+ T cells, and their functional activity (IFN-γ, TNFα, IL-2, perforin, granzyme and CD107a/b production) was induced by exposure to the TcTLE peptide. All patients tested had TcTLE-specific CD8+ T cells with frequencies ranging from 0.07–0.37%. Interestingly, seven of the twenty-eight patients had HLA-A homozygous alleles: A*24 (5 patients), A*23 (1 patient) and A*01 (1 patient), which belong to the A24 and A1 supertypes. In the remaining 21 patients with HLA-A heterozygous alleles, the most prominent alleles were A24 and A68. The most common allele sub-type was A*2402 (sixteen patients), which belongs to the A24 supertype, followed by A*6802 (six patients) from the A2 supertype. Additionally, the A*3002/A*3201 alleles from the A1 supertype were detected in one patient. All patients presented CD8+ T cells producing at least one cytokine after TcTLE peptide stimulation. Conclusion/Significance These results show that TcTLE is a promiscuous peptide that is presented by the A24 and A1 supertypes, in addition to the A2 supertype, suggesting its potential as a target for immunotherapy. PMID:26974162

  16. Retinol as a micronutrients related to cervical local immunity: The expression of tumor necrosis factor-alpha specifically stimulated with E6 epitope of human papillomavirus type-16 and ratio of CD4+/CD8+ T cell in natural history of cervical cancer

    NASA Astrophysics Data System (ADS)

    Utami, T. W.; Aziz, M. F.; Ibrahim, F.; Andrijono

    2017-08-01

    Retinol is one of the antioxidant micronutrients that plays essential roles in the immune system, by preventing the persistence of modulating CD4+ and CD8+ T cells and cytokines production. Tumor Necrosis Factor-Alpha (TNF-α) is an acute pro-inflammatory cytokine which has many crucial roles in controlling HPV. In contrast, when persistent infection occurs, TNF-α induces carcinogenesis. The ratio of CD4+ cells to CD8+ T cells and adequate TNF-α production in acute HPV infection are key points for clearance. The aim of this research is to analyze the sufficiency level of retinol deposit, the expression of TNF-α, and the ratio of CD4+: CD8+ T cells in a normal cervix, clearance and persistent HPV subclinical infection, and cervical cancer group. The sufficiency level of retinol deposit was analyzed from peripheral blood using the ELISA method. The cervico-vaginal secretions, which were incubated for 24 hours, were stimulated specifically by E6 epitope HPV type-16, measuring TNF-α expression semi-quantitatively by the ELISpot method and CD4+/CD8+ T cells quantitatively by flowcytometry method. The sufficient level of retinol deposit in a normal cervix, clearance HPV subclinical infection, persistent, and cervical cancer group was 85%, 75% (OR 1.89), 33.3% (OR 11.33), and 75% (OR 1.89), respectively. The expression of TNF-α in normal cervix group was 10%, while for cervical cancer it was 75% (OR 27.00; p < 0.001). There was no expression in clearance and persistent HPV subclinical infection groups. A high ratio of CD4+: CD8+ T cells in the normal cervix and cervical cancer group was 10% and 25% (OR 0.33). There was no high ratio of CD4+: CD8+ T cells in clearance (OR 1.22) and persistent (OR 0.95) HPV subclinical infection groups. This study was able to prove that the normal cervix group has the highest retinol deposit sufficiency level and the cervical cancer group has the highest TNF-α expression (OR 27; p < 0.001). The lowest of retinol deposit sufficiency

  17. The Crystal Structure of CD8alpha,Beta in Complex With YTS156.7.7 Fab And Interaction With Other CD8 Antibodies Define the Binding Mode of CD8alpha,Beta to MHC Class I

    SciTech Connect

    Shore, D.A.; Issafras, H.; Landais, E.; Teyton, L.; Wilson, I.A.

    2009-05-27

    The CD8{alpha}{beta} heterodimer interacts with class I pMHC on antigen-presenting cells as a co-receptor for TCR-mediated activation of cytotoxic T cells. To characterize this immunologically important interaction, we used monoclonal antibodies (mAbs) specific to either CD8{alpha} or CD8{beta} to probe the mechanism of CD8{alpha}{beta} binding to pMHCI. The YTS156.7 mAb inhibits this interaction and blocks T cell activation. To elucidate the molecular basis for this inhibition, the crystal structure of the CD8{alpha}{beta} immunoglobulin-like ectodomains were determined in complex with mAb YTS156.7 Fab at 2.7 {angstrom} resolution. The YTS156.7 epitope on CD8{beta} was identified and implies that residues in the CDR1 and CDR2-equivalent loops of CD8{beta} are occluded upon binding to class I pMHC. To further characterize the pMHCI/CD8{alpha}{beta} interaction, binding of class I tetramers to CD8{alpha}{beta} on the surface of T cells was assessed in the presence of anti-CD8 mAbs. In contrast to YTS156.7, mAb YTS105.18, which is specific for CD8{alpha}, does not inhibit binding of CD8{alpha}{beta} to class I tetramers, indicating the YTS105.18 epitope is not occluded in the pMHCI/CD8{alpha}{beta} complex. Together, these data indicate a model for the pMHCI/CD8{alpha}{beta} interaction similar to that observed for CD8{alpha}{alpha} in the CD8{alpha}{alpha}/pMHCI complex, but in which CD8{alpha} occupies the lower orientation (membrane proximal to the antigen presenting cell), and CD8{beta} occupies the upper position (membrane distal). The implication of this molecular assembly for the function of CD8{alpha}{beta} in T cell activation is discussed.

  18. Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using the PROFILER technology

    PubMed Central

    Cariccio, Veronica Lanza; Domina, Maria; Benfatto, Salvatore; Venza, Mario; Venza, Isabella; Faleri, Agnese; Bruttini, Marco; Bartolini, Erika; Giuliani, Marzia Monica; Santini, Laura; Brunelli, Brunella; Norais, Nathalie; Borgogni, Erica; Midiri, Angelina; Galbo, Roberta; Romeo, Letizia; Biondo, Carmelo; Masignani, Vega; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2016-01-01

    ABSTRACT There is a strong need for rapid and reliable epitope mapping methods that can keep pace with the isolation of increasingly larger numbers of mAbs. We describe here the identification of a conformational epitope using Phage-based Representation OF ImmunoLigand Epitope Repertoire (PROFILER), a recently developed high-throughput method based on deep sequencing of antigen-specific lambda phage-displayed libraries. A novel bactericidal monoclonal antibody (mAb 9F11) raised against Neisseria meningitidis adhesin A (NadA), an important component of the Bexsero® anti-meningococcal vaccine, was used to evaluate the technique in comparison with other epitope mapping methods. The PROFILER technology readily identified NadA fragments that were capable of fully recapitulating the reactivity of the entire antigen against mAb 9F11. Further analysis of these fragments using mutagenesis and hydrogen-deuterium exchange mass-spectrometry allowed us to identify the binding site of mAb 9F11 (A250-D274) and an adjoining sequence (V275-H312) that was also required for the full functional reconstitution of the epitope. These data suggest that, by virtue of its ability to detect a great variety of immunoreactive antigen fragments in phage-displayed libraries, the PROFILER technology can rapidly and reliably identify epitope-containing regions and provide, in addition, useful clues for the functional characterization of conformational mAb epitopes. PMID:26963435

  19. Multivalent display of minimal Clostridium difficile glycan epitopes mimics antigenic properties of larger glycans

    PubMed Central

    Broecker, Felix; Hanske, Jonas; Martin, Christopher E.; Baek, Ju Yuel; Wahlbrink, Annette; Wojcik, Felix; Hartmann, Laura; Rademacher, Christoph; Anish, Chakkumkal; Seeberger, Peter H.

    2016-01-01

    Synthetic cell-surface glycans are promising vaccine candidates against Clostridium difficile. The complexity of large, highly antigenic and immunogenic glycans is a synthetic challenge. Less complex antigens providing similar immune responses are desirable for vaccine development. Based on molecular-level glycan–antibody interaction analyses, we here demonstrate that the C. difficile surface polysaccharide-I (PS-I) can be resembled by multivalent display of minimal disaccharide epitopes on a synthetic scaffold that does not participate in binding. We show that antibody avidity as a measure of antigenicity increases by about five orders of magnitude when disaccharides are compared with constructs containing five disaccharides. The synthetic, pentavalent vaccine candidate containing a peptide T-cell epitope elicits weak but highly specific antibody responses to larger PS-I glycans in mice. This study highlights the potential of multivalently displaying small oligosaccharides to achieve antigenicity characteristic of larger glycans. The approach may result in more cost-efficient carbohydrate vaccines with reduced synthetic effort. PMID:27091615

  20. Multivalent display of minimal Clostridium difficile glycan epitopes mimics antigenic properties of larger glycans.

    PubMed

    Broecker, Felix; Hanske, Jonas; Martin, Christopher E; Baek, Ju Yuel; Wahlbrink, Annette; Wojcik, Felix; Hartmann, Laura; Rademacher, Christoph; Anish, Chakkumkal; Seeberger, Peter H

    2016-04-19

    Synthetic cell-surface glycans are promising vaccine candidates against Clostridium difficile. The complexity of large, highly antigenic and immunogenic glycans is a synthetic challenge. Less complex antigens providing similar immune responses are desirable for vaccine development. Based on molecular-level glycan-antibody interaction analyses, we here demonstrate that the C. difficile surface polysaccharide-I (PS-I) can be resembled by multivalent display of minimal disaccharide epitopes on a synthetic scaffold that does not participate in binding. We show that antibody avidity as a measure of antigenicity increases by about five orders of magnitude when disaccharides are compared with constructs containing five disaccharides. The synthetic, pentavalent vaccine candidate containing a peptide T-cell epitope elicits weak but highly specific antibody responses to larger PS-I glycans in mice. This study highlights the potential of multivalently displaying small oligosaccharides to achieve antigenicity characteristic of larger glycans. The approach may result in more cost-efficient carbohydrate vaccines with reduced synthetic effort.

  1. Tomato bushy stunt virus (TBSV), a versatile platform for polyvalent display of antigenic epitopes and vaccine design

    SciTech Connect

    Kumar, Shantanu; Ochoa, Wendy; Singh, Pratik; Hsu, Catherine; Schneemann, Anette; Manchester, Marianne; Olson, Mark; Reddy, Vijay

    2009-05-25

    Viruses-like particles (VLPs) are frequently being used as platforms for polyvalent display of foreign epitopes of interest on their capsid surface to improve their presentation enhancing the antigenicity and host immune response. In the present study, we used the VLPs of Tomato bushy stunt virus (TBSV), an icosahedral plant virus, as a platform to display 180 copies of 16 amino acid epitopes of ricin toxin fused to the C-terminal end of a modified TBSV capsid protein (NDELTA52). Expression of the chimeric recombinant protein in insect cells resulted in spontaneous assembly of VLPs displaying the ricin epitope. Cryo-electron microscopy and image reconstruction of the chimeric VLPs at 22 A resolution revealed the locations and orientation of the ricin epitope exposed on the TBSV capsid surface. Furthermore, injection of chimeric VLPs into mice generated antisera that detected the native ricin toxin. The ease of fusing of short peptides of 15-20 residues and their ability to form two kinds (T = 1, T = 3) of bio-nanoparticles that result in the display of 60 or 180 copies of less constrained and highly exposed antigenic epitopes makes TBSV an attractive and versatile display platform for vaccine design.

  2. The Breadth of Synthetic Long Peptide Vaccine-Induced CD8+ T Cell Responses Determines the Efficacy against Mouse Cytomegalovirus Infection

    PubMed Central

    Panagioti, Eleni; Redeker, Anke; van Duikeren, Suzanne; Franken, Kees LMC; Drijfhout, Jan Wouter; van der Burg, Sjoerd H.

    2016-01-01

    There is an ultimate need for efficacious vaccines against human cytomegalovirus (HCMV), which causes severe morbidity and mortality among neonates and immunocompromised individuals. In this study we explored synthetic long peptide (SLP) vaccination as a platform modality to protect against mouse CMV (MCMV) infection in preclinical mouse models. In both C57BL/6 and BALB/c mouse strains, prime-booster vaccination with SLPs containing MHC class I restricted epitopes of MCMV resulted in the induction of strong and polyfunctional (i.e., IFN-γ+, TNF+, IL-2+) CD8+ T cell responses, equivalent in magnitude to those induced by the virus itself. SLP vaccination initially led to the formation of effector CD8+ T cells (KLRG1hi, CD44hi, CD127lo, CD62Llo), which eventually converted to a mixed central and effector-memory T cell phenotype. Markedly, the magnitude of the SLP vaccine-induced CD8+ T cell response was unrelated to the T cell functional avidity but correlated to the naive CD8+ T cell precursor frequency of each epitope. Vaccination with single SLPs displayed various levels of long-term protection against acute MCMV infection, but superior protection occurred after vaccination with a combination of SLPs. This finding underlines the importance of the breadth of the vaccine-induced CD8+ T cell response. Thus, SLP-based vaccines could be a potential strategy to prevent CMV-associated disease. PMID:27637068

  3. Phage-displayed peptides that mimic epitopes of hepatitis E virus capsid.

    PubMed

    Larralde, Osmany; Petrik, Juraj

    2017-08-01

    Hepatitis E is an emerging zoonotic infection of increasing public health threat for the UK, especially for immunosuppressed individuals. A human recombinant vaccine has been licensed only in China and is not clear whether it protects against hepatitis E virus (HEV) genotype 3, the most prevalent in Europe. The aim of this study was to use phage display technology as a tool to identify peptides that mimic epitopes of HEV capsid (mimotopes). We identified putative linear and conformational mimotopes using sera from Scottish blood donors that have the immunological imprint of past HEV infection. Four mimotopes did not have homology with the primary sequence of HEV ORF2 capsid but competed effectively with a commercial HEV antigen for binding to anti-HEV reference serum. When the reactivity profile of each mimotope was compared with Wantai HEV-IgG ELISA, the most sensitive HEV immunoassay, mimotopes showed 95.2-100% sensitivity while the specificity ranged from 81.5 to 95.8%. PepSurf algorithm was used to map affinity-selected peptides onto the ORF2 crystal structure of HEV genotype 3, which predicted that these four mimototopes are clustered in the P domain of ORF2 capsid, near conformational epitopes of anti-HEV neutralising monoclonal antibodies. These HEV mimotopes may have potential applications in the design of structural vaccines and the development of new diagnostic tests.

  4. Expression and immunological characterization of cardamom mosaic virus coat protein displaying HIV gp41 epitopes.

    PubMed

    Damodharan, Subha; Gujar, Ravindra; Pattabiraman, Sathyamurthy; Nesakumar, Manohar; Hanna, Luke Elizabeth; Vadakkuppattu, Ramanathan D; Usha, Ramakrishnan

    2013-05-01

    The coat protein of cardamom mosaic virus (CdMV), a member of the genus Macluravirus, assembles into virus-like particles when expressed in an Escherichia coli expression system. The N and C-termini of the coat protein were engineered with the Kennedy peptide and the 2F5 and 4E10 epitopes of gp41 of HIV. The chimeric proteins reacted with sera from HIV positive persons and also stimulated secretion of cytokines by peripheral blood mononuclear cells from these persons. Thus, a system based on the coat protein of CdMV can be used to display HIV-1 antigens. © 2013 The Societies and Wiley Publishing Asia Pty Ltd.

  5. Memory CD8(+) T cells elicited by HIV-1 lipopeptide vaccines display similar phenotypic profiles but differences in term of magnitude and multifunctionality compared with FLU- or EBV-specific memory T cells in humans.

    PubMed

    Figueiredo, Suzanne; Charmeteau, Benedicte; Surenaud, Mathieu; Salmon, Dominique; Launay, Odile; Guillet, Jean-Gérard; Hosmalin, Anne; Gahery, Hanne

    2014-01-16

    Differentiation marker, multifunctionality and magnitude analyses of specific-CD8(+) memory T cells are crucial to improve development of HIV vaccines designed to generate cell-mediated immunity. Therefore, we fully characterized the HIV-specific CD8(+) T cell responses induced in volunteers vaccinated with HIV lipopeptide vaccines for phenotypic markers, tetramer staining, cytokine secretion, and cytotoxic activities. The frequency of ex vivo CD8(+) T cells elicited by lipopeptide vaccines is very rare and central-memory phenotype and functions of these cells were been shown to be important in AIDS immunity. So, we expanded them using specific peptides to compare the memory T cell responses induced in volunteers by HIV vaccines with responses to influenza (FLU) or Epstein Barr virus (EBV). By analyzing the differentiation state of IFN-γ-secreting CD8(+) T cells, we found a CCR7(-)CD45RA(-)CD28(+int)/CD28(-) profile (>85%) belonging to a subset of intermediate-differentiated effector T cells for HIV, FLU, and EBV. We then assessed the quality of the response by measuring various T cell functions. The percentage of single IFN-γ T cell producers in response to HIV was 62% of the total of secreting T cells compared with 35% for FLU and EBV, dual and triple (IFN-γ/IL-2/CD107a) T cell producers could also be detected but at lower levels (8% compared with 37%). Finally, HIV-specific T cells secreted IFN-γ and TNF-α, but not the dual combination like FLU- and EBV-specific T cells. Thus, we found that the functional profile and magnitude of expanded HIV-specific CD8(+) T precursors were more limited than those of to FLU- and EBV-specific CD8(+) T cells. These data show that CD8(+) T cells induced by these HIV vaccines have a similar differentiation profile to FLU and EBV CD8(+) T cells, but that the vaccine potency to induce multifunctional T cells needs to be increased in order to improve vaccination strategies. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Putative phage-display epitopes of the porcine epidemic diarrhea virus S1 protein and their anti-viral activity

    USDA-ARS?s Scientific Manuscript database

    Porcine epidemic diarrhea virus (PEDV) is a pathogen of swine that causes severe diarrhea and dehydration resulting in substantial morbidity and mortality in newborn piglets. Phage display is a technique with wide application, in particular, the identification of key antigen epitopes for the develop...

  7. Human influenza viruses and CD8(+) T cell responses.

    PubMed

    Grant, Emma J; Quiñones-Parra, Sergio M; Clemens, E Bridie; Kedzierska, Katherine

    2016-02-01

    Influenza A viruses (IAVs) cause significant morbidity and mortality worldwide, despite new strain-specific vaccines being available annually. As IAV-specific CD8(+) T cells promote viral control in the absence of neutralizing antibodies, and can mediate cross-reactive immunity toward distinct IAVs to drive rapid recovery from both mild and severe influenza disease, there is great interest in developing a universal T cell vaccine. However, despite detailed studies in mouse models of influenza virus infection, there is still a paucity of data on human epitope-specific CD8(+) T cell responses to IAVs. This review focuses on our current understanding of human CD8(+) T cell immunity against distinct IAVs and discusses the possibility of achieving a CD8(+) T cell mediated-vaccine that protects against multiple, distinct IAV strains across diverse human populations. We also review the importance of CD8(+) T cell immunity in individuals highly susceptible to severe influenza infection, including those hospitalised with influenza, the elderly and Indigenous populations. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Rabbit hemorrhagic disease virus capsid, a versatile platform for foreign B-cell epitope display inducing protective humoral immune responses.

    PubMed

    Moreno, Noelia; Mena, Ignacio; Angulo, Iván; Gómez, Yolanda; Crisci, Elisa; Montoya, María; Castón, José R; Blanco, Esther; Bárcena, Juan

    2016-08-23

    Virus-like particles (VLPs), comprised of viral structural proteins devoid of genetic material, are tunable nanoparticles that can be chemically or genetically engineered, to be used as platforms for multimeric display of foreign antigens. Here, we report the engineering of chimeric VLPs, derived from rabbit hemorrhagic disease virus (RHDV) for presentation of foreign B-cell antigens to the immune system. The RHDV capsid comprises 180 copies of a single capsid subunit (VP60). To evaluate the ability of chimeric RHDV VLPs to elicit protective humoral responses against foreign antigens, we tested two B-cell epitopes: a novel neutralizing B-cell epitope, derived from feline calicivirus capsid protein, and a well characterized B-cell epitope from the extracellular domain of influenza A virus M2 protein (M2e). We generated sets of chimeric RHDV VLPs by insertion of the foreign B-cell epitopes at three different locations within VP60 protein (which involved different levels of surface accessibility) and in different copy numbers per site. The immunogenic potential of the chimeric VLPs was analyzed in the mouse model. The results presented here indicated that chimeric RHDV VLPs elicit potent protective humoral responses against displayed foreign B-cell epitopes, demonstrated by both, in vitro neutralization and in vivo protection against a lethal challenge.

  9. Rabbit hemorrhagic disease virus capsid, a versatile platform for foreign B-cell epitope display inducing protective humoral immune responses

    PubMed Central

    Moreno, Noelia; Mena, Ignacio; Angulo, Iván; Gómez, Yolanda; Crisci, Elisa; Montoya, María; Castón, José R.; Blanco, Esther; Bárcena, Juan

    2016-01-01

    Virus-like particles (VLPs), comprised of viral structural proteins devoid of genetic material, are tunable nanoparticles that can be chemically or genetically engineered, to be used as platforms for multimeric display of foreign antigens. Here, we report the engineering of chimeric VLPs, derived from rabbit hemorrhagic disease virus (RHDV) for presentation of foreign B-cell antigens to the immune system. The RHDV capsid comprises 180 copies of a single capsid subunit (VP60). To evaluate the ability of chimeric RHDV VLPs to elicit protective humoral responses against foreign antigens, we tested two B-cell epitopes: a novel neutralizing B-cell epitope, derived from feline calicivirus capsid protein, and a well characterized B-cell epitope from the extracellular domain of influenza A virus M2 protein (M2e). We generated sets of chimeric RHDV VLPs by insertion of the foreign B-cell epitopes at three different locations within VP60 protein (which involved different levels of surface accessibility) and in different copy numbers per site. The immunogenic potential of the chimeric VLPs was analyzed in the mouse model. The results presented here indicated that chimeric RHDV VLPs elicit potent protective humoral responses against displayed foreign B-cell epitopes, demonstrated by both, in vitro neutralization and in vivo protection against a lethal challenge. PMID:27549017

  10. CD8(+) T Cell Immune Response in Immunocompetent Mice during Zika Virus Infection.

    PubMed

    Huang, Huarong; Li, Shihua; Zhang, Yongli; Han, Xiaojuan; Jia, Baoqian; Liu, Hongtao; Liu, Dandan; Tan, Shuguang; Wang, Qihui; Bi, Yuhai; Liu, William J; Hou, Baidong; Gao, George Fu; Zhang, Fuping

    2017-08-23

    Zika virus (ZIKV) infection cause neurologic complications, including Guillain-Barré syndrome (GBS) in adults and central nerve system (CNS) abnormalities in fetuses. We investigated the immune response, especially CD8(+) T cell response in C57BL/6 (B6) wild type (WT) mouse during ZIKV infection. We found that robust CD8(+) T cell response was elicited, MHC-I restricted CD8(+) T cell epitopes were identified and a tetramer that recognized ZIKV specific CD8(+) T cells was developed. Finally, virus specific memory CD8(+) T cells were generated in these mice. The CD8(+) T cells from these infected mice were functional, as evidenced by that adoptive transfer of ZIKV specific CD8(+) T cells could protect CNS against ZIKV infection and cross protective to Dengue virus (DENV) infection. Thus, our findings provided comprehensive insight into the immune responses against ZIKV and demonstrated that WT mouse could be a natural and easy access model for evaluating immune response to ZIKV infection.IMPORTANCE ZIKV infection cause severe clinical consequences, including Guillain-Barré syndrome (GBS) in adults, microcephaly, congenital malformations in fetuses and newborn infants, therefore, study of the immune response especially adaptive immune response to ZIKV infection is important to understand the disease caused by ZIKV infection. The importance of our finding is that we characterized the CD8(+) T cell immune response to ZIKV in a comprehensive manner, and identified ZIKV epitopes. Using the identified immunodominant epitope, we developed a tetramer that could recognize ZIKV specific CD8(+) T cells in vivo, which simplified detection and evaluation of ZIKV specific immune responses. In addition, the finding that a tetramer positive memory CD8(+) T cell responses were generated and that CD8(+) T cells could traffic to ZIKV infected brain greatly enhanced our understanding of ZIKV infection and will provide important insight for ZIKV vaccines design. Copyright © 2017

  11. Heterogeneous differentiation patterns of individual CD8+ T cells.

    PubMed

    Gerlach, Carmen; Rohr, Jan C; Perié, Leïla; van Rooij, Nienke; van Heijst, Jeroen W J; Velds, Arno; Urbanus, Jos; Naik, Shalin H; Jacobs, Heinz; Beltman, Joost B; de Boer, Rob J; Schumacher, Ton N M

    2013-05-03

    Upon infection, antigen-specific CD8(+) T lymphocyte responses display a highly reproducible pattern of expansion and contraction that is thought to reflect a uniform behavior of individual cells. We tracked the progeny of individual mouse CD8(+) T cells by in vivo lineage tracing and demonstrated that, even for T cells bearing identical T cell receptors, both clonal expansion and differentiation patterns are heterogeneous. As a consequence, individual naïve T lymphocytes contributed differentially to short- and long-term protection, as revealed by participation of their progeny during primary versus recall infections. The discordance in fate of individual naïve T cells argues against asymmetric division as a singular driver of CD8(+) T cell heterogeneity and demonstrates that reproducibility of CD8(+) T cell responses is achieved through population averaging.

  12. MimoPro: a more efficient Web-based tool for epitope prediction using phage display libraries

    PubMed Central

    2011-01-01

    Background A B-cell epitope is a group of residues on the surface of an antigen which stimulates humoral responses. Locating these epitopes on antigens is important for the purpose of effective vaccine design. In recent years, mapping affinity-selected peptides screened from a random phage display library to the native epitope has become popular in epitope prediction. These peptides, also known as mimotopes, share the similar structure and function with the corresponding native epitopes. Great effort has been made in using this similarity between such mimotopes and native epitopes in prediction, which has resulted in better outcomes than statistics-based methods can. However, it cannot maintain a high degree of satisfaction in various circumstances. Results In this study, we propose a new method that maps a group of mimotopes back to a source antigen so as to locate the interacting epitope on the antigen. The core of this method is a searching algorithm that is incorporated with both dynamic programming (DP) and branch and bound (BB) optimization and operated on a series of overlapping patches on the surface of a protein. These patches are then transformed to a number of graphs using an adaptable distance threshold (ADT) regulated by an appropriate compactness factor (CF), a novel parameter proposed in this study. Compared with both Pep-3D-Search and PepSurf, two leading graph-based search tools, on average from the results of 18 test cases, MimoPro, the Web-based implementation of our proposed method, performed better in sensitivity, precision, and Matthews correlation coefficient (MCC) than both did in epitope prediction. In addition, MimoPro is significantly faster than both Pep-3D-Search and PepSurf in processing. Conclusions Our search algorithm designed for processing well constructed graphs using an ADT regulated by CF is more sensitive and significantly faster than other graph-based approaches in epitope prediction. MimoPro is a viable alternative to both

  13. Human immunome, bioinformatic analyses using HLA supermotifs and the parasite genome, binding assays, studies of human T cell responses, and immunization of HLA-A*1101 transgenic mice including novel adjuvants provide a foundation for HLA-A03 restricted CD8+T cell epitope based, adjuvanted vaccine protective against Toxoplasma gondii

    PubMed Central

    2010-01-01

    Background Toxoplasmosis causes loss of life, cognitive and motor function, and sight. A vaccine is greatly needed to prevent this disease. The purpose of this study was to use an immmunosense approach to develop a foundation for development of vaccines to protect humans with the HLA-A03 supertype. Three peptides had been identified with high binding scores for HLA-A03 supertypes using bioinformatic algorhythms, high measured binding affinity for HLA-A03 supertype molecules, and ability to elicit IFN-γ production by human HLA-A03 supertype peripheral blood CD8+ T cells from seropositive but not seronegative persons. Results Herein, when these peptides were administered with the universal CD4+T cell epitope PADRE (AKFVAAWTLKAAA) and formulated as lipopeptides, or administered with GLA-SE either alone, or with Pam2Cys added, we found we successfully created preparations that induced IFN-γ and reduced parasite burden in HLA-A*1101(an HLA-A03 supertype allele) transgenic mice. GLA-SE is a novel emulsified synthetic TLR4 ligand that is known to facilitate development of T Helper 1 cell (TH1) responses. Then, so our peptides would include those expressed in tachyzoites, bradyzoites and sporozoites from both Type I and II parasites, we used our approaches which had identified the initial peptides. We identified additional peptides using bioinformatics, binding affinity assays, and study of responses of HLA-A03 human cells. Lastly, we found that immunization of HLA-A*1101 transgenic mice with all the pooled peptides administered with PADRE, GLA-SE, and Pam2Cys is an effective way to elicit IFN-γ producing CD8+ splenic T cells and protection. Immunizations included the following peptides together: KSFKDILPK (SAG1224-232); AMLTAFFLR (GRA6164-172); RSFKDLLKK (GRA7134-142); STFWPCLLR (SAG2C13-21); SSAYVFSVK(SPA250-258); and AVVSLLRLLK(SPA89-98). This immunization elicited robust protection, measured as reduced parasite burden using a luciferase transfected parasite

  14. Insert engineering and solubility screening improves recovery of virus-like particle subunits displaying hydrophobic epitopes

    PubMed Central

    Abidin, R S; Lua, L H L; Middelberg, A P J; Sainsbury, F

    2015-01-01

    The Polyomavirus coat protein, VP1 has been developed as an epitope presentation system able to provoke humoral immunity against a variety of pathogens, such as Influenza and Group A Streptococcus. The ability of the system to carry cytotoxic T cell epitopes on a surface-exposed loop and the impact on protein solubility has not been examined. Four variations of three selected epitopes were cloned into surface-exposed loops of VP1, and expressed in Escherichia coli. VP1 pentamers, also known as capsomeres, were purified via a glutathione-S-transferase tag. Size exclusion chromatography indicated severe aggregation of the recombinant VP1 during enzymatic tag removal resulting from the introduction the hydrophobic epitopes. Inserts were modified to possess double aspartic acid residues at each end of the hydrophobic epitopes and a high-throughput buffer condition screen was implemented with protein aggregation monitored during tag removal by spectrophotometry and dynamic light scattering. These analyses showed that the insertion of charged residues at the extremities of epitopes could improve solubility of capsomeres and revealed multiple windows of opportunity for further condition optimization. A combination of epitope design, pH optimization, and the additive l-arginine permitted the recovery of soluble VP1 pentamers presenting hydrophobic epitopes and their subsequent assembly into virus-like particles. PMID:26401641

  15. CD8+ T-cell response promotes evolution of hepatitis C virus nonstructural proteins.

    PubMed

    Ruhl, Marianne; Knuschke, Torben; Schewior, Kevin; Glavinic, Lejla; Neumann-Haefelin, Christoph; Chang, Dae-In; Klein, Marina; Heinemann, Falko M; Tenckhoff, Hannelore; Wiese, Manfred; Horn, Peter A; Viazov, Sergei; Spengler, Ulrich; Roggendorf, Michael; Scherbaum, Norbert; Nattermann, Jacob; Hoffmann, Daniel; Timm, Jörg

    2011-06-01

    Hepatitis C virus (HCV) acquires mutations that allow it to escape the CD8+ T-cell response, although the extent to which this process contributes to viral evolution at the population level is not clear. We studied viral adaptation using data from a large outbreak of HCV genotype 1b infection that occurred among women immunized with contaminated immunoglobulin from 1977 to 1978. The HCV nonstructural protein coding regions NS3-NS5B were sequenced from 78 patients, and mutations were mapped according to their location inside or outside previously described CD8+ T-cell epitopes. A statistical approach was developed to identify sites/regions under reproducible selection pressure associated with HLA class I. The frequency of nonsynonymous mutations was significantly higher inside previously described CD8+ T-cell epitopes than outside-particularly in NS3/4A and NS5B. We identified new regions that are under selection pressure, indicating that not all CD8+ T-cell epitopes have been identified; 6 new epitopes that interact with CD8+ T cells were identified and confirmed in vitro. In some CD8+ T-cell epitopes mutations were reproducibly identified in patients that shared the relevant HLA allele, indicating immune pressure at the population level. There was statistical support for selection of mutations in 18 individual epitopes. Interestingly, 14 of these were restricted by HLA-B allele. HLA class I-associated selection pressure on the nonstructural proteins and here predominantly on NS3/4A and NS5B promotes evolution of HCV. HLA-B alleles have a dominant effect in this selection process. Adaptation of HCV to the CD8+ T-cell response at the population level creates challenges for vaccine design. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

  16. Globular Head-Displayed Conserved Influenza H1 Hemagglutinin Stalk Epitopes Confer Protection against Heterologous H1N1 Virus

    PubMed Central

    Klausberger, Miriam; Tscheliessnig, Rupert; Neff, Silke; Nachbagauer, Raffael; Wohlbold, Teddy John; Wilde, Monika; Palmberger, Dieter; Krammer, Florian; Jungbauer, Alois; Grabherr, Reingard

    2016-01-01

    Significant genetic variability in the head region of the influenza A hemagglutinin, the main target of current vaccines, makes it challenging to develop a long-lived seasonal influenza prophylaxis. Vaccines based on the conserved hemagglutinin stalk domain might provide broader cross-reactive immunity. However, this region of the hemagglutinin is immunosubdominant to the head region. Peptide-based vaccines have gained much interest as they allow the immune system to focus on relevant but less immunogenic epitopes. We developed a novel influenza A hemagglutinin-based display platform for H1 hemagglutinin stalk peptides that we identified in an epitope mapping assay using human immune sera and synthetic HA peptides. Flow cytometry and competition assays suggest that the identified stalk sequences do not recapitulate the epitopes of already described broadly neutralizing stalk antibodies. Vaccine constructs displaying 25-mer stalk sequences provided up to 75% protection from lethal heterologous virus challenge in BALB/c mice and induced antibody responses against the H1 hemagglutinin. The developed platform based on a vaccine antigen has the potential to be either used as stand-alone or as prime-vaccine in combination with conventional seasonal or pandemic vaccines for the amplification of stalk-based cross-reactive immunity in humans or as platform to evaluate the relevance of viral peptides/epitopes for protection against influenza virus infection. PMID:27088239

  17. Oral administration of genetically modified Bifidobacterium displaying HCV-NS3 multi-epitope fusion protein could induce an HCV-NS3-specific systemic immune response in mice.

    PubMed

    Takei, Saki; Omoto, Chika; Kitagawa, Koichi; Morishita, Naoya; Katayama, Takane; Shigemura, Katsumi; Fujisawa, Masato; Kawabata, Masato; Hotta, Hak; Shirakawa, Toshiro

    2014-05-23

    More than 170 million people worldwide are chronic HCV (Hepatitis C virus) carriers, and about 30% of them will develop progressive liver disease, such as cirrhosis and hepatocellular carcinoma. A combination of pegylated interferon-α with ribavirin, the standard treatment for HCV infection, has been effective in fewer than 50% of patients infected with HCV genotype 1. A strong T cell response against the nonstructural protein 3 (NS3) is important for recovery from acute HCV infection, and an early multi-specific CD4+ helper and CD8+ cytotoxic T cell response is critical for HCV clearance. In the present study, we successfully constructed a genetically modified Bifidobacterium longum (B. longum) displaying recombinant HCV-NS3 peptides containing some CD4 and CD8 epitopes located in the HCV-NS3 region as an oral vaccine against chronic HCV infection. The oral administration of this vaccine could induce NS3-specific immune responses in mice through intestinal mucosal immunity. Our findings suggest that this novel oral vaccine has great potential as a novel oral vaccine against chronic HCV infection.

  18. Chimeric Virus-Like Particle Vaccines Displaying Conserved Enterovirus 71 Epitopes Elicit Protective Neutralizing Antibodies in Mice through Divergent Mechanisms

    PubMed Central

    Ye, Xiaohua; Ku, Zhiqiang; Liu, Qingwei; Wang, Xiaoli; Shi, Jinping; Zhang, Yunfang; Kong, Liangliang; Cong, Yao

    2014-01-01

    Enterovirus 71 (EV71) is a major causative agent of hand, food, and mouth disease, which frequently occurs in young children. Since there are 11 subgenotypes (A, B1 to B5, and C1 to C5) within EV71, an EV71 vaccine capable of protecting against all of these subgenotypes is desirable. We report here the vaccine potential and protective mechanism of two chimeric virus-like particles (VLPs) presenting conserved neutralizing epitopes of EV71. We show that fusions of hepatitis B core antigen (HBc) with the SP55 or SP70 epitope of EV71, designated HBcSP55 and HBcSP70, respectively, can be rapidly generated and self-assembled into VLPs with the epitopes displayed on the surface. Immunization with the chimeric VLPs induced carrier- and epitope-specific antibody responses in mice. Anti-HBcSP55 and anti-HBcSP70 sera, but not anti-HBc sera, were able to neutralize in vitro multiple genotypes and strains of EV71. Importantly, passive immunization with anti-HBcSP55 or anti-HBcSP70 sera protected neonatal mice against lethal EV71 infections. Interestingly, anti-HBcSP70 sera could inhibit EV71 attachment to susceptible cells, whereas anti-HBcSP55 sera could not. However, both antisera were able to neutralize EV71 infection in vitro at the postattachment stage. The divergent mechanism of neutralization and protection conferred by anti-SP70 and anti-SP55 sera is in part attributed to their respective ability to bind authentic viral particles. Collectively, our study not only demonstrates that chimeric VLPs displaying the SP55 and SP70 epitopes are promising candidates for a broad-spectrum EV71 vaccine but also reveals distinct mechanisms of neutralization by the SP55- and SP70-targeted antibodies. PMID:24131712

  19. High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum.

    PubMed

    Christiansen, Anders; Kringelum, Jens V; Hansen, Christian S; Bøgh, Katrine L; Sullivan, Eric; Patel, Jigar; Rigby, Neil M; Eiwegger, Thomas; Szépfalusi, Zsolt; de Masi, Federico; Nielsen, Morten; Lund, Ole; Dufva, Martin

    2015-08-06

    Phage display is a prominent screening technique with a multitude of applications including therapeutic antibody development and mapping of antigen epitopes. In this study, phages were selected based on their interaction with patient serum and exhaustively characterised by high-throughput sequencing. A bioinformatics approach was developed in order to identify peptide motifs of interest based on clustering and contrasting to control samples. Comparison of patient and control samples confirmed a major issue in phage display, namely the selection of unspecific peptides. The potential of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage display by (i) enabling the analysis of complex biological samples, (ii) circumventing the traditional laborious picking and functional testing of individual phage clones and (iii) reducing the number of selection rounds.

  20. Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes.

    PubMed

    Rossant, Christine J; Carroll, Danielle; Huang, Ling; Elvin, John; Neal, Frances; Walker, Edward; Benschop, Joris J; Kim, Eldar E; Barry, Simon T; Vaughan, Tristan J

    2014-01-01

    Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and β-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.

  1. Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes

    PubMed Central

    Rossant, Christine J; Carroll, Danielle; Huang, Ling; Elvin, John; Neal, Frances; Walker, Edward; Benschop, Joris J; Kim, Eldar E; Barry, Simon T; Vaughan, Tristan J

    2014-01-01

    Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and β-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins. PMID:25484064

  2. High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum

    PubMed Central

    Christiansen, Anders; Kringelum, Jens V.; Hansen, Christian S.; Bøgh, Katrine L.; Sullivan, Eric; Patel, Jigar; Rigby, Neil M.; Eiwegger, Thomas; Szépfalusi, Zsolt; Masi, Federico de; Nielsen, Morten; Lund, Ole; Dufva, Martin

    2015-01-01

    Phage display is a prominent screening technique with a multitude of applications including therapeutic antibody development and mapping of antigen epitopes. In this study, phages were selected based on their interaction with patient serum and exhaustively characterised by high-throughput sequencing. A bioinformatics approach was developed in order to identify peptide motifs of interest based on clustering and contrasting to control samples. Comparison of patient and control samples confirmed a major issue in phage display, namely the selection of unspecific peptides. The potential of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage display by (i) enabling the analysis of complex biological samples, (ii) circumventing the traditional laborious picking and functional testing of individual phage clones and (iii) reducing the number of selection rounds. PMID:26246327

  3. Duality of the murine CD8 compartment

    PubMed Central

    Genolet, Raphaël; Leignadier, Julie; Østerås, Magne; Farinelli, Laurent; Stevenson, Brian J.; Luescher, Immanuel F.

    2014-01-01

    CD8αβ plays crucial roles in the thymic selection, differentiation, and activation of some, but not all, CD8+ T cells, whereas CD8αα does not. To investigate these roles, we produced mice that expressed transgene P14 T-cell receptor β (TCRβ) chain and CD8β or did not (WT and KO mice, respectively). The primary CD8+ T-cell response to acute lymphocytic choriomeningitis virus (LCMV) infection was predominantly Db/GP33 specific and CD8 independent in KO mice and was mostly CD8 dependent in WT mice. Cytotoxic T lymphocytes (CTL) from KO mice failed to mobilize intracellular Ca2+ and to kill via perforin/granzyme. Their strong Fas/FasL-mediated cytotoxicity and IFN-γ response were signaled via a Ca2+-independent, PI3K-dependent pathway. This was also true for 15–20% of CD8-independent CTL found in WT mice. Conversely, the perforin/granzyme-mediated killing and IFN-γ response of CD8-dependent CTL were signaled via a Ca2+, p56lck, and nuclear factor of activated T cells-dependent pathway. Deep sequencing of millions of TCRα chain transcripts revealed that the TCR repertoires of preimmune CD8+ T cells were highly diverse, but those of LCMV Db/GP33-specific CTL, especially from KO mice, were narrow. The immune repertoires exhibited biased use of Vα segments that encoded different complementary-determining region 1α (CDR1α) and CDR2α sequences. We suggest that TCR from WT CD8-independent T cells may engage MHC–peptide complexes in a manner unfavorable for efficient CD8 engagement and Ca2+ signaling but permissive for Ca2+-independent, PI3K-dependent signaling. This duality of the CD8 compartment may provide organisms with broader protective immunity. PMID:24594598

  4. Specificity and Dynamics of Effector and Memory CD8 T Cell Responses in Human Tick-Borne Encephalitis Virus Infection

    PubMed Central

    Blom, Kim; Braun, Monika; Pakalniene, Jolita; Dailidyte, Laura; Béziat, Vivien; Lampen, Margit H.; Klingström, Jonas; Lagerqvist, Nina; Kjerstadius, Torbjörn; Michaëlsson, Jakob; Lindquist, Lars; Ljunggren, Hans-Gustaf; Sandberg, Johan K.; Mickiene, Aukse; Gredmark-Russ, Sara

    2015-01-01

    Tick-borne encephalitis virus (TBEV) is transferred to humans by ticks. The virus causes tick-borne encephalitis (TBE) with symptoms such as meningitis and meningoencephalitis. About one third of the patients suffer from long-lasting sequelae after clearance of the infection. Studies of the immune response during TBEV-infection are essential to the understanding of host responses to TBEV-infection and for the development of therapeutics. Here, we studied in detail the primary CD8 T cell response to TBEV in patients with acute TBE. Peripheral blood CD8 T cells mounted a considerable response to TBEV-infection as assessed by Ki67 and CD38 co-expression. These activated cells showed a CD45RA-CCR7-CD127- phenotype at day 7 after hospitalization, phenotypically defining them as effector cells. An immunodominant HLA-A2-restricted TBEV epitope was identified and utilized to study the characteristics and temporal dynamics of the antigen-specific response. The functional profile of TBEV-specific CD8 T cells was dominated by variants of mono-functional cells as the effector response matured. Antigen-specific CD8 T cells predominantly displayed a distinct Eomes+Ki67+T-bet+ effector phenotype at the peak of the response, which transitioned to an Eomes-Ki67-T-bet+ phenotype as the infection resolved and memory was established. These transcription factors thus characterize and discriminate stages of the antigen-specific T cell response during acute TBEV-infection. Altogether, CD8 T cells responded strongly to acute TBEV infection and passed through an effector phase, prior to gradual differentiation into memory cells with distinct transcription factor expression-patterns throughout the different phases. PMID:25611738

  5. Involvement and prognosis value of CD8(+) T cells in giant cell arteritis.

    PubMed

    Samson, Maxime; Ly, Kim Heang; Tournier, Benjamin; Janikashvili, Nona; Trad, Malika; Ciudad, Marion; Gautheron, Alexandrine; Devilliers, Hervé; Quipourt, Valérie; Maurier, François; Meaux-Ruault, Nadine; Magy-Bertrand, Nadine; Manckoundia, Patrick; Ornetti, Paul; Maillefert, Jean-Francis; Besancenot, Jean-François; Ferrand, Christophe; Mesturoux, Laura; Labrousse, François; Fauchais, Anne-Laure; Saas, Philippe; Martin, Laurent; Audia, Sylvain; Bonnotte, Bernard

    2016-08-01

    CD8(+) T cells participate in the pathogenesis of some vasculitides. However, little is known about their role in Giant Cell Arteritis (GCA). This study was conducted to investigate CD8(+) T cell involvement in the pathogenesis of GCA. Analyses were performed at diagnosis and after 3 months of glucocorticoid treatment in 34 GCA patients and 26 age-matched healthy volunteers. Percentages of CD8(+) T-cell subsets, spectratype analysis of the TCR Vβ families of CD8(+) T cells, levels of cytokines and chemokines and immunohistochemistry of temporal artery biopsies (TAB) were assessed. Among total CD8(+) T cells, percentages of circulating cytotoxic CD8 T lymphocytes (CTL, CD3(+)CD8(+)perforin(+)granzymeB(+)), Tc17 (CD3(+)CD8(+)IL-17(+)), CD63(+)CD8(+) T cells and levels of soluble granzymes A and B were higher in patients than in controls, whereas the percentage of Tc1 cells (CD3(+)CD8(+)IFN-γ(+)) was similar. Moreover, CD8(+) T cells displayed a restricted TCR repertoire in GCA patients. Percentages of circulating CTL, Tc17 and soluble levels of granzymes A and B decreased after treatment. CXCR3 expression on CD8(+) T cells and its serum ligands (CXCL9, -10, -11) were higher in patients. Analyses of TAB revealed high expression of CXCL9 and -10 associated with infiltration by CXCR3(+)CD8(+) T cells expressing granzyme B and TiA1. The intensity of the CD8 T-cell infiltrate in TAB was predictive of the severity of the disease. This study demonstrates the implication and the prognostic value of CD8(+) T-cells in GCA and suggests that CD8(+) T-cells are recruited within the vascular wall through an interaction between CXCR3 and its ligands. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. De novo generation of escape variant-specific CD8+ T-cell responses following cytotoxic T-lymphocyte escape in chronic human immunodeficiency virus type 1 infection.

    PubMed

    Allen, Todd M; Yu, Xu G; Kalife, Elizabeth T; Reyor, Laura L; Lichterfeld, Mathias; John, Mina; Cheng, Michael; Allgaier, Rachel L; Mui, Stanley; Frahm, Nicole; Alter, Galit; Brown, Nancy V; Johnston, Mary N; Rosenberg, Eric S; Mallal, Simon A; Brander, Christian; Walker, Bruce D; Altfeld, Marcus

    2005-10-01

    Human immunodeficiency virus type 1 (HIV-1) evades CD8(+) T-cell responses through mutations within targeted epitopes, but little is known regarding its ability to generate de novo CD8(+) T-cell responses to such mutants. Here we examined gamma interferon-positive, HIV-1-specific CD8(+) T-cell responses and autologous viral sequences in an HIV-1-infected individual for more than 6 years following acute infection. Fourteen optimal HIV-1 T-cell epitopes were targeted by CD8(+) T cells, four of which underwent mutation associated with dramatic loss of the original CD8(+) response. However, following the G(357)S escape in the HLA-A11-restricted Gag(349-359) epitope and the decline of wild-type-specific CD8(+) T-cell responses, a novel CD8(+) T-cell response equal in magnitude to the original response was generated against the variant epitope. CD8(+) T cells targeting the variant epitope did not exhibit cross-reactivity against the wild-type epitope but rather utilized a distinct T-cell receptor Vbeta repertoire. Additional studies of chronically HIV-1-infected individuals expressing HLA-A11 demonstrated that the majority of the subjects targeted the G(357)S escape variant of the Gag(349-359) epitope, while the wild-type consensus sequence was significantly less frequently recognized. These data demonstrate that de novo responses against escape variants of CD8(+) T-cell epitopes can be generated in chronic HIV-1 infection and provide the rationale for developing vaccines to induce CD8(+) T-cell responses directed against both the wild-type and variant forms of CD8 epitopes to prevent the emergence of cytotoxic T-lymphocyte escape variants.

  7. HIV-1-Specific CD8 T Cells Exhibit Limited Cross-Reactivity during Acute Infection.

    PubMed

    Du, Victor Y; Bansal, Anju; Carlson, Jonathan; Salazar-Gonzalez, Jesus F; Salazar, Maria G; Ladell, Kristin; Gras, Stephanie; Josephs, Tracy M; Heath, Sonya L; Price, David A; Rossjohn, Jamie; Hunter, Eric; Goepfert, Paul A

    2016-04-15

    Prior work has demonstrated that HIV-1-specific CD8 T cells can cross-recognize variant epitopes. However, most of these studies were performed in the context of chronic infection, where the presence of viral quasispecies makes it difficult to ascertain the true nature of the original antigenic stimulus. To overcome this limitation, we evaluated the extent of CD8 T cell cross-reactivity in patients with acute HIV-1 clade B infection. In each case, we determined the transmitted founder virus sequence to identify the autologous epitopes restricted by individual HLA class I molecules. Our data show that cross-reactive CD8 T cells are infrequent during the acute phase of HIV-1 infection. Moreover, in the uncommon instances where cross-reactive responses were detected, the variant epitopes were poorly recognized in cytotoxicity assays. Molecular analysis revealed that similar antigenic structures could be cross-recognized by identical CD8 T cell clonotypes mobilized in vivo, yet even subtle differences in a single TCR-accessible peptide residue were sufficient to disrupt variant-specific reactivity. These findings demonstrate that CD8 T cells are highly specific for autologous epitopes during acute HIV-1 infection. Polyvalent vaccines may therefore be required to provide optimal immune cover against this genetically labile pathogen.

  8. Identification of a Conserved B-cell Epitope on Reticuloendotheliosis Virus Envelope Protein by Screening a Phage-displayed Random Peptide Library

    PubMed Central

    Xue, Mei; Shi, Xingming; Zhang, Jing; Zhao, Yan; Cui, Hongyu; Hu, Shunlei; Gao, Hongbo; Cui, Xianlan; Wang, Yun-Feng

    2012-01-01

    Background The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported. Methods and Results This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched 213SVQYHPL219 of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that 213SVQYHPL219 was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group. Conclusions and Significance We identified 213SVQYHPL219 as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group. PMID:23185456

  9. Rapid Conformational Epitope Mapping of anti-gp120 Antibodies with a Designed Mutant Panel Displayed on Yeast

    PubMed Central

    Mata-Fink, Jordi; Kriegsman, Barry; Xin, Yu Hui; Zhu, Hanna; Hanson, Melissa; Irvine, Darrell J.; Wittrup, K. Dane

    2013-01-01

    gp120 is a substrate for protein engineering both for HIV immunogen design and as a bait for isolating anti-HIV antibodies from patient samples. In this work we describe the display of a stripped core gp120 on the yeast cell surface. Validation against a panel of neutralizing antibodies confirms that yeast-displayed gp120 presents the CD4 binding site in the correct conformation. We map the epitope of the broadly neutralizing anti-gp120 antibody VRC01 using both a random mutagenesis library and a defined mutant panel, and find the resultant epitope maps are consistent with one another and with the crystallographically identified contact residues. Mapping the VRC01-competitive antibodies b12 and b13 reveals energetic differences in their epitopes that are not obvious from existing crystal structures. These data suggest mutation sets that abrogate binding to broadly neutralizing antibodies with greater specificity than the canonical mutation D368R, useful in rapidly assessing the nature of a vaccine response. PMID:23159556

  10. Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity

    PubMed Central

    Vasconcelos, Jose R.; Motz, R. Geoffrey; Sullivan, Nicole L.; Gohara, David W.; Blase, Jennifer R.; Di Cera, Enrico; Hoft, Daniel F.

    2016-01-01

    Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4+ and CD8+ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8+ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8+ T cell epitope enhance subdominant pathogen-specific CD8+ T cell responses. More specifically, CD8+ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8+ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T

  11. Long-Term Immunity to Trypanosoma cruzi in the Absence of Immunodominant trans-Sialidase-Specific CD8+ T Cells

    PubMed Central

    Rosenberg, Charles S.; Zhang, Weibo; Bustamante, Juan M.

    2016-01-01

    Trypanosoma cruzi infection drives the expansion of remarkably focused CD8+ T cell responses targeting epitopes encoded by variant trans-sialidase (TS) genes. Infection of C57BL/6 mice with T. cruzi results in up to 40% of all CD8+ T cells committed to recognition of the dominant TSKB20 and subdominant TSKB18 TS epitopes. However, despite this enormous response, these mice fail to clear T. cruzi infection and subsequently develop chronic disease. One possible reason for the failure to cure T. cruzi infection is that immunodomination by these TS-specific T cells may interfere with alternative CD8+ T cell responses more capable of complete parasite elimination. To address this possibility, we created transgenic mice that are centrally tolerant to these immunodominant epitopes. Mice expressing TSKB20, TSKB18, or both epitopes controlled T. cruzi infection and developed effector CD8+ T cells that maintained an activated phenotype. Memory CD8+ T cells from drug-cured TSKB-transgenic mice rapidly responded to secondary T. cruzi infection. In the absence of the response to TSKB20 and TSKB18, immunodominance did not shift to other known subdominant epitopes despite the capacity of these mice to expand epitope-specific T cells specific for the model antigen ovalbumin expressed by engineered parasites. Thus, CD8+ T cell responses tightly and robustly focused on a few epitopes within variant TS antigens appear to neither contribute to, nor detract from, the ability to control T. cruzi infection. These data also indicate that the relative position of an epitope within a CD8+ immunodominance hierarchy does not predict its importance in pathogen control. PMID:27354447

  12. Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity.

    PubMed

    Eickhoff, Christopher S; Zhang, Xiuli; Vasconcelos, Jose R; Motz, R Geoffrey; Sullivan, Nicole L; O'Shea, Kelly; Pozzi, Nicola; Gohara, David W; Blase, Jennifer R; Di Cera, Enrico; Hoft, Daniel F

    2016-09-01

    Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4+ and CD8+ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8+ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8+ T cell epitope enhance subdominant pathogen-specific CD8+ T cell responses. More specifically, CD8+ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8+ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T

  13. Identification of atherosclerosis-associated conformational heat shock protein 60 epitopes by phage display and structural alignment.

    PubMed

    Perschinka, Hannes; Wellenzohn, Bernd; Parson, Walther; van der Zee, Ruurd; Willeit, Johann; Kiechl, Stefan; Wick, Georg

    2007-09-01

    Autoimmune reactions to HSP60 are believed to play a key role during development of early atherosclerosis. Due to the high degree of phylogenetic conservation between microbial and human HSP60, bacterial infections might be responsible for inducing cross-reactivity to self HSP60, which is expressed on the surface of arterial endothelial cells stressed by classical atherosclerosis risk factors. Conformational epitopes recognized by polyclonal anti-mycobacterial HSP60 antibodies from subjects with atherosclerosis were identified using a phage displayed random library of cyclic constrained 7mer peptides. After five rounds of selection, DNA sequencing of strongly binding clones revealed that one peptide motif (CIGSPSTNC) was present in 64% of all clones, and a second motif (CSFHYQNRC) in 14%. Using a newly developed method for structural alignment of small constrained peptides onto a protein surface, we located the motif present in 14% of all clones on the surface of mycobacterial HSP60. The motif present in 64% of all clones was found on the surface of mycobacterial HSP60 as well as in the homologous region of human HSP60, which makes this epitope a promising candidate for further investigations on cross-reactive epitopes involved in early atherogenesis.

  14. High-dimensional immunomonitoring models of HIV-1–specific CD8 T-cell responses accurately identify subjects achieving spontaneous viral control

    PubMed Central

    Ndhlovu, Zaza M.; Chibnik, Lori B.; Proudfoot, Jacqueline; Vine, Seanna; McMullen, Ashley; Cesa, Kevin; Porichis, Filippos; Jones, R. Brad; Alvino, Donna Marie; Hart, Meghan G.; Stampouloglou, Eleni; Piechocka-Trocha, Alicja; Kadie, Carl; Pereyra, Florencia; Heckerman, David; De Jager, Philip L.; Walker, Bruce D.

    2013-01-01

    The development of immunomonitoring models to determine HIV-1 vaccine efficacy is a major challenge. Studies suggest that HIV-1–specific CD8 T cells play a critical role in subjects achieving spontaneous viral control (HIV-1 controllers) and that they will be important in immune interventions. However, no single CD8 T-cell function is uniquely associated with controller status and the heterogeneity of responses targeting different epitopes further complicates the discovery of determinants of protective immunity. In the present study, we describe immunomonitoring models integrating multiple functions of epitope-specific CD8 T cells that distinguish controllers from subjects with treated or untreated progressive infection. Models integrating higher numbers of variables and trained with the least absolute shrinkage and selection operator (LASSO) variant of logistic regression and 10-fold cross-validation produce “diagnostic tests” that display an excellent capacity to delineate subject categories. The test accuracy reaches 75% area under the receiving operating characteristic curve in cohorts matched for prevalence of protective alleles. Linear mixed-effects model analyses show that the proliferative capacity, cytokine production, and kinetics of cytokine secretion are associated with HIV-1 control. Although proliferative capacity is the strongest single discriminant, integrated modeling of different dimensions of data leverages individual associations. This strategy may have important applications in predictive model development and immune monitoring of HIV-1 vaccine trials. PMID:23233659

  15. A protective role for dengue virus-specific CD8+ T cells.

    PubMed

    Yauch, Lauren E; Zellweger, Raphaël M; Kotturi, Maya F; Qutubuddin, Afrina; Sidney, John; Peters, Bjoern; Prestwood, Tyler R; Sette, Alessandro; Shresta, Sujan

    2009-04-15

    Infection with one of the four serotypes of dengue virus (DENV1-4) can result in a range of clinical manifestations in humans, from dengue fever to the more serious dengue hemorrhagic fever/dengue shock syndrome. Although T cells have been implicated in the immunopathogenesis of secondary infections with heterologous DENV serotypes, the role of T cells in protection against DENV is unknown. In this study, we used a mouse-passaged DENV2 strain, S221, to investigate the role of CD8(+) T cells in the immune response to primary DENV infection. S221 did not replicate well in wild-type mice, but did induce a CD8(+) T cell response, whereas viral replication and a robust CD8(+) T cell response were observed after infection of IFN-alpha/betaR(-/-) mice. Depletion of CD8(+) T cells from IFN-alpha/betaR(-/-) mice before infection resulted in significantly higher viral loads compared with undepleted mice. Mapping the specificity of the CD8(+) T cell response led to the identification of 12 epitopes derived from 6 of the 10 DENV proteins, with a similar immunodominance hierarchy observed in wild-type and IFN-alpha/betaR(-/-) mice. DENV-specific CD8(+) T cells produced IFN-gamma, TNF-alpha, expressed cell surface CD107a, and exhibited cytotoxic activity in vivo. Finally, immunization with four of the immunodominant CD8(+) T cell epitopes enhanced viral clearance. Collectively, our results reveal an important role for CD8(+) T cells in the host defense against DENV and demonstrate that the anti-DENV CD8(+) T cell response can be enhanced by immunization, providing rationale for designing DENV-specific vaccines that induce cell-mediated immunity.

  16. CD8 T Cell Response Maturation Defined by Anentropic Specificity and Repertoire Depth Correlates with SIVΔnef-induced Protection

    PubMed Central

    Adnan, Sama; Colantonio, Arnaud D.; Yu, Yi; Gillis, Jacqueline; Wong, Fay E.; Becker, Ericka A.; Reeves, R. Keith; Lifson, Jeffrey D.; O’Connor, Shelby L.; Johnson, R. Paul

    2015-01-01

    The live attenuated simian immunodeficiency virus (LASIV) vaccine SIVΔnef is one of the most effective vaccines in inducing protection against wild-type lentiviral challenge, yet little is known about the mechanisms underlying its remarkable protective efficacy. Here, we exploit deep sequencing technology and comprehensive CD8 T cell epitope mapping to deconstruct the CD8 T cell response, to identify the regions of immune pressure and viral escape, and to delineate the effect of epitope escape on the evolution of the CD8 T cell response in SIVΔnef-vaccinated animals. We demonstrate that the initial CD8 T cell response in the acute phase of SIVΔnef infection is mounted predominantly against more variable epitopes, followed by widespread sequence evolution and viral escape. Furthermore, we show that epitope escape expands the CD8 T cell repertoire that targets highly conserved epitopes, defined as anentropic specificity, and generates de novo responses to the escaped epitope variants during the vaccination period. These results correlate SIVΔnef-induced protection with expanded anentropic specificity and increased response depth. Importantly, these findings render SIVΔnef, long the gold standard in HIV/SIV vaccine research, as a proof-of-concept vaccine that highlights the significance of the twin principles of anentropic specificity and repertoire depth in successful vaccine design. PMID:25688559

  17. CD8 Raft localization is induced by its assembly into CD8alpha beta heterodimers, Not CD8alpha alpha homodimers.

    PubMed

    Pang, Dick John; Hayday, Adrian C; Bijlmakers, Marie-José

    2007-05-04

    The coreceptor CD8 is expressed as a CD8alphabeta heterodimer on major histocompatibility complex class I-restricted TCRalphabeta T cells, and as a CD8alphaalpha homodimer on subsets of memory T cells, intraepithelial lymphocytes, natural killer cells, and dendritic cells. Although the role of CD8alphaalpha is not well understood, it is increasingly clear that this protein is not a functional homologue of CD8alphabeta. On major histocompatibility complex class I-restricted T cells, CD8alphabeta is a more efficient TCR coreceptor than CD8alphaalpha. This property has for the mouse protein been attributed to the recruitment of CD8alphabeta into lipid rafts, which is dependent on CD8beta palmitoylation. Here, these divergent distributions of CD8alphabeta and CD8alphaalpha are demonstrated for the human CD8 proteins as well. However, although palmitoylation of both CD8alpha and CD8beta chains was detected, this modification did not contribute to raft localization. In contrast, arginines in the cytoplasmic domain are crucial for raft localization of CD8betabeta. Most strikingly, the assembly of a non-raft localized CD8beta chain with a non-raft localized CD8alpha chain resulted in raft-localized CD8alphabeta heterodimers. Using chimeric CD8 proteins, this property of the heterodimer was found to be determined by the assembly of CD8alpha and CD8beta extracellular regions. The presence of two CD8alpha extracellular regions, on the other hand, appears to preclude raft localization. Thus, heterodimer formation and raft association are intimately linked for CD8alphabeta. These results emphasize that lipid raft localization is a key feature of human CD8alphabeta that clearly distinguishes it from CD8alphaalpha.

  18. Optimized DNA Vaccines to Specifically Induce Therapeutic CD8 T Cell Responses Against Autochthonous Breast Tumors

    PubMed Central

    Cho, Hyun-Il; Niu, Guilian; Bradley, Norma; Celis, Esteban

    2008-01-01

    Vaccines capable of inducing CD8 T cell responses to antigens expressed by tumor cells are considered as attractive choices for the treatment and prevention of malignant diseases. Our group has previously reported that immunization with synthetic peptide corresponding to a CD8 T cell epitope derived from the rat neu oncogene administered together with a Toll-like receptor agonist as adjuvant, induced immune responses that translated into prophylactic and therapeutic benefit against autochthonous tumors in an animal model of breast cancer (BALB-neuT mice). DNA-based vaccines offer some advantages over peptide vaccines, such as the possibility of including multiple CD8 T cell epitopes in a single construct. Thus, we have evaluated the use of DNA vaccination for its ability to generate effective CD8 T cell responses against breast tumors expressing the rat neu oncogene. The results show that as with peptide vaccination, DNA-based vaccines were very effective in stimulating tumor-reactive CD8 T cell responses. Moreover, vaccination with modified DNA plasmids resulted in significant anti-tumor effects that were mediated by CD8 T cells without the requirement of generating antibodies to the product of rat neu. These results bear importance for the design of DNA vaccines for the treatment and prevention of cancer. PMID:18253731

  19. Familial CD8 deficiency due to a mutation in the CD8α gene

    PubMed Central

    de la Calle-Martin, Oscar; Hernandez, Manuel; Ordi, Jose; Casamitjana, Natalia; Arostegui, Juan I.; Caragol, Isabel; Ferrando, Monserrat; Labrador, Moises; Rodriguez-Sanchez, Jose L.; Espanol, Teresa

    2001-01-01

    CD8 glycoproteins play an important role in both the maturation and function of MHC class I-restricted T lymphocytes. A 25-year-old man, from a consanguineous family, with recurrent bacterial infections and total absence of CD8+ cells, was studied. Ab deficiencies and ZAP-70 and TAP defects were ruled out. A missense mutation (gly90→ser) in both alleles of the immunoglobulin domain of the CD8α gene was shown to correlate with the absence of CD8 expression found in the patient and two sisters. Conversely, high percentages of CD4–CD8–TCRαβ+ T cells were found in the three siblings. A novel autosomal recessive immunologic defect characterized by absence of CD8+ cells is described. These findings may help to further understanding of the role of CD8 molecules in human immune response. PMID:11435463

  20. Nuclear location of an endogenously expressed antigen, EBNA1, restricts access to macroautophagy and the range of CD4 epitope display.

    PubMed

    Leung, Carol S; Haigh, Tracey A; Mackay, Laura K; Rickinson, Alan B; Taylor, Graham S

    2010-02-02

    Whereas exogenously acquired proteins are the major source of antigens feeding the MHC class II pathway in antigen-presenting cells, some endogenously expressed antigens also access that pathway but the rules governing such access are poorly understood. Here we address this using Epstein-Barr virus (EBV)-coded nuclear antigen EBNA1, a protein naturally expressed in EBV-infected B lymphoblastoid cell lines (LCLs) and a source of multiple CD4(+) T cell epitopes. Using CD4(+) T cell clones against three indicator epitopes, we find that two epitopes are weakly displayed on the LCL surface whereas the third is undetectable, a pattern of limited epitope presentation that is maintained even when nuclear expression of EBNA1 is induced to high supraphysiological levels. Inhibitor and siRNA studies show that, of the two epitopes weakly presented under these conditions, one involves macroautophagy, and the second involves antigen delivery to the MHC II pathway by another endogenous route. In contrast, when EBNA1 is expressed as a cytoplasmic protein, all three CD4 epitopes are processed and presented much more efficiently, and all involve macroautophagy. We conclude that EBNA1's nuclear location limits its accessibility to the macroautophagy pathway and, in consequence, limits the level and range of EBNA1 CD4 epitopes naturally displayed on the infected cell surface.

  1. Expanded CD8 T-cell sharing between periphery and CNS in multiple sclerosis

    PubMed Central

    Salou, Marion; Garcia, Alexandra; Michel, Laure; Gainche-Salmon, Anne; Loussouarn, Delphine; Nicol, Bryan; Guillot, Flora; Hulin, Philippe; Nedellec, Steven; Baron, Daniel; Ramstein, Gérard; Soulillou, Jean-Paul; Brouard, Sophie; Nicot, Arnaud B; Degauque, Nicolas; Laplaud, David A

    2015-01-01

    Objective In multiple sclerosis (MS), central nervous system (CNS), cerebrospinal fluid (CSF), and blood display TCR clonal expansions of CD8+ T cells. These clones have been assumed – but never demonstrated – to be similar in the three compartments. Addressing this key question is essential to infer the implication of peripheral clonally expanded CD8+ T cells in the disease. Methods For the first time, TCR Vβ repertoire from paired blood (purified CD8+ and CD4+ T cells), CSF and CNS (22 lesions, various inflammatory and demyelination statuses) samples from three MS patients was studied using complementary determining region 3 (CDR3) spectratyping and high-throughput sequencing. In parallel, blood and CNS clonally expanded CD8+ T cells were characterized by fluorescent staining. Results TCR Vβ repertoire analysis revealed strong sharing of predominant T-cell clones between CNS lesions, CSF, and blood CD8+ T cells. In parallel, we showed that blood oligoclonal CD8+ T cells exhibit characteristics of pathogenic cells, as they displayed a bias toward a memory phenotype in MS patients, with increased expression of CCR5, CD11a and Granzyme B (GZM-B) compared to non oligoclonal counterparts. CNS-infiltrating T cells were mainly CD8 expressing CD11a and GZM-B. Interpretation This study highlights the predominant implication of CD8+ T cells in MS pathophysiology and demonstrates that potentially aggressive CD8+ T cells can be easily identified and characterized from blood and CSF samples. PMID:26125037

  2. Multi-scale modeling of the CD8 immune response

    SciTech Connect

    Barbarroux, Loic; Michel, Philippe; Adimy, Mostafa; Crauste, Fabien

    2016-06-08

    During the primary CD8 T-Cell immune response to an intracellular pathogen, CD8 T-Cells undergo exponential proliferation and continuous differentiation, acquiring cytotoxic capabilities to address the infection and memorize the corresponding antigen. After cleaning the organism, the only CD8 T-Cells left are antigen-specific memory cells whose role is to respond stronger and faster in case they are presented this very same antigen again. That is how vaccines work: a small quantity of a weakened pathogen is introduced in the organism to trigger the primary response, generating corresponding memory cells in the process, giving the organism a way to defend himself in case it encounters the same pathogen again. To investigate this process, we propose a non linear, multi-scale mathematical model of the CD8 T-Cells immune response due to vaccination using a maturity structured partial differential equation. At the intracellular scale, the level of expression of key proteins is modeled by a delay differential equation system, which gives the speeds of maturation for each cell. The population of cells is modeled by a maturity structured equation whose speeds are given by the intracellular model. We focus here on building the model, as well as its asymptotic study. Finally, we display numerical simulations showing the model can reproduce the biological dynamics of the cell population for both the primary response and the secondary responses.

  3. Multi-scale modeling of the CD8 immune response

    NASA Astrophysics Data System (ADS)

    Barbarroux, Loic; Michel, Philippe; Adimy, Mostafa; Crauste, Fabien

    2016-06-01

    During the primary CD8 T-Cell immune response to an intracellular pathogen, CD8 T-Cells undergo exponential proliferation and continuous differentiation, acquiring cytotoxic capabilities to address the infection and memorize the corresponding antigen. After cleaning the organism, the only CD8 T-Cells left are antigen-specific memory cells whose role is to respond stronger and faster in case they are presented this very same antigen again. That is how vaccines work: a small quantity of a weakened pathogen is introduced in the organism to trigger the primary response, generating corresponding memory cells in the process, giving the organism a way to defend himself in case it encounters the same pathogen again. To investigate this process, we propose a non linear, multi-scale mathematical model of the CD8 T-Cells immune response due to vaccination using a maturity structured partial differential equation. At the intracellular scale, the level of expression of key proteins is modeled by a delay differential equation system, which gives the speeds of maturation for each cell. The population of cells is modeled by a maturity structured equation whose speeds are given by the intracellular model. We focus here on building the model, as well as its asymptotic study. Finally, we display numerical simulations showing the model can reproduce the biological dynamics of the cell population for both the primary response and the secondary responses.

  4. Description of an elasmobranch TCR coreceptor: CD8α from Rhinobatos productus

    USGS Publications Warehouse

    Hansen, J.D.; Farrugia, T.J.; Woodson, J.; Laing, K.J.

    2011-01-01

    Cell-mediated immunity plays an essential role for the control and eradication of intracellular pathogens. To learn more about the evolutionary origins of the first signal (Signal 1) for T-cell activation, we cloned CD8α from an elasmobranch, Rhinobatos productus. Similar to full-length CD8α cDNAs from other vertebrates, Rhpr-CD8α (1800 bp) encodes a 219 amino acid open reading frame composed of a signal peptide, an extracellular IgSF V domain and a stalk/hinge region followed by a well-conserved transmembrane domain and cytoplasmic tail. Overall, the mature Rhpr-CD8α protein (201 aa) displays ~30% amino acid identity with mammalian CD8α including absolute conservation of cysteine residues involved in the IgSf V domain fold and dimerization of CD8αα and CD8αβ. One prominent feature is the absence of the LCK association motif (CXC) that is needed for achieving signal 1 in tetrapods. Both elasmobranch and teleost CD8α protein sequences possess a similar but distinctly different motif (CXH) in the cytoplasmic tail. The overall genomic structure of CD8α has been conserved during the course of vertebrate evolution both for the number of exons and phase of splicing. Finally, quantitative RTPCR demonstrated that elasmobranch CD8α is expressed in lymphoid-rich tissues similar to CD8 in other vertebrates. The results from this study indicate the existence of CD8 prior to the emergence of the gnathostomes (>450 MYA) while providing evidence that the canonical LCK association motif in mammals is likely a derived characteristic of tetrapod CD8α, suggesting potential differences for T-cell education and activation in the various gnathostomes.

  5. Damage to the blood-brain barrier during experimental cerebral malaria results from synergistic effects of CD8+ T cells with different specificities.

    PubMed

    Poh, Chek Meng; Howland, Shanshan W; Grotenbreg, Gijsbert M; Rénia, Laurent

    2014-11-01

    CD8(+) T cells play a pathogenic role in the development of murine experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA (PbA) infection in C57BL/6 mice. Only a limited number of CD8(+) epitopes have been described. Here, we report the identification of a new epitope from the bergheilysin protein recognized by PbA-specific CD8(+) T cells. Induction and functionality of these specific CD8(+) T cells were investigated in parallel with previously reported epitopes, using new tools such as tetramers and reporter cell lines that were developed for this study. We demonstrate that CD8(+) T cells of diverse specificities induced during PbA infection share many characteristics. They express cytolytic markers (gamma interferon [IFN-γ], granzyme B) and chemokine receptors (CXCR3, CCR5) and damage the blood-brain barrier in vivo. Our earlier finding that brain microvessels in mice infected with PbA, but not with non-ECM-causing strains, cross-presented a shared epitope was generalizable to these additional epitopes. Suppressing the induction of specific CD8(+) T cells through tolerization with a high-dose peptide injection was unable to confer protection against ECM, suggesting that CD8(+) T cells of other specificities participate in this process. The tools that we developed can be used to further investigate the heterogeneity of CD8(+) T cell responses that are involved in ECM. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  6. Identification of a new epitope for HIV-neutralizing antibodies in the gp41 membrane proximal external region by an Env-tailored phage display library.

    PubMed

    Zhou, Mingkui; Meyer, Torsten; Koch, Stefanie; Koch, Joachim; von Briesen, Hagen; Benito, José M; Soriano, Vincent; Haberl, Annette; Bickel, Markus; Dübel, Stefan; Hust, Michael; Dietrich, Ursula

    2013-02-01

    HIV controllers are a valuable source for the identification of HIV-neutralizing antibodies, as chronic infection over decades allows extensive affinity maturation of antibodies for improved Ag recognition. We analyzed a small cohort of elite controllers (ECs) for HIV-neutralizing antibodies using a panel of standardized HIV-1 pseudovirions on TZM-bl cells. An HIV-1 Env-tailored phage display library was generated to select epitopes targeted by neutralizing antibodies in the EC26 plasma sample showing the broadest neutralizing activity. Selected Env fragments were mostly allocated to the membrane proximal external region of gp41. After preabsorbing the EC26 plasma with the selected phage EC26-2A4, we achieved 50% depletion of its neutralizing activity. Furthermore, antibodies affinity-purified with the EC26-2A4 epitope from EC26 plasma showed neutralizing activity, proving that the selected phage indeed contains an epitope targeted by neutralizing plasma antibodies. Epitope fine mapping of the purified plasma antibodies on peptide arrays identified a new epitope overlapping, but clearly distinct, from the prominent 2F5 epitope. Of note, the purified antibodies did not show autoreactivity with cardiolipin, whereas low reactivity with phosphatidylserine comparable to mAb 2F5 was observed. Thus, this new epitope represents a promising candidate for further analysis in view of HIV vaccine development. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Salmonella enteritidis fimbriae displaying a heterologous epitope reveal a uniquely flexible structure and assembly mechanism.

    PubMed

    White, A P; Collinson, S K; Banser, P A; Dolhaine, D J; Kay, W W

    2000-02-18

    Two distinct Salmonella fimbrins, AgfA and SefA, comprising thin aggregative fimbriae SEF17 and SEF14, respectively, were each genetically engineered to carry PT3, an alpha-helical 16-amino acid Leishmania T-cell epitope derived from the metalloprotease gp63. To identify regions within AgfA and SefA fimbrins amenable to replacement with this epitope, PCR-generated chimeric fimbrin genes were constructed and used to replace the native chromosomal agfA and sefA genes in Salmonella enteritidis. Immunoblot analysis using anti-SEF17 and anti-PT3 sera demonstrated that all ten AgfA chimeric fimbrin proteins were expressed by S. enteritidis under normal growth conditions. Immunoelectron microscopy confirmed that eight of the AgfA::PT3 proteins were effectively assembled into cell surface-exposed fimbriae. The PT3 replacements in AgfA altered Congo red (CR) binding, cell-cell adhesion and cell surface properties of S. enteritidis to varying degrees. However, these chimeric fimbriae were still highly stable, being resistant to proteinase K digestion and requiring harsh formic acid treatment for depolymerization. In marked contrast to AgfA, none of the chimeric SefA proteins were expressed or assembled into fimbriae. Since each PT3 replacement constituted over 10% of the AgfA amino acid sequence and all ten replacements collectively represented greater than 75% of the entire AgfA primary sequence, the ability of AgfA to accept large sequence substitutions and still assemble into fibers is unique among fimbriae and other structural proteins. This structural flexibility may be related to the novel fivefold repeating sequence of AgfA and its recently proposed structure Proper formation of chimeric fimbrial fibers suggests an unusual assembly mechanism for thin aggregative fimbriae which tolerates aberrant structures. This study opens a range of possibilities for Salmonella thin aggregative fimbriae as a carrier of heterologous epitopes and as an experimental model for studies

  8. Increased CD4+/CD8+ Double-Positive T Cells in Chronic Chagasic Patients

    PubMed Central

    Giraldo, Nicolas A.; Bolaños, Natalia I.; Cuellar, Adriana; Guzman, Fanny; Uribe, Ana Maria; Bedoya, Astrid; Olaya, Natalia; Cucunubá, Zulma M.; Roa, Nubia; Rosas, Fernando; Velasco, Víctor; Puerta, Concepción J.; González, John M.

    2011-01-01

    Background CD4+/CD8+ double positive (DP) T cells have been described in healthy individuals as well as in patients with autoimmune and chronic infectious diseases. In chronic viral infections, this cell subset has effector memory phenotype and displays antigen specificity. No previous studies of double positive T cells in parasite infections have been carried out. Methodology/Principal Findings Seventeen chronic chagasic patients (7 asymptomatic and 10 symptomatic) and 24 non-infected donors, including 12 healthy and 12 with non-chagasic cardiomyopathy donors were analyzed. Peripheral blood was stained for CD3, CD4, CD8, HLA-DR and CD38, and lymphocytes for intracellular perforin. Antigen specificity was assessed using HLA*A2 tetramers loaded with T. cruzi K1 or influenza virus epitopes. Surface expression of CD107 and intracellular IFN-γ production were determined in K1-specific DP T cells from 11 chagasic donors. Heart tissue from a chronic chagasic patient was stained for both CD8 and CD4 by immunochemistry. Chagasic patients showed higher frequencies of DP T cells (2.1%±0.9) compared with healthy (1.1%±0.5) and non-chagasic cardiomyopathy (1.2%±0.4) donors. DP T cells from Chagasic patients also expressed more HLA-DR, CD38 and perforin and had higher frequencies of T. cruzi K1-specific cells. IFN-γ production in K1-specific cells was higher in asymptomatic patients after polyclonal stimulation, while these cells tended to degranulate more in symptomatic donors. Immunochemistry revealed that double positive T cells infiltrate the cardiac tissue of a chagasic donor. Conclusions Chagasic patients have higher percentages of circulating double positive T cells expressing activation markers, potential effector molecules and greater class I antigenic specificity against T. cruzi. Although K1 tetramer positive DP T cell produced little IFN-γ, they displayed degranulation activity that was increased in symptomatic patients. Moreover, K1-specific DP T cells can

  9. A Conserved Epitope Mapped with a Monoclonal Antibody against the VP3 Protein of Goose Parvovirus by Using Peptide Screening and Phage Display Approaches

    PubMed Central

    Li, Chenxi; Liu, Hongyu; Li, Jinzhe; Liu, Dafei; Meng, Runze; Zhang, Qingshan; Shaozhou, Wulin; Bai, Xiaofei; Zhang, Tingting; Liu, Ming; Zhang, Yun

    2016-01-01

    Background Waterfowl parvovirus (WPV) infection causes high mortality and morbidity in both geese (Anser anser) and Muscovy ducks (Cairina moschata), resulting in significant losses to the waterfowl industries. The VP3 protein of WPV is a major structural protein that induces neutralizing antibodies in the waterfowl. However, B-cell epitopes on the VP3 protein of WPV have not been characterized. Methods and Results To understand the antigenic determinants of the VP3 protein, we used the monoclonal antibody (mAb) 4A6 to screen a set of eight partially expressed overlapping peptides spanning VP3. Using western blotting and an enzyme-linked immunosorbent assay (ELISA), we localized the VP3 epitope between amino acids (aa) 57 and 112. To identify the essential epitope residues, a phage library displaying 12-mer random peptides was screened with mAb 4A6. Phage clone peptides displayed a consensus sequence of YxRFHxH that mimicked the sequence 82Y/FNRFHCH88, which corresponded to amino acid residues 82 to 88 of VP3 protein of WPVs. mAb 4A6 binding to biotinylated fragments corresponding to amino acid residues 82 to 88 of the VP3 protein verified that the 82FxRFHxH88 was the VP3 epitope and that amino acids 82F is necessary to retain maximal binding to mAb 4A6. Parvovirus-positive goose and duck sera reacted with the epitope peptide by dot blotting assay, revealing the importance of these amino acids of the epitope in antibody-epitope binding reactivity. Conclusions and Significance We identified the motif FxRFHxH as a VP3-specific B-cell epitope that is recognized by the neutralizing mAb 4A6. This finding might be valuable in understanding of the antigenic topology of VP3 of WPV. PMID:27191594

  10. A Conserved Epitope Mapped with a Monoclonal Antibody against the VP3 Protein of Goose Parvovirus by Using Peptide Screening and Phage Display Approaches.

    PubMed

    Li, Chenxi; Liu, Hongyu; Li, Jinzhe; Liu, Dafei; Meng, Runze; Zhang, Qingshan; Shaozhou, Wulin; Bai, Xiaofei; Zhang, Tingting; Liu, Ming; Zhang, Yun

    2016-01-01

    Waterfowl parvovirus (WPV) infection causes high mortality and morbidity in both geese (Anser anser) and Muscovy ducks (Cairina moschata), resulting in significant losses to the waterfowl industries. The VP3 protein of WPV is a major structural protein that induces neutralizing antibodies in the waterfowl. However, B-cell epitopes on the VP3 protein of WPV have not been characterized. To understand the antigenic determinants of the VP3 protein, we used the monoclonal antibody (mAb) 4A6 to screen a set of eight partially expressed overlapping peptides spanning VP3. Using western blotting and an enzyme-linked immunosorbent assay (ELISA), we localized the VP3 epitope between amino acids (aa) 57 and 112. To identify the essential epitope residues, a phage library displaying 12-mer random peptides was screened with mAb 4A6. Phage clone peptides displayed a consensus sequence of YxRFHxH that mimicked the sequence 82Y/FNRFHCH88, which corresponded to amino acid residues 82 to 88 of VP3 protein of WPVs. mAb 4A6 binding to biotinylated fragments corresponding to amino acid residues 82 to 88 of the VP3 protein verified that the 82FxRFHxH88 was the VP3 epitope and that amino acids 82F is necessary to retain maximal binding to mAb 4A6. Parvovirus-positive goose and duck sera reacted with the epitope peptide by dot blotting assay, revealing the importance of these amino acids of the epitope in antibody-epitope binding reactivity. We identified the motif FxRFHxH as a VP3-specific B-cell epitope that is recognized by the neutralizing mAb 4A6. This finding might be valuable in understanding of the antigenic topology of VP3 of WPV.

  11. Phenotypic and functional characteristics of IL-21-expressing CD8+ T cells in human nasal polyps

    PubMed Central

    Xiao, Li; Jia, Lei; Bai, Lu; He, Long; Yang, Binyan; Wu, Changyou; Li, Huabin

    2016-01-01

    Although CD4+ T cells are recognized to play an important role in the inflammatory response of nasal polyps (NPs), the biological functions of CD8+ T cells in polypogenesis remain unclear. In this study, we analyzed cell markers, cytokine expression and transcription factors in IL-21-expressing CD8+ T cells in polyp tissues of NP patients. The results showed that the majority of IL-21-producing CD8+ T cells were effector memory cells and they co-expressed IFN-γ. IL-21-expressing CD8+ T cells in polyp tissues expressed higher CXCR5, PD-1, and ICOS levels than cells in control tissues and showed significantly higher T-bet and Bcl-6 expression levels compared with IL-21−CD8+ T cells. Purified polyp CD8+ T cells promoted IgG production from isolated polyp B cells in vitro, and recombinant IL-12 modulated the expression of IL-21, IFN-γ and CD40L in purified polyp CD8+ T cells. Moreover, the percentage of IL-21+CD8+ T cells in polyp tissues was positively correlated with endoscopic and CT scan scores in NP patients. These findings indicated that polyp CD8+ T cells, by co-expressing IL-21 and IFN-γ and other markers, display a Tfh cell functionality, which is associated with the clinical severity of NP patients. PMID:27468819

  12. Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection

    PubMed Central

    Nunes-Alves, Cláudio; Booty, Matthew G.; Carpenter, Stephen M.; Rothchild, Alissa C.; Martin, Constance J.; Desjardins, Danielle; Steblenko, Katherine; Kløverpris, Henrik N.; Madansein, Rajhmun; Ramsuran, Duran; Leslie, Alasdair; Correia-Neves, Margarida; Behar, Samuel M.

    2015-01-01

    The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRβ bias. Using a retrogenic model of TB10.44-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity. PMID:25945999

  13. Characterization and functional analyses of a novel chicken CD8a variant X1 (CD8a1)

    USDA-ARS?s Scientific Manuscript database

    We provide the first description of cloning, as well as structural and functional analysis of a novel variant in the chicken CD8alpha family, termed the CD8-alpha X1 (CD8alpha1) gene. Multiple alignment of CD8alpha1 with known CD8alpha and beta sequences of other species revealed relatively low con...

  14. Extensive CD4 and CD8 T-cell cross-reactivity between alphaherpesviruses1

    PubMed Central

    Dong, Lichun; Russell, Ronnie M.; Barlow, Russell S.; Haas, Juergen G.; Ramchandani, Meena S.; Johnston, Christine; Buus, Soren; Redwood, Alec J.; White, Katie D.; Mallal, Simon A.; Phillips, Elizabeth J.; Posavad, Christine M.; Wald, Anna; Koelle, David M.

    2015-01-01

    The alphaherpesvirinae subfamily includes HSV types 1 and 2 and the sequence-divergent pathogen varicella zoster virus (VZV). T cells, controlled by TCR and HLA molecules that tolerate limited epitope amino acid variation, might cross-react between these microbes. We show that memory PBMC expansion with either HSV or VZV enriches for CD4 T cell lines that recognize the other agent at the whole virus, protein, and peptide levels, consistent with bi-directional cross-reactivity. HSV-specific CD4 T cells recovered from HSV seronegative persons can be partially explained by such VZV cross-reactivity. HSV-1-reactive CD8 T cells also cross-react with VZV-infected cells, full-length VZV proteins, and VZV peptides, and kill VZV-infected dermal fibroblasts. Mono- and cross-reactive CD8 T cells use distinct TCRB CDR3 sequences. Cross-reactivity to VZV is reconstituted by cloning and expressing TCRA/TCRB receptors from T-cells that are initially isolated using HSV reagents. Overall, we define 13 novel CD4 and CD8 HSV-VZV cross-reactive epitopes and strongly imply additional cross-reactive peptide sets. Viral proteins can harbor both CD4 and CD8 HSV/VZV cross-reactive epitopes. Quantitative estimates of HSV/VZV cross-reactivity for both CD4 and CD8 T cells vary from 10-50%. Based on these findings, we hypothesize host herpesvirus immune history may influence the pathogenesis and clinical outcome of subsequent infections or vaccinations for related pathogens, and that cross-reactive epitopes and TCRs may be useful for multi-alphaherpesvirus vaccine design and adoptive cellular therapy. PMID:26810224

  15. Comparison of influenza and SIV specific CD8 T cell responses in macaques.

    PubMed

    Jegaskanda, Sinthujan; Reece, Jeanette C; De Rose, Robert; Stambas, John; Sullivan, Lucy; Brooks, Andrew G; Kent, Stephen J; Sexton, Amy

    2012-01-01

    Macaques are a potentially useful non-human primate model to compare memory T-cell immunity to acute virus pathogens such as influenza virus and effector T-cell responses to chronic viral pathogens such as SIV. However, immunological reagents to study influenza CD8(+) T-cell responses in the macaque model are limited. We recently developed an influenza-SIV vaccination model of pigtail macaques (Macaca nemestrina) and used this to study both influenza-specific and SIV-specific CD8(+) T-cells in 39 pigtail macaques expressing the common Mane-A*10(+) (Mane-A01*084) MHC-I allele. To perform comparative studies between influenza and SIV responses a common influenza nucleoprotein-specific CD8(+) T-cell response was mapped to a minimal epitope (termed RA9), MHC-restricted to Mane-A*10 and an MHC tetramer developed to study this response. Influenza-specific memory CD8(+) T-cell response maintained a highly functional profile in terms of multitude of effector molecule expression (CD107a, IFN-γ, TNF-α, MIP-1β and IL-2) and showed high avidity even in the setting of SIV infection. In contrast, within weeks following active SIV infection, SIV-specific CD8(+) effector T-cells expressed fewer cytokines/degranulation markers and had a lower avidity compared to influenza specific CD8(+) T-cells. Further, the influenza specific memory CD8 T-cell response retained stable expression of the exhaustion marker programmed death-marker-1 (PD-1) and co-stimulatory molecule CD28 following infection with SIV. This contrasted with the effector SIV-specific CD8(+) T-cells following SIV infection which expressed significantly higher amounts of PD-1 and lower amounts of CD28. Our results suggest that strategies to maintain a more functional CD8(+) T-cell response, profile may assist in controlling HIV disease.

  16. Display of the Viral Epitopes on Lactococcus lactis: A Model for Food Grade Vaccine against EV71.

    PubMed

    Varma, Nadimpalli Ravi S; Toosa, Haryanti; Foo, Hooi Ling; Alitheen, Noorjahan Banu Mohamed; Nor Shamsudin, Mariana; Arbab, Ali S; Yusoff, Khatijah; Abdul Rahim, Raha

    2013-01-01

    In this study, we have developed a system for display of antigens of Enterovirus type 71 (EV71) on the cell surface of L. lactis. The viral capsid protein (VP1) gene from a local viral isolate was utilized as the candidate vaccine for the development of oral live vaccines against EV71 using L. lactis as a carrier. We expressed fusion proteins in E. coli and purified fusion proteins were incubated with L. lactis. We confirmed that mice orally fed with L. lactis displaying these fusion proteins on its surface were able to mount an immune response against the epitopes of EV71. This is the first example of an EV71 antigen displayed on the surface of a food grade organism and opens a new perspective for alternative vaccine strategies against the EV71. We believe that the method of protein docking utilized in this study will allow for more flexible presentations of short peptides and proteins on the surface of L. lactis to be useful as a delivery vehicle.

  17. Display of the Viral Epitopes on Lactococcus lactis: A Model for Food Grade Vaccine against EV71

    PubMed Central

    Varma, Nadimpalli Ravi S.; Toosa, Haryanti; Foo, Hooi Ling; Alitheen, Noorjahan Banu Mohamed; Nor Shamsudin, Mariana; Arbab, Ali S.; Yusoff, Khatijah; Abdul Rahim, Raha

    2013-01-01

    In this study, we have developed a system for display of antigens of Enterovirus type 71 (EV71) on the cell surface of L. lactis. The viral capsid protein (VP1) gene from a local viral isolate was utilized as the candidate vaccine for the development of oral live vaccines against EV71 using L. lactis as a carrier. We expressed fusion proteins in E. coli and purified fusion proteins were incubated with L. lactis. We confirmed that mice orally fed with L. lactis displaying these fusion proteins on its surface were able to mount an immune response against the epitopes of EV71. This is the first example of an EV71 antigen displayed on the surface of a food grade organism and opens a new perspective for alternative vaccine strategies against the EV71. We believe that the method of protein docking utilized in this study will allow for more flexible presentations of short peptides and proteins on the surface of L. lactis to be useful as a delivery vehicle. PMID:23476790

  18. Channel catfish CD8a and CD8ß co-receptors characterization expression and polymorphism

    USDA-ARS?s Scientific Manuscript database

    In this study we report the identification and characterization of channel catfish, Ictalurus punctatus CD8a and CD8ß genes. Both genes encode predicted proteins containing a leader, a immunoglobulin superfamily V domain, a stalk/hinge region, a transmembrane region and a positively charged cytoplas...

  19. Induction of a Cross-Reactive CD8+ T Cell Response following Foot-and-Mouth Disease Virus Vaccination▿

    PubMed Central

    Guzman, Efrain; Taylor, Geraldine; Charleston, Bryan; Ellis, Shirley A.

    2010-01-01

    Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. Current inactivated FMDV vaccines generate short-term, serotype-specific protection, mainly through neutralizing antibody. An improved understanding of the mechanisms of protective immunity would aid design of more effective vaccines. We have previously reported the presence of virus-specific CD8+ T cells in FMDV-vaccinated and -infected cattle. In the current study, we aimed to identify CD8+ T cell epitopes in FMDV recognized by cattle vaccinated with inactivated FMDV serotype O. Analysis of gamma interferon (IFN-γ)-producing CD8+ T cells responding to stimulation with FMDV-derived peptides revealed one putative CD8+ T cell epitope present within the structural protein P1D, comprising residues 795 to 803 of FMDV serotype O UKG/2001. The restricting major histocompatibility complex (MHC) class I allele was N*02201, expressed by the A31 haplotype. This epitope induced IFN-γ release, proliferation, and target cell killing by αβ CD8+ T cells, but not CD4+ T cells. A protein alignment of representative samples from each of the 7 FMDV serotypes showed that the putative epitope is highly conserved. CD8+ T cells from FMDV serotype O-vaccinated A31+ cattle recognized antigen-presenting cells (APCs) loaded with peptides derived from all 7 FMDV serotypes, suggesting that CD8+ T cells recognizing the defined epitope are cross-reactive to equivalent peptides derived from all of the other FMDV serotypes. PMID:20861264

  20. Characterization of adhesive epitopes with the OmpS display system.

    PubMed

    Lång, H; Mäki, M; Rantakari, A; Korhonen, T K

    2000-01-01

    OmpS is an outer membrane protein of Vibrio cholerae where it forms trimeric pores that function in the uptake of maltose and maltodextrins. Based on sequence similarity to LamB proteins, a model of OmpS folding in the outer membrane has been constructed. According to this model, OmpS contains 18 transmembrane beta-strands and nine surface-accessible loops. Adhesive epitopes can, when inserted into surface-accessible loop 4 (L4) and expressed in Escherichia coli, retain their functional characteristics. We inserted three D-repeats from the Staphylococcus aureus fibronectin-binding protein FnBPA into L4 of OmpS and showed that E. coli cells expressing these hybrids bind fibronectin. DNA fragments covering the N-terminal half of the globoside-binding P-fimbrial adhesin class II PapG of E. coli were cloned into the same surface accessible loop (L4) of OmpS. Fragments of papG encoding 53 or 186 amino acids from the N-terminal end of class II PapG adhesin were found to confer bacterial adhesiveness to globoside. Removal of 23 amino acids from the N-terminus of PapG did not affect receptor binding, but removal of 31 amino acids abolished it. The newly developed night sky image technique was also used to demonstrate the binding properties of membrane vesicles carrying the hybrid proteins. We raised antibodies against the purified hybrid protein containing 53 amino acids from PapG. This antiserum recognized the P-fimbriae on E. coli cells. These data provide evidence that the N-terminal first 53 amino acids of class II PapG contain the receptor-binding domain.

  1. Epitope mapping and key amino acid identification of anti-CD22 immunotoxin CAT-8015 using hybrid β-lactamase display

    PubMed Central

    Bannister, D.; Popovic, B.; Sridharan, S.; Giannotta, F.; Filée, P.; Yilmaz, N.; Minter, R.

    2011-01-01

    Monoclonal antibodies are a commercially successful class of drug molecules and there are now a growing number of antibodies coupled to toxic payloads, which demonstrate clinical efficacy. Determining the precise epitope of therapeutic antibodies is beneficial in understanding the structure–activity relationship of the drug, but in many cases is not done due to the structural complexity of, in particular, conformational protein epitopes. Using the immunotoxin CAT-8015 as a test case, this study demonstrates that a new methodology, hybrid β-lactamase display, can be employed to elucidate a complex epitope on CD22. Following insertion of random CD22 gene fragments into a permissive site within β-lactamase, proteins expressed in Escherichia coli were first screened for correct folding by resistance to ampicillin and then selected by phage display for affinity to CAT-8015. The optimal protein region recognised by CAT-8015 could then be used as a tool for fine epitope mapping, using alanine-scanning analysis, demonstrating that this technology is well suited to the rapid characterisation of antibody epitopes. PMID:21159620

  2. Epitope mapping and key amino acid identification of anti-CD22 immunotoxin CAT-8015 using hybrid β-lactamase display.

    PubMed

    Bannister, D; Popovic, B; Sridharan, S; Giannotta, F; Filée, P; Yilmaz, N; Minter, R

    2011-04-01

    Monoclonal antibodies are a commercially successful class of drug molecules and there are now a growing number of antibodies coupled to toxic payloads, which demonstrate clinical efficacy. Determining the precise epitope of therapeutic antibodies is beneficial in understanding the structure-activity relationship of the drug, but in many cases is not done due to the structural complexity of, in particular, conformational protein epitopes. Using the immunotoxin CAT-8015 as a test case, this study demonstrates that a new methodology, hybrid β-lactamase display, can be employed to elucidate a complex epitope on CD22. Following insertion of random CD22 gene fragments into a permissive site within β-lactamase, proteins expressed in Escherichia coli were first screened for correct folding by resistance to ampicillin and then selected by phage display for affinity to CAT-8015. The optimal protein region recognised by CAT-8015 could then be used as a tool for fine epitope mapping, using alanine-scanning analysis, demonstrating that this technology is well suited to the rapid characterisation of antibody epitopes.

  3. The Adaptor Molecule MyD88 Directly Promotes CD8 T cell Responses to Vaccinia Virus

    PubMed Central

    Zhao, Yuan; Trez, Carl De; Flynn, Rachel; Ware, Carl F.; Croft, Michael; Salek-Ardakani, Shahram

    2009-01-01

    Vaccinia Virus (VACV) elicits a robust CD8 T cell response that plays an important role in host resistance. To date, there is little information on the molecules that are essential to generate large pools of VACV-specific effector CD8 T cells. Here, we show that the adaptor molecule MyD88 is critical for the magnitude of primary CD8 T cell responses to both dominant and subdominant VACV epitopes. MyD88−/− mice exhibit profound reduction in CD8 T cell expansion and anti-viral cytokine production. Surprisingly, the defect was not due to impaired antigen-presenting cell function, as MyD88−/− DC matured normally and were able to promote strong CD8 T cell priming following VACV infection. Rather, adoptive transfer experiments demonstrated that intrinsic MyD88-dependent pathways in CD8 T cells were critical. MyD88-deficient CD8 T cells failed to accumulate in wild-type hosts, and poor expansion of MyD88-deficient VACV-specific CD8 T cells resulted after virus infection. In contrast, no defect was evident in the absence of TRIF, TLR2, TLR4, TLR9, and IL-1R. Together, our results highlight an important role for MyD88 in initial anti-viral CD8 T cell responses and suggest that targeting this pathway may be useful in promoting and sustaining anti-VACV immunity. PMID:19414781

  4. Intracellular staining for TNF and IFN-gamma detects different frequencies of antigen-specific CD8(+) T cells.

    PubMed

    Badovinac, V P; Harty, J T

    2000-04-21

    CD8(+) T lymphocytes are important mediators of adaptive immunity against certain viral, protozoan and bacterial pathogens. Activated CD8(+) T cells are able to induce cytolysis of infected cells (perforin and CD95-CD95L mediated pathways) and also elaborate cytokines, including IFN-gamma and TNF after appropriate MHC class I-peptide recognition. New technologies for the detection of antigen-specific CD8(+) T cells, including tetrameric MHC class I-peptide complexes, intracellular IFN-gamma staining and IFN-gamma ELISPOT analysis have revised our understanding of the magnitude of the CD8(+) T cell response to infection. Here, using intracellular cytokine staining, we compare detection of IFN-gamma and TNF in the analysis of pathogen-specific CD8(+) T cell lines and CD8(+) T cells after primary viral infection (LCMV) or secondary bacterial infection (Listeria monocytogenes). Under multiple conditions and with multiple epitopes, we find that staining for intracellular IFN-gamma consistently detects a higher frequency of antigen-specific CD8(+) T cells than detection of intracellular TNF. However, (a) intracellular staining for TNF can be used to detect antigen-specific CD8(+) T cell responses and (b) intracellular staining for cytokines is a useful approach for in vitro characterization of antigen-specific CD8(+) T cell lines.

  5. Identification of two linear B-cell epitopes from West Nile virus NS1 by screening a phage-displayed random peptide library

    PubMed Central

    2011-01-01

    Background The West Nile virus (WNV) nonstructural protein 1 (NS1) is an important antigenic protein that elicits protective antibody responses in animals and can be used for the serological diagnosis of WNV infection. Although previous work has demonstrated the vital role of WNV NS1-specific antibody responses, the specific epitopes in the NS1 have not been identified. Results The present study describes the identification of two linear B-cell epitopes in WNV NS1 through screening a phage-displayed random 12-mer peptide library with two monoclonal antibodies (mAbs) 3C7 and 4D1 that directed against the NS1. The mAbs 3C7 and 4D1 recognized phages displaying peptides with the consensus motifs LTATTEK and VVDGPETKEC, respectively. Exact sequences of both motifs were found in the NS1 (895LTATTEK901 and 925VVDGPETKEC934). Further identification of the displayed B cell epitopes were conducted using a set of truncated peptides expressed as MBP fusion proteins. The data indicated that 896TATTEK901 and925VVDGPETKEC934 are minimal determinants of the linear B cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. Antibodies present in the serum of WNV-positive horses recognized the minimal linear epitopes in Western blot analysis, indicating that the two peptides are antigenic in horses during infection. Furthermore, we found that the epitope recognized by 3C7 is conserved only among WNV strains, whereas the epitope recognized by 4D1 is a common motif shared among WNV and other members of Japanese encephalitis virus (JEV) serocomplex. Conclusions We identified TATTEK and VVDGPETKEC as NS1-specific linear B-cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against WNV and other viruses of JEV serocomplex. PMID:21729328

  6. Measuring the diaspora for virus-specific CD8+ T cells.

    PubMed

    Marshall, D R; Turner, S J; Belz, G T; Wingo, S; Andreansky, S; Sangster, M Y; Riberdy, J M; Liu, T; Tan, M; Doherty, P C

    2001-05-22

    The CD8(+) T cell diaspora has been analyzed after secondary challenge with an influenza A virus that replicates only in the respiratory tract. Numbers of D(b)NP(366)- and D(b)PA(224)-specific CD8(+) T cells were measured by tetramer staining at the end of the recall response, then followed sequentially in the lung, lymph nodes, spleen, blood, and other organs. The extent of clonal expansion did not reflect the sizes of the preexisting memory T cell pools. Although the high-frequency CD8(+) tetramer(+) populations in the pneumonic lung and mediastinal lymph nodes fell rapidly from peak values, the "whole mouse" virus-specific CD8(+) T cell counts decreased only 2-fold over the 4 weeks after infection, then subsided at a fairly steady rate to reach a plateau at about 2 months. The largest numbers were found throughout in the spleen, then the bone marrow. The CD8(+)D(b)NP(366)+ and CD8(+)D(b)PA(224)+ sets remained significantly enlarged for at least 4 months, declining at equivalent rates while retaining the nucleoprotein > acid polymerase immunodominance hierarchy characteristic of the earlier antigen-driven phase. Lowest levels of the CD69 "activation marker" were detected consistently on virus-specific CD8(+) T cells in the blood, then the spleen. Those in the bone marrow and liver were intermediate, and CD69(hi) T cells were very prominent in the regional lymph nodes and the nasal-associated lymphoid tissue. Any population of "resting" CD8(+) memory T cells is thus phenotypically heterogeneous, widely dispersed, and subject to broad homeostatic and local environmental effects irrespective of epitope specificity or magnitude.

  7. CD8 T-cell recognition of acquired alloantigen promotes acute allograft rejection

    PubMed Central

    Harper, Simon J. F.; Ali, Jason M.; Wlodek, Elizabeth; Negus, Marg C.; Harper, Ines G.; Chhabra, Manu; Qureshi, M. Saeed; Mallik, Mekhola; Bolton, Eleanor; Bradley, J. Andrew; Pettigrew, Gavin J.

    2015-01-01

    Adaptive CD8 T-cell immunity is the principal arm of the cellular alloimmune response, but its development requires help. This can be provided by CD4 T cells that recognize alloantigen “indirectly,” as self-restricted allopeptide, but this process remains unexplained, because the target epitopes for CD4 and CD8 T-cell recognition are “unlinked” on different cells (recipient and donor antigen presenting cells (APCs), respectively). Here, we test the hypothesis that the presentation of intact and processed MHC class I alloantigen by recipient dendritic cells (DCs) (the “semidirect” pathway) allows linked help to be delivered by indirect-pathway CD4 T cells for generating destructive cytotoxic CD8 T-cell alloresponses. We show that CD8 T-cell–mediated rejection of murine heart allografts that lack hematopoietic APCs requires host secondary lymphoid tissue (SLT). SLT is necessary because within it, recipient dendritic cells can acquire MHC from graft parenchymal cells and simultaneously present it as intact protein to alloreactive CD8 T cells and as processed peptide alloantigen for recognition by indirect-pathway CD4 T cells. This enables delivery of essential help for generating cytotoxic CD8 T-cell responses that cause rapid allograft rejection. In demonstrating the functional relevance of the semidirect pathway to transplant rejection, our findings provide a solution to a long-standing conundrum as to why SLT is required for CD8 T-cell allorecognition of graft parenchymal cells and suggest a mechanism by which indirect-pathway CD4 T cells provide help for generating effector cytotoxic CD8 T-cell alloresponses at late time points after transplantation. PMID:26420874

  8. Identification of an Immunogenic Mimic of a Conserved Epitope on the Plasmodium falciparum Blood Stage Antigen AMA1 Using Virus-Like Particle (VLP) Peptide Display

    PubMed Central

    Crossey, Erin; Frietze, Kathryn; Narum, David L.; Peabody, David S.; Chackerian, Bryce

    2015-01-01

    We have developed a peptide display platform based on VLPs of the RNA bacteriophage MS2 that combines the high immunogenicity of VLP display with affinity selection capabilities. Random peptides can be displayed on the VLP surface by genetically inserting sequences into a surface-exposed loop of the viral coat protein. VLP-displayed peptides can then be isolated by selection using antibodies, and the VLP selectants can then be used directly as immunogens. Here, we investigated the ability of this platform to identify mimotopes of a highly conserved conformational epitope present on the Plasmodium falciparum blood-stage protein AMA1. Using 4G2, a monoclonal antibody that binds to this epitope and is a potent inhibitor of erythrocyte invasion, we screened three different VLP-peptide libraries and identified specific VLPs that bound strongly to the selecting mAb. We then tested the ability of a handful of selected VLPs to elicit anti-AMA1 antibody responses in mice. Most of the selected VLPs failed to reliably elicit AMA1 specific antibodies. However, one VLP consistently induced antibodies that cross-reacted with AMA1. Surprisingly, this VLP bound to 4G2 more weakly than the other selectants we identified. Taken together, these data demonstrate that VLP-peptide display can identify immunogenic mimics of a complex conformational epitope and illustrate the promise and challenges of this approach. PMID:26147502

  9. Complementary Dendritic Cell–activating Function of CD8+ and CD4+ T Cells

    PubMed Central

    Mailliard, Robbie B.; Egawa, Shinichi; Cai, Quan; Kalinska, Anna; Bykovskaya, Svetlana N.; Lotze, Michael T.; Kapsenberg, Martien L.; Storkus, Walter J.; Kalinski, Pawel

    2002-01-01

    Dendritic cells (DCs) activated by CD40L-expressing CD4+ T cells act as mediators of “T helper (Th)” signals for CD8+ T lymphocytes, inducing their cytotoxic function and supporting their long-term activity. Here, we show that the optimal activation of DCs, their ability to produce high levels of bioactive interleukin (IL)-12p70 and to induce Th1-type CD4+ T cells, is supported by the complementary DC-activating signals from both CD4+ and CD8+ T cells. Cord blood– or peripheral blood–isolated naive CD8+ T cells do not express CD40L, but, in contrast to naive CD4+ T cells, they are efficient producers of IFN-γ at the earliest stages of the interaction with DCs. Naive CD8+ T cells cooperate with CD40L-expressing naive CD4+ T cells in the induction of IL-12p70 in DCs, promoting the development of primary Th1-type CD4+ T cell responses. Moreover, the recognition of major histocompatibility complex class I–presented epitopes by antigen-specific CD8+ T cells results in the TNF-α– and IFN-γ–dependent increase in the activation level of DCs and in the induction of type-1 polarized mature DCs capable of producing high levels of IL-12p70 upon a subsequent CD40 ligation. The ability of class I–restricted CD8+ T cells to coactivate and polarize DCs may support the induction of Th1-type responses against class I–presented epitopes of intracellular pathogens and contact allergens, and may have therapeutical implications in cancer and chronic infections. PMID:11854360

  10. Modulating DNA methylation in activated CD8+ T cells inhibits regulatory T cell-induced binding of Foxp3 to the CD8+ T Cell IL-2 promoter.

    PubMed

    Miller, Michelle M; Akaronu, Nnenna; Thompson, Elizabeth M; Hood, Sylvia F; Fogle, Jonathan E

    2015-02-01

    We have previously demonstrated that CD4(+)CD25(+) regulatory T cells (Tregs) activated during the course of feline immunodeficiency virus (FIV) infection suppress CD8(+) CTL function in a TGF-β-dependent fashion, inhibiting IFN-γ and IL-2 production and inducing G1 cell-cycle arrest. In this article, we describe the molecular events occurring at the IL-2 promoter leading to suppression of IL-2 production. These experiments demonstrate that Foxp3 induced by lentivirus-activated Tregs in the CD8(+) target cells binds to the IL-2 promoter, actively repressing IL-2 transcription. We further demonstrate that the chronic activation of CD8(+) T cells during FIV infection results in chromatin remodeling at the IL-2 promoter, specifically, demethylation of CpG residues. These DNA modifications occur during active transcription and translation of IL-2; however, these changes render the IL-2 promoter permissive to Foxp3-induced transcriptional repression. These data help explain, in part, the seemingly paradoxical observations that CD8(+) T cells displaying an activation phenotype exhibit altered antiviral function. Further, we demonstrate that blocking demethylation of CpG residues at the IL-2 promoter inhibits Foxp3 binding, suggesting a potential mechanism for rescue and/or reactivation of CD8(+) T cells. Using the FIV model for lentiviral persistence, these studies provide a framework for understanding how immune activation combined with Treg-mediated suppression may affect CD8(+) T cell IL-2 transcription, maturation, and antiviral function.

  11. Cloning, protein expression and display of synthetic multi-epitope mycobacterial antigens on Salmonella typhi Ty21a cell surface.

    PubMed

    Sarhan, Mohammed A A; Musa, Mustaffa; Zainuddin, Zainul F

    2011-09-01

    Expressing proteins of interest as fusion to proteins of bacterial envelope is a powerful technique for biotechnological and medical applications. The synthetic gene (VacII) encoding for T-cell epitopes of selected genes of Mycobacterium tuberculosis namely, ESAT6, MTP40, 38 kDa, and MPT64 was fused with N- terminus of Pseudomonas syringae ice nucleation protein (INP) outer membrane protein. The fused genes were cloned into a bacterial expression vector pKK223-3. The recombinant protein was purified by Ni-NAT column. VacII gene was displayed on the cell surface of Salmonella typhi Ty21a using N-terminal region of ice nucleation proteins (INP) as an anchoring motif. Glycine method confirmed that VacII was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INP- VacII fusion protein of the expected size (52 kDa).

  12. Antigen-specific therapy of EAE via intranasal delivery of filamentous phage displaying a myelin immunodominant epitope.

    PubMed

    Rakover, Idan S; Zabavnik, Natalia; Kopel, Rela; Paz-Rozner, Miri; Solomon, Beka

    2010-08-25

    The presence of anti-myelin antibodies (Abs) in patients with early multiple sclerosis (MS) and in MS animal models has led to renewed interest in the role of B cells, plasma cells and their products in the pathogenesis of the disease, and in their therapeutic potential. Here, we present a novel strategy based on filamentous phage display of the myelin oligodendrocyte glycoprotein immunodominant epitope (MOG 36-44) fused to the main coat protein. Filamentous phages are well characterized, both structurally and genetically. We found that the fibrous shape of the phage (1000 nm long and 6 nm wide) enables penetration into the central nervous system (CNS) when administered nasally. Thus, intranasal treatment of experimental autoimmune encephalomyelitis (EAE) in mice, with phage MOG, showed improved neuronal function, reduced levels of proinflammatory cytokines, particularly monocyte chemoattractant protein 1 (MCP-1), interferon gamma (IFN-gamma) and IL-6, but no change in IL-10 or IL-12 levels. Moreover, the treatment induced depletion of the autoantibodies against MOG and prevented demyelination resulting in improved clinical scores and the reduced inflammation in the CNS and periphery in EAE mice compared to untreated sick animals. Copyright 2010 Elsevier B.V. All rights reserved.

  13. Dengue virus protein recognition by virus-specific murine CD8+ cytotoxic T lymphocytes.

    PubMed Central

    Rothman, A L; Kurane, I; Lai, C J; Bray, M; Falgout, B; Men, R; Ennis, F A

    1993-01-01

    The identification of the protein targets for dengue virus-specific T lymphocytes may be useful for planning the development of subunit vaccines against dengue. We studied the recognition by murine dengue virus-specific major histocompatibility complex class I-restricted, CD8+ cytotoxic T lymphocytes (CTL) of dengue virus proteins using recombinant vaccinia viruses containing segments of the dengue virus genome. CTL from H-2k mice recognized a single serotype-cross-reactive epitope on the nonstructural (NS) protein NS3. CTL from H-2b mice recognized a serotype-cross-reactive epitope that was localized to NS4a or NS4b. CTL from H-2d mice recognized at least three epitopes: a serotype-specific epitope on one of the structural proteins, a serotype-cross-reactive epitope on NS3, and a serotype-cross-reactive epitope on NS1 or NS2a. Our findings demonstrate the limited recognition of dengue virus proteins by CTL from three inbred mouse strains and the predominance of CTL epitopes on dengue virus nonstructural proteins, particularly NS3. Since human dengue virus-specific CTL show similar patterns of recognition, these findings suggest that nonstructural proteins should be considered in designing vaccines against dengue. PMID:7678307

  14. Non-hematopoietic cells in lymph nodes drive memory CD8 T cell inflation during murine cytomegalovirus infection.

    PubMed

    Torti, Nicole; Walton, Senta M; Brocker, Thomas; Rülicke, Thomas; Oxenius, Annette

    2011-10-01

    During human and murine cytomegalovirus (MCMV) infection an exceptionally large virus-specific CD8 T cell pool is maintained in the periphery lifelong. This anomalous response is only seen for specific subsets of MCMV-specific CD8 T cells which are referred to as 'inflationary T cells'. How memory CD8 T cell inflation is induced and maintained is unclear, though their activated phenotype strongly suggests an involvement of persistent antigen encounter during MCMV latency. To dissect the cellular and molecular requirements for memory CD8 T cell inflation, we have generated a transgenic mouse expressing an MHC class I-restricted T cell receptor specific for an immunodominant inflationary epitope of MCMV. Through a series of adoptive transfer experiments we found that memory inflation was completely dependent on antigen presentation by non-hematopoietic cells, which are also the predominant site of MCMV latency. In particular, non-hematopoietic cells selectively induced robust proliferation of inflationary CD8 T cells in lymph nodes, where a majority of the inflationary CD8 T cells exhibit a central-memory phenotype, but not in peripheral tissues, where terminally differentiated inflationary T cells accumulate. These results indicate that continuous restimulation of central memory CD8 T cells in the lymph nodes by infected non-hematopoietic cells ensures the maintenance of a functional effector CD8 T pool in the periphery, providing protection against viral reactivation events.

  15. CD8+ T cells specific for the islet autoantigen IGRP are restricted in their T cell receptor chain usage

    PubMed Central

    Fuchs, Yannick F.; Eugster, Anne; Dietz, Sevina; Sebelefsky, Christian; Kühn, Denise; Wilhelm, Carmen; Lindner, Annett; Gavrisan, Anita; Knoop, Jan; Dahl, Andreas; Ziegler, Anette-G.; Bonifacio, Ezio

    2017-01-01

    CD8+ T cells directed against beta cell autoantigens are considered relevant for the pathogenesis of type 1 diabetes. Using single cell T cell receptor sequencing of CD8+ T cells specific for the IGRP265-273 epitope, we examined whether there was expansion of clonotypes and sharing of T cell receptor chains in autoreactive CD8+ T cell repertoires. HLA-A*0201 positive type 1 diabetes patients (n = 19) and controls (n = 18) were analysed. TCR α- and β-chain sequences of 418 patient-derived IGRP265-273-multimer+ CD8+ T cells representing 48 clonotypes were obtained. Expanded populations of IGRP265-273-specific CD8+ T cells with dominant clonotypes that had TCR α-chains shared across patients were observed. The SGGSNYKLTF motif corresponding to TRAJ53 was contained in 384 (91.9%) cells, and in 20 (41.7%) patient-derived clonotypes. TRAJ53 together with TRAV29/DV5 was found in 15 (31.3%) clonotypes. Using next generation TCR α-chain sequencing, we found enrichment of one of these TCR α-chains in the memory CD8+ T cells of patients as compared to healthy controls. CD8+ T cell clones bearing the enriched motifs mediated antigen-specific target cell lysis. We provide the first evidence for restriction of T cell receptor motifs in the alpha chain of human CD8+ T cells with specificity to a beta cell antigen. PMID:28300170

  16. Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

    PubMed Central

    Han, Thomas; Sui, Jianhua; Bennett, Andrew S.; Liddington, Robert C.; Donis, Ruben O.; Zhu, Quan; Marasco, Wayne A.

    2011-01-01

    Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines. PMID:21569761

  17. Prophylactic vaccination with phage-displayed epitope of C. albicans elicits protective immune responses against systemic candidiasis in C57BL/6 mice.

    PubMed

    Yang, Qiong; Wang, Li; Lu, Da-ning; Gao, Rui-juan; Song, Jin-na; Hua, Pan-yu; Yuan, Da-wei

    2005-07-01

    Epitope LKVIRK on 47 kDa of heat shock protein (Hsp) 90 of Candida albicans, corresponding to residues 386-391 of the Hsp90, is recognized by patients recovering from invasive candidiasis. The efficacy of hybrid phage displaying epitope LKVIRK in the N-terminal region of the major coat protein (pVIII) in inducing anti-invasive candidiasis immune response was studied in C57BL/6 mice. Indirect phage-ELISA results demonstrated that the mice immunized with hybrid phage had significantly higher titers of epitope LKVIRK-specific serum IgG as compared to those immunized with heat-killed C. albicans (HK-CA). C57BL/6 mice immunized either with hybrid phage or with wild-type phage also developed significant levels of delayed-type hypersensitivity (DTH) response and splenocyte proliferation, as well as with HK-CA. In addition, high levels of IFN-gamma in the CD4(+) splenocytes from phage-immunized mice were detected as well during 1 week post-inoculation. Furthermore, mice immunized with hybrid phage acquired a resistance to systemic C. albicans infection as confirmed by fewer C. albicans cells in the kidneys, and had a longer lifespan compared to control groups following intravenous challenge with C. albicans. These results indicate that hybrid phage displaying epitope LKVIRK may serve as a potential vaccine conferring a resistance to systemic candidiasis.

  18. Variable HIV peptide stability in human cytosol is critical to epitope presentation and immune escape

    PubMed Central

    Lazaro, Estibaliz; Kadie, Carl; Stamegna, Pamela; Zhang, Shao Chong; Gourdain, Pauline; Lai, Nicole Y.; Zhang, Mei; Martinez, Sergio A.; Heckerman, David; Le Gall, Sylvie

    2011-01-01

    Induction of virus-specific CD8+ T cell responses is critical for the success of vaccines against chronic viral infections. Despite the large number of potential MHC-I–restricted epitopes located in viral proteins, MHC-I–restricted epitope generation is inefficient, and factors defining the production and presentation of MHC-I–restricted viral epitopes are poorly understood. Here, we have demonstrated that the half-lives of HIV-derived peptides in cytosol from primary human cells were highly variable and sequence dependent, and significantly affected the efficiency of cell recognition by CD8+ T cells. Furthermore, multiple clinical isolates of HLA-associated HIV epitope variants displayed reduced half-lives relative to consensus sequence. This decreased cytosolic peptide stability diminished epitope presentation and CTL recognition, illustrating a mechanism of immune escape. Chaperone complexes including Hsp90 and histone deacetylase HDAC6 enhanced peptide stability by transient protection from peptidase degradation. Based on empirical results with 166 peptides, we developed a computational approach utilizing a sequence-based algorithm to estimate the cytosolic stability of antigenic peptides. Our results identify sequence motifs able to alter the amount of peptide available for loading onto MHC-I, suggesting potential new strategies to modulate epitope production from vaccine immunogens. PMID:21555856

  19. Understanding the Failure of CD8+ T-Cell Vaccination against Simian/Human Immunodeficiency Virus▿

    PubMed Central

    De Boer, Rob J.

    2007-01-01

    Although CD8+ T cells play an important role in controlling viral infections, boosting specific CD8+ T cells by prophylactic vaccination with simian immunodeficiency virus (SIV) epitopes fails to provide sterilizing immunity. Viral replication rates and viral contraction rates after the peak viremia hardly depend on the presence of memory CD8+ T cells. To study these paradoxical findings, we parameterize novel mathematical models for acute SIV and human immunodeficiency virus infection. These models explain that failure of vaccination is due to the fact that effector/target ratios are too low during the viral expansion phase. Because CD8+ T cells require cell-to-cell contacts, immune protection requires high effector/target ratios at the primary site of infection. Effector/target ratios become favorable for immune control at the time of the peak in the viral load when the virus becomes limited by other factors, such as the availability of uninfected target cells. At the viral set point, effector/target ratios are much higher, and perturbations of the number of CD8+ effector cells have a large impact on the viral load. Such protective effector/target ratios are difficult to achieve with nucleic acid- or protein-based vaccines. PMID:17202215

  20. Altered Function in CD8+ T Cells following Paramyxovirus Infection of the Respiratory Tract

    PubMed Central

    Gray, Peter M.; Arimilli, Subhashini; Palmer, Ellen M.; Parks, Griffith D.; Alexander-Miller, Martha A.

    2005-01-01

    For many respiratory pathogens, CD8+ T cells have been shown to play a critical role in clearance. However, there are still many unanswered questions with regard to the factors that promote the most efficacious immune response and the potential for immunoregulation of effector cells at the local site of infection. We have used infection of the respiratory tract with the model paramyxovirus simian virus 5 (SV5) to study CD8+ T-cell responses in the lung. For the present study, we report that over time a population of nonresponsive, virus-specific CD8+ T cells emerged in the lung, culminating in a lack of function in ∼85% of cells specific for the immunodominant epitope from the viral matrix (M) protein by day 40 postinfection. Concurrent with the induction of nonresponsiveness, virus-specific cells that retained function at later times postinfection exhibited an increased requirement for CD8 engagement. This change was coupled with a nearly complete loss of functional phosphoprotein-specific cells, a response previously shown to be almost exclusively CD8 independent. These studies add to the growing evidence for immune dysregulation following viral infection of the respiratory tract. PMID:15731228

  1. Anti-CD8 conjugated nanoparticles to target mammalian cells expressing CD8.

    PubMed

    Bicho, A; Peça, Inês N; Roque, A C A; Cardoso, M Margarida

    2010-10-31

    This work aimed at the development of targeted drug delivery systems using nanoparticles fused with antibodies. The antibody anti-human CD8 was coupled onto PLGA nanoparticles, and the ability of these particles to specifically target cells expressing CD8 was studied. The obtained particles were found to be of spherical shape exhibiting a size between 350 and 600 nm. In vitro experiments with different cellular cultures (TE671, CHO and HEK293) using unmodified nanoparticles containing rhodamine have shown that particles were present on their surface within 48 h of incubation. In vitro tests using anti-CD8 conjugated nanoparticles in CHO cell cultures indicated that all transfected cells which express CD8 show these particles on their surface within 1h of incubation. These results demonstrated that, in a shorter time, the produced particles can target cells expressing CD8 on their surface which offers the ability to reduce drug side effects. The antibody-coupled nanoparticles represent a promising approach to improve the efficacy of active targeting for lymphoblastic leukaemia therapy. Copyright © 2010 Elsevier B.V. All rights reserved.

  2. Discriminating protective from non-protective Plasmodium-specific CD8+ T cell responses

    PubMed Central

    Doll, Katherine L.; Pewe, Lecia L.; Kurup, Samarchith P.; Harty, John T.

    2016-01-01

    Despite decades of research, malaria remains a global health crisis. Current subunit vaccine approaches do not provide efficient long-term, sterilizing immunity against Plasmodium infections in humans. Conversely, whole parasite vaccinations with their larger array of target antigens have conferred long lasting sterilizing protection to humans. Similar studies in rodent models of malaria reveal that CD8+ T cells play a critical role in liver-stage immunity after whole parasite vaccination. However, it is unknown whether all CD8+ T cell specificities elicited by whole parasite vaccination contribute to protection, an issue of great relevance for enhanced subunit vaccination. Here we show that robust CD8+ T cell responses of similar phenotype are mounted following prime-boost immunization against Plasmodium berghei GAP5041-48, S20318-325, TRAP130-138 or CSP252-260 protein-derived epitopes in mice, but only CSP252-260- and TRAP130-138-specific CD8+ T cells provide sterilizing immunity and reduce liver parasite burden following sporozoite challenge. Further, CD8+ T cells specific to sporozoite surface-expressed CSP and TRAP proteins, but not the intracellular GAP50 and S20 proteins, are efficiently recognized by sporozoite-infected hepatocytes in vitro. These results suggest that 1) protection-relevant antigenic targets, regardless of their immunogenic potential, must be efficiently presented by infected hepatocytes for CD8+ T cells to eliminate liver-stage Plasmodium infection and 2) proteins expressed on the surface of sporozoites may be good target antigens for protective CD8+ T cells. PMID:27084099

  3. High-affinity interactions between peptides and heat shock protein 70 augment CD8+ T lymphocyte immune responses.

    PubMed

    Flechtner, Jessica B; Cohane, Kenya Prince; Mehta, Sunil; Slusarewicz, Paul; Leonard, Alexis Kays; Barber, Brian H; Levey, Daniel L; Andjelic, Sofija

    2006-07-15

    Exogenously delivered antigenic peptides complexed to heat shock proteins (HSPs) are able to enter the endogenous Ag-processing pathway and prime CD8+ CTL. It was determined previously that a hybrid peptide containing a MHC class I-binding epitope and HSP70-binding sequence Javelin (J0) in complex with HSP70 could induce cytotoxic T cell responses in vivo that were more robust than those induced by the minimal epitope complexed with HSP70. The present study introduces a novel, higher-affinity HSP70-binding sequence (J1) that significantly enhances binding of various antigenic peptides to HSP70. A competition binding assay revealed a dissociation constant that was 15-fold lower for the H2-K(b) OVA epitope SIINFEKL-J1 compared with SIINFEKL-J0, indicating a substantially higher affinity for HSP70. Further, modifying the orientation of the hybrid epitope and introducing a cleavable linker sequence between the Javelin and the epitope results in even greater immunogenicity, presumably by greater efficiency of epitope processing. The enhanced immunogenicity associated with Javelin J1 and the cleavable linker is consistently observed with multiple mouse and human epitopes. Thus, by creating a series of epitopes with uniform, high-affinity binding to HSP70, successful multiple epitope immunizations are possible, with equal delivery of each antigenic epitope to the immune system via HSP70. These modified epitopes have the potential for creating successful multivalent vaccines for immunotherapy of both infectious disease and cancer.

  4. DOCK8 deficiency impairs CD8 T cell survival and function in humans and mice

    PubMed Central

    Randall, Katrina L.; Chan, Stephanie S.-Y.; Ma, Cindy S.; Fung, Ivan; Mei, Yan; Yabas, Mehmet; Tan, Andy; Arkwright, Peter D.; Al Suwairi, Wafaa; Lugo Reyes, Saul Oswaldo; Yamazaki-Nakashimada, Marco A.; de la Luz Garcia-Cruz, Maria; Smart, Joanne M.; Picard, Capucine; Okada, Satoshi; Jouanguy, Emmanuelle; Casanova, Jean-Laurent; Lambe, Teresa; Cornall, Richard J.; Russell, Sarah; Oliaro, Jane; Tangye, Stuart G.; Bertram, Edward M.

    2011-01-01

    In humans, DOCK8 immunodeficiency syndrome is characterized by severe cutaneous viral infections. Thus, CD8 T cell function may be compromised in the absence of DOCK8. In this study, by analyzing mutant mice and humans, we demonstrate a critical, intrinsic role for DOCK8 in peripheral CD8 T cell survival and function. DOCK8 mutation selectively diminished the abundance of circulating naive CD8 T cells in both species, and in DOCK8-deficient humans, most CD8 T cells displayed an exhausted CD45RA+CCR7− phenotype. Analyses in mice revealed the CD8 T cell abnormalities to be cell autonomous and primarily postthymic. DOCK8 mutant naive CD8 T cells had a shorter lifespan and, upon encounter with antigen on dendritic cells, exhibited poor LFA-1 synaptic polarization and a delay in the first cell division. Although DOCK8 mutant T cells underwent near-normal primary clonal expansion after primary infection with recombinant influenza virus in vivo, they showed greatly reduced memory cell persistence and recall. These findings highlight a key role for DOCK8 in the survival and function of human and mouse CD8 T cells. PMID:22006977

  5. Akt signaling is critical for memory CD8(+) T-cell development and tumor immune surveillance.

    PubMed

    Rogel, Anne; Willoughby, Jane E; Buchan, Sarah L; Leonard, Henry J; Thirdborough, Stephen M; Al-Shamkhani, Aymen

    2017-02-14

    Memory CD8(+) T cells confer long-term immunity against tumors, and anticancer vaccines therefore should maximize their generation. Multiple memory CD8(+) T-cell subsets with distinct functional and homing characteristics exist, but the signaling pathways that regulate their development are ill defined. Here we examined the role of the serine/threonine kinase Akt in the generation of protective immunity by CD8(+) T cells. Akt is known to be activated by the T-cell antigen receptor and the cytokine IL-2, but its role in T-cell immunity in vivo has not been explored. Using CD8(+) T cells from pdk1(K465E/K465E) knockin mice, we found that decreased Akt activity inhibited the survival of T cells during the effector-to-memory cell transition and abolished their differentiation into C-X-C chemokine receptor 3 (CXCR3)(lo)CD43(lo) effector-like memory cells. Consequently, antitumor immunity by CD8(+) T cells that display defective Akt signaling was substantially diminished during the memory phase. Reduced memory T-cell survival and altered memory cell differentiation were associated with up-regulation of the proapoptotic protein Bim and the T-box transcription factor eomesodermin, respectively. These findings suggest an important role for effector-like memory CD8(+) T cells in tumor immune surveillance and identify Akt as a key signaling node in the development of protective memory CD8(+) T-cell responses.

  6. Divide, Conquer, and Sense: CD8(+)CD28(-) T Cells in Perspective.

    PubMed

    Arosa, Fernando A; Esgalhado, André J; Padrão, Carolina A; Cardoso, Elsa M

    2016-01-01

    Understanding the rationale for the generation of a pool of highly differentiated effector memory CD8(+) T cells displaying a weakened capacity to scrutinize for peptides complexed with major histocompatibility class I molecules via their T cell receptor, lacking the "signal 2" CD28 receptor, and yet expressing a highly diverse array of innate receptors, from natural killer receptors, interleukin receptors, and damage-associated molecular pattern receptors, among others, is one of the most challenging issues in contemporary human immunology. The prevalence of these differentiated CD8(+) T cells, also known as CD8(+)CD28(-), CD8(+)KIR(+), NK-like CD8(+) T cells, or innate CD8(+) T cells, in non-lymphoid organs and tissues, in peripheral blood of healthy elderly, namely centenarians, but also in stressful and chronic inflammatory conditions suggests that they are not merely end-of-the-line dysfunctional cells. These experienced CD8(+) T cells are highly diverse and capable of sensing a variety of TCR-independent signals, which enables them to respond and fine-tune tissue homeostasis.

  7. Divide, Conquer, and Sense: CD8+CD28− T Cells in Perspective

    PubMed Central

    Arosa, Fernando A.; Esgalhado, André J.; Padrão, Carolina A.; Cardoso, Elsa M.

    2017-01-01

    Understanding the rationale for the generation of a pool of highly differentiated effector memory CD8+ T cells displaying a weakened capacity to scrutinize for peptides complexed with major histocompatibility class I molecules via their T cell receptor, lacking the “signal 2” CD28 receptor, and yet expressing a highly diverse array of innate receptors, from natural killer receptors, interleukin receptors, and damage-associated molecular pattern receptors, among others, is one of the most challenging issues in contemporary human immunology. The prevalence of these differentiated CD8+ T cells, also known as CD8+CD28−, CD8+KIR+, NK-like CD8+ T cells, or innate CD8+ T cells, in non-lymphoid organs and tissues, in peripheral blood of healthy elderly, namely centenarians, but also in stressful and chronic inflammatory conditions suggests that they are not merely end-of-the-line dysfunctional cells. These experienced CD8+ T cells are highly diverse and capable of sensing a variety of TCR-independent signals, which enables them to respond and fine-tune tissue homeostasis. PMID:28096804

  8. Akt signaling is critical for memory CD8+ T-cell development and tumor immune surveillance

    PubMed Central

    Rogel, Anne; Willoughby, Jane E.; Buchan, Sarah L.; Leonard, Henry J.; Thirdborough, Stephen M.; Al-Shamkhani, Aymen

    2017-01-01

    Memory CD8+ T cells confer long-term immunity against tumors, and anticancer vaccines therefore should maximize their generation. Multiple memory CD8+ T-cell subsets with distinct functional and homing characteristics exist, but the signaling pathways that regulate their development are ill defined. Here we examined the role of the serine/threonine kinase Akt in the generation of protective immunity by CD8+ T cells. Akt is known to be activated by the T-cell antigen receptor and the cytokine IL-2, but its role in T-cell immunity in vivo has not been explored. Using CD8+ T cells from pdk1K465E/K465E knockin mice, we found that decreased Akt activity inhibited the survival of T cells during the effector-to-memory cell transition and abolished their differentiation into C-X-C chemokine receptor 3 (CXCR3)loCD43lo effector-like memory cells. Consequently, antitumor immunity by CD8+ T cells that display defective Akt signaling was substantially diminished during the memory phase. Reduced memory T-cell survival and altered memory cell differentiation were associated with up-regulation of the proapoptotic protein Bim and the T-box transcription factor eomesodermin, respectively. These findings suggest an important role for effector-like memory CD8+ T cells in tumor immune surveillance and identify Akt as a key signaling node in the development of protective memory CD8+ T-cell responses. PMID:28137869

  9. A Numerically Subdominant CD8 T Cell Response to Matrix Protein of Respiratory Syncytial Virus Controls Infection with Limited Immunopathology

    PubMed Central

    Liu, Jie; Haddad, Elias K.; Marceau, Joshua; Morabito, Kaitlyn M.; Rao, Srinivas S.; Filali-Mouhim, Ali; Sekaly, Rafick-Pierre; Graham, Barney S.

    2016-01-01

    CD8 T cells are involved in pathogen clearance and infection-induced pathology in respiratory syncytial virus (RSV) infection. Studying bulk responses masks the contribution of individual CD8 T cell subsets to protective immunity and immunopathology. In particular, the roles of subdominant responses that are potentially beneficial to the host are rarely appreciated when the focus is on magnitude instead of quality of response. Here, by evaluating CD8 T cell responses in CB6F1 hybrid mice, in which multiple epitopes are recognized, we found that a numerically subdominant CD8 T cell response against DbM187 epitope of the virus matrix protein expressed high avidity TCR and enhanced signaling pathways associated with CD8 T cell effector functions. Each DbM187 T effector cell lysed more infected targets on a per cell basis than the numerically dominant KdM282 T cells, and controlled virus replication more efficiently with less pulmonary inflammation and illness than the previously well-characterized KdM282 T cell response. Our data suggest that the clinical outcome of viral infections is determined by the integrated functional properties of a variety of responding CD8 T cells, and that the highest magnitude response may not necessarily be the best in terms of benefit to the host. Understanding how to induce highly efficient and functional T cells would inform strategies for designing vaccines intended to provide T cell-mediated immunity. PMID:26943673

  10. Effective Treatment of Established GL261 Murine Gliomas through Picornavirus Vaccination-Enhanced Tumor Antigen-Specific CD8+ T Cell Responses.

    PubMed

    Renner, Danielle N; Jin, Fang; Litterman, Adam J; Balgeman, Alexis J; Hanson, Lisa M; Gamez, Jeffrey D; Chae, Michael; Carlson, Brett L; Sarkaria, Jann N; Parney, Ian F; Ohlfest, John R; Pirko, Istvan; Pavelko, Kevin D; Johnson, Aaron J

    2015-01-01

    Glioblastoma (GBM) is among the most invasive and lethal of cancers, frequently infiltrating surrounding healthy tissue and giving rise to rapid recurrence. It is therefore critical to establish experimental model systems and develop therapeutic approaches that enhance anti-tumor immunity. In the current study, we have employed a newly developed murine glioma model to assess the efficacy of a novel picornavirus vaccination approach for the treatment of established tumors. The GL261-Quad system is a variation of the GL261 syngeneic glioma that has been engineered to expresses model T cell epitopes including OVA257-264. MRI revealed that both GL261 and GL261-Quad tumors display characteristic features of human gliomas such as heterogeneous gadolinium leakage and larger T2 weighted volumes. Analysis of brain-infiltrating immune cells demonstrated that GL261-Quad gliomas generate detectable CD8+ T cell responses toward the tumor-specific Kb:OVA257-264 antigen. Enhancing this response via a single intracranial or peripheral vaccination with picornavirus expressing the OVA257-264 antigen increased anti-tumor CD8+ T cells infiltrating the brain, attenuated progression of established tumors, and extended survival of treated mice. Importantly, the efficacy of the picornavirus vaccination is dependent on functional cytotoxic activity of CD8+ T cells, as the beneficial response was completely abrogated in mice lacking perforin expression. Therefore, we have developed a novel system for evaluating mechanisms of anti-tumor immunity in vivo, incorporating the GL261-Quad model, 3D volumetric MRI, and picornavirus vaccination to enhance tumor-specific cytotoxic CD8+ T cell responses and track their effectiveness at eradicating established gliomas in vivo.

  11. Maternal CD4+ and CD8+ T Cell Tolerance Towards a Fetal Minor Histocompatibility Antigen in T Cell Receptor Transgenic Mice1

    PubMed Central

    Perchellet, Antoine L.; Jasti, Susmita; Petroff, Margaret G.

    2013-01-01

    ABSTRACT Tolerance of the maternal immune system in pregnancy is important for successful pregnancy because the semiallogeneic fetus may be subject to antifetal responses. We examined maternal tolerance to the fetus using a murine system in which a model paternally inherited antigen, ovalbumin (OVA), is expressed exclusively in the fetus and placenta. By employing T cell receptor (TCR) transgenic mice specific for major histocompatibility complex class I- or class II-restricted epitopes of OVA (OT-I and OT-II) as mothers, we investigated the fate of fetus-specific CD8+ and CD4+ T cells, respectively, during gestation. Both OVA-specific CD8+ and CD4+ T cells displayed an activated phenotype in the peripheral lymphoid tissues of OVA-bred OT-I and OT-II mice, consistent with their encounter of fetal antigen. Whereas a small percentage of OVA-specific CD4+ T cells were deleted in the periphery and thymus of OVA-bred OT-II mice, with evidence of TCR downregulation in the remaining T cells, deletion and TCR downregulation were not observed in OVA-bred OT-I mice. Both CD4+ and CD8+ T cells upregulated inducible costimulator expression in response to the fetal antigen, but only CD4+ T cells consistently upregulated the inhibitory receptors programmed cell death 1 and cytotoxic T lymphocyte antigen-4. More regulatory T cells (Tregs) were present in pregnant OVA-bred than in WT-bred OT-II mice, revealing that Tregs expanded specifically in response to the fetal antigen. These data indicate that several mechanisms tolerize fetal antigen-specific maternal CD4+ T cells, whereas tolerance of fetal antigen-specific CD8+ T cells is less effective. The importance of these mechanisms is underscored by the finding that fetal loss occurs in OVA-bred OT-I but not OT-II mice. PMID:24025737

  12. The use of hybrid phage displaying antigen epitope and recombinant protein in the diagnosis of systemic Candida albicans infection in rabbits and cancer patients.

    PubMed

    Quanping, Su; Yanyan, Huai; Yicun, Wang; Zhigang, Ju; Yuling, Geng; Li, Wang

    2010-12-01

    Hsp90 and Sap2 are 2 immunodominant antigens of Candida albicans. Both of them can induce the production of antibody. In this article, systemically infected rabbits were used to study the Hsp90 and Sap2 antibody production. Also, pET28a-Hsp90 protein, pET28a-Sap2 protein, hybrid phage displaying LKVIRK epitope, and hybrid phage displaying VKYTS epitope were used for diagnosis of the antibody in cancer patients. The results showed that the Sap2 antibody appeared earlier than Hsp90 antibody in systemically infected rabbits. Meanwhile, both of the antibodies can perform protection in rabbits. The conclusion is that Sap2 antibody, which appears at early stage in systemic candidiasis, may be better than Hsp90 antibody for the diagnosis of invasive candidiasis. For 141 sera of cancer patients, 52 sera were detected Sap2 antibody and 57 sera were detected Hsp90 antibody. Only 14 sera contained both the 2 antibodies. Although recombinant protein was slightly more sensitive than hybrid phage, there was no significant difference between them. For its easy preparation, less expensive hybrid phage displaying antigen epitope may be a better agent for diagnosis of candidiasis.

  13. The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response.

    PubMed

    Akondy, Rama S; Monson, Nathan D; Miller, Joseph D; Edupuganti, Srilatha; Teuwen, Dirk; Wu, Hong; Quyyumi, Farah; Garg, Seema; Altman, John D; Del Rio, Carlos; Keyserling, Harry L; Ploss, Alexander; Rice, Charles M; Orenstein, Walter A; Mulligan, Mark J; Ahmed, Rafi

    2009-12-15

    The live yellow fever vaccine (YF-17D) offers a unique opportunity to study memory CD8(+) T cell differentiation in humans following an acute viral infection. We have performed a comprehensive analysis of the virus-specific CD8(+) T cell response using overlapping peptides spanning the entire viral genome. Our results showed that the YF-17D vaccine induces a broad CD8(+) T cell response targeting several epitopes within each viral protein. We identified a dominant HLA-A2-restricted epitope in the NS4B protein and used tetramers specific for this epitope to track the CD8(+) T cell response over a 2 year period. This longitudinal analysis showed the following. 1) Memory CD8(+) T cells appear to pass through an effector phase and then gradually down-regulate expression of activation markers and effector molecules. 2) This effector phase was characterized by down-regulation of CD127, Bcl-2, CCR7, and CD45RA and was followed by a substantial contraction resulting in a pool of memory T cells that re-expressed CD127, Bcl-2, and CD45RA. 3) These memory cells were polyfunctional in terms of degranulation and production of the cytokines IFN-gamma, TNF-alpha, IL-2, and MIP-1beta. 4) The YF-17D-specific memory CD8(+) T cells had a phenotype (CCR7(-)CD45RA(+)) that is typically associated with terminally differentiated cells with limited proliferative capacity (T(EMRA)). However, these cells exhibited robust proliferative potential showing that expression of CD45RA may not always associate with terminal differentiation and, in fact, may be an indicator of highly functional memory CD8(+) T cells generated after acute viral infections.

  14. Vaccine generated immunity targets an HPV16 E7 HLA-A2.1-restricted CD8(+) T cell epitope relocated to an early gene or a late gene of the cottontail rabbit papillomavirus (CRPV) genome in HLA-A2.1 transgenic rabbits.

    PubMed

    Bounds, Callie E; Hu, Jiafen; Cladel, Nancy M; Balogh, Karla; Christensen, Neil D

    2011-02-01

    The newly established HLA-A2.1 transgenic rabbit model has proven useful for testing the immunogenicity of well known and computer-predicted A2-restricted epitopes. In the current study we compared the protective immunity induced to a preferred HPV16 E7 A2-restricted epitope that has been relocated to positions within the CRPV E7 gene and the CRPV L2 gene. Epitope expression from both the E7 protein and the L2 protein resulted in increased protection against viral DNA challenge of the HLA-A2.1 transgenic rabbits as compared to control-vaccinated rabbit groups. These data indicate that proteins expressed at both early and late time points during a natural papillomavirus infection can be targeted by epitope-specific immunity and indicate this immunity is increased to early rather than late expressed proteins of papillomaviruses. This study also highlights the broad utility of the HLAA2.1 transgenic rabbit model for testing numerous immunological factors involved in vaccine generated protective immunity.

  15. Protection of rhesus macaques against disease progression from pathogenic SHIV-89.6PD by vaccination with phage-displayed HIV-1 epitopes.

    PubMed

    Chen, X; Scala, G; Quinto, I; Liu, W; Chun, T W; Justement, J S; Cohen, O J; vanCott, T C; Iwanicki, M; Lewis, M G; Greenhouse, J; Barry, T; Venzon, D; Fauci, A S

    2001-11-01

    The antigenic polymorphism of HIV-1 is a major obstacle in developing an effective vaccine. Accordingly, we screened random peptide libraries (RPLs) displayed on phage with antibodies from HIV-infected individuals and identified an array of HIV-specific epitopes that behave as antigenic mimics of conformational epitopes of gp120 and gp41 proteins. We report that the selected epitopes are shared by a collection of HIV-1 isolates of clades A-F. The phage-borne epitopes are immunogenic in rhesus macaques, where they elicit envelope-specific antibody responses. Upon intravenous challenge with 60 MID50 of pathogenic SHIV-89.6PD, all monkeys became infected; however, in contrast to the naive and mock-immunized monkeys, four of five mimotope-immunized monkeys experienced lower levels of peak viremia, followed by viral set points of undetectable or transient levels of viremia and a mild decline of CD4+ T cells, and were protected from progression to AIDS-like illness. These results provide a new approach to the design of broadly protective HIV-1 vaccines.

  16. Identification of a Conserved Linear B-Cell Epitope of Streptococcus dysgalactiae GapC Protein by Screening Phage-Displayed Random Peptide Library

    PubMed Central

    Fan, Ziyao; Zhou, Xue; Yu, Liquan; Sun, Hunan; Wu, Zhijun; Yu, Yongzhong; Song, Baifen; Ma, Jinzhu; Tong, Chunyu; Wang, Xintong; Zhu, Zhanbo; Cui, Yudong

    2015-01-01

    The GapC of Streptococcus dysgalactiae (S. dysgalactiae) is a highly conserved surface protein that can induce protective humoral immune response in animals. However, B-cell epitopes on the S. dysgalactiae GapC have not been well identified. In this study, a monoclonal antibody (mAb5B7) against the GapC1-150 protein was prepared. After passive transfer, mAb5B7 could partially protect mice against S. dysgalactiae infection. Eleven positive phage clones recognized by mAb5B7 were identified by screening phage-displayed random 12-peptide library, most of which matched the consensus motif DTTQGRFD. The motif sequence exactly matches amino acids 48-55 of the S. dysgalactiae GapC protein. In addition, the motif 48DTTQGRFD55 shows high homology among various streptococcus species. Site-directed mutagenic analysis further confirmed that residues D48, T50, Q51, G52 and F54 formed the core motif of 48DTTQGRFD55. This motif was the minimal determinant of the B-cell epitope recognized by the mAb5B7. As expected, epitope-peptide evoked protective immune response against S. dysgalactiae infection in immunized mice. Taken together, this identified conserved B-cell epitope within S. dysgalactiae GapC could provide very valuable insights for vaccine design against S. dysgalactiae infection. PMID:26121648

  17. Identification of the immunogenic epitopes of the whole venom component of the Hemiscorpius lepturus scorpion using the phage display peptide library.

    PubMed

    Jahdasani, Roghaye; Jamnani, Fatemeh Rahimi; Behdani, Mahdi; Habibi-Anbouhi, Mahdi; Yardehnavi, Najmeh; Shahbazzadeh, Delavar; Kazemi-Lomedasht, Fatemeh

    2016-12-15

    The venom of the Hemiscorpius lepturus scorpion contains mixtures of bioactive compounds that disturb biochemical and physiological functions of the victims. Hemiscorpius lepturus envenomation is recognized as a serious health concern in tropical regions. So far, there is no preventive procedure, and the main focus is on treatment of victims with an antiserum purified from hyper-immunized horses. Although antisera can neutralize the venom, they, in some cases, lead to anaphylactic shock and even death. Selection of peptides mimicking antigenic and immunogenic epitopes of toxins from random peptide libraries is a novel approach for the development of recombinant toxins and poly-epitopic vaccine. To achieve this aim, a phage display peptide library and three rounds of biopanning were performed on immobilized antibodies (IgGs) purified from the sera of hyper-immunized horses. The results show that the highest binding of the phage to immobilized horse antibodies occurred in the third round of biopanning. Over 125 individual clones carrying mimotopes of Hemiscorpius lepturus toxins were selected and subjected for sequencing. The sequencing results identified unique peptides mimicking the antigenic and immunogenic epitopes of Hemiscorpius lepturus toxins. The results of this study provide a basis for further studies and the development of a putative epitopic vaccine and a recombinant toxin. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Phenotypic Alterations Involved in CD8+ Treg Impairment in Systemic Sclerosis.

    PubMed

    Negrini, Simone; Fenoglio, Daniela; Parodi, Alessia; Kalli, Francesca; Battaglia, Florinda; Nasi, Giorgia; Curto, Monica; Tardito, Samuele; Ferrera, Francesca; Filaci, Gilberto

    2017-01-01

    Systemic sclerosis (SSc) is a connective tissue disease characterized by tissue fibrosis, vasculopathy, and autoimmunity. Although the exact pathogenetic mechanisms behind SSc remain to be fully elucidated, a great deal of evidence suggests the existence of an unbalanced ratio between the effector and regulatory arms of the immune system. With regard to the T regulatory (Treg) compartment, we observed that CD8+ Treg subsets display functional defects in SSc-affected patients. Since CD127 down-modulation and CD39 upregulation have been observed on Treg subsets, the phenotypic expression of these molecules was analyzed on the CD8+CD28- Treg precursors and on CD8+ Treg cells generated in vitro through interleukin-10 commitment. Immunophenotypic data from SSc patients were compared to those obtained from healthy subjects. The analyses performed on ex vivo-isolated CD8+CD28- Treg precursors did not show any significant differences in CD39 or CD127 expression as compared to values obtained from healthy donors. On the contrary, in vitro-generated CD8+ Tregs obtained from SSc patients displayed reduced expression of the CD39 molecule as compared to controls. Moreover, the percentage of CD127+ cells was significantly higher in in vitro-generated CD8+ Tregs from SSc patients compared to CD8+ Tregs obtained from healthy donors. Taken together, these findings may indicate an impairment of maturation processes affecting CD8+ Treg cells in SSc patients. This impairment of maturation involves phenotypic alterations that are mainly characterized by a deficient CD39 upregulation and a lack of down-modulation of the CD127 molecule.

  19. Phenotypic Alterations Involved in CD8+ Treg Impairment in Systemic Sclerosis

    PubMed Central

    Negrini, Simone; Fenoglio, Daniela; Parodi, Alessia; Kalli, Francesca; Battaglia, Florinda; Nasi, Giorgia; Curto, Monica; Tardito, Samuele; Ferrera, Francesca; Filaci, Gilberto

    2017-01-01

    Systemic sclerosis (SSc) is a connective tissue disease characterized by tissue fibrosis, vasculopathy, and autoimmunity. Although the exact pathogenetic mechanisms behind SSc remain to be fully elucidated, a great deal of evidence suggests the existence of an unbalanced ratio between the effector and regulatory arms of the immune system. With regard to the T regulatory (Treg) compartment, we observed that CD8+ Treg subsets display functional defects in SSc-affected patients. Since CD127 down-modulation and CD39 upregulation have been observed on Treg subsets, the phenotypic expression of these molecules was analyzed on the CD8+CD28− Treg precursors and on CD8+ Treg cells generated in vitro through interleukin-10 commitment. Immunophenotypic data from SSc patients were compared to those obtained from healthy subjects. The analyses performed on ex vivo-isolated CD8+CD28− Treg precursors did not show any significant differences in CD39 or CD127 expression as compared to values obtained from healthy donors. On the contrary, in vitro-generated CD8+ Tregs obtained from SSc patients displayed reduced expression of the CD39 molecule as compared to controls. Moreover, the percentage of CD127+ cells was significantly higher in in vitro-generated CD8+ Tregs from SSc patients compared to CD8+ Tregs obtained from healthy donors. Taken together, these findings may indicate an impairment of maturation processes affecting CD8+ Treg cells in SSc patients. This impairment of maturation involves phenotypic alterations that are mainly characterized by a deficient CD39 upregulation and a lack of down-modulation of the CD127 molecule. PMID:28154567

  20. Systematic identification of immunodominant CD8+ T-cell responses to influenza A virus in HLA-A2 individuals

    PubMed Central

    Wu, Chao; Zanker, Damien; Valkenburg, Sophie; Kedzierska, Katherine; Zou, Quan Ming; Doherty, Peter C.; Chen, Weisan

    2011-01-01

    Immunodominant T-cell responses are important for virus clearance. However, the identification of immunodominant T-cell peptide + HLA glycoprotein epitopes has been hindered by the extent of HLA polymorphism and the limitations of predictive algorithms. A simple, systematic approach has been used here to screen for immunodominant CD8+ T-cell specificities. The analysis targeted healthy HLA-A2+ donors to allow comparison with responses to the well-studied influenza matrix protein 1 epitope. Although influenza matrix protein 1 was consistently detected in all individual samples in our study, the response to this epitope was only immunodominant in three of eight, whereas for the other five, prominent CD8+ T-cell responses tended to focus on various peptides from the influenza nucleoprotein that were not presented by HLA-A2. Importantly, with the four immunodominant T-cell epitopes identified here, only one would have been detected by the current prediction programs. The other three peptides would have been either considered too long or classified as not containing typical HLA binding motifs. Our data stress the importance of systematic analysis for discovering HLA-dependent, immunodominant CD8+ T-cell epitopes derived from viruses and tumors. Focusing on HLA-A2 and predictive algorithms may be too limiting as we seek to develop targeted immunotherapy and vaccine strategies that depend on T cell-mediated immunity. PMID:21562214

  1. Trypanosoma cruzi Subverts Host Cell Sialylation and May Compromise Antigen-specific CD8+ T Cell Responses*

    PubMed Central

    Freire-de-Lima, Leonardo; Alisson-Silva, Frederico; Carvalho, Sebastião T.; Takiya, Christina M.; Rodrigues, Maurício M.; DosReis, George A.; Mendonça-Previato, Lucia; Previato, José O.; Todeschini, Adriane R.

    2010-01-01

    Upon activation, cytotoxic CD8+ T lymphocytes are desialylated exposing β-galactose residues in a physiological change that enhances their effector activity and that can be monitored on the basis of increased binding of the lectin peanut agglutinin. Herein, we investigated the impact of sialylation mediated by trans-sialidase, a specific and unique Trypanosoma transglycosylase for sialic acid, on CD8+ T cell response of mice infected with T. cruzi. Our data demonstrate that T. cruzi uses its trans-sialidase enzyme to resialylate the CD8+ T cell surface, thereby dampening antigen-specific CD8+ T cell response that might favor its own persistence in the mammalian host. Binding of the monoclonal antibody S7, which recognizes sialic acid-containing epitopes on the 115-kDa isoform of CD43, was augmented on CD8+ T cells from ST3Gal-I-deficient infected mice, indicating that CD43 is one sialic acid acceptor for trans-sialidase activity on the CD8+ T cell surface. The cytotoxic activity of antigen-experienced CD8+ T cells against the immunodominant trans-sialidase synthetic peptide IYNVGQVSI was decreased following active trans-sialidase- mediated resialylation in vitro and in vivo. Inhibition of the parasite's native trans-sialidase activity during infection strongly decreased CD8+ T cell sialylation, reverting it to the glycosylation status expected in the absence of parasite manipulation increasing mouse survival. Taken together, these results demonstrate, for the first time, that T. cruzi subverts sialylation to attenuate CD8+ T cell interactions with peptide-major histocompatibility complex class I complexes. CD8+ T cell resialylation may represent a sophisticated strategy to ensure lifetime host parasitism. PMID:20106975

  2. Frequency, Private Specificity, and Cross-Reactivity of Preexisting Hepatitis C Virus (HCV)-Specific CD8+ T Cells in HCV-Seronegative Individuals: Implications for Vaccine Responses.

    PubMed

    Zhang, Shihong; Bakshi, Rakesh K; Suneetha, Pothakamuri Venkata; Fytili, Paraskevi; Antunes, Dinler A; Vieira, Gustavo F; Jacobs, Roland; Klade, Christoph S; Manns, Michael P; Kraft, Anke R M; Wedemeyer, Heiner; Schlaphoff, Verena; Cornberg, Markus

    2015-08-01

    T cell responses play a critical role in controlling or clearing viruses. Therefore, strategies to prevent or treat infections include boosting T cell responses. T cells specific for various pathogens have been reported in unexposed individuals and an influence of such cells on the response toward vaccines is conceivable. However, little is known about their frequency, repertoire, and impact on vaccination. We performed a detailed characterization of CD8(+) T cells specific to a hepatitis C virus (HCV) epitope (NS3-1073) in 121 HCV-seronegative individuals. We show that in vitro HCV NS3-1073-specific CD8(+) T cell responses were rather abundantly detectable in one-third of HCV-seronegative individuals irrespective of risk factors for HCV exposure. Ex vivo, these NS3-1073-specific CD8(+) T cells were found to be both naive and memory cells. Importantly, recognition of various peptides derived from unrelated viruses by NS3-1073-specific CD8(+) T cells showed a considerable degree of T cell cross-reactivity, suggesting that they might in part originate from previous heterologous infections. Finally, we further provide evidence that preexisting NS3-1073-specific CD8(+) T cells can impact the T cell response toward peptide vaccination. Healthy, vaccinated individuals who showed an in vitro response toward NS3-1073 already before vaccination displayed a more vigorous and earlier response toward the vaccine. Preventive and therapeutic vaccines are being developed for many viral infections and often aim on inducing T cell responses. Despite effective antiviral drugs against HCV, there is still a need for a preventive vaccine. However, the responses to vaccines can be highly variable among different individuals. Preexisting T cells in unexposed individuals could be one reason that helps to explain the variable T cell responses to vaccines. Based on our findings, we suggest that HCV CD8(+) T cells are abundant in HCV-seronegative individuals but that their repertoire is

  3. Frequency, Private Specificity, and Cross-Reactivity of Preexisting Hepatitis C Virus (HCV)-Specific CD8+ T Cells in HCV-Seronegative Individuals: Implications for Vaccine Responses

    PubMed Central

    Zhang, Shihong; Bakshi, Rakesh K.; Suneetha, Pothakamuri Venkata; Fytili, Paraskevi; Antunes, Dinler A.; Vieira, Gustavo F.; Jacobs, Roland; Klade, Christoph S.; Manns, Michael P.; Kraft, Anke R. M.; Wedemeyer, Heiner; Schlaphoff, Verena

    2015-01-01

    ABSTRACT T cell responses play a critical role in controlling or clearing viruses. Therefore, strategies to prevent or treat infections include boosting T cell responses. T cells specific for various pathogens have been reported in unexposed individuals and an influence of such cells on the response toward vaccines is conceivable. However, little is known about their frequency, repertoire, and impact on vaccination. We performed a detailed characterization of CD8+ T cells specific to a hepatitis C virus (HCV) epitope (NS3-1073) in 121 HCV-seronegative individuals. We show that in vitro HCV NS3-1073-specific CD8+ T cell responses were rather abundantly detectable in one-third of HCV-seronegative individuals irrespective of risk factors for HCV exposure. Ex vivo, these NS3-1073-specific CD8+ T cells were found to be both naive and memory cells. Importantly, recognition of various peptides derived from unrelated viruses by NS3-1073-specific CD8+ T cells showed a considerable degree of T cell cross-reactivity, suggesting that they might in part originate from previous heterologous infections. Finally, we further provide evidence that preexisting NS3-1073-specific CD8+ T cells can impact the T cell response toward peptide vaccination. Healthy, vaccinated individuals who showed an in vitro response toward NS3-1073 already before vaccination displayed a more vigorous and earlier response toward the vaccine. IMPORTANCE Preventive and therapeutic vaccines are being developed for many viral infections and often aim on inducing T cell responses. Despite effective antiviral drugs against HCV, there is still a need for a preventive vaccine. However, the responses to vaccines can be highly variable among different individuals. Preexisting T cells in unexposed individuals could be one reason that helps to explain the variable T cell responses to vaccines. Based on our findings, we suggest that HCV CD8+ T cells are abundant in HCV-seronegative individuals but that their repertoire

  4. Differential in vivo clearance and response to secondary heterologous infections by H2(b)-restricted dengue virus-specific CD8+ T cells.

    PubMed

    Beaumier, Coreen M; Jaiswal, Smita; West, Kim Y; Friberg, Heather; Mathew, Anuja; Rothman, Alan L

    2010-10-01

    Cytotoxic T lymphocytes (CTL) are hypothesized to play a role in clearance during primary dengue virus (DENV) infections, and contribute to immunopathology during secondary heterologous infections in humans. We previously reported skewed T-cell responses to secondary DENV infection in BALB/c (H-2(d)) mice, reproducing characteristics of human DENV infection. To set the stage for using widely available transgenic and knockout mice, we extended these studies to identify DENV-specific T-cell responses in C57BL/6 (H-2(b)) mice. We identified dominant CD8+ T-cell responses to H-2D(b)-restricted epitopes on the DENV NS4a (aa 249-265) and NS5 (aa 521-537) proteins. High frequencies of IFN-γ- and TNF-α-producing T cells directed at both epitopes were detected following primary infection with all four DENV serotypes, and were augmented by secondary DENV infections. In vivo cytotoxicity assays demonstrated rapid clearance of target cells pulsed with the NS4a peptide; in contrast, NS5 peptide-pulsed target cells were poorly cleared in vivo. These data characterize two H-2(b)-restricted T-cell epitopes displaying divergent in vivo function. These results should facilitate further studies of the in vivo effects of DENV-specific T cells, including the use of genetically modified mouse strains.

  5. The HIV-1 antisense protein (ASP) induces CD8 T cell responses during chronic infection.

    PubMed

    Bet, Anne; Maze, Emmanuel Atangana; Bansal, Anju; Sterrett, Sarah; Gross, Antoine; Graff-Dubois, Stéphanie; Samri, Assia; Guihot, Amélie; Katlama, Christine; Theodorou, Ioannis; Mesnard, Jean-Michel; Moris, Arnaud; Goepfert, Paul A; Cardinaud, Sylvain

    2015-02-10

    CD8+ T cells recognize HIV-1 epitopes translated from a gene's primary reading frame (F1) and any one of its five alternative reading frames (ARFs) in the forward (F2, F3) or reverse (R1-3) directions. The 3' end of HIV-1's proviral coding strand contains a conserved sequence that is directly overlapping but antiparallel to the env gene (ARF R2) and encodes for a putative antisense HIV-1 protein called ASP. ASP expression has been demonstrated in vitro using HIV-transfected cell lines or infected cells. Although antibodies to ASP were previously detected in patient sera, T cell recognition of ASP-derived epitopes has not been evaluated. We therefore investigated the ex vivo and in vitro induction of ASP-specific T cell responses as a measure of immune recognition and protein expression during HIV-1 infection. A panel of overlapping peptides was initially designed from the full-length ASP sequence to perform a global assessment of T cell responses. Recognition of ASP-derived antigens was evaluated in an IFN-γELISpot assay using PBMCs from HIV-1 seropositive and seronegative individuals. Eight of 25 patients had positive responses to ASP antigens and none of the seronegative donors responded. As a complimentary approach, a second set of antigens was designed using HLA-I binding motifs and affinities. Two ASP-derived peptides with high predicted binding affinities for HLA-A*02 (ASP-YL9) and HLA-B*07 (ASP-TL10) were tested using PBMCs from HIV-1 seropositive and seronegative individuals who expressed the matching HLA-I-restricting allele. We found that HLA-I-restricted ASP peptides were only recognized by CD8+ T cells from patients with the relevant HLA-I and did not induce responses in any of the seronegative donors or patients who do not express the restrictive HLA alleles. Further, ASP-YL9-specific CD8+ T cells had functional profiles that were similar to a previously described HLA-A*02-restricted epitope (Gag-SL9). Specific recognition of ASP-YL9 by CD8+ T cells

  6. Short-Lived Effector CD8 T Cells Induced by Genetically Attenuated Malaria Parasite Vaccination Express CD11c

    PubMed Central

    Cooney, Laura A.; Gupta, Megha; Thomas, Sunil; Mikolajczak, Sebastian; Choi, Kimberly Y.; Gibson, Claire; Jang, Ihn K.; Danziger, Sam; Aitchison, John; Gardner, Malcolm J.; Kappe, Stefan H. I.

    2013-01-01

    Vaccination with a single dose of genetically attenuated malaria parasites can induce sterile protection against sporozoite challenge in the rodent Plasmodium yoelii model. Protection is dependent on CD8+ T cells, involves perforin and gamma interferon (IFN-γ), and is correlated with the expansion of effector memory CD8+ T cells in the liver. Here, we have further characterized vaccine-induced changes in the CD8+ T cell phenotype and demonstrated significant upregulation of CD11c on CD3+ CD8b+ T cells in the liver, spleen, and peripheral blood. CD11c+ CD8+ T cells are predominantly CD11ahi CD44hi CD62L−, indicative of antigen-experienced effector cells. Following in vitro restimulation with malaria-infected hepatocytes, CD11c+ CD8+ T cells expressed inflammatory cytokines and cytotoxicity markers, including IFN-γ, tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), perforin, and CD107a. CD11c− CD8+ T cells, on the other hand, expressed negligible amounts of all inflammatory cytokines and cytotoxicity markers tested, indicating that CD11c marks multifunctional effector CD8+ T cells. Coculture of CD11c+, but not CD11c−, CD8+ T cells with sporozoite-infected primary hepatocytes significantly inhibited liver-stage parasite development. Tetramer staining for the immunodominant circumsporozoite protein (CSP)-specific CD8+ T cell epitope demonstrated that approximately two-thirds of CSP-specific cells expressed CD11c at the peak of the CD11c+ CD8+ T cell response, but CD11c expression was lost as the CD8+ T cells entered the memory phase. Further analyses showed that CD11c+ CD8+ T cells are primarily KLRG1+ CD127− terminal effectors, whereas all KLRG1− CD127+ memory precursor effector cells are CD11c− CD8+ T cells. Together, these results suggest that CD11c marks a subset of highly inflammatory, short-lived, antigen-specific effector cells, which may play an important role in eliminating infected hepatocytes. PMID:23980113

  7. Short-lived effector CD8 T cells induced by genetically attenuated malaria parasite vaccination express CD11c.

    PubMed

    Cooney, Laura A; Gupta, Megha; Thomas, Sunil; Mikolajczak, Sebastian; Choi, Kimberly Y; Gibson, Claire; Jang, Ihn K; Danziger, Sam; Aitchison, John; Gardner, Malcolm J; Kappe, Stefan H I; Wang, Ruobing

    2013-11-01

    Vaccination with a single dose of genetically attenuated malaria parasites can induce sterile protection against sporozoite challenge in the rodent Plasmodium yoelii model. Protection is dependent on CD8(+) T cells, involves perforin and gamma interferon (IFN-γ), and is correlated with the expansion of effector memory CD8(+) T cells in the liver. Here, we have further characterized vaccine-induced changes in the CD8(+) T cell phenotype and demonstrated significant upregulation of CD11c on CD3(+) CD8b(+) T cells in the liver, spleen, and peripheral blood. CD11c(+) CD8(+) T cells are predominantly CD11a(hi) CD44(hi) CD62L(-), indicative of antigen-experienced effector cells. Following in vitro restimulation with malaria-infected hepatocytes, CD11c(+) CD8(+) T cells expressed inflammatory cytokines and cytotoxicity markers, including IFN-γ, tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), perforin, and CD107a. CD11c(-) CD8(+) T cells, on the other hand, expressed negligible amounts of all inflammatory cytokines and cytotoxicity markers tested, indicating that CD11c marks multifunctional effector CD8(+) T cells. Coculture of CD11c(+), but not CD11c(-), CD8(+) T cells with sporozoite-infected primary hepatocytes significantly inhibited liver-stage parasite development. Tetramer staining for the immunodominant circumsporozoite protein (CSP)-specific CD8(+) T cell epitope demonstrated that approximately two-thirds of CSP-specific cells expressed CD11c at the peak of the CD11c(+) CD8(+) T cell response, but CD11c expression was lost as the CD8(+) T cells entered the memory phase. Further analyses showed that CD11c(+) CD8(+) T cells are primarily KLRG1(+) CD127(-) terminal effectors, whereas all KLRG1(-) CD127(+) memory precursor effector cells are CD11c(-) CD8(+) T cells. Together, these results suggest that CD11c marks a subset of highly inflammatory, short-lived, antigen-specific effector cells, which may play an important role in eliminating infected

  8. Broadening CD4(+) and CD8(+) T Cell Responses against Hepatitis C Virus by Vaccination with NS3 Overlapping Peptide Panels in Cross-Priming Liposomes.

    PubMed

    Filskov, Jonathan; Mikkelsen, Marianne; Hansen, Paul R; Christensen, Jan P; Thomsen, Allan R; Andersen, Peter; Bukh, Jens; Agger, Else Marie

    2017-07-15

    Despite the introduction of effective drugs to treat patients with chronic hepatitis C virus (HCV) infection, a vaccine would be the only means to substantially reduce the worldwide disease burden. An incomplete understanding of how HCV interacts with its human host and evades immune surveillance has hampered vaccine development. It is generally accepted that in infected individuals, a narrow repertoire of exhausted T cells is a hallmark of persistent infection, whereas broad, vigorous CD4(+) and CD8(+) T cell responses are associated with control of acute hepatitis C. We employed a vaccine approach based on a mixture of peptides (pepmix) spanning the entire sequence of HCV nonstructural protein 3 (NS3) in cross-priming cationic liposomes (CAF09) to facilitate a versatile presentation of all possible T cell epitopes, regardless of the HLA background of the vaccine recipient. Here, we demonstrate that vaccination of mice with NS3 pepmix broadens the repertoire of epitope-specific T cells compared to the corresponding recombinant protein (rNS3). Moreover, vaccination with rNS3 induced only CD4(+) T cells, whereas the NS3 pepmix induced a far more vigorous CD4(+) T cell response and was as potent a CD8(+) T cell inducer as an adenovirus-vectored vaccine expressing NS3. Importantly, the cellular responses are dominated by multifunctional T cells, such as gamma interferon-positive (IFN-γ(+)) tumor necrosis factor alpha-positive (TNF-α(+)) coproducers, and displayed cytotoxic capacity in mice. In conclusion, we present a novel vaccine approach against HCV, inducing a broadened T cell response targeting both immunodominant and potential subdominant epitopes, which may be key elements to counter T cell exhaustion and prevent chronicity.IMPORTANCE With at least 700,000 annual deaths, development of a vaccine against hepatitis C virus (HCV) has high priority, but the tremendous ability of the virus to dodge the human immune system poses great challenges. Furthermore, many

  9. Structure-based design of a protein immunogen that displays an HIV-1 gp41 neutralizing epitope.

    PubMed

    Stanfield, Robyn L; Julien, Jean-Philippe; Pejchal, Robert; Gach, Johannes S; Zwick, Michael B; Wilson, Ian A

    2011-12-02

    Antibody Z13e1 is a relatively broadly neutralizing anti-human immunodeficiency virus type 1 antibody that recognizes the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Based on the crystal structure of an MPER epitope peptide in complex with Z13e1 Fab, we identified an unrelated protein, interleukin (IL)-22, with a surface-exposed region that is structurally homologous in its backbone to the gp41 Z13e1 epitope. By grafting the gp41 Z13e1 epitope sequence onto the structurally homologous region in IL-22, we engineered a novel protein (Z13-IL22-2) that contains the MPER epitope sequence for use as a potential immunogen and as a reagent for the detection of Z13e1-like antibodies. The Z13-IL22-2 protein binds Fab Z13e1 with a K(d) of 73 nM. The crystal structure of Z13-IL22-2 in complex with Fab Z13e1 shows that the epitope region is faithfully replicated in the Fab-bound scaffold protein; however, isothermal calorimetry studies indicate that Fab binding to Z13-IL22-2 is not a lock-and-key event, leaving open the question of whether conformational changes upon binding occur in the Fab, in Z13-IL-22, or in both.

  10. Structure-Based Design of a Protein Immunogen that Displays an HIV-1 gp41 Neutralizing Epitope

    SciTech Connect

    Stanfield, Robyn L.; Julien, Jean-Philippe; Pejchal, Robert; Gach, Johannes S.; Zwick, Michael B.; Wilson, Ian A.

    2012-06-27

    Antibody Z13e1 is a relatively broadly neutralizing anti-human immunodeficiency virus type 1 antibody that recognizes the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Based on the crystal structure of an MPER epitope peptide in complex with Z13e1 Fab, we identified an unrelated protein, interleukin (IL)-22, with a surface-exposed region that is structurally homologous in its backbone to the gp41 Z13e1 epitope. By grafting the gp41 Z13e1 epitope sequence onto the structurally homologous region in IL-22, we engineered a novel protein (Z13-IL22-2) that contains the MPER epitope sequence for use as a potential immunogen and as a reagent for the detection of Z13e1-like antibodies. The Z13-IL22-2 protein binds Fab Z13e1 with a K{sub d} of 73 nM. The crystal structure of Z13-IL22-2 in complex with Fab Z13e1 shows that the epitope region is faithfully replicated in the Fab-bound scaffold protein; however, isothermal calorimetry studies indicate that Fab binding to Z13-IL22-2 is not a lock-and-key event, leaving open the question of whether conformational changes upon binding occur in the Fab, in Z13-IL-22, or in both.

  11. Unique transcriptional profile of liver-resident memory CD8+ T cells induced by immunization with malaria sporozoites

    PubMed Central

    Tse, Sze-Wah; Cockburn, Ian A.; Zhang, Hao; Scott, Alan L.; Zavala, Fidel

    2013-01-01

    Sterile immunity against live Plasmodium infection can be achieved by immunization with radiation attenuated sporozoites. This protection is known to be mediated in part by antigen-specific memory CD8+ T cells, presumably those residing in the liver. We characterized and compared the transcriptional profile of parasite-specific memory CD8+ T cells residing in the liver and spleen after immunization of mice with irradiated sporozoites. Microarray-based expression analysis of these memory CD8+ T cells indicated that liver resident memory cells display a distinct gene expression profile. We found major differences in the expression of immune function genes as well as genes involved in the cell cycle, cell trafficking, transcription and intracellular signaling. Importantly, the malaria parasite-induced liver resident CD8+ T cells display a transcriptional profile different to that described for CD8+ T cells following other microbial challenges. PMID:23594961

  12. Transcriptional regulator Id2 mediates CD8+ T cell immunity.

    PubMed

    Cannarile, Michael A; Lind, Nicholas A; Rivera, Richard; Sheridan, Alison D; Camfield, Kristin A; Wu, Bei Bei; Cheung, Kitty P; Ding, Zhaoqing; Goldrath, Ananda W

    2006-12-01

    Transcriptional programs that initiate and sustain the proliferation, differentiation and survival of CD8(+) T cells during immune responses are not completely understood. Here we show that inhibitor of DNA binding 2 (Id2), an antagonist of E protein transcription factors, was upregulated in CD8(+) T cells during infection and that expression of Id2 was maintained in memory CD8(+) T cells. Although Id2-deficient naive CD8(+) T cells recognized antigen and proliferated normally early after infection, effector CD8(+) T cells did not accumulate because the cells were highly susceptible to apoptosis. Id2-deficient CD8(+) T cells responding to infection had changes in the expression of genes that influence survival and had altered memory formation. Our data emphasize the importance of Id2 in regulating gene expression by CD8(+) T cells and the magnitude of effector responses, suggesting a mechanism involving Id protein- and E protein-mediated survival and differentiation of mature T cells.

  13. Peripheral CD4(+)CD8(+) T cells are differentiated effector memory cells with antiviral functions.

    PubMed

    Nascimbeni, Michelina; Shin, Eui-Cheol; Chiriboga, Luis; Kleiner, David E; Rehermann, Barbara

    2004-07-15

    Although an increased frequency of CD4(+)CD8(+) T cells has been observed in the peripheral blood during viral infections, their role, function, and biologic significance are still poorly understood. Here we demonstrate that the circulating CD4(+)CD8(+) T-cell population contains mature effector memory lymphocytes specific for antigens of multiple past, latent, and high-level persistent viral infections. Upon in vitro antigenic challenge, a higher frequency of CD4(+)CD8(+) than single-positive cells displayed a T helper 1/T cytotoxic 1 (Th1/Tc1) cytokine profile and proliferated. Ex vivo, more double-positive than single-positive cells exhibited a differentiated phenotype. Accordingly, their lower T-cell receptor excision circles (TREC) content and shorter telomeres proved they had divided more frequently than single-positive cells. Consistent with expression of the tissue-homing marker CXCR3, CD4(+)CD8(+) T cells were demonstrated in situ at the site of persistent viral infection (ie, in the liver during chronic hepatitis C). Finally, a prospective analysis of hepatitis C virus (HCV) infection in a chimpanzee, the only animal model for HCV infection, showed a close correlation between the frequency of activated CD4(+)CD8(+) T cells and viral kinetics. Collectively, these findings demonstrate that peripheral CD4(+)CD8(+) T cells take part in the adaptive immune response against infectious pathogens and broaden the perception of the T-cell populations involved in antiviral immune responses.

  14. Invariant NKT Cells Regulate the CD8 T Cell Response during Theiler's Virus Infection

    PubMed Central

    Mars, Lennart T.; Mas, Magali; Beaudoin, Lucie; Bauer, Jan; Leite-de-Moraes, Maria; Lehuen, Agnès; Bureau, Jean-Francois; Liblau, Roland S.

    2014-01-01

    Invariant NKT cells are innate lymphocytes with a broad tissue distribution. Here we demonstrate that iNKT cells reside in the central nervous system (CNS) in the absence of inflammation. Their presence in the CNS dramatically augments following inoculation of C57Bl/6 mice with the neurotropic Theiler's murine encephalomyelitis virus (TMEV). At the peak of inflammation the cellular infiltrate comprises 45 000 iNKT cells for 1 250 CD8 T cells specific for the immunodominant TMEV epitope. To study the interaction between these two T cell subsets, we infected both iNKT cell deficient Jα18-/- mice and iNKT cell enriched Vα14 transgenic mice with TMEV. The CD8 T cell response readily cleared TMEV infection in the iNKT cell deficient mice. However, in the iNKT cell enriched mice TMEV infection persisted and was associated with significant mortality. This was caused by the inhibition of the CD8 T cell response in the cervical lymph nodes and spleen after T cell priming. Taken together we demonstrate that iNKT cells reside in the CNS in the absence of inflammation and that their enrichment is associated with the inhibition of the anti-viral CD8 T cell response and an augmented mortality during acute encephalomyelitis. PMID:24498175

  15. Gene Deletions in Mycobacterium bovis BCG Stimulate Increased CD8+ T Cell Responses

    PubMed Central

    Panas, Michael W.; Sixsmith, Jaimie D.; White, KeriAnn; Korioth-Schmitz, Birgit; Shields, Shana T.; Moy, Brian T.; Lee, Sunhee; Schmitz, Joern E.; Jacobs, William R.; Porcelli, Steven A.; Haynes, Barton F.; Letvin, Norman L.

    2014-01-01

    Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host's immune response. It has been suggested that mycobacteria may contain genes that reduce the host's ability to elicit CD8+ T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8+ T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8+ T cell responses in vivo. This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8+ T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors. PMID:25287928

  16. Multifaceted role of BTLA in the control of CD8+ T cell fate after antigen encounter.

    PubMed

    Ritthipichai, Krit; Haymaker, Cara; Martinez-Paniagua, Melisa; Aschenbrenner, Andrew; Yi, Xiaohui; Zhang, Minying; Kale, Charuta; Hailemichael, Yared; Overwijk, Willem W; Vence, Luis; Roszik, Jason; Varadarajan, Navin; Nurieva, Roza; Radvanyi, Laszlo G; Hwu, Patrick; Bernatchez, Chantale

    2017-07-28

    Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown an overall clinical response rate 40-50% in metastatic melanoma patients. BTLA (B-and-T lymphocyte attenuator) expression on transferred CD8(+) TIL was associated with better clinical outcome. The suppressive function of the ITIM and ITSM motifs of BTLA is well described. Here, we sought to determine the functional characteristics of the CD8(+)BTLA(+)TIL subset and define the contribution of the Grb2 motif of BTLA in T cell co-stimulation. 

    Experimental Design: We determined the functional role and downstream signal of BTLA in both human CD8(+) TIL and mouse CD8(+) T cells. Functional assays were used including single cell analysis, Reverse Phase Protein Array (RPPA), antigen-specific vaccination models with adoptively transferred TCR-transgenic T cells as well as Patient-Derived Xenograft (PDX) model using Immunodeficient NOD-scid IL2Rgamma(null) (NSG) tumor-bearing mice treated with autologous TIL.

    Results: CD8(+)BTLA(-) TIL could not control tumor growth in vivo as well as their BTLA(+) counterpart and antigen-specific CD8(+)BTLA(-) T cells had impaired recall response to a vaccine. However CD8(+)BTLA(+) TIL displayed improved survival following the killing of a tumor target and heightened "serial killing" capacity. Using mutants of BTLA signaling motifs we uncovered a costimulatory function mediated by Grb2 through enhancing the secretion of IL-2 and the activation of Src after TCR stimulation. 

    Conclusions:Our data portrays BTLA as a molecule with the singular ability to provide both co-stimulatory and co-inhibitory signals to activated CD8(+) T cells, resulting in extended survival, improved tumor control and the development of a functional recall response. Copyright ©2017, American Association for Cancer Research.

  17. Intrinsic transgene immunogenicity gears CD8(+) T-cell priming after rAAV-mediated muscle gene transfer.

    PubMed

    Carpentier, Maxime; Lorain, Stéphanie; Chappert, Pascal; Lalfer, Mélanie; Hardet, Romain; Urbain, Dominique; Peccate, Cécile; Adriouch, Sahil; Garcia, Luis; Davoust, Jean; Gross, David-Alexandre

    2015-04-01

    Antitransgene CD8(+) T-cell responses are an important hurdle after recombinant adeno-associated virus (rAAV) vector-mediated gene transfer. Indeed, depending on the mutational genotype of the host, transgene amino-acid sequences of foreign origin can elicit deleterious cellular and humoral responses. We compared here two different major histocompatibility complex (MHC) class I epitopes of an engineered ovalbumin transgene delivered in muscle tissue by rAAV1 vector and found very different strength of CD8 responses, muscle destruction being correlated with the course of the immunodominant response. We further demonstrate that robust CD8(+) T-cell priming can occur through the cross-presentation pathway but requires the presence of either a strong MHC class II epitope or antibodies to the transgene product. Finally, manipulating transgene subcellular localization, we found that provided we avoid transgene expression in antigen presenting cells, the poorly accessible cytosolic form of ovalbumin transgene lacking strong MHC II epitope, evades CD8(+) T-cell priming and remains permanently expressed in muscle with no immune cell infiltration. Our results demonstrate that the intrinsic immunogenicity of transgenes delivered with rAAV vector in muscle can be manipulated in a rational manner to avoid adverse immune responses.

  18. Intrinsic Transgene Immunogenicity Gears CD8(+) T-cell Priming After rAAV-Mediated Muscle Gene Transfer.

    PubMed

    Carpentier, Maxime; Lorain, Stéphanie; Chappert, Pascal; Lalfer, Mélanie; Hardet, Romain; Urbain, Dominique; Peccate, Cécile; Adriouch, Sahil; Garcia, Luis; Davoust, Jean; Gross, David-Alexandre

    2015-04-01

    Antitransgene CD8(+) T-cell responses are an important hurdle after recombinant adeno-associated virus (rAAV) vector-mediated gene transfer. Indeed, depending on the mutational genotype of the host, transgene amino-acid sequences of foreign origin can elicit deleterious cellular and humoral responses. We compared here two different major histocompatibility complex (MHC) class I epitopes of an engineered ovalbumin transgene delivered in muscle tissue by rAAV1 vector and found very different strength of CD8 responses, muscle destruction being correlated with the course of the immunodominant response. We further demonstrate that robust CD8(+) T-cell priming can occur through the cross-presentation pathway but requires the presence of either a strong MHC class II epitope or antibodies to the transgene product. Finally, manipulating transgene subcellular localization, we found that provided we avoid transgene expression in antigen presenting cells, the poorly accessible cytosolic form of ovalbumin transgene lacking strong MHC II epitope, evades CD8(+) T-cell priming and remains permanently expressed in muscle with no immune cell infiltration. Our results demonstrate that the intrinsic immunogenicity of transgenes delivered with rAAV vector in muscle can be manipulated in a rational manner to avoid adverse immune responses.

  19. Mycobacterium tuberculosis Directs Immunofocusing of CD8+ T Cell Responses Despite Vaccination

    PubMed Central

    Woodworth, Joshua S.; Shin, Daniel; Volman, Mattijs; Nunes-Alves, Cláudio; Fortune, Sarah M.; Behar, Samuel M.

    2011-01-01

    Vaccines that elicit T cell responses try to mimic protective memory T cell immunity after infection by increasing the frequency of Ag-specific T cells in the immune repertoire. However, the factors that determine immunodominance during infection and after vaccination and the relation between immunodominance and protection are incompletely understood. We previously identified TB10.4(20–28) as an immunodominant epitope recognized by H2-Kd–restricted CD8+ T cells after M. tuberculosis infection. Here we report a second epitope, EspA(150–158), that is recognized by a substantial number of pulmonary CD8+ T cells. The relative abundance of these T cells in the naive repertoire only partially predicts their relative frequency after M. tuberculosis infection. Furthermore, although vaccination with recombinant vaccinia virus expressing these epitopes changes their relative immunodominance in the preinfection T cell repertoire, this change is transient after challenge with M. tuberculosis. We speculate that factors intrinsic to the chronic nature of M. tuberculosis infection establishes the hierarchy of immunodominance and may explain the failure of some vaccines to provide protection. PMID:21178003

  20. Dominant influence of an HLA-B27 restricted CD8+ T cell response in mediating HCV clearance and evolution.

    PubMed

    Neumann-Haefelin, Christoph; McKiernan, Susan; Ward, Scott; Viazov, Sergei; Spangenberg, Hans Christian; Killinger, Thomas; Baumert, Thomas F; Nazarova, Natalja; Sheridan, Isabelle; Pybus, Oliver; von Weizsäcker, Fritz; Roggendorf, Michael; Kelleher, Dermot; Klenerman, Paul; Blum, Hubert E; Thimme, Robert

    2006-03-01

    Virus-specific CD8+ T cell responses play an important role in the natural course of infection; however, the impact of certain CD8+ T cell responses in determining clinical outcome has not been fully defined. A well-defined cohort of women inoculated with HCV from a single source showed that HLA-B27 has a strong association with spontaneous clearance. The immunological basis for this association is unknown. However, the finding is especially significant because HLA-B27 has also been shown to have a protective role in HIV infection. We report the identification of an HLA-B27 restricted hepatitis C virus (HCV)-specific CD8+ T cell epitope that is recognized in the majority of recovered HLA-B27 positive women. In chronically HCV-infected individuals, analysis of the corresponding viral sequence showed a strong association between sequence variations within this epitope and expression of HLA-B27, indicating allele-specific selection pressure at the population level. Functional analysis in 3 chronically HCV-infected patients showed that the emerging variant viral epitopes represent escape mutations. In conclusion, our results suggest a dominant role of HLA-B27 in mediating spontaneous viral clearance as well as viral evolution in HCV infection and mechanistically link both associations to a dominant novel CD8+ T cell epitope. These results support the central role of virus-specific CD8+ T cells and the genetically determined restriction of the virus-specific T cell repertoire in HCV infection.

  1. Multiple specificities in the murine CD4+ and CD8+ T-cell response to dengue virus.

    PubMed Central

    Rothman, A L; Kurane, I; Ennis, F A

    1996-01-01

    The target epitopes, serotype specificity, and cytolytic function of dengue virus-specific T cells may influence their theoretical roles in protection against secondary infection as well as the immunopathogenesis of dengue hemorrhagic fever. To study these factors in an experimental system, we isolated dengue virus-specific CD4+ and CD8+ T-cell clones from dengue-2 virus-immunized BALB/c mice. The T-cell response to dengue virus in this mouse strain was heterogeneous; we identified at least five different CD4+ phenotypes and six different CD8+ phenotypes. Individual T-cell clones recognized epitopes on the dengue virus pre-M, E, NSl/NS2A, and NS3 proteins and were restricted by the I-Ad, I-Ed, Ld, and Kd antigens. Both serotype-specific and serotype-cross-reactive clones were isolated in the CD4+ and CD8+ subsets; among CD8+ clones, those that recognized the dengue virus structural proteins were serotype specific whereas those that recognized the nonstructural proteins were serotype cross-reactive. All of the CD8+ and one of five CD4+ clones lysed dengue virus-infected target cells. Using synthetic peptides, we identified an Ld-restricted epitope on the E protein (residues 331 to 339, SPCKIPFEI) and a Kd-restricted epitope on the NS3 protein (residues 296 to 310, ARGYISTRVEM GEAA). These data parallel previous findings of studies using human dengue virus-specific T-cell clones. This experimental mouse system may be useful for studying the role of the virus serotype and HLA haplotype on T-cell responses after primary dengue virus infection. PMID:8794288

  2. Private specificities of CD8 T cell responses control patterns of heterologous immunity

    PubMed Central

    Kim, Sung-Kwon; Cornberg, Markus; Wang, Xiaoting Z.; Chen, Hong D.; Selin, Liisa K.; Welsh, Raymond M.

    2005-01-01

    CD8 T cell cross-reactivity between viruses can play roles in protective heterologous immunity and damaging immunopathology. This cross-reactivity is sometimes predictable, such as between lymphocytic choriomeningitis virus (LCMV) and Pichinde virus, where cross-reactive epitopes share six out of eight amino acids. Here, however, we demonstrate more subtle and less predictable cross-reactivity between LCMV and the unrelated vaccinia virus (VV). Epitope-specific T cell receptor usage differed between individual LCMV-infected C57BL/6 mice, even though the mice had similar epitope-specific T cell hierarchies. LCMV-immune mice challenged with VV showed variations, albeit in a distinct hierarchy, in proliferative expansions of and down-regulation of IL-7Rα by T cells specific to different LCMV epitopes. T cell responses to a VV-encoded epitope that is cross-reactive with LCMV fluctuated greatly in VV-infected LCMV-immune mice. Adoptive transfers of splenocytes from individual LCMV-immune donors resulted in nearly identical VV-induced responses in each of several recipients, but responses differed depending on the donor. This indicates that the specificities of T cell responses that are not shared between individuals may influence cross-reactivity with other antigens and play roles in heterologous immunity upon encounter with another pathogen. This variability in cross-reactive T cell expansion that is unique to the individual may underlie variation in the pathogenesis of infectious diseases. PMID:15710651

  3. Determinants of Human Immunodeficiency Virus Type 1 Escape from the Primary CD8+ Cytotoxic T Lymphocyte Response

    PubMed Central

    Jones, Nicola A.; Wei, Xiping; Flower, Darren R.; Wong, MaiLee; Michor, Franziska; Saag, Michael S.; Hahn, Beatrice H.; Nowak, Martin A.; Shaw, George M.; Borrow, Persephone

    2004-01-01

    CD8+ cytotoxic T lymphocytes (CTLs) play an important role in containment of virus replication in primary human immunodeficiency virus (HIV) infection. HIV's ability to mutate to escape from CTL pressure is increasingly recognized; but comprehensive studies of escape from the CD8 T cell response in primary HIV infection are currently lacking. Here, we have fully characterized the primary CTL response to autologous virus Env, Gag, and Tat proteins in three patients, and investigated the extent, kinetics, and mechanisms of viral escape from epitope-specific components of the response. In all three individuals, we observed variation beginning within weeks of infection at epitope-containing sites in the viral quasispecies, which conferred escape by mechanisms including altered peptide presentation/recognition and altered antigen processing. The number of epitope-containing regions exhibiting evidence of early CTL escape ranged from 1 out of 21 in a subject who controlled viral replication effectively to 5 out of 7 in a subject who did not. Evaluation of the extent and kinetics of HIV-1 escape from >40 different epitope-specific CD8 T cell responses enabled analysis of factors determining escape and suggested that escape is restricted by costs to intrinsic viral fitness and by broad, codominant distribution of CTL-mediated pressure on viral replication. PMID:15545352

  4. Comprehensive mapping of functional epitopes on dengue virus glycoprotein E DIII for binding to broadly neutralizing antibodies 4E11 and 4E5A by phage display.

    PubMed

    Frei, Julia C; Kielian, Margaret; Lai, Jonathan R

    2015-11-01

    Here we investigated the binding of Dengue virus envelope glycoprotein domain III (DIII) by two broadly neutralizing antibodies (bNAbs), 4E11 and 4E5A. There are four serotypes of Dengue virus (DENV-1 to -4), whose DIII sequences vary by up to 49%. We used combinatorial alanine scanning mutagenesis, a phage display approach, to map functional epitopes (those residues that contribute most significantly to the energetics of antibody-antigen interaction) on these four serotypes. Our results showed that 4E11, which binds strongly to DENV-1, -2, and -3, and moderately to DENV-4, recognized a common conserved core functional epitope involving DIII residues K310, L/I387, L389, and W391. There were also unique recognition features for each serotype, suggesting that 4E11 has flexible recognition requirements. Similar scanning studies for the related bNAb 4E5A, which binds more tightly to DENV-4, identified broader functional epitopes on DENV-1. These results provide useful information for immunogen and therapeutic antibody design.

  5. Differential targeting of viral components by CD4+ versus CD8+ T lymphocytes in dengue virus infection.

    PubMed

    Rivino, Laura; Kumaran, Emmanuelle A P; Jovanovic, Vojislav; Nadua, Karen; Teo, En Wei; Pang, Shyue Wei; Teo, Guo Hui; Gan, Victor Chih Hao; Lye, David C; Leo, Yee Sin; Hanson, Brendon J; Smith, Kenneth G C; Bertoletti, Antonio; Kemeny, David M; MacAry, Paul A

    2013-03-01

    Dengue virus (DENV) is the principal arthropod-borne viral pathogen afflicting human populations. While repertoires of antibodies to DENV have been linked to protection or enhanced infection, the role of T lymphocytes in these processes remains poorly defined. This study provides a comprehensive overview of CD4(+) and CD8(+) T cell epitope reactivities against the DENV 2 proteome in adult patients experiencing secondary DENV infection. Dengue virus-specific T cell responses directed against an overlapping 15mer peptide library spanning the DENV 2 proteome were analyzed ex vivo by enzyme-linked immunosorbent spot assay, and recognition of individual peptides was further characterized in specific T cell lines. Thirty novel T cell epitopes were identified, 9 of which are CD4(+) and 21 are CD8(+) T cell epitopes. We observe that whereas CD8(+) T cell epitopes preferentially target nonstructural proteins (NS3 and NS5), CD4(+) epitopes are skewed toward recognition of viral components that are also targeted by B lymphocytes (envelope, capsid, and NS1). Consistently, a large proportion of dengue virus-specific CD4(+) T cells have phenotypic characteristics of circulating follicular helper T cells (CXCR5 expression and production of interleukin-21 or gamma interferon), suggesting that they are interacting with B cells in vivo. This study shows that during a dengue virus infection, the protein targets of human CD4(+) and CD8(+) T cells are largely distinct, thus highlighting key differences in the immunodominance of DENV proteins for these two cell types. This has important implications for our understanding of how the two arms of the human adaptive immune system are differentially targeted and employed as part of our response to DENV infection.

  6. Distinct Kinetics of Effector CD8+ Cytotoxic T Cells after Infection with Trypanosoma cruzi in Naïve or Vaccinated Mice

    PubMed Central

    Tzelepis, Fanny; de Alencar, Bruna C. G.; Penido, Marcus L. O.; Gazzinelli, Ricardo T.; Persechini, Pedro M.; Rodrigues, Mauricio M.

    2006-01-01

    The kinetics of effector CD8+-T-cell responses to specific Trypanosoma cruzi epitopes was investigated after challenge. Our results suggest that the delayed kinetics differs from that observed in other microbial infections and facilitates the establishment of the disease in naïve mice. In contrast, in vaccinated mice, the swift CD8+-T-cell response helps host survival after challenge. PMID:16552083

  7. Extensive Polymorphism and Evidence of Immune Selection in a Highly Dominant Antigen Recognized by Bovine CD8 T Cells Specific for Theileria annulata▿†‖

    PubMed Central

    MacHugh, Niall D.; Weir, William; Burrells, Alison; Lizundia, Regina; Graham, Simon P.; Taracha, Evans L.; Shiels, Brian R.; Langsley, Gordon; Morrison, W. Ivan

    2011-01-01

    Although parasite strain-restricted CD8 T cell responses have been described for several protozoa, the precise role of antigenic variability in immunity is poorly understood. The tick-borne protozoan parasite Theileria annulata infects leukocytes and causes an acute, often fatal lymphoproliferative disease in cattle. Building on previous evidence of strain-restricted CD8 T cell responses to T. annulata, this study set out to identify and characterize the variability of the target antigens. Three antigens were identified by screening expressed parasite cDNAs with specific CD8 T cell lines. In cattle expressing the A10 class I major histocompatibility complex haplotype, A10-restricted CD8 T cell responses were shown to be focused entirely on a single dominant epitope in one of these antigens (Ta9). Sequencing of the Ta9 gene from field isolates of T. annulata demonstrated extensive sequence divergence, resulting in amino acid polymorphism within the A10-restricted epitope and a second A14-restricted epitope. Statistical analysis of the allelic sequences revealed evidence of positive selection for amino acid substitutions within the region encoding the CD8 T cell epitopes. Sequence differences in the A10-restricted epitope were shown to result in differential recognition by individual CD8 T cell clones, while clones also differed in their ability to recognize different alleles. Moreover, the representation of these clonal specificities within the responding CD8 T cell populations differed between animals. As well as providing an explanation for incomplete protection observed after heterologous parasite challenge of vaccinated cattle, these results have important implications for the choice of antigens for the development of novel subunit vaccines. PMID:21300773

  8. Variable epitope library carrying heavily mutated survivin-derived CTL epitope variants as a new class of efficient vaccine immunogen tested in a mouse model of breast cancer.

    PubMed

    NoeDominguez-Romero, Allan; Zamora-Alvarado, Rubén; Servín-Blanco, Rodolfo; Pérez-Hernández, Erendira G; Castrillon-Rivera, Laura E; Munguia, Maria Elena; Acero, Gonzalo; Govezensky, Tzipe; Gevorkian, Goar; Manoutcharian, Karen

    2014-01-01

    The antigenic variability of tumor cells leading to dynamic changes in cancer epitope landscape along with escape from immune surveillance by down-regulating tumor antigen expression/presentation and immune tolerance are major obstacles for the design of effective vaccines. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response as well as HIV-neutralizing antibodies. In this proof-of-concept study, we tested immunogenic properties and anti-tumor effects of the VELs bearing survivin-derived CTL epitope (GWEPDDNPI) variants in an aggressive metastatic mouse 4T1 breast tumor model. The constructed VELs had complexities of 10,500 and 8,000 individual members, generated as combinatorial M13 phage display and synthetic peptide libraries, respectively, with structural composition GWXPXDXPI, where X is any of 20 natural amino acids. Statistically significant tumor growth inhibition was observed in BALB/c mice immunized with the VELs in both prophylactic and therapeutic settings. Vaccinated mice developed epitope-specific spleen cell and CD8+ IFN-γ+ T-cell responses that recognize more than 50% of the panel of 87 mutated epitope variants, as demonstrated in T-cell proliferation assays and FACS analysis. These data indicate the feasibility of the application of this new class of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against cancer.

  9. Variable epitope library carrying heavily mutated survivin-derived CTL epitope variants as a new class of efficient vaccine immunogen tested in a mouse model of breast cancer

    PubMed Central

    NoeDominguez-Romero, Allan; Zamora-Alvarado, Rubén; Servín-Blanco, Rodolfo; Pérez-Hernández, Erendira G; Castrillon-Rivera, Laura E; Munguia, Maria Elena; Acero, Gonzalo; Govezensky, Tzipe; Gevorkian, Goar; Manoutcharian, Karen

    2014-01-01

    The antigenic variability of tumor cells leading to dynamic changes in cancer epitope landscape along with escape from immune surveillance by down-regulating tumor antigen expression/presentation and immune tolerance are major obstacles for the design of effective vaccines. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response as well as HIV-neutralizing antibodies. In this proof-of-concept study, we tested immunogenic properties and anti-tumor effects of the VELs bearing survivin-derived CTL epitope (GWEPDDNPI) variants in an aggressive metastatic mouse 4T1 breast tumor model. The constructed VELs had complexities of 10,500 and 8,000 individual members, generated as combinatorial M13 phage display and synthetic peptide libraries, respectively, with structural composition GWXPXDXPI, where X is any of 20 natural amino acids. Statistically significant tumor growth inhibition was observed in BALB/c mice immunized with the VELs in both prophylactic and therapeutic settings. Vaccinated mice developed epitope-specific spleen cell and CD8+ IFN-γ+ T-cell responses that recognize more than 50% of the panel of 87 mutated epitope variants, as demonstrated in T-cell proliferation assays and FACS analysis. These data indicate the feasibility of the application of this new class of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against cancer. PMID:25483665

  10. CD8 T cells and Mycobacterium tuberculosis infection.

    PubMed

    Lin, Philana Ling; Flynn, JoAnne L

    2015-05-01

    Tuberculosis is primarily a respiratory disease that is caused by Mycobacterium tuberculosis. M. tuberculosis can persist and replicate in macrophages in vivo, usually in organized cellular structures called granulomas. There is substantial evidence for the importance of CD4 T cells in control of tuberculosis, but the evidence for a requirement for CD8 T cells in this infection has not been proven in humans. However, animal model data support a non-redundant role for CD8 T cells in control of M. tuberculosis infection. In humans, infection with this pathogen leads to generation of specific CD8 T cell responses. These responses include classical (MHC Class I restricted) and non-classical CD8 T cells. Here, we discuss the potential roles of CD8 T cells in defense against tuberculosis, and our current understanding of the wide range of CD8 T cell types seen in M. tuberculosis infection.

  11. Selection of peptide ligands for the antimucin core antibody C595 using phage display technology: definition of candidate epitopes for a cancer vaccine.

    PubMed

    Laing, P; Tighe, P; Kwiatkowski, E; Milligan, J; Price, M; Sewell, H

    1995-06-01

    Aims-To further define the specificity of the antimucin core antibody C595 by fitting it with a family of hexapeptide ligands by immunoselection of filamentous bacteriophage from a gene III display library of approximately 6.4 x 10(7) random hexapeptides.Methods-Three rounds of immuno-selection were used to enrich for C595 binding phage. DNA sequencing revealed the hexapeptides expressed. Bacteriophage and corresponding synthetic hexapeptides were used in ELISA assay to determine binding affinities.Results-Twenty nine clones from this selected population were analysed. Seven contained the natural epitope RPAP, encoded by two different DNA sequences; 17/29 contained the motif RLPP. In all, 28/29 clones contained the motif RXXP and one clone (RVRPAP) contained the motif RXXP in two peptidic registers; 24/28 clones (6/8 DNA sequences) contained a hydrophobic residue (V or I) at position 1 relative to the RXXP motif. In addition the proximity of RXXP to glycine (position 5) suggests that this contributes in the natural epitope to antibody/antigen binding, which was not detected by chemical synthetic methods. One clone, KSKAGV, bears no obvious relationship to the natural epitope and therefore qualifies as a weakly binding mimotope.Conclusions-This approach has rapidly defined the specificity of this antibody in unprecedented detail, and provides a more comprehensive molecular basis for exploring the immune recognition of the MUC1 mucin by the C595 antibody. Importantly, the novel but related epitopes seen provide peptide specificities and a strategy which may prove useful in generating cancer vaccine candidates.

  12. Killing of Targets by CD8+ T Cells in the Mouse Spleen Follows the Law of Mass Action

    PubMed Central

    Ganusov, Vitaly V.; Barber, Daniel L.; De Boer, Rob J.

    2011-01-01

    It has been difficult to correlate the quality of CD8 T cell responses with protection against viral infections. To investigate the relationship between efficacy and magnitude of T cell responses, we quantify the rate at which individual CD8 effector and memory T cells kill target cells in the mouse spleen. Using mathematical modeling, we analyze recent data on the loss of target cells pulsed with three different peptides from the mouse lymphocytic choriomeningitis virus (LCMV) in mouse spleens with varying numbers of epitope-specific CD8 T cells. We find that the killing of targets follows the law of mass-action, i.e., the death rate of individual target cells remains proportional to the frequency (or the total number) of specific CD8 T cells in the spleen despite the fact that effector cell densities and effector to target ratios vary about a 1000-fold. The killing rate of LCMV-specific CD8 T cells is largely independent of T cell specificity and differentiation stage. Our results thus allow one to calculate the critical T cell concentration at which growth of a virus with a given replication rate can be prevented from the start of infection by memory CD8 T cell response. PMID:21283669

  13. Full-Breadth Analysis of CD8+ T-Cell Responses in Acute Hepatitis C Virus Infection and Early Therapy

    PubMed Central

    Lauer, Georg M.; Lucas, Michaela; Timm, Joerg; Ouchi, Kei; Kim, Arthur Y.; Day, Cheryl L.; zur Wiesch, Julian Schulze; Paranhos-Baccala, Glaucia; Sheridan, Isabelle; Casson, Deborah R.; Reiser, Markus; Gandhi, Rajesh T.; Li, Bin; Allen, Todd M.; Chung, Raymond T.; Klenerman, Paul; Walker, Bruce D.

    2005-01-01

    Multispecific CD8+ T-cell responses are thought to be important for the control of acute hepatitis C virus (HCV) infection, but to date little information is actually available on the breadth of responses at early time points. Additionally, the influence of early therapy on these responses and their relationships to outcome are controversial. To investigate this issue, we performed comprehensive analysis of the breadth and frequencies of virus-specific CD8+ T-cell responses on the single epitope level in eight acutely infected individuals who were all started on early therapy. During the acute phase, responses against up to five peptides were identified. During therapy, CD8+ T-cell responses decreased rather than increased as virus was controlled, and no new specificities emerged. A sustained virological response following completion of treatment was independent of CD8+ T-cell responses, as well as CD4+ T-cell responses. Rapid recrudescence also occurred despite broad CD8+ T-cell responses. Importantly, in vivo suppression of CD3+ T cells using OKT3 in one subject did not result in recurrence of viremia. These data suggest that broad CD8+ T-cell responses alone may be insufficient to contain HCV replication, and also that early therapy is effective independent of such responses. PMID:16189000

  14. Unsupervised learning techniques reveal heterogeneity in memory CD8(+) T cell differentiation following acute, chronic and latent viral infections.

    PubMed

    Liu, Mingyong; Barton, Erik S; Jennings, Ryan N; Oldenburg, Darby G; Whirry, Juliann M; White, Douglas W; Grayson, Jason M

    2017-09-01

    CD8(+) T lymphocytes are critical for the control of gammaherpesvirus latency. To determine how memory CD8(+) T cells generated during latency differ from those primed during acute or chronic viral infection, we adoptively transferred naive P14 CD8(+) T cells into uninfected recipients, and examined surface proteins, cytokines and transcription factors following infection with the Armstrong (acute) or Clone 13 (chronic) strains of lymphocytic choriomeningitis virus (LCMV), or murine gammaherpesvirus 68 (MHV68) expressing the LCMV epitope D(b)GP33-41. By performing k-means clustering and generating self organizing maps (SOM), we observed increased short-lived effector-like, CD27(l)(o) CD62L(l)(o) and Bcl-6(l)(o) CD8(+) T cells following latent infection. In addition, we found that memory CD8(+) T cells from latent primed mice underwent less expansion following adoptive transfer and antigen rechallenge. Data from cluster models were combined and visualized by principal component analysis (PCA) demonstrating memory CD8(+) T cells from latent infection occupy an intermediate differentiation space. Copyright © 2017. Published by Elsevier Inc.

  15. IL-2 complex treatment amplifies CD8(+) T cell mediated immunity following herpes simplex virus-1 infection.

    PubMed

    Rajasagi, Naveen K; Rouse, Barry T

    2016-12-01

    CD8(+) T cells play an important role in controlling numerous virus infections and some tumors and therefore several strategies have been adopted to modulate CD8(+) T cell responses. One such approach includes treatment with IL-2 bound to a monoclonal antibody against IL-2 (IL-2 complex) which was shown to enhance CD8(+) T cell responses and provide protection against some cancers and pathogens. This report analyses the value of IL-2 complex therapy to protect against a cutaneous virus infection as occurs with herpes simplex virus-1 (HSV-1) infection. Treatment with IL-2 complex after infection reduced virus levels and lesion severity in a zosteriform model of HSV infection in mice. Furthermore, IL-2 complex treatment expanded HSV-1-gB epitope-specific CD8(+) T cells, IFN-γ and TNF-α producing CD8(+) T cells as well as cells that produced more than one cytokine. In addition, IL-2 complex therapy recipients showed enhanced cytolytic activity of CD8(+) T cells as shown by increased granzyme B expression and lytic granule release. Taken, together, these studies demonstrate that IL-2 complex therapy can be useful to boost protection against a cutaneous virus infection. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  16. Cytomegalovirus vector expressing RAE-1γ induces enhanced anti-tumor capacity of murine CD8(+) T cells.

    PubMed

    Tršan, Tihana; Vuković, Kristina; Filipović, Petra; Brizić, Ana Lesac; Lemmermann, Niels A W; Schober, Kilian; Busch, Dirk H; Britt, William J; Messerle, Martin; Krmpotić, Astrid; Jonjić, Stipan

    2017-08-01

    Designing CD8(+) T-cell vaccines, which would provide protection against tumors is still considered a great challenge in immunotherapy. Here we show the robust potential of cytomegalovirus (CMV) vector expressing the NKG2D ligand RAE-1γ as CD8(+) T cell-based vaccine against malignant tumors. Immunization with the CMV vector expressing RAE-1γ, delayed tumor growth or even provided complete protection against tumor challenge in both prophylactic and therapeutic settings. Moreover, a potent tumor control in mice vaccinated with this vector can be further enhanced by blocking the immune checkpoints TIGIT and PD-1. CMV vector expressing RAE-1γ potentiated expansion of KLRG1(+) CD8(+) T cells with enhanced effector properties. This vaccination was even more efficient in neonatal mice, resulting in the expansion and long-term maintenance of epitope-specific CD8(+) T cells conferring robust resistance against tumor challenge. Our data show that immunomodulation of CD8(+) T-cell responses promoted by herpesvirus expressing a ligand for NKG2D receptor can provide a powerful platform for the prevention and treatment of CD8(+) T-cell sensitive tumors. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Prolonged presence of effector-memory CD8 T cells in the central nervous system after dengue virus encephalitis.

    PubMed

    van der Most, Robbert G; Murali-Krishna, Kaja; Ahmed, Rafi

    2003-01-01

    Dengue virus infection in the central nervous system (CNS) of immunized mice results in a strong influx of CD8 T cells into the brain. Whereas the kinetics of the splenic antiviral response are conventional, i.e. expansion followed by a rapid drop in the frequency of specific CD8 T cells, dengue virus-specific CD8 T cells are retained in the CNS at a high frequency. These CD8 T cells display a partially activated phenotype (CD69(high), Ly-6A/E(high), CD62L(low)), characteristic for effector-memory T cells. CD43 expression, visualized by staining with the 1B11 mAb, decreased in time, suggesting that these persisting CD8 T cells differentiated into memory cells. These data add to the growing evidence implicating the CNS as a non-lymphoid tissue capable of supporting prolonged T cell survival/maintenance.

  18. Consensus nomenclature for CD8+ T cell phenotypes in cancer

    PubMed Central

    Apetoh, Lionel; Smyth, Mark J.; Drake, Charles G.; Abastado, Jean-Pierre; Apte, Ron N.; Ayyoub, Maha; Blay, Jean-Yves; Bonneville, Marc; Butterfield, Lisa H.; Caignard, Anne; Castelli, Chiara; Cavallo, Federica; Celis, Esteban; Chen, Lieping; Colombo, Mario P.; Comin-Anduix, Begoña; Coukos, Georges; Dhodapkar, Madhav V.; Dranoff, Glenn; Frazer, Ian H.; Fridman, Wolf-Hervé; Gabrilovich, Dmitry I.; Gilboa, Eli; Gnjatic, Sacha; Jäger, Dirk; Kalinski, Pawel; Kaufman, Howard L.; Kiessling, Rolf; Kirkwood, John; Knuth, Alexander; Liblau, Roland; Lotze, Michael T.; Lugli, Enrico; Marincola, Francesco; Melero, Ignacio; Melief, Cornelis J.; Mempel, Thorsten R.; Mittendorf, Elizabeth A.; Odun, Kunle; Overwijk, Willem W.; Palucka, Anna Karolina; Parmiani, Giorgio; Ribas, Antoni; Romero, Pedro; Schreiber, Robert D.; Schuler, Gerold; Srivastava, Pramod K.; Tartour, Eric; Valmori, Danila; van der Burg, Sjoerd H.; van der Bruggen, Pierre; van den Eynde, Benoît J.; Wang, Ena; Zou, Weiping; Whiteside, Theresa L.; Speiser, Daniel E.; Pardoll, Drew M.; Restifo, Nicholas P.; Anderson, Ana C.

    2015-01-01

    Whereas preclinical investigations and clinical studies have established that CD8+ T cells can profoundly affect cancer progression, the underlying mechanisms are still elusive. Challenging the prevalent view that the beneficial effect of CD8+ T cells in cancer is solely attributable to their cytotoxic activity, several reports have indicated that the ability of CD8+ T cells to promote tumor regression is dependent on their cytokine secretion profile and their ability to self-renew. Evidence has also shown that the tumor microenvironment can disarm CD8+ T cell immunity, leading to the emergence of dysfunctional CD8+ T cells. The existence of different types of CD8+ T cells in cancer calls for a more precise definition of the CD8+ T cell immune phenotypes in cancer and the abandonment of the generic terms “pro-tumor” and “antitumor.” Based on recent studies investigating the functions of CD8+ T cells in cancer, we here propose some guidelines to precisely define the functional states of CD8+ T cells in cancer. PMID:26137416

  19. Consensus nomenclature for CD8(+) T cell phenotypes in cancer.

    PubMed

    Apetoh, Lionel; Smyth, Mark J; Drake, Charles G; Abastado, Jean-Pierre; Apte, Ron N; Ayyoub, Maha; Blay, Jean-Yves; Bonneville, Marc; Butterfield, Lisa H; Caignard, Anne; Castelli, Chiara; Cavallo, Federica; Celis, Esteban; Chen, Lieping; Colombo, Mario P; Comin-Anduix, Begoña; Coukos, Georges; Dhodapkar, Madhav V; Dranoff, Glenn; Frazer, Ian H; Fridman, Wolf-Hervé; Gabrilovich, Dmitry I; Gilboa, Eli; Gnjatic, Sacha; Jäger, Dirk; Kalinski, Pawel; Kaufman, Howard L; Kiessling, Rolf; Kirkwood, John; Knuth, Alexander; Liblau, Roland; Lotze, Michael T; Lugli, Enrico; Marincola, Francesco; Melero, Ignacio; Melief, Cornelis J; Mempel, Thorsten R; Mittendorf, Elizabeth A; Odun, Kunle; Overwijk, Willem W; Palucka, Anna Karolina; Parmiani, Giorgio; Ribas, Antoni; Romero, Pedro; Schreiber, Robert D; Schuler, Gerold; Srivastava, Pramod K; Tartour, Eric; Valmori, Danila; van der Burg, Sjoerd H; van der Bruggen, Pierre; van den Eynde, Benoît J; Wang, Ena; Zou, Weiping; Whiteside, Theresa L; Speiser, Daniel E; Pardoll, Drew M; Restifo, Nicholas P; Anderson, Ana C

    2015-04-01

    Whereas preclinical investigations and clinical studies have established that CD8(+) T cells can profoundly affect cancer progression, the underlying mechanisms are still elusive. Challenging the prevalent view that the beneficial effect of CD8(+) T cells in cancer is solely attributable to their cytotoxic activity, several reports have indicated that the ability of CD8(+) T cells to promote tumor regression is dependent on their cytokine secretion profile and their ability to self-renew. Evidence has also shown that the tumor microenvironment can disarm CD8(+) T cell immunity, leading to the emergence of dysfunctional CD8(+) T cells. The existence of different types of CD8(+) T cells in cancer calls for a more precise definition of the CD8(+) T cell immune phenotypes in cancer and the abandonment of the generic terms "pro-tumor" and "antitumor." Based on recent studies investigating the functions of CD8(+) T cells in cancer, we here propose some guidelines to precisely define the functional states of CD8(+) T cells in cancer.

  20. CD8+ T Cells in Leishmania Infections: Friends or Foes?

    PubMed Central

    Stäger, Simona; Rafati, Sima

    2012-01-01

    Host protection against several intracellular pathogens requires the induction of CD8+ T cell responses. CD8+ T cells are potent effector cells that can produce high amounts of pro-inflammatory cytokines and kill infected target cells efficiently. However, a protective role for CD8+ T cells during Leishmania infections is still controversial and largely depends on the infection model. In this review, we discuss the role of CD8+ T cells during various types of Leishmania infections, following vaccination, and as potential immunotherapeutic targets. PMID:22566891

  1. Definition of the HLA-A2 restricted peptides recognized by human CD8+ effector T cells by flow-assisted sorting of the CD8+ CD45RA+ CD28- T cell subpopulation.

    PubMed

    Höhn, H; Jülch, M; Pilch, H; Kortsik, C; Tully, G; Neukirch, C; Freitag, K; Maeurer, M

    2003-01-01

    In response to antigenic stimulation, naive MHC-class I restricted and antigen-specific CD8+ CD45RA+ CD28+ T cells undergo clonal expansion, differentiate into CD8+ CD45RO+ memory T cells and convert to CD8+ CD45RA+ CD28- T cells displaying potent immune effector functions upon re-encounter with the nominal antigen. We show that the effector CD8+ CD45RA+ CD28- T cell subset is expanded in peripheral blood lymphocytes (PBL) from patients with human papilloma virus (HPV)+ cervical lesions as well as in PBL from patients with pulmonary tuberculosis. Flow-cytometric cell sorted CD8+ CD45RA+ CD28- and CD8+ CD45RA+ CD28- T cells were tested for recognition of HLA-A2 restricted peptides derived either from the human papillomavirus (HPV)16-E7 gene product, or from M. tuberculosis antigens. Mostly CD8+ CD45+ CD28- T cells define antigen/peptide-specific and MHC-restricted responses. These data were confirmed in PBL from patients with tuberculosis using HLA-A2 tetramer-complexes loaded with a peptide from the M. tuberculosis Ag85b antigen by flow cytometry. The sorting of this T cell subset enables to determine the fine specificity of CD8+ effector T cells without the need for in vitro manipulation.

  2. Impaired functional responses in follicular lymphoma CD8(+)TIM-3(+) T lymphocytes following TCR engagement.

    PubMed

    Gravelle, Pauline; Do, Catherine; Franchet, Camille; Mueller, Sabina; Oberic, Lucie; Ysebaert, Loïc; Larocca, Luigi Maria; Hohaus, Stefan; Calmels, Marie-Noëlle; Frenois, François-Xavier; Kridel, Robert; Gascoyne, Randy D; Laurent, Guy; Brousset, Pierre; Valitutti, Salvatore; Laurent, Camille

    2016-01-01

    Upregulation of T cell immunoglobulin-3 (TIM-3) has been associated with negative regulation of the immune response in chronic infection and cancer, including lymphoma. Here, we investigated the possible correlation between TIM-3 expression by ex vivo cytotoxic T cells (CTL) from follicular lymphoma (FL) biopsies and their functional unresponsiveness that could limit the favorable impact of CTL on disease progression. We report a high percentage of CD8(+)TIM-3(+)T cells in lymph nodes of FL patients. When compared to their CD8(+)TIM-3(-) counterparts, CD8(+)TIM-3(+) T cells exhibited defective cytokine production following TCR engagement. Furthermore, CD8(+)TIM-3(+) T cells display ex vivo markers of lytic granule release and remain unresponsive to further TCR-induced activation of the lytic machinery. Although confocal microscopy showed that TIM-3 expression on CD8(+) T cells correlated with minor alterations of immunological synapse, a selective reduction of ERK signaling in CD8(+)TIM-3(+)T cells was observed by phospho-flow analysis. Finally, short relapse-free survival despite rituximab(R)-chemotherapy was observed in patients with high content of TIM-3(+) cells and a poor infiltrate of granzyme B(+) T cells in FL lymph nodes. Together, our data indicate that, besides selective TCR early signaling defects, TIM-3 expression correlates with unresponsiveness of ex vivo CD8(+) T cells in FL. They show that scores based on the combination of exhaustion and cytolytic markers in FL microenvironment might be instrumental to identify patients at early risk of relapses following R-chemotherapy.

  3. CD8+ T Cells Specific to Apoptosis-Associated Antigens Predict the Response to Tumor Necrosis Factor Inhibitor Therapy in Rheumatoid Arthritis

    PubMed Central

    Citro, Alessandra; Scrivo, Rossana; Martini, Helene; Martire, Carmela; De Marzio, Paolo; Vestri, Anna Rita; Sidney, John; Sette, Alessandro; Barnaba, Vincenzo; Valesini, Guido

    2015-01-01

    CD8+ T cells specific to caspase-cleaved antigens derived from apoptotic T cells (apoptotic epitopes) represent a principal player in chronic immune activation, which is known to amplify immunopathology in various inflammatory diseases. The purpose of the present study was to investigate the relationship involving these autoreactive T cells, the rheumatoid arthritis immunopathology, and the response to tumor necrosis factor-α inhibitor therapy. The frequency of autoreactive CD8+ T cells specific to various apoptotic epitopes, as detected by both enzyme-linked immunospot assay and dextramers of major histocompatibility complex class I molecules complexed with relevant apoptotic epitopes, was longitudinally analyzed in the peripheral blood of rheumatoid arthritis patients who were submitted to etanercept treatment (or other tumor necrosis factor inhibitors as a control). The percentage of apoptotic epitope-specific CD8+ T cells was significantly higher in rheumatoid arthritis patients than in healthy donors, and correlated with the disease activity. More important, it was significantly more elevated in responders to tumor necrosis factor-α inhibitor therapy than in non-responders before the start of therapy; it significantly dropped only in the former following therapy. These data indicate that apoptotic epitope-specific CD8+ T cells may be involved in rheumatoid arthritis immunopathology through the production of inflammatory cytokines and that they may potentially represent a predictive biomarker of response to tumor necrosis factor-α inhibitor therapy to validate in a larger cohort of patients. PMID:26061065

  4. mRNA Structural Constraints on EBNA1 Synthesis Impact on In Vivo Antigen Presentation and Early Priming of CD8+ T Cells

    PubMed Central

    Lekieffre, Lea; Bhat, Purnima; Martinez, Michelle; Croft, Nathan P.; Kaplan, Warren; Tellam, Ross L.; Khanna, Rajiv

    2014-01-01

    Recent studies have shown that virally encoded mRNA sequences of genome maintenance proteins from herpesviruses contain clusters of unusual structural elements, G-quadruplexes, which modulate viral protein synthesis. Destabilization of these G-quadruplexes can override the inhibitory effect on self-synthesis of these proteins. Here we show that the purine-rich repetitive mRNA sequence of Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) comprising G-quadruplex structures, limits both the presentation of MHC class I-restricted CD8+ T cell epitopes by CD11c+ dendritic cells in draining lymph nodes and early priming of antigen-specific CD8+ T-cells. Destabilization of the G-quadruplex structures through codon-modification significantly enhanced in vivo antigen presentation and activation of virus-specific T cells. Ex vivo imaging of draining lymph nodes by confocal microscopy revealed enhanced antigen-specific T-cell trafficking and APC-CD8+ T-cell interactions in mice primed with viral vectors encoding a codon-modified EBNA1 protein. More importantly, these antigen-specific T cells displayed enhanced expression of the T-box transcription factor and superior polyfunctionality consistent with the qualitative impact of translation efficiency. These results provide an important insight into how viruses exploit mRNA structure to down regulate synthesis of their viral maintenance proteins and delay priming of antigen-specific T cells, thereby establishing a successful latent infection in vivo. Furthermore, targeting EBNA1 mRNA rather than protein by small molecules or antisense oligonucleotides will enhance EBNA1 synthesis and the early priming of effector T cells, to establish a more rapid immune response and prevent persistent infection. PMID:25299404

  5. mRNA Structural constraints on EBNA1 synthesis impact on in vivo antigen presentation and early priming of CD8+ T cells.

    PubMed

    Tellam, Judy T; Zhong, Jie; Lekieffre, Lea; Bhat, Purnima; Martinez, Michelle; Croft, Nathan P; Kaplan, Warren; Tellam, Ross L; Khanna, Rajiv

    2014-10-01

    Recent studies have shown that virally encoded mRNA sequences of genome maintenance proteins from herpesviruses contain clusters of unusual structural elements, G-quadruplexes, which modulate viral protein synthesis. Destabilization of these G-quadruplexes can override the inhibitory effect on self-synthesis of these proteins. Here we show that the purine-rich repetitive mRNA sequence of Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) comprising G-quadruplex structures, limits both the presentation of MHC class I-restricted CD8(+) T cell epitopes by CD11c(+) dendritic cells in draining lymph nodes and early priming of antigen-specific CD8(+) T-cells. Destabilization of the G-quadruplex structures through codon-modification significantly enhanced in vivo antigen presentation and activation of virus-specific T cells. Ex vivo imaging of draining lymph nodes by confocal microscopy revealed enhanced antigen-specific T-cell trafficking and APC-CD8(+) T-cell interactions in mice primed with viral vectors encoding a codon-modified EBNA1 protein. More importantly, these antigen-specific T cells displayed enhanced expression of the T-box transcription factor and superior polyfunctionality consistent with the qualitative impact of translation efficiency. These results provide an important insight into how viruses exploit mRNA structure to down regulate synthesis of their viral maintenance proteins and delay priming of antigen-specific T cells, thereby establishing a successful latent infection in vivo. Furthermore, targeting EBNA1 mRNA rather than protein by small molecules or antisense oligonucleotides will enhance EBNA1 synthesis and the early priming of effector T cells, to establish a more rapid immune response and prevent persistent infection.

  6. Perforin and IL-2 Upregulation Define Qualitative Differences among Highly Functional Virus-Specific Human CD8+ T Cells

    PubMed Central

    Makedonas, George; Hutnick, Natalie; Haney, Danielle; Amick, Alexandra C.; Gardner, Jay; Cosma, Gabriela; Hersperger, Adam R.; Dolfi, Douglas; Wherry, E. John; Ferrari, Guido; Betts, Michael R.

    2010-01-01

    The prevailing paradigm of T lymphocyte control of viral replication is that the protective capacity of virus-specific CD8+ T cells is directly proportional to the number of functions they can perform, with IL-2 production capacity considered critical. Having recently defined rapid perforin upregulation as a novel effector function of antigen-specific CD8+ T cells, here we sought to determine whether new perforin production is a component of polyfunctional CD8+ T cell responses that contributes to the control of several human viral infections: cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza (flu), and adenovirus (Ad). We stimulated normal human donor PBMC with synthetic peptides whose amino acid sequences correspond to defined CTL epitopes in the aforementioned viruses, and then used polychromatic flow cytometry to measure the functional capacity and the phenotype of the responding CD8+ T cells. While EBV and flu-specific CD8+ T cells rarely upregulate perforin, CMV-specific cells often do and Ad stimulates an exceptionally strong perforin response. The differential propensity of CD8+ T cells to produce either IL-2 or perforin is in part related to levels of CD28 and the transcription factor T-bet, as CD8+ T cells that rapidly upregulate perforin harbor high levels of T-bet and those producing IL-2 express high amounts of CD28. Thus, “polyfunctional” profiling of antigen-specific CD8+ T cells must not be limited to simply the number of functions the cell can perform, or one particular memory phenotype, but should actually define which combinations of memory markers and functions are relevant in each pathogenic context. PMID:20221423

  7. Immunization with a mimotope of GD2 ganglioside induces CD8+ T cells that recognize cell adhesion molecules on tumor cells.

    PubMed

    Wierzbicki, Andrzej; Gil, Margaret; Ciesielski, Michael; Fenstermaker, Robert A; Kaneko, Yutaro; Rokita, Hanna; Lau, Joseph T; Kozbor, Danuta

    2008-11-01

    The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.

  8. Masked Selection: A Straightforward and Flexible Approach for the Selection of Binders Against Specific Epitopes and Differentially Expressed Proteins by Phage Display*

    PubMed Central

    Even-Desrumeaux, Klervi; Nevoltris, Damien; Lavaut, Marie Noelle; Alim, Karima; Borg, Jean-Paul; Audebert, Stéphane; Kerfelec, Brigitte; Baty, Daniel; Chames, Patrick

    2014-01-01

    Phage display is a well-established procedure to isolate binders against a wide variety of antigens that can be performed on purified antigens, but also on intact cells. As selection steps are performed in vitro, it is possible to focus the outcome of the selection on relevant epitopes by performing some additional steps, such as depletion or competitive elutions. However in practice, the efficiency of these steps is often limited and can lead to inconsistent results. We have designed a new selection method named masked selection, based on the blockade of unwanted epitopes to favor the targeting of relevant ones. We demonstrate the efficiency and flexibility of this method by selecting single-domain antibodies against a specific portion of a fusion protein, by selecting binders against several members of the seven transmembrane receptor family using transfected HEK cells, or by selecting binders against unknown breast cancer markers not expressed on normal samples. The relevance of this approach for antibody-based therapies was further validated by the identification of four of these markers, Epithelial cell adhesion molecule, Transferrin receptor 1, Metastasis cell adhesion molecule, and Sushi containing domain 2, using immunoprecipitation and mass spectrometry. This new phage display strategy can be applied to any type of antibody fragments or alternative scaffolds, and is especially suited for the rapid discovery and identification of cell surface markers. PMID:24361863

  9. Naturally-occurring human serum antibodies to inner core lipopolysaccharide epitopes of Neisseria meningitidis protect against invasive meningococcal disease caused by isolates displaying homologous inner core structures.

    PubMed

    Jäkel, Anne; Plested, Joyce S; Hoe, J Claire; Makepeace, Katherine; Gidney, Margaret-Anne J; Lacelle, Suzanne; St Michael, Frank; Cox, Andrew D; Richards, James C; Moxon, E Richard

    2008-12-02

    Sera from healthy infants (under 1 year old), toddlers (3-4 years) and adults (18-65 years) were assayed for their ability to bind to inner core (ic) lipopolysaccharide (LPS) epitopes of Neisseria meningitidis. Antibodies (Abs) reacting to inner core structures, including different substitutions of the first heptose (HepI) and second heptose (HepII) residues of the LPS backbone, truncated and fully extended LPS glycoforms, were detected and for each structure, these inner core antibodies showed an age-related pattern of acquisition. A novel column-based methodology was used to affinity purify IgG antibodies in which purified inner core LPS (derived from a mutant MC58) was covalently linked to Sepharose 4B. Comparison of reactivity before and after affinity purification of the pooled sera showed that the purified Abs bound to the surface of N. meningitidis organisms displaying truncated and extended LPS with a homologous inner core region, promoted the deposition of C3b, were opsonophagocytic in vitro and decreased bacteraemia when used to passively protect infants rats. In addition, the purified Abs were bactericidal in vitro against the mutant strain displaying truncated LPS with a homologous inner core region. These results demonstrate that naturally occurring serum human antibodies to N. meningitidis LPS can access inner core epitopes of encapsulated organisms with a fully extended LPS.

  10. Targeted suppression of autoreactive CD8(+) T-cell activation using blocking anti-CD8 antibodies.

    PubMed

    Clement, Mathew; Pearson, James A; Gras, Stephanie; van den Berg, Hugo A; Lissina, Anya; Llewellyn-Lacey, Sian; Willis, Mark D; Dockree, Tamsin; McLaren, James E; Ekeruche-Makinde, Julia; Gostick, Emma; Robertson, Neil P; Rossjohn, Jamie; Burrows, Scott R; Price, David A; Wong, F Susan; Peakman, Mark; Skowera, Ania; Wooldridge, Linda

    2016-10-17

    CD8(+) T-cells play a role in the pathogenesis of autoimmune diseases such as multiple sclerosis and type 1 diabetes. However, drugs that target the entire CD8(+) T-cell population are not desirable because the associated lack of specificity can lead to unwanted consequences, most notably an enhanced susceptibility to infection. Here, we show that autoreactive CD8(+) T-cells are highly dependent on CD8 for ligand-induced activation via the T-cell receptor (TCR). In contrast, pathogen-specific CD8(+) T-cells are relatively CD8-independent. These generic differences relate to an intrinsic dichotomy that segregates self-derived and exogenous antigen-specific TCRs according to the monomeric interaction affinity with cognate peptide-major histocompatibility complex class I (pMHCI). As a consequence, "blocking" anti-CD8 antibodies can suppress autoreactive CD8(+) T-cell activation in a relatively selective manner. These findings provide a rational basis for the development and in vivo assessment of novel therapeutic strategies that preferentially target disease-relevant autoimmune responses within the CD8(+) T-cell compartment.

  11. Targeted suppression of autoreactive CD8+ T-cell activation using blocking anti-CD8 antibodies

    PubMed Central

    Clement, Mathew; Pearson, James A.; Gras, Stephanie; van den Berg, Hugo A.; Lissina, Anya; Llewellyn-Lacey, Sian; Willis, Mark D.; Dockree, Tamsin; McLaren, James E.; Ekeruche-Makinde, Julia; Gostick, Emma; Robertson, Neil P.; Rossjohn, Jamie; Burrows, Scott R.; Price, David A.; Wong, F. Susan; Peakman, Mark; Skowera, Ania; Wooldridge, Linda

    2016-01-01

    CD8+ T-cells play a role in the pathogenesis of autoimmune diseases such as multiple sclerosis and type 1 diabetes. However, drugs that target the entire CD8+ T-cell population are not desirable because the associated lack of specificity can lead to unwanted consequences, most notably an enhanced susceptibility to infection. Here, we show that autoreactive CD8+ T-cells are highly dependent on CD8 for ligand-induced activation via the T-cell receptor (TCR). In contrast, pathogen-specific CD8+ T-cells are relatively CD8-independent. These generic differences relate to an intrinsic dichotomy that segregates self-derived and exogenous antigen-specific TCRs according to the monomeric interaction affinity with cognate peptide-major histocompatibility complex class I (pMHCI). As a consequence, “blocking” anti-CD8 antibodies can suppress autoreactive CD8+ T-cell activation in a relatively selective manner. These findings provide a rational basis for the development and in vivo assessment of novel therapeutic strategies that preferentially target disease-relevant autoimmune responses within the CD8+ T-cell compartment. PMID:27748447

  12. Initial HIV-1 antigen-specific CD8+ T cells in acute HIV-1 infection inhibit transmitted/founder virus replication.

    PubMed

    Freel, Stephanie A; Picking, Ralph A; Ferrari, Guido; Ding, Haitao; Ochsenbauer, Christina; Kappes, John C; Kirchherr, Jennifer L; Soderberg, Kelly A; Weinhold, Kent J; Cunningham, Coleen K; Denny, Thomas N; Crump, John A; Cohen, Myron S; McMichael, Andrew J; Haynes, Barton F; Tomaras, Georgia D

    2012-06-01

    CD8-mediated virus inhibition can be detected in HIV-1-positive subjects who naturally control virus replication. Characterizing the inhibitory function of CD8(+) T cells during acute HIV-1 infection (AHI) can elucidate the nature of the CD8(+) responses that can be rapidly elicited and that contribute to virus control. We examined the timing and HIV-1 antigen specificity of antiviral CD8(+) T cells during AHI. Autologous and heterologous CD8(+) T cell antiviral functions were assessed longitudinally during AHI in five donors from the CHAVI 001 cohort using a CD8(+) T cell-mediated virus inhibition assay (CD8 VIA) and transmitted/founder (T/F) viruses. Potent CD8(+) antiviral responses against heterologous T/F viruses appeared during AHI at the first time point sampled in each of the 5 donors (Fiebig stages 1/2 to 5). Inhibition of an autologous T/F virus was durable to 48 weeks; however, inhibition of heterologous responses declined concurrent with the resolution of viremia. HIV-1 viruses from 6 months postinfection were more resistant to CD8(+)-mediated virus inhibition than cognate T/F viruses, demonstrating that the virus escapes early from CD8(+) T cell-mediated inhibition of virus replication. CD8(+) T cell antigen-specific subsets mediated inhibition of T/F virus replication via soluble components, and these soluble responses were stimulated by peptide pools that include epitopes that were shown to drive HIV-1 escape during AHI. These data provide insights into the mechanisms of CD8-mediated virus inhibition and suggest that functional analyses will be important for determining whether similar antigen-specific virus inhibition can be induced by T cell-directed vaccine strategies.

  13. Specific mutation of a gammaherpesvirus-expressed antigen in response to CD8 T cell selection in vivo.

    PubMed

    Loh, Joy; Popkin, Daniel L; Droit, Lindsay; Braaten, Douglas C; Zhao, Guoyan; Zhang, Xin; Vachharajani, Punit; Myers, Nancy; Hansen, Ted H; Virgin, Herbert W

    2012-03-01

    Herpesviruses are thought to be highly genetically stable, and their use as vaccine vectors has been proposed. However, studies of the human gammaherpesvirus, Epstein-Barr virus, have found viral isolates containing mutations in HLA class I-restricted epitopes. Using murine gammaherpesvirus 68 expressing ovalbumin (OVA), we examined the stability of a gammaherpesvirus antigenic locus under strong CD8 T cell selection in vivo. OVA-specific CD8 T cells selected viral isolates containing mutations in the OVA locus but minimal alterations in other genomic regions. Thus, a CD8 T cell response to a gammaherpesvirus-expressed antigen that is not essential for replication or pathogenesis can result in selective mutation of that antigen in vivo. This finding may have relevance for the use of herpesvirus vectors for chronic antigen expression in vivo.

  14. EBNA1 antigen-specific CD8+ T cells in cerebrospinal fluid of patients with multiple sclerosis.

    PubMed

    Erdur, Hebun; Scholz, Veronika; Streitz, Mathias; Hammer, Markus; Meisel, Christian; Schönemann, Constanze; Wandinger, Klaus-Peter; Rosche, Berit

    2016-05-15

    Epidemiological data suggests that Epstein-Barr virus may be involved in the pathogenesis of Multiple Sclerosis (MS). We aimed to determine the frequency of CD8+ T cells specific for one EBNA1-derived epitope (HPVGEADYFEY) in cerebrospinal fluid (CSF) and blood of patients with MS and other inflammatory neurological diseases (OIND). The frequency of specific CD8+ T cells was assessed by HLA-class-I-binding pentamers restricted to HLA-B35. The frequency of HPVGEADYFEY-specific CD8+ T cells did neither differ significantly in blood nor CSF in MS compared to OIND, but was consistently higher in CSF compared to blood regardless of diagnosis. Copyright © 2016. Published by Elsevier B.V.

  15. Comprehensive analysis of dengue virus-specific responses supports an HLA-linked protective role for CD8+ T cells

    PubMed Central

    Weiskopf, Daniela; Angelo, Michael A.; de Azeredo, Elzinandes L.; Sidney, John; Greenbaum, Jason A.; Fernando, Anira N.; Broadwater, Anne; Kolla, Ravi V.; De Silva, Aruna D.; de Silva, Aravinda M.; Mattia, Kimberly A.; Doranz, Benjamin J.; Grey, Howard M.; Shresta, Sujan; Peters, Bjoern; Sette, Alessandro

    2013-01-01

    The role of CD8+ T cells in dengue virus infection and subsequent disease manifestations is not fully understood. According to the original antigenic sin theory, skewing of T-cell responses induced by primary infection with one serotype causes less effective response upon secondary infection with a different serotype, predisposing individuals to severe disease. A comprehensive analysis of CD8+ responses in the general population from the Sri Lankan hyperendemic area, involving the measurement of ex vivo IFNγ responses associated with more than 400 epitopes, challenges the original antigenic sin theory. Although skewing of responses toward primary infecting viruses was detected, this was not associated with impairment of responses either qualitatively or quantitatively. Furthermore, we demonstrate higher magnitude and more polyfunctional responses for HLA alleles associated with decreased susceptibility to severe disease, suggesting that a vigorous response by multifunctional CD8+ T cells is associated with protection from dengue virus disease. PMID:23580623

  16. Comprehensive analysis of dengue virus-specific responses supports an HLA-linked protective role for CD8+ T cells.

    PubMed

    Weiskopf, Daniela; Angelo, Michael A; de Azeredo, Elzinandes L; Sidney, John; Greenbaum, Jason A; Fernando, Anira N; Broadwater, Anne; Kolla, Ravi V; De Silva, Aruna D; de Silva, Aravinda M; Mattia, Kimberly A; Doranz, Benjamin J; Grey, Howard M; Shresta, Sujan; Peters, Bjoern; Sette, Alessandro

    2013-05-28

    The role of CD8(+) T cells in dengue virus infection and subsequent disease manifestations is not fully understood. According to the original antigenic sin theory, skewing of T-cell responses induced by primary infection with one serotype causes less effective response upon secondary infection with a different serotype, predisposing individuals to severe disease. A comprehensive analysis of CD8(+) responses in the general population from the Sri Lankan hyperendemic area, involving the measurement of ex vivo IFNγ responses associated with more than 400 epitopes, challenges the original antigenic sin theory. Although skewing of responses toward primary infecting viruses was detected, this was not associated with impairment of responses either qualitatively or quantitatively. Furthermore, we demonstrate higher magnitude and more polyfunctional responses for HLA alleles associated with decreased susceptibility to severe disease, suggesting that a vigorous response by multifunctional CD8(+) T cells is associated with protection from dengue virus disease.

  17. CD8+ T cells from HLA-B*57 elite suppressors effectively suppress replication of HIV-1 escape mutants.

    PubMed

    Pohlmeyer, Christopher W; Buckheit, Robert W; Siliciano, Robert F; Blankson, Joel N

    2013-12-12

    Elite Controllers or Suppressors (ES) are HIV-1 positive individuals who maintain plasma viral loads below the limit of detection of standard clinical assays without antiretroviral therapy. Multiple lines of evidence suggest that the control of viral replication in these patients is due to a strong and specific cytotoxic T lymphocyte (CTL) response. The ability of CD8+ T cells to control HIV-1 replication is believed to be impaired by the development of escape mutations. Surprisingly, viruses amplified from the plasma of ES have been shown to contain multiple escape mutations, and it is not clear how immunologic control is maintained in the face of virologic escape. We investigated the effect of escape mutations within HLA*B-57-restricted Gag epitopes on the CD8+ T cell mediated suppression of HIV-1 replication. Using site directed mutagenesis, we constructed six NL4-3 based viruses with canonical escape mutations in one to three HLA*B-57-restricted Gag epitopes. Interestingly, similar levels of CTL-mediated suppression of replication in autologous primary CD4+ T cells were observed for all of the escape mutants. Intracellular cytokine staining was performed in order to determine the mechanisms involved in the suppression of the escape variants. While low baseline CD8+ T cells responses to wild type and escape variant peptides were seen, stimulation of PBMC with either wild type or escape variant peptides resulted in increased IFN-γ and perforin expression. These data presented demonstrate that CD8+ T cells from ES are capable of suppressing replication of virus harboring escape mutations in HLA-B*57-restricted Gag epitopes. Additionally, our data suggest that ES CD8+ T cells are capable of generating effective de novo responses to escape mutants.

  18. CD8+ T cells from HLA-B*57 elite suppressors effectively suppress replication of HIV-1 escape mutants

    PubMed Central

    2013-01-01

    Background Elite Controllers or Suppressors (ES) are HIV-1 positive individuals who maintain plasma viral loads below the limit of detection of standard clinical assays without antiretroviral therapy. Multiple lines of evidence suggest that the control of viral replication in these patients is due to a strong and specific cytotoxic T lymphocyte (CTL) response. The ability of CD8+ T cells to control HIV-1 replication is believed to be impaired by the development of escape mutations. Surprisingly, viruses amplified from the plasma of ES have been shown to contain multiple escape mutations, and it is not clear how immunologic control is maintained in the face of virologic escape. Results We investigated the effect of escape mutations within HLA*B-57-restricted Gag epitopes on the CD8+ T cell mediated suppression of HIV-1 replication. Using site directed mutagenesis, we constructed six NL4-3 based viruses with canonical escape mutations in one to three HLA*B-57-restricted Gag epitopes. Interestingly, similar levels of CTL-mediated suppression of replication in autologous primary CD4+ T cells were observed for all of the escape mutants. Intracellular cytokine staining was performed in order to determine the mechanisms involved in the suppression of the escape variants. While low baseline CD8+ T cells responses to wild type and escape variant peptides were seen, stimulation of PBMC with either wild type or escape variant peptides resulted in increased IFN-γ and perforin expression. Conclusions These data presented demonstrate that CD8+ T cells from ES are capable of suppressing replication of virus harboring escape mutations in HLA-B*57-restricted Gag epitopes. Additionally, our data suggest that ES CD8+ T cells are capable of generating effective de novo responses to escape mutants. PMID:24330837

  19. Infection history determines the differentiation state of human CD8+ T cells.

    PubMed

    van Aalderen, Michiel C; Remmerswaal, Ester B M; Verstegen, Niels J M; Hombrink, Pleun; ten Brinke, Anja; Pircher, Hanspeter; Kootstra, Neeltje A; ten Berge, Ineke J M; van Lier, René A W

    2015-05-01

    After the resolution of the acute phase of infection, otherwise quiescent antigen-experienced CD8(+) T cells confer rapid protection upon reinfection with viral pathogens or, in the case of persistent viruses, help to maintain control of the infection. Depending on the type of virus, antigen-specific CD8(+) T cells have distinct traits, ranging from typical memory cell properties in the case of rapidly cleared viruses to immediate effector functions for persistent viruses. We here show that both the differentiation stage, defined by the expression of cell surface markers, such as CD45RA, CCR7, CD28, and CD27, and distinct expression levels of T-bet and eomesodermin (Eomes) predict the functional profile of antigen-experienced CD8(+) T cells. Furthermore, virus-specific CD8(+) T cells targeting different respiratory syncytial virus-, influenza A virus-, Epstein-Barr virus (EBV)-, human cytomegalovirus (hCMV)-, and HIV-1-specific epitopes adopt distinct T-bet and Eomes expression patterns that appear to be installed early during the primary response. Importantly, the associations between surface phenotype, T-bet/Eomes expression levels, and the expression of markers that predict CD8(+) T-cell function change according to viral infection history, particularly against the background of HIV-1 and, to lesser extent, of human cytomegalovirus and/or Epstein-Barr virus infection. Thus, the functionality of human antigen-experienced CD8(+) T cells follows at least two dimensions, one outlined by the surface phenotype and another by the T-bet/Eomes expression levels, which are determined by previous or persistent viral challenges. Functional human CD8(+) T-cell subsets have been defined using surface markers like CD45RA, CCR7, CD28, and CD27. However, the induction of function-defining traits, like granzyme B expression, is controlled by transcription factors like T-bet and Eomes. Here, we describe how T-bet and Eomes levels distinctly relate to the expression of molecules

  20. Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

    PubMed

    Eikawa, Shingo; Kakimi, Kazuhiro; Isobe, Midori; Kuzushima, Kiyotaka; Luescher, Immanuel; Ohue, Yoshihiro; Ikeuchi, Kazuhiro; Uenaka, Akiko; Nishikawa, Hiroyoshi; Udono, Heiichiro; Oka, Mikio; Nakayama, Eiichi

    2013-01-15

    Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response. Copyright © 2012 UICC.

  1. Active evasion of CTL mediated killing and low quality responding CD8+ T cells contribute to persistence of brucellosis.

    PubMed

    Durward, Marina; Radhakrishnan, Girish; Harms, Jerome; Bareiss, Claire; Magnani, Diogo; Splitter, Gary A

    2012-01-01

    Brucellosis is a common zoonotic disease that remains endemic in many parts of the world. Dissecting the host immune response during this disease provides insight as to why brucellosis is often difficult to resolve. We used a Brucella epitope specific in vivo killing assay to investigate the ability of CD8+ T cells to kill targets treated with purified pathogenic protein. Importantly, we found the pathogenic protein TcpB to be a novel effector of adaptive immune evasion by inhibiting CD8+ T cell killing of Brucella epitope specific target cells in mice. Further, BALB/c mice show active Brucella melitensis infection beyond one year, many with previously unreported focal infection of the urogenital area. A fraction of CD8+ T cells show a CD8+ Tmem phenotype of LFA-1hi, CD127hi, KLRG-1lo during the course of chronic brucellosis, while the CD8+ T cell pool as a whole had a very weak polyfunctional cytokine response with diminished co-expression of IFN-γ with TNFα and/or IL-2, a hallmark of exhaustion. When investigating the expression of these 3 cytokines individually, we observed significant IFN-γ expression at 90 and 180 days post-infection. TNFα expression did not significantly exceed or fall below background levels at any time. IL-2 expression did not significantly exceeded background, but, interestingly, did fall significantly below that of uninfected mice at 180 days post-infection. Brucella melitensis evades and blunts adaptive immunity during acute infection and our findings provide potential mechanisms for the deficit observed in responding CD8+ T cells during chronic brucellosis.

  2. Virus-Specific CD8+ T-Cell Responses Better Define HIV Disease Progression than HLA Genotype ▿ †

    PubMed Central

    Dinges, Warren L.; Richardt, Julia; Friedrich, David; Jalbert, Emilie; Liu, Yi; Stevens, Claire E.; Maenza, Janine; Collier, Ann C.; Geraghty, Daniel E.; Smith, Jeremy; Moodie, Zoe; Mullins, James I.; McElrath, M. Juliana; Horton, Helen

    2010-01-01

    HLA alleles B57/58, B27, and B35 have the strongest genetic associations with HIV-1 disease progression. The mechanisms of these relationships may be host control of HIV-1 infection via CD8+ T-cell responses. We examined these immune responses in subjects from the Seattle Primary Infection Cohort with these alleles. CD8+ T-cell responses to conserved HIV epitopes within B57/58 alleles (TW10 and KF11) and B27 alleles (KK10 and FY10) delayed declines in CD4+ T-cell counts (4 to 8 times longer), while responses to variable epitopes presented by B35 alleles (DL9 and IL9) resulted in more rapid progression. The plasma viral load was higher in B57/58+ and B27+ subjects lacking the conserved B57/58- and B27-restricted responses. The presence of certain B57/58-, B27-, and B35-restricted HIV-specific CD8+ T-cell responses after primary HIV-1 infection better defined disease progression than the HLA genotype alone, suggesting that it is the HIV-specific CD8+ T cells and not the presence of a particular HLA allele that determine disease progression. Further, the most effective host CD8+ T-cell responses to HIV-1 were prevalent within an HLA allele, represented a high total allele fraction of the host CD8+ T-cell response, and targeted conserved regions of HIV-1. These data suggest that vaccine immunogens should contain only conserved regions of HIV-1. PMID:20147397

  3. Pulmonary Dendritic Cell Subsets Shape the Respiratory Syncytial Virus-Specific CD8+ T Cell Immunodominance Hierarchy in Neonates.

    PubMed

    Malloy, Allison M W; Ruckwardt, Tracy J; Morabito, Kaitlyn M; Lau-Kilby, Annie W; Graham, Barney S

    2017-01-01

    Young infants are generally more susceptible to viral infections and experience more severe disease than do adults. CD8(+) T cells are important for viral clearance, and although often ineffective in neonates they can be protective when adequately stimulated. Using a murine CB6F1/J hybrid model of respiratory syncytial virus (RSV) infection, we previously demonstrated that the CD8(+) T cell immunodominance hierarchy to two RSV-derived epitopes, K(d)M282-90 and D(b)M187-195, was determined by the age at infection. To determine whether age-dependent RSV-specific CD8(+) T cell responses could be modified through enhanced innate signaling, we used TLR4 or TLR9 agonist treatment at the time of infection, which remarkably changed the neonatal codominant response to an adult-like K(d)M282-90 CD8(+) T cell immunodominant response. This shift was associated with an increase in the number of conventional dendritic cells, CD11b(+) and CD103(+) dendritic cells, in the lung-draining lymph node, as well as increased expression of the costimulatory molecule CD86. The magnitude of the K(d)M282-90 CD8(+) T cell response in TLR agonist-treated neonates could be blocked with Abs against CD80 and CD86. These studies demonstrate the age-dependent function of conventional dendritic cells, their role in determining immunodominance hierarchy, and epitope-specific CD8(+) T cell requirements for costimulation, all of which influence the immune response magnitude. The unique impact of TLR agonists on neonatal T cell responses is important to consider for RSV vaccines designed for young infants.

  4. Identification of immediate early gene products of bovine herpes virus 1 (BHV-1) as dominant antigens recognized by CD8 T cells in immune cattle.

    PubMed

    Hart, Jane; MacHugh, Niall D; Sheldrake, Tara; Nielsen, Morten; Morrison, W Ivan

    2017-07-01

    In common with other herpes viruses, bovine herpes virus 1 (BHV-1) induces strong virus-specific CD8 T-cell responses. However, there is a paucity of information on the antigenic specificity of the responding T-cells. The development of a system to generate virus-specific CD8 T-cell lines from BHV-1-immune cattle, employing Theileria-transformed cell lines for antigen presentation, has enabled us to address this issue. Use of this system allowed the study to screen for CD8 T-cell antigens that are efficiently presented on the surface of virus-infected cells. Screening of a panel of 16 candidate viral gene products with CD8 T-cell lines from 3 BHV-1-immune cattle of defined MHC genotypes identified 4 antigens, including 3 immediate early (IE) gene products (ICP4, ICP22 and Circ) and a tegument protein (UL49). Identification of the MHC restriction specificities revealed that the antigens were presented by two or three class I MHC alleles in each animal. Six CD8 T-cell epitopes were identified in the three IE proteins by screening of synthetic peptides. Use of an algorithm (NetMHCpan) that predicts the peptide-binding characteristics of restricting MHC alleles confirmed and, in some cases refined, the identity of the epitopes. Analyses of the epitope specificity of the CD8 T-cell lines showed that a large component of the response is directed against these IE epitopes. The results indicate that these IE gene products are dominant targets of the CD8 T-cell response in BHV-I-immune cattle and hence are prime-candidate antigens for the generation of a subunit vaccine.

  5. Broadly targeted CD8+ T cell responses restricted by major histocompatibility complex E

    DOE PAGES

    Hansen, Scott G.; Wu, Helen L.; Burwits, Benjamin J.; ...

    2016-02-12

    Major histocompatibility complex (MHC)-E is a highly conserved, ubiquitously expressed, nonclassical, MHC-Ib molecule with limited polymorphism primarily involved in regulation of NK cell reactivity via interaction with NKG2/CD94 receptors. We found that vaccination of rhesus macaques with Rh157.5/.4 gene-deleted rhesus Cytomegalovirus (RhCMV) vectors uniquely diverts MHC-E function to presentation of highly diverse peptide epitopes to CD8α/β+ T cells, approximately 4 distinct epitopes per 100 amino acids, in all tested protein antigens. Computational structural analysis revealed that a relatively stable, open binding groove in MHC-E attains broad peptide binding specificity by imposing a similar backbone configuration on bound peptides with fewmore » restrictions based on amino acid side chains. Since MHC-E is up-regulated on cells infected with HIV/SIV and other persistent viruses to evade NK cell activity, MHC-E-restricted CD8+ T cell responses have the potential to exploit pathogen immune evasion adaptations, a capability that might endow these unconventional responses with superior efficacy.« less

  6. Broadly targeted CD8+ T cell responses restricted by major histocompatibility complex E

    SciTech Connect

    Hansen, Scott G.; Wu, Helen L.; Burwits, Benjamin J.; Hughes, Colette M.; Hammond, Katherine B.; Ventura, Abigail B.; Reed, Jason S.; Gilbride, Roxanne M.; Ainslie, Emily; Morrow, David W.; Ford, Julia C.; Selseth, Andrea N.; Pathak, Reesab; Malouli, Daniel; Legasse, Alfred W.; Axthelm, Michael K.; Nelson, Jay A.; Gillespie, Geraldine M.; Walters, Lucy C.; Brackenridge, Simon; Sharpe, Hannah R.; Lopez, Cesar Augusto; Fruh, Klaus; Korber, Bette Tina; McMichael, Andrew J.; Gnanakaran, Sandrasegaram; Sacha, Jonah B.; Picker, Louis J.

    2016-02-12

    Major histocompatibility complex (MHC)-E is a highly conserved, ubiquitously expressed, nonclassical, MHC-Ib molecule with limited polymorphism primarily involved in regulation of NK cell reactivity via interaction with NKG2/CD94 receptors. We found that vaccination of rhesus macaques with Rh157.5/.4 gene-deleted rhesus Cytomegalovirus (RhCMV) vectors uniquely diverts MHC-E function to presentation of highly diverse peptide epitopes to CD8α/β+ T cells, approximately 4 distinct epitopes per 100 amino acids, in all tested protein antigens. Computational structural analysis revealed that a relatively stable, open binding groove in MHC-E attains broad peptide binding specificity by imposing a similar backbone configuration on bound peptides with few restrictions based on amino acid side chains. Since MHC-E is up-regulated on cells infected with HIV/SIV and other persistent viruses to evade NK cell activity, MHC-E-restricted CD8+ T cell responses have the potential to exploit pathogen immune evasion adaptations, a capability that might endow these unconventional responses with superior efficacy.

  7. Defining CD8+ T cell determinants during human viral infection in populations of Asian ethnicity.

    PubMed

    Rivino, Laura; Tan, Anthony T; Chia, Adeline; Kumaran, Emmanuelle A P; Grotenbreg, Gijsbert M; MacAry, Paul A; Bertoletti, Antonio

    2013-10-15

    The identification of virus-specific CD8(+) T cell determinants is a fundamental requirement for our understanding of viral disease pathogenesis. T cell epitope mapping strategies increasingly rely on algorithms that predict the binding of peptides to MHC molecules. There is, however, little information on the reliability of predictive algorithms in the context of human populations, in particular, for those expressing HLA class I molecules for which there are limited experimental data available. In this study, we evaluate the ability of NetMHCpan to predict antiviral CD8(+) T cell epitopes that we identified with a traditional approach in patients of Asian ethnicity infected with Dengue virus, hepatitis B virus, or severe acute respiratory syndrome coronavirus. We experimentally demonstrate that the predictive power of algorithms defining peptide-MHC interaction directly correlates with the amount of training data on which the predictive algorithm has been constructed. These results highlight the limited applicability of the NetMHCpan algorithm for populations expressing HLA molecules for which there are little or no experimental binding data, such as those of Asian ethnicity.

  8. Killing of targets by effector CD8 T cells in the mouse spleen follows the law of mass action

    SciTech Connect

    Ganusov, Vitaly V

    2009-01-01

    In contrast with antibody-based vaccines, it has been difficult to measure the efficacy of T cell-based vaccines and to correlate the efficacy of CD8 T cell responses with protection again viral infections. In part, this difficulty is due to poor understanding of the in vivo efficacy of CD8 T cells produced by vaccination. Using a: recently developed experimental method of in vivo cytotoxicity we have investigated quantitative aspects of killing of peptide-pulsed targets by effector and memory CD8 T cells, specific to three epitopes of lymphocytic choriomeningitis virus (LCMV), in the mouse spleen. By analyzing data on killing of targets with varying number of epitope-specific effector and memory CD8 T cells, we find that killing of targets by effectors follows the law of mass-action, that is the death rate of peptide-pulsed targets is proportional to the frequency of CTLs in the spleen. In contrast, killing of targets by memory CD8 T cells does not follow the mass action law because the death rate of targets saturates at high frequencies of memory CD8 T cells. For both effector and memory cells, we also find little support for the killing term that includes the decrease of the death rate of targets with target cell density. Interestingly, our analysis suggests that at low CD8 T cell frequencies, memory CD8 T cells on the per capita basis are more efficient at killing peptide-pulsed targets than effectors, but at high frequencies, effectors are more efficient killers than memory T cells. Comparison of the estimated killing efficacy of effector T cells with the value that is predicted from theoretical physics and based on motility of T cells in lymphoid tissues, suggests that limiting step in the killing of peptide-pulsed targets is delivering the lethal hit and not finding the target. Our results thus form a basis for quantitative understanding of the process of killing of virus-infected cells by T cell responses in tissues and can be used to correlate the

  9. A new model for CD8+ T cell memory inflation based upon a recombinant adenoviral vector1

    PubMed Central

    Bolinger, Beatrice; Sims, Stuart; O’Hara, Geraldine; de Lara, Catherine; Tchilian, Elma; Firner, Sonja; Engeler, Daniel; Ludewig, Burkhard; Klenerman, Paul

    2013-01-01

    CD8+ T cell memory inflation, first described in murine cytomegalovirus (MCMV) infection, is characterized by the accumulation of high-frequency, functional antigen-specific CD8+ T cell pools with an effector-memory phenotype and enrichment in peripheral organs. Although persistence of antigen is considered essential, the rules underpinning memory inflation are still unclear. The MCMV model is, however, complicated by the virus’s low-level persistence, and stochastic reactivation. We developed a new model of memory inflation based upon a βgal-recombinant adenovirus vector (Ad-LacZ). After i.v. administration in C57BL/6 mice we observe marked memory inflation in the βgal96 epitope, while a second epitope, βgal497, undergoes classical memory formation. The inflationary T cell responses show kinetics, distribution, phenotype and functions similar to those seen in MCMV and are reproduced using alternative routes of administration. Memory inflation in this model is dependent on MHC Class II. As in MCMV, only the inflating epitope showed immunoproteasome-independence. These data define a new model for memory inflation, which is fully replication-independent, internally controlled and reproduces the key immunologic features of the CD8+ T cell response. This model provides insight into the mechanisms responsible for memory inflation, and since it is based on a vaccine vector, also is relevant to novel T cell-inducing vaccines in humans. PMID:23509359

  10. Mosaic vaccines elicit CD8+ T lymphocyte responses that confer enhanced immune coverage of diverse HIV strains in monkeys.

    PubMed

    Santra, Sampa; Liao, Hua-Xin; Zhang, Ruijin; Muldoon, Mark; Watson, Sydeaka; Fischer, Will; Theiler, James; Szinger, James; Balachandran, Harikrishnan; Buzby, Adam; Quinn, David; Parks, Robert J; Tsao, Chun-Yen; Carville, Angela; Mansfield, Keith G; Pavlakis, George N; Felber, Barbara K; Haynes, Barton F; Korber, Bette T; Letvin, Norman L

    2010-03-01

    An effective HIV vaccine must elicit immune responses that recognize genetically diverse viruses. It must generate CD8+ T lymphocytes that control HIV replication and CD4+ T lymphocytes that provide help for the generation and maintenance of both cellular and humoral immune responses against the virus. Creating immunogens that can elicit cellular immune responses against the genetically varied circulating isolates of HIV presents a key challenge for creating an HIV vaccine. Polyvalent mosaic immunogens derived by in silico recombination of natural strains of HIV are designed to induce cellular immune responses that recognize genetically diverse circulating virus isolates. Here we immunized rhesus monkeys by plasmid DNA prime and recombinant vaccinia virus boost with vaccine constructs expressing either consensus or polyvalent mosaic proteins. As compared to consensus immunogens, the mosaic immunogens elicited CD8+ T lymphocyte responses to more epitopes of each viral protein than did the consensus immunogens and to more variant sequences of CD8+ T lymphocyte epitopes. This increased breadth and depth of epitope recognition may contribute both to protection against infection by genetically diverse viruses and to the control of variant viruses that emerge as they mutate away from recognition by cytotoxic T lymphocytes.

  11. T-bet and Eomes Are Differentially Linked to the Exhausted Phenotype of CD8+ T Cells in HIV Infection

    PubMed Central

    Buggert, Marcus; Tauriainen, Johanna; Yamamoto, Takuya; Frederiksen, Juliet; Ivarsson, Martin A.; Michaëlsson, Jakob; Lund, Ole; Hejdeman, Bo; Jansson, Marianne; Sönnerborg, Anders; Koup, Richard A.; Betts, Michael R.; Karlsson, Annika C.

    2014-01-01

    CD8+ T cell exhaustion represents a major hallmark of chronic HIV infection. Two key transcription factors governing CD8+ T cell differentiation, T-bet and Eomesodermin (Eomes), have previously been shown in mice to differentially regulate T cell exhaustion in part through direct modulation of PD-1. Here, we examined the relationship between these transcription factors and the expression of several inhibitory receptors (PD-1, CD160, and 2B4), functional characteristics and memory differentiation of CD8+ T cells in chronic and treated HIV infection. The expression of PD-1, CD160, and 2B4 on total CD8+ T cells was elevated in chronically infected individuals and highly associated with a T-betdimEomeshi expressional profile. Interestingly, both resting and activated HIV-specific CD8+ T cells in chronic infection were almost exclusively T-betdimEomeshi cells, while CMV-specific CD8+ T cells displayed a balanced expression pattern of T-bet and Eomes. The T-betdimEomeshi virus-specific CD8+ T cells did not show features of terminal differentiation, but rather a transitional memory phenotype with poor polyfunctional (effector) characteristics. The transitional and exhausted phenotype of HIV-specific CD8+ T cells was longitudinally related to persistent Eomes expression after antiretroviral therapy (ART) initiation. Strikingly, these characteristics remained stable up to 10 years after ART initiation. This study supports the concept that poor human viral-specific CD8+ T cell functionality is due to an inverse expression balance between T-bet and Eomes, which is not reversed despite long-term viral control through ART. These results aid to explain the inability of HIV-specific CD8+ T cells to control the viral replication post-ART cessation. PMID:25032686

  12. Contribution of herpesvirus specific CD8 T cells to anti-viral T cell response in humans.

    PubMed

    Sandalova, Elena; Laccabue, Diletta; Boni, Carolina; Tan, Anthony T; Fink, Katja; Ooi, Eng Eong; Chua, Robert; Shafaeddin Schreve, Bahar; Ferrari, Carlo; Bertoletti, Antonio

    2010-08-19

    Herpesviruses infect most humans. Their infections can be associated with pathological conditions and significant changes in T cell repertoire but evidences of symbiotic effects of herpesvirus latency have never been demonstrated. We tested the hypothesis that HCMV and EBV-specific CD8 T cells contribute to the heterologous anti-viral immune response. Volume of activated/proliferating virus-specific and total CD8 T cells was evaluated in 50 patients with acute viral infections: 20 with HBV, 12 with Dengue, 12 with Influenza, 3 with Adenovirus infection and 3 with fevers of unknown etiology. Virus-specific (EBV, HCMV, Influenza) pentamer+ and total CD8 T cells were analyzed for activation (CD38/HLA-DR), proliferation (Ki-67/Bcl-2(low)) and cytokine production. We observed that all acute viral infections trigger an expansion of activated/proliferating CD8 T cells, which differs in size depending on the infection but is invariably inflated by CD8 T cells specific for persistent herpesviruses (HCMV/EBV). CD8 T cells specific for other non-related non persistent viral infection (i.e. Influenza) were not activated. IL-15, which is produced during acute viral infections, is the likely contributing mechanism driving the selective activation of herpesvirus specific CD8 T cells. In addition we were able to show that herpesvirus specific CD8 T cells displayed an increased ability to produce the anti-viral cytokine interferon-gamma during the acute phase of heterologous viral infection. Taken together, these data demonstrated that activated herpesvirus specific CD8 T cells inflate the activated/proliferating CD8 T cells population present during acute viral infections in human and can contribute to the heterologous anti-viral T cell response.

  13. HIV-TB coinfection impairs CD8(+) T-cell differentiation and function while dehydroepiandrosterone improves cytotoxic antitubercular immune responses.

    PubMed

    Suarez, Guadalupe V; Angerami, Matías T; Vecchione, María B; Laufer, Natalia; Turk, Gabriela; Ruiz, Maria J; Mesch, Viviana; Fabre, Bibiana; Maidana, Patricia; Ameri, Diego; Cahn, Pedro; Sued, Omar; Salomón, Horacio; Bottasso, Oscar A; Quiroga, María F

    2015-09-01

    Tuberculosis (TB) is the leading cause of death among HIV-positive patients. The decreasing frequencies of terminal effector (TTE ) CD8(+) T cells may increase reactivation risk in persons latently infected with Mycobacterium tuberculosis (Mtb). We have previously shown that dehydroepiandrosterone (DHEA) increases the protective antitubercular immune responses in HIV-TB patients. Here, we aimed to study Mtb-specific cytotoxicity, IFN-γ secretion, memory status of CD8(+) T cells, and their modulation by DHEA during HIV-TB coinfection. CD8(+) T cells from HIV-TB patients showed a more differentiated phenotype with diminished naïve and higher effector memory and TTE T-cell frequencies compared to healthy donors both in total and Mtb-specific CD8(+) T cells. Notably, CD8(+) T cells from HIV-TB patients displayed higher Terminal Effector (TTE ) CD45RA(dim) proportions with lower CD45RA expression levels, suggesting a not fully differentiated phenotype. Also, PD-1 expression levels on CD8(+) T cells from HIV-TB patients increased although restricted to the CD27(+) population. Interestingly, DHEA plasma levels positively correlated with TTE in CD8(+) T cells and in vitro DHEA treatment enhanced Mtb-specific cytotoxic responses and terminal differentiation in CD8(+) T cells from HIV-TB patients. Our data suggest that HIV-TB coinfection promotes a deficient CD8(+) T-cell differentiation, whereas DHEA may contribute to improving antitubercular immunity by enhancing CD8(+) T-cell functions during HIV-TB coinfection. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2.

    PubMed

    Tumban, Ebenezer; Peabody, Julianne; Peabody, David S; Chackerian, Bryce

    2011-01-01

    Current human papillomavirus (HPV) vaccines that are based on virus-like particles (VLPs) of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin. L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV) encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV. VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts.

  15. Induction of Specific CD8+ T Cells against Intracellular Bacteria by CD8+ T-Cell-Oriented Immunization Approaches

    PubMed Central

    Nagata, Toshi; Koide, Yukio

    2010-01-01

    For protection against intracellular bacteria such as Mycobacterium tuberculosis and Listeria monocytogenes, the cellular arm of adaptive immunity is necessary. A variety of immunization methods have been evaluated and are reported to induce specific CD8+ T cells against intracellular bacterial infection. Modified BCG vaccines have been examined to enhance CD8+ T-cell responses. Naked DNA vaccination is a promising strategy to induce CD8+ T cells. In addition to this strategy, live attenuated intracellular bacteria such as Shigella, Salmonella, and Listeria have been utilized as carriers of DNA vaccines in animal models. Vaccination with dendritic cells pulsed with antigenic peptides or the cells introduced antigen genes by virus vectors such as retroviruses is also a powerful strategy. Furthermore, vaccination with recombinant lentivirus has been attempted to induce specific CD8+ T cells. Combinations of these strategies (prime-boost immunization) have been studied for the efficient induction of intracellular bacteria-specific CD8+ T cells. PMID:20508818

  16. Contribution of TCR signaling strength to CD8+ T cell peripheral tolerance mechanisms.

    PubMed

    Smith, Trevor R F; Verdeil, Gregory; Marquardt, Kristi; Sherman, Linda A

    2014-10-01

    Peripheral tolerance mechanisms are in place to prevent T cells from mediating aberrant immune responses directed against self and environmental Ags. Mechanisms involved in the induction of peripheral tolerance include T cell-intrinsic pathways, such as anergy or deletion, or exogenous tolerance mediated by regulatory T cells. We have previously shown that the density of peptide-MHC class I recognized by the TCR determines whether CD8(+) T cells undergo anergy or deletion. Specifically, using a TCR-transgenic CD8(+) T cell model, we demonstrated that persistent peripheral exposure to low- or high-dose peptides in the absence of inflammatory signals resulted in clonal deletion or anergy of the T cell, respectively. In this study, by altering the affinity of the peptide-MHC tolerogen for TCR, we have confirmed that this mechanism is dependent on the level of TCR signaling that the CD8(+) T cell receives. Using altered peptide ligands (APLs) displaying high TCR affinities, we show that increasing the TCR signaling favors anergy induction. Conversely, using APLs displaying a decreased TCR affinity tilted our system in the direction of deletional tolerance. We demonstrate how differential peripheral CD8(+) T cell tolerance mechanisms are controlled by both the potency and density of MHC class I-peptide tolerogen.

  17. Enhancing Human Immunodeficiency Virus-Specific CD8+ T Cell Responses with Heteroclitic Peptides

    PubMed Central

    Adegoke, Adeolu Oyemade; Grant, Michael David

    2015-01-01

    Human immunodeficiency virus (HIV)-specific CD8+ T cells play a critical role in containing HIV replication and delaying disease progression. However, HIV-specific CD8+ T cells become progressively more “exhausted” as chronic HIV infection proceeds. Symptoms of T cell exhaustion range from expression of inhibitory receptors and selective loss of cytokine production capacity through reduced proliferative potential, impaired differentiation into effector cells and increased susceptibility to apoptosis. While effective combination antiretroviral therapy (cART) durably reduces HIV viremia to undetectable levels, this alone does not restore the full pluripotency of HIV-specific CD8+ T cells. In a number of studies, a subset of peptide epitope variants categorized as heteroclitic, restimulated more potent cellular immune responses in vitro than did the native, immunizing peptides themselves. This property of heteroclitic peptides has been exploited in experimental cancer and chronic viral infection models to promote clearance of transformed cells and persistent viruses. In this review, we consider the possibility that heteroclitic peptides could improve the efficacy of therapeutic vaccines as part of HIV immunotherapy or eradication strategies. We review literature on heteroclitic peptides and illustrate their potential to beneficially modulate the nature of HIV-specific T cell responses toward those found in the small minority of HIV-infected, aviremic cART-naïve persons termed elite controllers or long-term non-progressors. Our review suggests that the efficacy of HIV vaccines could be improved by identification, testing, and incorporation of heteroclitic variants of native HIV peptide epitopes. PMID:26257743

  18. Associations of HLA-A, HLA-B and HLA-C Alleles Frequency with Prevalence of Herpes Simplex Virus Infections and Diseases Across Global Populations: Implication for the Development of an Universal CD8+ T-Cell Epitope-Based Vaccine

    PubMed Central

    Samandary, Sarah; Kridane-Miledi, Hédia; Sandoval, Jacqueline S.; Choudhury, Zareen; Langa-Vives, Francina; Spencer, Doran; Chentoufi, Aziz A.; Lemonnier, François A.; BenMohamed, Lbachir

    2014-01-01

    A significant portion of the world’s population is infected with herpes simplex virus type 1 and/or type 2 (HSV-1 and/or HSV-2), that cause a wide range of diseases including genital herpes, oro-facial herpes, and the potentially blinding ocular herpes. While the global prevalence and distribution of HSV-1 and HSV-2 infections cannot be exactly established, the general trends indicate that: (i) HSV-1 infections are much more prevalent globally than HSV-2; (ii) Over half billion people worldwide are infected with HSV-2; (iii) the sub-Saharan African populations account for a disproportionate burden of genital herpes infections and diseases; (iv) the dramatic differences in the prevalence of herpes infections between regions of the world appear to be associated with differences in the frequencies of human leukocyte antigen (HLA) alleles. The present report: (i) analyzes the prevalence of HSV-1 and HSV-2 infections across various regions of the world; (ii) analyzes potential associations of common HLA-A, HLA-B and HLA-C alleles with the prevalence of HSV-1 and HSV-2 infections in the Caucasoid, Oriental, Hispanic and Black major populations; and (iii) discusses how our recently developed HLA-A, HLA-B, and HLA-C transgenic/H-2 class I null mice will help validate HLA/herpes prevalence associations. Overall, high prevalence of herpes infection and disease appears to be associated with high frequency of HLA-A*24, HLA-B*27, HLA-B*53 and HLA-B*58 alleles. In contrast, low prevalence of herpes infection and disease appears to be associated with high frequency of HLA-B*44 allele. The finding will aid in developing a T-cell epitope-based universal herpes vaccine and immunotherapy. PMID:24798939

  19. Associations of HLA-A, HLA-B and HLA-C alleles frequency with prevalence of herpes simplex virus infections and diseases across global populations: implication for the development of an universal CD8+ T-cell epitope-based vaccine.

    PubMed

    Samandary, Sarah; Kridane-Miledi, Hédia; Sandoval, Jacqueline S; Choudhury, Zareen; Langa-Vives, Francina; Spencer, Doran; Chentoufi, Aziz A; Lemonnier, François A; BenMohamed, Lbachir

    2014-08-01

    A significant portion of the world's population is infected with herpes simplex virus type 1 and/or type 2 (HSV-1 and/or HSV-2), that cause a wide range of diseases including genital herpes, oro-facial herpes, and the potentially blinding ocular herpes. While the global prevalence and distribution of HSV-1 and HSV-2 infections cannot be exactly established, the general trends indicate that: (i) HSV-1 infections are much more prevalent globally than HSV-2; (ii) over a half billion people worldwide are infected with HSV-2; (iii) the sub-Saharan African populations account for a disproportionate burden of genital herpes infections and diseases; (iv) the dramatic differences in the prevalence of herpes infections between regions of the world appear to be associated with differences in the frequencies of human leukocyte antigen (HLA) alleles. The present report: (i) analyzes the prevalence of HSV-1 and HSV-2 infections across various regions of the world; (ii) analyzes potential associations of common HLA-A, HLA-B and HLA-C alleles with the prevalence of HSV-1 and HSV-2 infections in the Caucasoid, Oriental, Hispanic and Black major populations; and (iii) discusses how our recently developed HLA-A, HLA-B, and HLA-C transgenic/H-2 class I null mice will help validate HLA/herpes prevalence associations. Overall, high prevalence of herpes infection and disease appears to be associated with high frequency of HLA-A(∗)24, HLA-B(∗)27, HLA-B(∗)53 and HLA-B(∗)58 alleles. In contrast, low prevalence of herpes infection and disease appears to be associated with high frequency of HLA-B(∗)44 allele. The finding will aid in developing a T-cell epitope-based universal herpes vaccine and immunotherapy.

  20. NFAT2 Regulates Generation of Innate-Like CD8+ T Lymphocytes and CD8+ T Lymphocytes Responses

    PubMed Central

    Pachulec, Emilia; Neitzke-Montinelli, Vanessa; Viola, João P. B.

    2016-01-01

    Nuclear factor of activated T cells (NFAT) 2 null mutant mice die in utero of cardiac failure, precluding analysis of the role of NFAT2 in lymphocyte responses. Only the NFAT2−/−/Rag-1−/− chimeric mice model gave insight into the role of NFAT2 transcription factor in T lymphocyte development, activation, and differentiation. As reports are mainly focused on the role of NFAT2 in CD4+ T lymphocytes activation and differentiation, we decided to investigate NFAT2’s impact on CD8+ T lymphocyte responses. We report that NFAT2 is phosphorylated and inactive in the cytoplasm of naive CD8+ T cells, and upon TCR stimulation, it is dephosphorylated and translocated into the nucleus. To study the role of NFAT2 in CD8+ T responses, we employed NFAT2fl/flCD4-Cre mice with NFAT2 deletion specifically in T cells. Interestingly, the absence of NFAT2 in T cells resulted in increased percentage of non-conventional innate-like CD8+ T cells. These cells were CD122+, rapid producer of interferon gamma (IFN-γ) and had characteristics of conventional memory CD8+ T cells. We also observed an expansion of PLZF+ expressing CD3+ thymocyte population in the absence of NFAT2 and increased IL-4 production. Furthermore, we found that CD8+ T lymphocytes deficient in NFAT2 had reduced activation, proliferation, and IFN-γ and IL-2 production at suboptimal TCR strength. NFAT2 absence did not significantly influence differentiation of CD8+ T cells into cytotoxic effector cells but reduced their IFN-γ production. This work documents NFAT2 as a negative regulator of innate-like CD8+ T cells development. NFAT2 is required for complete CD8+ T cell responses at suboptimal TCR stimulation and regulates IFN-γ production by cytotoxic CD8+ T cells in vitro. PMID:27766099

  1. A brief history of CD8 T cells.

    PubMed

    Masopust, David; Vezys, Vaiva; Wherry, E John; Ahmed, Rafi

    2007-11-01

    CD8 T cells comprise a powerful branch of the adaptive immune system, yet were not formally recognized until long after the discovery of antibody. CD8 T cells contribute to the eradication of intracellular infections and to the control of many chronic infections. There is tempered optimism that CD8 T cell memory elicited via vaccination may hold the key to manufacturing protective immunity against pathogens that cause chronic infections in humans. The specificity and destructive capabilities of CD8 T cells may also be harnessed for the eradication of tumors. However, CD8 T cells also contribute to a variety of clinical difficulties such as immune mediated pathology, rejection of organ transplants, and autoimmunity. Clearly, learning how to safely generate protective long-lived CD8 T cell memory and how to control or eliminate specific unwanted responses could deliver substantial clinical rewards, and there is a great need for continued research on the biology and therapeutic potential of these cells. Herein, we recount the historical developments leading to the discovery of CD8 T cells, highlight a few of the important discoveries that have followed, and discuss some of the critical issues on the horizon.

  2. A conformational epitope mapped in the bovine herpesvirus type 1 envelope glycoprotein B by phage display and the HSV-1 3D structure.

    PubMed

    Almeida, Greyciele R; Goulart, Luiz Ricardo; Cunha-Junior, Jair P; Bataus, Luiz A M; Japolla, Greice; Brito, Wilia M E D; Campos, Ivan T N; Ribeiro, Cristina; Souza, Guilherme R L

    2015-08-01

    The selected dodecapeptide (1)DRALYGPTVIDH(12) from a phage-displayed peptide library and the crystal structure of the envelope glycoprotein B (Env gB) from Herpes Simplex Virus type 1 (HSV-1) led us to the identification of a new discontinuous epitope on the Bovine herpesvirus type 1 (BoHV-1) Env gB. In silico analysis revealed a short BoHV-1 gB motif ((338)YKRD(341)) within a epitope region, with a high similarity to the motifs shared by the dodecapeptide N-terminal region ((5)YxARD(1)) and HSV-1 Env gB ((326)YARD(329)), in which the (328)Arg residue is described to be a neutralizing antibody target. Besides the characterization of an antibody-binding site of the BoHV-1 Env gB, we have demonstrated that the phage-fused peptide has the potential to be used as a reagent for virus diagnosis by phage-ELISA assay, which discriminated BoHV-1 infected serum samples from negative ones.

  3. Discovering neutralizing antibodies targeting the stem epitope of H1N1 influenza hemagglutinin with synthetic phage-displayed antibody libraries.

    PubMed

    Tung, Chao-Ping; Chen, Ing-Chien; Yu, Chung-Ming; Peng, Hung-Pin; Jian, Jhih-Wei; Ma, Shiou-Hwa; Lee, Yu-Ching; Jan, Jia-Tsrong; Yang, An-Suei

    2015-10-12

    Broadly neutralizing antibodies developed from the IGHV1-69 germline gene are known to bind to the stem region of hemagglutinin in diverse influenza viruses but the sequence determinants for the antigen recognition, including neutralization potency and binding affinity, are not clearly understood. Such understanding could inform designs of synthetic antibody libraries targeting the stem epitope on hemagglutinin, leading to artificially designed antibodies that are functionally advantageous over antibodies from natural antibody repertoires. In this work, the sequence space of the complementarity determining regions of a broadly neutralizing antibody (F10) targeting the stem epitope on the hemagglutinin of a strain of H1N1 influenza virus was systematically explored; the elucidated antibody-hemagglutinin recognition principles were used to design a phage-displayed antibody library, which was then used to discover neutralizing antibodies against another strain of H1N1 virus. More than 1000 functional antibody candidates were selected from the antibody library and were shown to neutralize the corresponding strain of influenza virus with up to 7 folds higher potency comparing with the parent F10 antibody. The antibody library could be used to discover functionally effective antibodies against other H1N1 influenza viruses, supporting the notion that target-specific antibody libraries can be designed and constructed with systematic sequence-function information.

  4. CD8+ T-Cell Responses in Hepatitis B and C: The (HLA-) A, B, and C of Hepatitis B and C.

    PubMed

    Nitschke, Katja; Luxenburger, Hendrik; Kiraithe, Muthamia M; Thimme, Robert; Neumann-Haefelin, Christoph

    Approximately 500 million people are chronically infected with the hepatitis B virus (HBV) or hepatitis C virus (HCV) worldwide and are thus at high risk of progressive liver disease, leading to liver fibrosis, cirrhosis and ultimately hepatocellular cancer. Virus-specific CD8+ T-cells play a major role in viral clearance in >90% of adult patients who clear HBV and in approximately 30% of patients who clear HCV in acute infection. However, several mechanisms contribute to the failure of the adaptive CD8+ T-cell response in those patients who progress to chronic infection. These include viral mutations leading to escape from the CD8+ T-cell response as well as exhaustion and dysfunction of virus-specific CD8+ T-cells. Antiviral efficacy of the virus-specific CD8+ T-cell response also strongly depends on its restriction by specific human leukocyte antigens (HLA) class I alleles. Our review will summarize the role of HLA-A, B and C-restricted CD8+ T-cells in HBV and HCV infection. Due to the current lack of a comprehensive database of HBV- and HCV-specific CD8+ T-cell epitopes, we also provide a summary of the repertoire of currently well-described HBV- and HCV-specific CD8+ T-cell epitopes. A better understanding of the factors that contribute to the success or failure of virus-specific CD8+ T-cells may help to develop new therapeutic options for HBV eradication in patients with chronic HBV infection (therapeutic vaccination and/or immunomodulation) as well as a prophylactic vaccine against HCV infection.

  5. Heteroclitic Peptides Increase Proliferation and Reduce Evidence of Human Immunodeficiency Virus-Specific CD8⁺ T Cell Dysfunction.

    PubMed

    Adegoke, Adeolu; Gladney, Krista; Gallant, Maureen; Grant, Michael

    2015-10-01

    Human immunodeficiency virus (HIV)-specific CD8(+) T cell dysfunction parallels disease progression; therefore, restoring potent HIV-specific CD8(+) T cell responses is a key therapeutic goal. Certain CD8(+) T cell peptide epitope variants, termed heteroclitic, enhance cytokine production by the HIV-specific CD8(+) T cells of some individuals. In this study, we investigated whether heteroclitic peptides that enhance cytokine production by HIV-specific CD8(+) T cells also reduce functional and phenotypic evidence of HIV-specific CD8(+) T cell exhaustion in those instances. Twenty-four variant peptides of human histocompatibility-linked leukocyte antigen (HLA)-A2-restricted reference HIV peptide epitopes designated as A2-7; Nef 83→91, A2-8; Nef 135→143, A2-Gag; Gag 77→85 and A2-9; Gag 433→440 were synthesized with conservative and semiconservative amino acid substitutions at positions 3, 5, and 7 or 3, 5, and 8 of Gag 433→440. Variants that enhanced interferon-gamma (IFN-γ) and/or interleukin-2 (IL-2) production in enzyme-linked immunospot assays (29 cases overall) were subsequently tested by 7-day in vitro peptide stimulation for their effects on HIV-specific CD8(+) T cell proliferation and programmed death-1 (PD-1) expression. Heteroclitic variants enhanced HIV-specific CD8(+) T cell proliferation by >20% in 13/29 cases tested, reduced PD-1 expression on proliferating cells by 15-50% in 10 cases, and reduced PD-1 expression on proliferating cells by >50% in 3 cases. In five cases, the same heteroclitic peptide increased proliferation by >20% and reduced PD-1 expression by >15%. These data demonstrate that heteroclitic peptides can alter the magnitude and character of HIV-specific CD8(+) cell responses relative to reference peptides and may have a unique immunotherapeutic value in therapeutic vaccines.

  6. Self-specific CD8+ T cells maintain a semi-naive state following lymphopenia-induced proliferation.

    PubMed

    Johnson, Lisa D S; Jameson, Stephen C

    2010-05-15

    Upon transfer into T cell-deficient hosts, naive CD8(+) T cells typically undergo lymphopenia-induced proliferation (LIP, also called homeostatic proliferation) and develop the phenotypic and functional characteristics of memory CD8(+) T cells. However, the capacity of T cells with self-peptide/MHC specificity to respond in this way has not been intensively studied. We examined pmel-1 TCR transgenic CD8(+) T cells that are specific for an epitope from gp100, a protein expressed by melanoma cells and normal melanocytes. Despite their self-specificity, naive pmel-1 cells were inefficient at LIP in typical lymphopenic hosts. In CD132 (common gamma-chain)-deficient hosts, pmel-1 CD8(+) T cells underwent extensive proliferation, but, surprisingly, the majority of these cells retained certain naive phenotypic traits (CD44(low), CD122(low)) rather than acquiring the expected central-memory phenotype. Following LIP, pmel-1 T cells acquired the capacity to control B16F10 tumor growth, but only in common gamma-chain-deficient host mice. Together, these data suggest that LIP does not always favor expansion of self-specific CD8 T cells and that sustained extensive lymphopenia is required for such cells to exhibit tumor control.

  7. Prolonged antigen presentation by immune complex-binding dendritic cells programs the proliferative capacity of memory CD8 T cells.

    PubMed

    León, Beatriz; Ballesteros-Tato, André; Randall, Troy D; Lund, Frances E

    2014-07-28

    The commitment of naive CD8 T cells to effector or memory cell fates can occur after a single day of antigenic stimulation even though virus-derived antigens (Ags) are still presented by DCs long after acute infection is resolved. However, the effects of extended Ag presentation on CD8 T cells are undefined and the mechanisms that regulate prolonged Ag presentation are unknown. We showed that the sustained presentation of two different epitopes from influenza virus by DCs prevented the premature contraction of the primary virus-specific CD8 T cell response. Although prolonged Ag presentation did not alter the number of memory CD8 T cells that developed, it was essential for programming the capacity of these cells to proliferate, produce cytokines, and protect the host after secondary challenge. Importantly, prolonged Ag presentation by DCs was dependent on virus-specific, isotype-switched antibodies (Abs) that facilitated the capture and cross-presentation of viral Ags by FcγR-expressing DCs. Collectively, our results demonstrate that B cells and Abs can regulate the quality and functionality of a subset of antiviral CD8 T cell memory responses and do so by promoting sustained Ag presentation by DCs during the contraction phase of the primary T cell response.

  8. Lack of variant specific CD8+ T-cell response against mutant and pre-existing variants leads to outgrowth of particular clones in acute hepatitis C

    PubMed Central

    2013-01-01

    Background CTL escape mutations have been described during acute hepatitis C in patients who developed chronic disease later on. Our aim was to investigate the mutual relationship between HCV specific CD8+ T cells and evolution of the viral sequence during early acute HCV infection. Results We sequenced multiple clones of NS3 1406 epitope in 4 HLA-A*02 patients with acute hepatitis C genotype 1b infection. Pentamers specific for the variants were used to monitor the corresponding CD8+ T cell response. We observed outgrowth of mutations, which induced only a weak and thus potentially insufficient CD8+ T cell response. In one patient we observed outgrowth of variant epitopes with similarities to a different genotype rather than de novo mutations most probably due to a lack of responsiveness to these likely pre-existing variants. We could show that in acute hepatitis C CTL escape mutations occur much earlier than demonstrated in previous studies. Conclusions The adaption of the virus to a new host is characterized by a high and rapid variability in epitopes under CD8+ T cell immune pressure. This adaption takes place during the very early phase of acute infection and strikingly some sequences were reduced below the limit of detection at some time points but were detected at high frequency again at later time points. Independent of the observed variability, HCV-specific CD8+ T cell responses decline and no adaption to different or new antigens during the course of infection could be detected. PMID:24073713

  9. Peripheral blood CD8αα+CD11c+MHC-II+CD3- cells attenuate autoimmune glomerulonephritis in rats.

    PubMed

    Wu, Jean; Zhou, Cindy; Robertson, Julie; Carlock, Colin; Lou, Ya-Huan

    2014-05-01

    In an anti-glomerular basement membrane (GBM) glomerulonephritis (GN) model, GN-resistant Lewis rats naturally recover from early glomerular inflammation. Here we investigated recovery mechanisms for development of a potential immunotherapy for autoimmune GN. Our previous studies suggested that glomeruli-infiltrating leukocytes with a phenotype of CD8αα+CD11c+MHC-II+CD3- (GIL CD8αα+ cells) were responsible for recovery through induction of T-cell apoptosis. Now, we identified peripheral blood CD8αα+CD11c+MHC-II+CD3- cells (PBMC CD8αα+CD3- cells), which shared 9 markers with GIL CD8αα+ cells. Upon incubation, PBMC CD8αα+CD3- cells displayed a morphology resembling that of dendritic cells. Similar to GIL CD8αα+ cells, PBMC CD8αα+CD3- cells were capable of inducing T-cell apoptosis in vitro. Hence, PBMC CD8αα+CD3- cells were likely the precursor of GIL CD8αα+ cells. We next tested their potential in vivo function. PBMC CD8αα+CD3- cells were able to infiltrate inflamed but not normal glomeruli. Isolated PBMC CD8αα+CD3- cells of Lewis rats were transferred into GN-prone Wistar-Kyoto rats at early inflammatory stage (days 17-25). When examined at day 45, both histopathology and blood urea nitrogen/serum creatinine level showed significantly attenuated GN in 80% of cell recipient Wistar-Kyoto rats. Separate experiments verified infiltration of transferred Lewis PBMC CD8αα+CD3- into the glomeruli, accompanied with apoptotic CD4+ T cells in the glomeruli of the recipient Wistar-Kyoto rats. Thus, PBMC CD8αα+CD3- cells of Lewis rats were able to terminate ongoing autoimmune inflammation in the glomeruli.

  10. Identification of antigenic epitopes of the SapA protein of Campylobacter fetus using a phage display peptide library.

    PubMed

    Zhao, Hailing; Yu, Shenye; Liu, Huifang; Si, Wei; Wang, Chunlai; Liu, Siguo

    2012-12-01

    In this study, we immunized mice with prokaryotically expressed recombinant surface layer protein, SapA, of Campylobacter fetus, generated hybridomas secreting mouse monoclonal antibodies (mAb) targeting SapA, and purified the mAb A2D5 from mouse ascites using saturated ammonium sulfate solution. The mAb A2D5, coated onto ELISA plates, was used to screen the phage random 12-peptide library through three rounds of panning. Following panning, 15 phage clones were randomly chosen and tested for reactivity with mAb A2D5 by indirect ELISA. Single-stranded DNA from positive clones was sequenced and compared with the sequence of SapA to predict the key epitope. ELISA and/or Western blot analyses further validated that synthetic peptides and recombinant peptide mimotopes all interact with mAb A2D5. Nine of ten positive phage clones identified by screening were sequenced successfully. Seven clones shared the same sequence HYDRHNYHWWHT; one had the sequence LSKNLPLTALGN; and the final one had the sequence SGMKEPELRSYS. These three sequences shared high homology with SapA J05577 in the region GNEKDFVTKIYSIALGNTSDVDGINYW, in which the underlined amino acids may serve as key residues in the epitope. ELISA and/or Western blot analyses showed that mAb A2D5 not only interacted with the four synthetic peptide mimotopes, but also with 14 prokaryotically expressed recombinant peptide mimotopes. The mimotopes identified in this study will aid future studies into the pathological processes and immune mechanisms of the SapA protein of C. fetus. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Phenotypical and functional evaluation of CD8+/S6F1+ T lymphocytes in haemophiliac individuals with HIV-1 infection.

    PubMed Central

    Cavallin, F; Traldi, A; Zambello, R

    1993-01-01

    In this study we investigated the distribution of the S6F1 antigen, an epitope of the lymphocyte function-associated antigen, on CD8+ T lymphocytes in a series of 15 HIV-1+ and 20 HIV-1- haemophiliac patients. MoAbs recognizing the S6F1 antigen have been claimed to distinguish between killer effectors (brightly S6F1+ stained) and suppressor cells (dimly S6F1+ stained) within the CD8+ lymphoid population. In addition, we tried to find a correlation between the spontaneous in vitro immunoglobulin synthesis from patients' peripheral blood lymphocytes and the pattern of S6F1 expression. Although the total number of double-positive CD8+/S6F1+ cells was similar in both HIV-1+ and HIV-1- haemophiliac patients, a significant increase in the CD8+/S6F1+ population bright versus dim was documented in HIV-1-infected with respect to HIV-1- haemophiliacs (bright/dim ratio 3.97 +/- 0.61 versus 0.75 +/- 0.1, respectively, P < 0.005). This finding was correlated to a significant increase in spontaneous in vitro immunoglobulin production in HIV-1+ subjects compared with control haemophiliacs (P < 0.005). Purified CD8+ lymphocytes from HIV-1+ subjects showed a reduced suppressor activity on mitogen-induced immunoglobulin production. Taken together, these data suggest that HIV-1 infection favours the generation of CD8+/S6F1+ bright cells with putative cytotoxic-associated function, leading to a progressive reduction in the number of CD8+/S6F1+ dim suppressor lymphocytes. This phenomenon may contribute to the polyclonal hypergammaglobulinaemia present in HIV-1+ haemophiliac patients. PMID:8324903

  12. Mapping and Role of the CD8(+) T Cell Response During Primary Zika Virus Infection in Mice.

    PubMed

    Elong Ngono, Annie; Vizcarra, Edward A; Tang, William W; Sheets, Nicholas; Joo, Yunichel; Kim, Kenneth; Gorman, Matthew J; Diamond, Michael S; Shresta, Sujan

    2017-01-11

    CD8(+) T cells may play a dual role in protection against and pathogenesis of flaviviruses, including Zika virus (ZIKV). We evaluated the CD8(+) T cell response in ZIKV-infected LysMCre(+)IFNAR(fl/fl) C57BL/6 (H-2(b)) mice lacking the type I interferon receptor in a subset of myeloid cells. In total, 26 and 15 CD8(+) T cell-reactive peptides for ZIKV African (MR766) and Asian (FSS13025) lineage strains, respectively, were identified and validated. CD8(+) T cells from infected mice were polyfunctional and mediated cytotoxicity. Adoptive transfer of ZIKV-immune CD8(+) T cells reduced viral burdens, whereas their depletion led to higher tissue burdens, and CD8(-/-) mice displayed higher mortality with ZIKV infection. Collectively, these results demonstrate that CD8(+) T cells protect against ZIKV infection. Further, this study provides a T cell competent mouse model for investigating ZIKV-specific T cell responses. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. A role for the histone H2A deubiquitinase MYSM1 in maintenance of CD8(+) T cells.

    PubMed

    Förster, Michael; Boora, Rupinder K; Petrov, Jessica C; Fodil, Nassima; Albanese, Isabella; Kim, Jamie; Gros, Philippe; Nijnik, Anastasia

    2017-05-01

    Several previous studies outlined the importance of the histone H2A deubiquitinase MYSM1 in the regulation of stem cell quiescence and haematopoiesis. In this study we investigated the role of MYSM1 in T-cell development. Using mouse models that allow conditional Mysm1 ablation at late stages of thymic development, we found that MYSM1 is intricately involved in the maintenance, activation and survival of CD8(+) T cells. Mysm1 ablation resulted in a twofold reduction in CD8(+) T-cell numbers, and also led to a hyperactivated CD8(+) T-cell state accompanied by impaired proliferation and increased pro-inflammatory cytokine production after ex vivo stimulation. These phenotypes coincided with an increased apoptosis and preferential up-regulation of p53 tumour suppressor protein in CD8(+) T cells. Lastly, we examined a model of experimental cerebral malaria, in which pathology is critically dependent on CD8(+) T cells. In the mice conditionally deleted for Mysm1 in the T-cell compartment, CD8(+) T-cell numbers remained reduced following infection, both in the periphery and in the brain, and the mice displayed improved survival after parasite challenge. Collectively, our data identify MYSM1 as a novel factor for CD8(+) T cells in the immune system, increasing our understanding of the role of histone H2A deubiquitinases in cytotoxic T-cell biology.

  14. Identification of a conserved JEV serocomplex B-cell epitope by screening a phage-display peptide library with a mAb generated against West Nile virus capsid protein

    PubMed Central

    2011-01-01

    Background The West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins, encapsidates the viral RNA to form the nucleocapsid, and is necessary for nuclear and nucleolar localization. The antigenic sites on C protein that are targeted by humoral immune responses have not been studied thoroughly, and well-defined B-cell epitopes on the WNV C protein have not been reported. Results In this study, we generated a WNV C protein-specific monoclonal antibody (mAb) and defined the linear epitope recognized by the mAb by screening a 12-mer peptide library using phage-display technology. The mAb, designated as 6D3, recognized the phages displaying a consensus motif consisting of the amino acid sequence KKPGGPG, which is identical to an amino acid sequence present in WNV C protein. Further fine mapping was conducted using truncated peptides expressed as MBP-fusion proteins. We found that the KKPGGPG motif is the minimal determinant of the linear epitope recognized by the mAb 6D3. Western blot (WB) analysis demonstrated that the KKPGGPG epitope could be recognized by antibodies contained in WNV- and Japanese encephalitis virus (JEV)-positive equine serum, but was not recognized by Dengue virus 1-4 (DENV1-4)-positive mice serum. Furthermore, we found that the epitope recognized by 6D3 is highly conserved among the JEV serocomplex of the Family Flaviviridae. Conclusion The KKPGGPG epitope is a JEV serocomplex-specific linear B-cell epitope recognized by the 6D3 mAb generated in this study. The 6D3 mAb may serve as a novel reagent in development of diagnostic tests for JEV serocomplex infection. Further, the identification of the B-cell epitope that is highly conserved among the JEV serocomplex may support the rationale design of vaccines against viruses of the JEV serocomplex. PMID:21375771

  15. Superior induction and maintenance of protective CD8 T cells in mice infected with mouse cytomegalovirus vector expressing RAE-1γ

    PubMed Central

    Tršan, Tihana; Busche, Andreas; Abram, Maja; Wensveen, Felix M.; Lemmermann, Niels A.; Arapović, Maja; Babić, Marina; Tomić, Adriana; Golemac, Mijo; Brinkmann, Melanie M.; Jäger, Wiebke; Oxenius, Annette; Polić, Bojan; Krmpotić, Astrid; Messerle, Martin; Jonjić, Stipan

    2013-01-01

    Due to a unique pattern of CD8 T-cell response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors for a number of clinically relevant infections and tumors. NKG2D is one of the most important activating NK cell receptors that plays a role in costimulation of CD8 T cells. Here we demonstrate that the expression of CD8 T-cell epitope of Listeria monocytogenes by a recombinant mouse CMV (MCMV) expressing the NKG2D ligand retinoic acid early-inducible protein 1-gamma (RAE-1γ) dramatically enhanced the effectiveness and longevity of epitope-specific CD8 T-cell response and conferred protection against a subsequent challenge infection with Listeria monocytogenes. Unexpectedly, the attenuated growth in vivo of the CMV vector expressing RAE-1γ and its capacity to enhance specific CD8 T-cell response were preserved even in mice lacking NKG2D, implying additional immune function for RAE-1γ beyond engagement of NKG2D. Thus, vectors expressing RAE-1γ represent a promising approach in the development of CD8 T-cell–based vaccines. PMID:24052528

  16. Superior induction and maintenance of protective CD8 T cells in mice infected with mouse cytomegalovirus vector expressing RAE-1γ.

    PubMed

    Trsan, Tihana; Busche, Andreas; Abram, Maja; Wensveen, Felix M; Lemmermann, Niels A; Arapovic, Maja; Babic, Marina; Tomic, Adriana; Golemac, Mijo; Brinkmann, Melanie M; Jäger, Wiebke; Oxenius, Annette; Polic, Bojan; Krmpotic, Astrid; Messerle, Martin; Jonjic, Stipan

    2013-10-08

    Due to a unique pattern of CD8 T-cell response induced by cytomegaloviruses (CMVs), live attenuated CMVs are attractive candidates for vaccine vectors for a number of clinically relevant infections and tumors. NKG2D is one of the most important activating NK cell receptors that plays a role in costimulation of CD8 T cells. Here we demonstrate that the expression of CD8 T-cell epitope of Listeria monocytogenes by a recombinant mouse CMV (MCMV) expressing the NKG2D ligand retinoic acid early-inducible protein 1-gamma (RAE-1γ) dramatically enhanced the effectiveness and longevity of epitope-specific CD8 T-cell response and conferred protection against a subsequent challenge infection with Listeria monocytogenes. Unexpectedly, the attenuated growth in vivo of the CMV vector expressing RAE-1γ and its capacity to enhance specific CD8 T-cell response were preserved even in mice lacking NKG2D, implying additional immune function for RAE-1γ beyond engagement of NKG2D. Thus, vectors expressing RAE-1γ represent a promising approach in the development of CD8 T-cell-based vaccines.

  17. CD8+ T-cell specificity is compromised at a defined MHCI/CD8 affinity threshold

    PubMed Central

    Dockree, Tamsin; Holland, Christopher J; Clement, Mathew; Ladell, Kristin; McLaren, James E; van den Berg, Hugo A; Gostick, Emma; L Miners, Kelly; Llewellyn-Lacey, Sian; Bridgeman, John S; Man, Stephen; Bailey, Mick; Burrows, Scott R; Price, David A; Wooldridge, Linda

    2017-01-01

    The CD8 co-receptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR)-binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8+ T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5-fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~10-fold). In this study, we used a panel of MHCI mutants with altered CD8-binding properties to show that TCR-mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T-cell transfer irrespective of antigen specificity. PMID:27670790

  18. CD8(+) T-cell specificity is compromised at a defined MHCI/CD8 affinity threshold.

    PubMed

    Dockree, Tamsin; Holland, Christopher J; Clement, Mathew; Ladell, Kristin; McLaren, James E; van den Berg, Hugo A; Gostick, Emma; L Miners, Kelly; Llewellyn-Lacey, Sian; Bridgeman, John S; Man, Stephen; Bailey, Mick; Burrows, Scott R; Price, David A; Wooldridge, Linda

    2017-01-01

    The CD8 co-receptor engages peptide-major histocompatibility complex class I (pMHCI) molecules at a largely invariant site distinct from the T-cell receptor (TCR)-binding platform and enhances the sensitivity of antigen-driven activation to promote effective CD8(+) T-cell immunity. A small increase in the strength of the pMHCI/CD8 interaction (~1.5-fold) can disproportionately amplify this effect, boosting antigen sensitivity by up to two orders of magnitude. However, recognition specificity is lost altogether with more substantial increases in pMHCI/CD8 affinity (~10-fold). In this study, we used a panel of MHCI mutants with altered CD8-binding properties to show that TCR-mediated antigen specificity is delimited by a pMHCI/CD8 affinity threshold. Our findings suggest that CD8 can be engineered within certain biophysical parameters to enhance the therapeutic efficacy of adoptive T-cell transfer irrespective of antigen specificity.

  19. Recombinant λ bacteriophage displaying nanobody towards third domain of HER-2 epitope inhibits proliferation of breast carcinoma SKBR-3 cell line.

    PubMed

    Shoae-Hassani, Alireza; Mortazavi-Tabatabaei, Seyed Abdolreza; Sharif, Shiva; Madadi, Shabnam; Rezaei-Khaligh, Hamidreza; Verdi, Javad

    2013-02-01

    Phage display of many nanobodies via filamentous phage in combination with helper phage has been reported by many scientists. The aim of this study was to produce lambda (λ) bacteriophage displaying high-affinity nanobody against HER-2 expressing breast carcinoma cells. Bacteriophage λ is a temperate phage with inherent biological safety in mammalian cells. Here we report the construction of a recombinant λ phage that efficiently expresses specific nanobody towards third domain of HER-2 target on SKBR-3 and MCF-7 cell lines in vitro. We constructed recombinant λ phage particles containing a mammalian expression cassette, C-Myc tagged, encoding VHH gene of camelid anti HER-2 third domain epitope using λ ZAP-cytomegalic virus (CMV) vector. The SKBR-3, MCF-7 and human endometrial stem cells were treated by the nanobody displayed recombinant λ phage. The cell growth inhibition assay was performed by MTT Cell Viability Assay Kit. After the fourth round of biopanning there was a significant enrichment in the phage specifically binding to the antigen. The ratio of targeted phage increased approximately 1,000-fold in the fifth round. The nanobody expressed by λ ZAP-CMV-VHH phagemid cloned in λ bioparticles significantly inhibited the proliferation of HER-2 positive SKBR-3 and MCF-7 cells. Recombinant bacteriophage λ ZAP-CMV-VHH-cDNA could be used efficiently for construction of nanobodies to mortify HER-2 positive breast carcinoma cells as a nanomedical therapeutic.

  20. Measles Virus Epitope Presentation by HLA: Novel Insights into Epitope Selection, Dominance, and Microvariation

    PubMed Central

    Schellens, Ingrid M.; Meiring, Hugo D.; Hoof, Ilka; Spijkers, Sanne N.; Poelen, Martien C. M.; van Gaans-van den Brink, Jacqueline A. M.; Costa, Ana I.; Vennema, Harry; Keşmir, Can; van Baarle, Debbie; van Els, Cécile A. C. M.

    2015-01-01

    Immunity to infections with measles virus (MV) can involve vigorous human leukocyte antigen (HLA) class I-restricted CD8+ cytotoxic T cell (CTL) responses. MV, albeit regarded monotypic, is known to undergo molecular evolution across its RNA genome. To address which regions of the MV proteome are eligible for recognition by CD8+ CTLs and how different HLA class I loci contribute to the epitope display, we interrogated the naturally processed and presented MV peptidome extracted from cell lines expressing in total a broad panel of 16 different common HLA-A, -B, and -C molecules. The repertoire and abundance of MV peptides were bona fide identified by nanoHPLC–MS/MS. ­Eighty-nine MV peptides were discovered and assignment to an HLA-A, -B, or -C allele, based on HLA-peptide affinity prediction, was in most cases successful. Length variation and presentation by multiple HLA class I molecules was common in the MV peptidome. More than twice as many unique MV epitopes were found to be restricted by HLA-B than by HLA-A, while MV peptides with supra-abundant expression rates (>5,000 cc) were rather associated with HLA-A and HLA-C. In total, 59 regions across the whole MV proteome were identified as targeted by HLA class I. Sequence coverage by epitopes was highest for internal proteins transcribed from the MV-P/V/C and -M genes and for hemagglutinin. At the genome level, the majority of the HLA class I-selected MV epitopes represented codons having a higher non-synonymous mutation rate than silent mutation rate, as established by comparison of a set of 58 unique full length MV genomes. Interestingly, more molecular variation was seen for the epitopes expressed at rates ≥1,000 cc. These data for the first time indicate that HLA class I broadly samples the MV proteome and that CTL pressure may contribute to the genomic evolution of MV. PMID:26579122

  1. An inducible transgenic mouse breast cancer model for the analysis of tumor antigen specific CD8+ T-cell responses.

    PubMed

    Bruns, Michael; Wanger, Jara; Utermöhlen, Olaf; Deppert, Wolfgang

    2015-11-17

    In Simian virus 40 (SV40) transgenic BALB/c WAP-T mice tumor development and progression is driven by SV40 tumor antigens encoded by inducible transgenes. WAP-T mice constitute a well characterized mouse model for breast cancer with strong similarities to the corresponding human disease. BALB/c mice mount only a weak cellular immune response against SV40 T-antigen (T-Ag). For studying tumor antigen specific CD8+ T-cell responses against transgene expressing cells, we created WAP-TNP mice, in which the transgene additionally codes for the NP118-126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), the immune-dominant T-cell epitope in BALB/c mice. We then investigated in WAP-TNP mice the immune responses against SV40 tumor antigens and the NP-epitope within the chimeric T-Ag/NP protein (T-AgNP). Analysis of the immune-reactivity against T-Ag in WAP-T and of T-AgNP in WAP-TNP mice revealed that, in contrast to wild type (wt) BALB/c mice, WAP-T and WAP-TNP mice were non-reactive against T-Ag. However, like wtBALB/c mice, WAP-T as well as WAP-TNP mice were highly reactive against the immune-dominant LCMV NP-epitope, thereby allowing the analysis of NP-epitope specific cellular immune responses in WAP-TNP mice. LCMV infection of WAP-TNP mice induced a strong, LCMV NP-epitope specific CD8+ T-cell response, which was able to specifically eliminate T-AgNP expressing mammary epithelial cells both prior to tumor formation (i.e. in cells of lactating mammary glands), as well as in invasive tumors. Elimination of tumor cells, however, was only transient, even after repeated LCMV infections. Further studies showed that already non-infected WAP-TNP tumor mice contained LCMV NP-epitope specific CD8+ T-cells, albeit with strongly reduced, though measurable activity. Functional impairment of these 'endogenous' NP-epitope specific T-cells seems to be caused by expression of the programmed death-1 protein (PD1), as anti-PD1 treatment of

  2. An inducible transgenic mouse breast cancer model for the analysis of tumor antigen specific CD8+ T-cell responses

    PubMed Central

    Bruns, Michael; Wanger, Jara; Utermöhlen, Olaf; Deppert, Wolfgang

    2015-01-01

    In Simian virus 40 (SV40) transgenic BALB/c WAP-T mice tumor development and progression is driven by SV40 tumor antigens encoded by inducible transgenes. WAP-T mice constitute a well characterized mouse model for breast cancer with strong similarities to the corresponding human disease. BALB/c mice mount only a weak cellular immune response against SV40 T-antigen (T-Ag). For studying tumor antigen specific CD8+ T-cell responses against transgene expressing cells, we created WAP-TNP mice, in which the transgene additionally codes for the NP118–126-epitope contained within the nucleoprotein of lymphocytic choriomeningitis virus (LCMV), the immune-dominant T-cell epitope in BALB/c mice. We then investigated in WAP-TNP mice the immune responses against SV40 tumor antigens and the NP-epitope within the chimeric T-Ag/NP protein (T-AgNP). Analysis of the immune-reactivity against T-Ag in WAP-T and of T-AgNP in WAP-TNP mice revealed that, in contrast to wild type (wt) BALB/c mice, WAP-T and WAP-TNP mice were non-reactive against T-Ag. However, like wtBALB/c mice, WAP-T as well as WAP-TNP mice were highly reactive against the immune-dominant LCMV NP-epitope, thereby allowing the analysis of NP-epitope specific cellular immune responses in WAP-TNP mice. LCMV infection of WAP-TNP mice induced a strong, LCMV NP-epitope specific CD8+ T-cell response, which was able to specifically eliminate T-AgNP expressing mammary epithelial cells both prior to tumor formation (i.e. in cells of lactating mammary glands), as well as in invasive tumors. Elimination of tumor cells, however, was only transient, even after repeated LCMV infections. Further studies showed that already non-infected WAP-TNP tumor mice contained LCMV NP-epitope specific CD8+ T-cells, albeit with strongly reduced, though measurable activity. Functional impairment of these ‘endogenous’ NP-epitope specific T-cells seems to be caused by expression of the programmed death-1 protein (PD1), as anti-PD1 treatment of

  3. NADPH oxidase deficiency underlies dysfunction of aged CD8+ Tregs

    PubMed Central

    Wen, Zhenke; Shimojima, Yasuhiro; Shirai, Tsuyoshi; Li, Yinyin; Ju, Jihang; Yang, Zhen; Tian, Lu; Goronzy, Jörg J.

    2016-01-01

    Immune aging results in progressive loss of both protective immunity and T cell–mediated suppression, thereby conferring susceptibility to a combination of immunodeficiency and chronic inflammatory disease. Here, we determined that older individuals fail to generate immunosuppressive CD8+CCR7+ Tregs, a defect that is even more pronounced in the age-related vasculitic syndrome giant cell arteritis. In young, healthy individuals, CD8+CCR7+ Tregs are localized in T cell zones of secondary lymphoid organs, suppress activation and expansion of CD4 T cells by inhibiting the phosphorylation of membrane-proximal signaling molecules, and effectively inhibit proliferative expansion of CD4 T cells in vitro and in vivo. We identified deficiency of NADPH oxidase 2 (NOX2) as the molecular underpinning of CD8 Treg failure in the older individuals and in patients with giant cell arteritis. CD8 Tregs suppress by releasing exosomes that carry preassembled NOX2 membrane clusters and are taken up by CD4 T cells. Overexpression of NOX2 in aged CD8 Tregs promptly restored suppressive function. Together, our data support NOX2 as a critical component of the suppressive machinery of CD8 Tregs and suggest that repairing NOX2 deficiency in these cells may protect older individuals from tissue-destructive inflammatory disease, such as large-vessel vasculitis. PMID:27088800

  4. CD39 Expression Identifies Terminally Exhausted CD8+ T Cells.

    PubMed

    Gupta, Prakash K; Godec, Jernej; Wolski, David; Adland, Emily; Yates, Kathleen; Pauken, Kristen E; Cosgrove, Cormac; Ledderose, Carola; Junger, Wolfgang G; Robson, Simon C; Wherry, E John; Alter, Galit; Goulder, Philip J R; Klenerman, Paul; Sharpe, Arlene H; Lauer, Georg M; Haining, W Nicholas

    2015-10-01

    Exhausted T cells express multiple co-inhibitory molecules that impair their function and limit immunity to chronic viral infection. Defining novel markers of exhaustion is important both for identifying and potentially reversing T cell exhaustion. Herein, we show that the ectonucleotidse CD39 is a marker of exhausted CD8+ T cells. CD8+ T cells specific for HCV or HIV express high levels of CD39, but those specific for EBV and CMV do not. CD39 expressed by CD8+ T cells in chronic infection is enzymatically active, co-expressed with PD-1, marks cells with a transcriptional signature of T cell exhaustion and correlates with viral load in HIV and HCV. In the mouse model of chronic Lymphocytic Choriomeningitis Virus infection, virus-specific CD8+ T cells contain a population of CD39high CD8+ T cells that is absent in functional memory cells elicited by acute infection. This CD39high CD8+ T cell population is enriched for cells with the phenotypic and functional profile of terminal exhaustion. These findings provide a new marker of T cell exhaustion, and implicate the purinergic pathway in the regulation of T cell exhaustion.

  5. Imaging CD8+ T cells during diverse viral infections

    PubMed Central

    Hickman, Heather D

    2015-01-01

    CD8+ T cells play a critical role in host defense against pathogens and tumors. Much of our current knowledge of the activation and subsequent effector activities of CD8+ T cells has been gained using ex vivo approaches examining the T cell population en masse for surface phenotype, activation status and the production of effector molecules. Thus, the precise behaviors and diversity of individual CD8+ T cells responding to virus infection in vivo have not been extensively explored, leaving many unanswered questions relevant to the rational design of antiviral vaccines and therapeutics. Recently, intravital multiphoton microscopy (MPM) has been used to image CD8+ T cell priming after infection with disparate viral pathogens ranging from small RNA viruses encoding few proteins to DNA viruses producing hundreds of viral proteins (many immunomodulatory). After priming, effector CD8+ T cells have been visualized in virus-infected tissue, both during primary infection and after transitioning to tissue resident memory cells (TRM). Here, I highlight recent advances in our understanding of antiviral CD8+ T cell responses revealed through intravital MPM. PMID:28243513

  6. Simian immunodeficiency virus infection of CD8+ lymphocytes in vivo.

    PubMed Central

    Dean, G A; Reubel, G H; Pedersen, N C

    1996-01-01

    To determine the lymphoid target cells of simian immunodeficiency virus (SIV) in vivo, peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL) were positively selected (>97% purity) for surface expression of CD4, CD8, or CD20 and then analyzed for SIV provirus using semiquantitative DNA amplification. We found provirus in CD4+ and CD8+ lymphocytes but none in CD20+ lymphocytes. During acute SIV infection (< or = 214 days postinoculation), the percentage of PBL and LNL CD4+ cells containing proviral DNA ranged from 0.2 to 20% and from 0.2 to 2%, respectively. Proviral burden in the CD8+ population of either PBL or LNL ranged from 0.01 to 0.2%. Virus isolation by cocultivation was positive for both CD4+ and CD8+ purified populations. No difference in proviral burden was observed between PBL and LNL subsets during acute SIV infection. Up to 19.4% of positively selected CD8+ cells also expressed CD4, and thus the provirus may reside within a dual-positive population. This dual-positive population may represent activated lymphocytes that are particularly susceptible to infection and may provide an opportunity for virus entry into the CD8+ CD4- lymphocytes in vivo. PMID:8764081

  7. Cell-autonomous CCL5 transcription by memory CD8 T cells is regulated by IL-4.

    PubMed

    Marçais, Antoine; Coupet, Charles-Antoine; Walzer, Thierry; Tomkowiak, Martine; Ghittoni, Raffaella; Marvel, Jacqueline

    2006-10-01

    Immunological memory is associated with the display of improved effector functions. The maintenance by CD8 memory cells of high levels of untranslated CCL5 mRNA allows these cells to immediately secrete this chemokine upon Ag stimulation. Untranslated mRNA storage is a newly described process supporting the immediate display of an effector function by memory lymphocytes. We have tested the capacity of different cytokines to regulate the memorization of CCL5 by memory CD8 T cells. We found that IL-4 treatment of murine CD8 T cells impairs immediate CCL5 secretion capacity by inhibiting CCL5 mRNA transcription through a STAT6-dependent pathway. The inhibition by IL-4 is reversible, as memory CD8 T cells reconstitute their CCL5 mRNA stores and reacquire their immediate CCL5 secretion capacity when IL-4 is withdrawn. This recovery is cell autonomous because it proceeds in culture medium in the absence of exogenous growth factors, suggesting that CCL5 expression by memory CD8 T cells is a default process. Overall, these results indicate that the expression of CCL5 is an intrinsic property acquired by memory CD8 T cells that is regulated by environmental factors.

  8. RSV-specific airway resident memory CD8+ T cells and differential disease severity after experimental human infection.

    PubMed

    Jozwik, Agnieszka; Habibi, Maximillian S; Paras, Allan; Zhu, Jie; Guvenel, Aleks; Dhariwal, Jaideep; Almond, Mark; Wong, Ernie H C; Sykes, Annemarie; Maybeno, Matthew; Del Rosario, Jerico; Trujillo-Torralbo, Maria-Belen; Mallia, Patrick; Sidney, John; Peters, Bjoern; Kon, Onn Min; Sette, Alessandro; Johnston, Sebastian L; Openshaw, Peter J; Chiu, Christopher

    2015-12-21

    In animal models, resident memory CD8+ T (Trm) cells assist in respiratory virus elimination but their importance in man has not been determined. Here, using experimental human respiratory syncytial virus (RSV) infection, we investigate systemic and local virus-specific CD8+ T-cell responses in adult volunteers. Having defined the immunodominance hierarchy, we analyse phenotype and function longitudinally in blood and by serial bronchoscopy. Despite rapid clinical recovery, we note surprisingly extensive lower airway inflammation with persistent viral antigen and cellular infiltrates. Pulmonary virus-specific CD8+ T cells display a CD69+CD103+ Trm phenotype and accumulate to strikingly high frequencies into convalescence without continued proliferation. While these have a more highly differentiated phenotype, they express fewer cytotoxicity markers than in blood. Nevertheless, their abundance before infection correlates with reduced symptoms and viral load, implying that CD8+ Trm cells in the human lung can confer protection against severe respiratory viral disease when humoral immunity is overcome.

  9. Heightened self-reactivity associated with selective survival, but not expansion, of naïve virus-specific CD8+ T cells in aged mice

    PubMed Central

    Quinn, Kylie M.; Zaloumis, Sophie G.; Cukalac, Tania; Kan, Wan-Ting; Sng, Xavier Y. X.; Mirams, Michiko; Watson, Katherine A.; Doherty, Peter C.; Thomas, Paul G.; Handel, Andreas; La Gruta, Nicole L.

    2016-01-01

    In advanced age, decreased CD8+ cytotoxic T-lymphocyte (CTL) responses to novel pathogens and cancer is paralleled by a decline in the number and function of naïve CTL precursors (CTLp). Although the age-related fall in CD8+ T-cell numbers is well established, neither the underlying mechanisms nor the extent of variation for different epitope specificities have been defined. Furthermore, naïve CD8+ T cells expressing high levels of CD44 accumulate with age, but it is unknown whether this accumulation reflects their preferential survival or an age-dependent driver of CD8+ T-cell proliferation. Here, we track the number and phenotype of four influenza A virus (IAV)-specific CTLp populations in naïve C57BL/6 (B6) mice during aging, and compare T-cell receptor (TCR) clonal diversity for the CD44hi and CD44lo subsets of one such population. We show differential onset of decline for several IAV-specific CD8+ T-cell populations with advanced age that parallel age-associated changes in the B6 immunodominance hierarchy, suggestive of distinct impacts of aging on different epitope-specific populations. Despite finding no evidence of clonal expansions in an aged, epitope-specific TCR repertoire, nonrandom alterations in TCR usage were observed, along with elevated CD5 and CD8 coreceptor expression. Collectively, these data demonstrate that naïve CD8+ T cells expressing markers of heightened self-recognition are selectively retained, but not clonally expanded, during aging. PMID:26787864

  10. CD8+ T Cell-Mediated Immunity during Trypanosoma cruzi Infection: A Path for Vaccine Development?

    PubMed Central

    dos Santos Virgilio, Fernando; Pontes, Camila; Dominguez, Mariana Ribeiro; Ersching, Jonatan; Rodrigues, Mauricio Martins; Vasconcelos, José Ronnie

    2014-01-01

    MHC-restricted CD8+ T cells are important during infection with the intracellular protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. Experimental studies performed in the past 25 years have elucidated a number of features related to the immune response mediated by these T cells, which are important for establishing the parasite/host equilibrium leading to chronic infection. CD8+ T cells are specific for highly immunodominant antigens expressed by members of the trans-sialidase family. After infection, their activation is delayed, and the cells display a high proliferative activity associated with high apoptotic rates. Although they participate in parasite control and elimination, they are unable to clear the infection due to their low fitness, allowing the parasite to establish the chronic phase when these cells then play an active role in the induction of heart immunopathology. Vaccination with a number of subunit recombinant vaccines aimed at eliciting specific CD8+ T cells can reverse this path, thereby generating a productive immune response that will lead to the control of infection, reduction of symptoms, and reduction of disease transmission. Due to these attributes, activation of CD8+ T lymphocytes may constitute a path for the development of a veterinarian or human vaccine. PMID:25104879

  11. CD8(+) T cell-mediated immunity during Trypanosoma cruzi infection: a path for vaccine development?

    PubMed

    Dos Santos Virgilio, Fernando; Pontes, Camila; Dominguez, Mariana Ribeiro; Ersching, Jonatan; Rodrigues, Mauricio Martins; Vasconcelos, José Ronnie

    2014-01-01

    MHC-restricted CD8(+) T cells are important during infection with the intracellular protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. Experimental studies performed in the past 25 years have elucidated a number of features related to the immune response mediated by these T cells, which are important for establishing the parasite/host equilibrium leading to chronic infection. CD8(+) T cells are specific for highly immunodominant antigens expressed by members of the trans-sialidase family. After infection, their activation is delayed, and the cells display a high proliferative activity associated with high apoptotic rates. Although they participate in parasite control and elimination, they are unable to clear the infection due to their low fitness, allowing the parasite to establish the chronic phase when these cells then play an active role in the induction of heart immunopathology. Vaccination with a number of subunit recombinant vaccines aimed at eliciting specific CD8(+) T cells can reverse this path, thereby generating a productive immune response that will lead to the control of infection, reduction of symptoms, and reduction of disease transmission. Due to these attributes, activation of CD8(+) T lymphocytes may constitute a path for the development of a veterinarian or human vaccine.

  12. Assessment of CD8+ T Cell Differentiation in Trypanosoma cruzi-Infected Children

    PubMed Central

    Albareda, María Cecilia; Olivera, Gabriela Carina; De Rissio, Ana María; Postan, Miriam

    2010-01-01

    We previously reported that the T cell compartment in chronically Trypanosoma cruzi-infected adult subjects display functional and phenotypic signs of immune senescence. This study aimed to investigate the differentiation and the senescent profile of the overall CD8+ T cell compartment in T. cruzi-infected children at the early stage of the disease. We found a lower percentage of naive (CD27+CD28+CD45RA+) and early antigen-experienced (CD45RA−CD27+CD28+), and higher percentages of late differentiated antigen-experienced (CD45RA−CD27−CD28−) CD8+ T cells in T. cruzi-infected children as compared with age-matched uninfected controls. The expression of the interleukin (IL)-7R is also decreased on naive and on antigen-experienced total CD8+ T cells with various degrees of differentiation. Conversely, the expression of HLA-DR, caspase-3, and CD57 did not vary on the total CD8+ T cell compartment. These findings suggest that the duration of the infection is relevant in the process of immune senescent that this parasite can induce. PMID:20439967

  13. Protective Efficacy of Individual CD8+ T Cell Specificities in Chronic Viral Infection §§

    PubMed Central

    Johnson, Susan; Bergthaler, Andreas; Graw, Frederik; Flatz, Lukas; Bonilla, Weldy V.; Siegrist, Claire-Anne; Lambert, Paul-Henri; Regoes, Roland R.; Pinschewer, Daniel D.

    2014-01-01

    Specific CD8+ T cells (CTLs) play an important role in resolving protracted infection with hepatitis B and C virus in humans and lymphocytic choriomeningitis virus (LCMV) in mice. The contribution of individual CTL specificities to chronic virus control, as well as epitope-specific patterns in timing and persistence of antiviral selection pressure remain, however, incompletely defined. To monitor and characterize the antiviral efficacy of individual CTL specificities throughout the course of chronic infection, we co-inoculated mice with a mixture of wildtype LCMV and genetically engineered CTL epitope-deficient mutant virus. A quantitative longitudinal assessment of viral competition revealed that mice continuously exerted CTL selection pressure on the persisting virus population. The timing of selection pressure characterized individual epitope specificities, and its magnitude varied considerably between individual mice. This longitudinal assessment of “antiviral efficacy” provides a novel parameter to characterize CTL responses in chronic viral infection. It demonstrates remarkable perseverance of all antiviral CTL specificities studied, thus raising hope for therapeutic vaccination in the treatment of persistent viral diseases. PMID:25567678

  14. Protective Role of Cross-Reactive CD8 T Cells Against Dengue Virus Infection.

    PubMed

    Elong Ngono, Annie; Chen, Hui-Wen; Tang, William W; Joo, Yunichel; King, Kevin; Weiskopf, Daniela; Sidney, John; Sette, Alessandro; Shresta, Sujan

    2016-11-01

    Infection with one of the four dengue virus serotypes (DENV1-4) presumably leads to lifelong immunity against the infecting serotype but not against heterotypic reinfection, resulting in a greater risk of developing Dengue Hemorrhagic Fever/Dengue Shock Syndrome (DHF/DSS) during secondary infection. Both antibodies and T cell responses have been implicated in DHF/DSS pathogenesis. According to the T cell-based hypothesis termed "original antigenic sin," secondary DENV infection is dominated by non-protective, cross-reactive T cells that elicit an aberrant immune response. The goal of our study was to compare the roles of serotype-specific and cross-reactive T cells in protection vs. pathogenesis during DENV infection in vivo. Specifically, we utilized IFN-α/βR(-/-) HLA*B0702 transgenic mice in the context of peptide vaccination with relevant human CD8 T cell epitopes. IFN-α/βR(-/-) HLA*B0702 transgenic mice were immunized with DENV serotype 2 (DENV2)-specific epitopes or variants found in any of the other three serotypes (DENV1, DENV3 or DENV4), followed by challenge with DENV. Although cross-reactive T cell responses were lower than responses elicited by serotype-specific T cells, immunization with either serotype-specific or variant peptide epitopes enhanced viral clearance, demonstrating that both serotype-specific and cross-reactive T cells can contribute to protection in vivo against DENV infection.

  15. Anti-CD8 antibodies can trigger CD8+ T cell effector function in the absence of TCR engagement and improve peptide-MHCI tetramer staining.

    PubMed

    Clement, Mathew; Ladell, Kristin; Ekeruche-Makinde, Julia; Miles, John J; Edwards, Emily S J; Dolton, Garry; Williams, Tamsin; Schauenburg, Andrea J A; Cole, David K; Lauder, Sarah N; Gallimore, Awen M; Godkin, Andrew J; Burrows, Scott R; Price, David A; Sewell, Andrew K; Wooldridge, Linda

    2011-07-15

    CD8(+) T cells recognize immunogenic peptides presented at the cell surface bound to MHCI molecules. Ag recognition involves the binding of both TCR and CD8 coreceptor to the same peptide-MHCI (pMHCI) ligand. Specificity is determined by the TCR, whereas CD8 mediates effects on Ag sensitivity. Anti-CD8 Abs have been used extensively to examine the role of CD8 in CD8(+) T cell activation. However, as previous studies have yielded conflicting results, it is unclear from the literature whether anti-CD8 Abs per se are capable of inducing effector function. In this article, we report on the ability of seven monoclonal anti-human CD8 Abs to activate six human CD8(+) T cell clones with a total of five different specificities. Six of seven anti-human CD8 Abs tested did not activate CD8(+) T cells. In contrast, one anti-human CD8 Ab, OKT8, induced effector function in all CD8(+) T cells examined. Moreover, OKT8 was found to enhance TCR/pMHCI on-rates and, as a consequence, could be used to improve pMHCI tetramer staining and the visualization of Ag-specific CD8(+) T cells. The anti-mouse CD8 Abs, CT-CD8a and CT-CD8b, also activated CD8(+) T cells despite opposing effects on pMHCI tetramer staining. The observed heterogeneity in the ability of anti-CD8 Abs to trigger T cell effector function provides an explanation for the apparent incongruity observed in previous studies and should be taken into consideration when interpreting results generated with these reagents. Furthermore, the ability of Ab-mediated CD8 engagement to deliver an activation signal underscores the importance of CD8 in CD8(+) T cell signaling.

  16. A distinct immunogenic region of glutamic acid decarboxylase 65 is naturally processed and presented by human islet cells to cytotoxic CD8 T cells

    PubMed Central

    Knight, R R; Dolton, G; Kronenberg-Versteeg, D; Eichmann, M; Zhao, M; Huang, G C; Beck, K; Cole, D K; Sewell, A K; Skowera, A; Peakman, M

    2015-01-01

    CD8 T cells specific for islet autoantigens are major effectors of β cell damage in type 1 diabetes, and measurement of their number and functional characteristics in blood represent potentially important disease biomarkers. CD8 T cell reactivity against glutamic acid decarboxylase 65 (GAD65) in HLA-A*0201 subjects has been reported to focus on an immunogenic region 114–123 (VMNILLQYVV), with studies demonstrating both 114–123 and 114–122 epitopes being targeted. However, the fine specificity of this response is unclear and the key question as to which epitope(s) β cells naturally process and present and, therefore, the pathogenic potential of CD8 T cells with different specificities within this region has not been addressed. We generated human leucocyte antigen (HLA)-A*0201-restricted CD8 T cell clones recognizing either 114–122 alone or both 114–122 and 114–123. Both clone types show potent and comparable effector functions (cytokine and chemokine secretion) and killing of indicator target cells externally pulsed with cognate peptide. However, only clones recognizing 114–123 kill target cells transfected with HLA-A*0201 and GAD2 and HLA-A*0201+ human islet cells. We conclude that the endogenous pathway of antigen processing by HLA-A*0201-expressing cells generates GAD65114–123 as the predominant epitope in this region. These studies highlight the importance of understanding β cell epitope presentation in the design of immune monitoring for potentially pathogenic CD8 T cells. PMID:25112375

  17. Comprehensive Analysis of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific Gamma Interferon-Secreting CD8+ T Cells in Primary HIV-1 Infection

    PubMed Central

    Cao, Jianhong; McNevin, John; Holte, Sarah; Fink, Lisa; Corey, Lawrence; McElrath, M. Juliana

    2003-01-01

    Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells provide an important defense in controlling HIV-1 replication, particularly following acquisition of infection. To delineate the breadth and potency of these responses in patients upon initial presentation and before treatment, we determined the fine specificities and frequencies of gamma interferon (IFN-γ)-secreting CD8+ T cells recognizing all HIV-1 proteins in patients with primary infection. In these subjects, the earliest detected responses were directed predominantly against Nef, Tat, Vpr, and Env. Tat- and Vpr-specific CD8+ T cells accounted for the greatest frequencies of mean IFN-γ spot-forming cells (SFC). Nef-specific responses (10 of 21) were more commonly detected. A mean of 2.3 epitopes were recognized with various avidities per subject, and the number increased with the duration of infection (R = 0.47, P = 0.031). The mean frequency of CD8+ T cells (985 SFC/106 peripheral blood mononuclear cells) correlated with the number of epitopes recognized (R = 0.84, P < 0.0001) and the number of HLA-restricting alleles (R = 0.79, P < 0.0001). Neither the total SFC frequencies nor the number of epitopes recognized correlated with the concurrent plasma viral load. Seventeen novel epitopes were identified, four of which were restricted to HLA alleles (A23 and B72) that are common among African descendents. Thus, primary HIV-1 infection induces strong CD8+-T-cell immunity whose specificities broaden over time, but their frequencies and breadth do not correlate with HIV-1 containment when examined concurrently. Many novel epitopes, particularly directed to Nef, Tat, and Env, and frequently with unique HLA restrictions, merit further consideration in vaccine design. PMID:12768006

  18. Mosaic HIV-1 Gag antigens can be Processed and Presented to human HIV-specific CD8+ T cells

    PubMed Central

    Ndhlovu, Zaza M; Piechocka-Trocha, Alicja; Vine, Seanna; McMullen, Ashley; Koofhethile, Kegakilwe C; Goulder, Phillip JR; Ndung’u, Thumbi; Barouch, Dan H; Walker, Bruce D

    2011-01-01

    Polyvalent “mosaic” HIV immunogens offer a potential solution for generating vaccines that can elicit immune responses against genetically diverse viruses. However, it is unclear whether key T cell epitopes can be processed and presented from these synthetic antigens and recognized by epitope-specific human T cells. Here we tested the ability of mosaic HIV immunogens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors to process and present major HIV clade B and clade C CD8 T cell epitopes in human cells. A bivalent mosaic vaccine expressing HIV Gag sequences was used to transduce PBMC from 12 HIV-1-infected individuals from the US and 10 HIV-1-infected individuals from South Africa, and intracellular cytokine staining together with tetramer staining was used to assess the ability of mosaic Gag antigens to stimulate pre-existing memory responses compared to natural clade B and C vectors. Mosaic Gag antigens expressed all 8 clade B epitopes tested in 12 US subjects and all 5 clade C epitopes tested in 10 South African subjects. Overall, the magnitude of cytokine production induced by stimulation with mosaic antigens was comparable to clade B and clade C antigens tested, but the mosaic antigens elicited greater cross-clade recognition. Additionally, mosaic antigens also induced HIV-specific CD4 T cell responses. Our studies demonstrate that mosaic antigens express major clade B and clade C viral T cell epitopes in human cells, and support the evaluation of mosaic HIV-1 vaccines in humans. PMID:21576505

  19. Mosaic HIV-1 Gag antigens can be processed and presented to human HIV-specific CD8+ T cells.

    PubMed

    Ndhlovu, Zaza M; Piechocka-Trocha, Alicja; Vine, Seanna; McMullen, Ashley; Koofhethile, Kegakilwe C; Goulder, Phillip J R; Ndung'u, Thumbi; Barouch, Dan H; Walker, Bruce D

    2011-06-15

    Polyvalent mosaic HIV immunogens offer a potential solution for generating vaccines that can elicit immune responses against genetically diverse viruses. However, it is unclear whether key T cell epitopes can be processed and presented from these synthetic Ags and recognized by epitope-specific human T cells. In this study, we tested the ability of mosaic HIV immunogens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors to process and present major HIV clade B and clade C CD8 T cell epitopes in human cells. A bivalent mosaic vaccine expressing HIV Gag sequences was used to transduce PBMCs from 12 HIV-1-infected individuals from the United States and 10 HIV-1-infected individuals from South Africa; intracellular cytokine staining, together with tetramer staining, was used to assess the ability of mosaic Gag Ags to stimulate pre-existing memory responses compared with natural clade B and C vectors. Mosaic Gag Ags expressed all eight clade B epitopes tested in 12 United States subjects and all 5 clade C epitopes tested in 10 South African subjects. Overall, the magnitude of cytokine production induced by stimulation with mosaic Ags was comparable to clade B and clade C Ags tested, but the mosaic Ags elicited greater cross-clade recognition. Additionally, mosaic Ags induced HIV-specific CD4 T cell responses. Our studies demonstrate that mosaic Ags express major clade B and clade C viral T cell epitopes in human cells, as well as support the evaluation of mosaic HIV-1 vaccines in humans.

  20. Tumor antigen-specific CD8(+) T cells are negatively regulated by PD-1 and Tim-3 in human gastric cancer.

    PubMed

    Lu, Xu; Yang, Lin; Yao, Daxing; Wu, Xuan; Li, Jingpo; Liu, Xuesong; Deng, Lijuan; Huang, Caiting; Wang, Yue; Li, Dan; Liu, Jingwei

    2017-03-01

    Cytotoxic CD8 T lymphocytes that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally important in eliciting tumor rejection. NY-ESO-1 is a major target of CD8(+) T cell recognition in gastric cancer (GC) and is among the most immunogenic tumor antigens defined to date. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8(+) T cell dysfunction may reveal effective strategies for immunotherapy. In an effort to understand in vivo tolerance mechanisms, we assessed the phenotype and function of NY-ESO-1-specific CD8(+) T cells derived from peripheral blood lymphocytes (PBLs) and tumor-associated lymphocytes (TALs) of GC patients. Here, we report that Tim-3 expression defines a subpopulation of PD-1(+) exhausted NY-ESO-1-specific CD8(+) T cell and PD-1(+)Tim-3(+) CD8(+) T cells represented the largest subset of NY-ESO-1-specific CD8(+) T cells in GC patients. Functionally, CD8(+)PD-1(+)Tim-3(+) T cells were more impaired in IFN-γ, TNF-α and IL-2 production compared with PD-1(+)Tim-3(-) or PD-1(-)Tim-3(-) subsets. Dual blockade of Tim-3 and PD-1 during T-cell priming efficiently augmented proliferation and cytokine production by NY-ESO-1-specific CD8(+) T cells could potentially be improved by therapeutic targeting of these inhibitory receptors, indicating that antitumor function of NY-ESO-1-specific CD8(+) T cells could potentially be improved by therapeutic targeting of these inhibitory receptors.