In silico prediction of immunogenic T cell epitopes for HLA-DQ8

Background HLA-DQ alleles are involved in the pathogenesis of hypersensitivity reactions, with HLA-DQ8 associated with several human autoimmune disorders. Limited success has been achieved using sequence-based computational techniques for predicting HLA-DQ8-restricted T cell epitopes while accuracy and efficiency of recently developed structure-based models need to be improved. Results We describe a combined structure-based prediction approach for DQ8-restricted T cell epitope prediction using a recently developed fast and accurate docking protocol, pDOCK, and molecular surface electrostatic potential (MSEP)-based clustering of pMHC binding interfaces. The prediction model was rigorously trained, tested and validated using experimentally verified DQ8 binding and non-binding peptides. High prediction accuracy (average area under the ROC curve, average AROC>0.94) is validated against experimental data. Our model also predicts all binding registers correctly and known T cell activators with 77% accuracy. We also studied the patterns of DQ8-binding peptides and reassure the existence of epitopes not conforming to binding motifs. Conclusions We have developed a model that can be successfully applied as a generic protocol for easy in silico identification of potential immunogenic T cell epitopes. The current model is therefore applicable for screening vaccine candidates irrespective of sequence motifs. We have also illustrated efficient discrimination of different categories of binders from non-binders as well as different categories of pMHC agonists from non-agonists, while accurately predicting the binding registers of DQ8-restricted peptides. This combined approach provides a set of sensitive and specific computational tools to facilitate high-throughput screening of peptides for immunotherapeutic applications such as controlling allergic and autoimmune responses.     

Allospecific T cell recognition of HLA-A2 antigens: Evidence for group-specific and subgroup-specific epitopes

Interleukin 2-dependent alloreactive cytotoxic T cell lines, with activity predominantly directed against the HLA-A2 antigen, have been generated in vitro by stimulating blood mononuclear cells from donors nonimmune to the Epstein-Barr (EB) virus with appropriate numbers of EB virus-transformed B cells from A2-homozygous individuals. Such effector cells were tested against a panel of EB virus-transformed target cell lines all expressing the serologically defined A2 antigen but typed into common A2 and variant A2 subgroups on the basis of their recognition by A2-restricted EB virus-specific cytotoxic T cells. Variant A2 responder cells cocultivated with common A2-bearing stimulators gave rise to effector T cell lines which recognized only the common A2-bearing subgroup of targets. By contrast, responder cells from A2-negative donors stimulated with common A2-bearing cells produced effector T cell lines in which the strong lysis of common A2-bearing targets was accompanied by a lower, but still significant, lysis directed against all targets within the variant A2 subgroup. In both cases, lysis of the target cells was blocked equally well by the anti-A2-specific monoclonal antibody MA2.1 as by the monoclonal antibody W6/32 specific for HLA-A, -B, and -C determinants. This suggests that HLA-A2 molecules possess at least two distinct sets of epitopes capable of inducing alloreactive T cell cytotoxicity: first, epitopes probably associated with T cell-restricting sites, which generate subgroup-specific responses, and second, epitopes shared by all A2 molecules, and perhaps associated with serologically defined sites, which generate pan A2 group-specific responses.Abbreviations used in this paper EB Epstein-Barr - IL-2 Interleukin 2 - UM unfractionated mononuclear - AET aminoethylisothiouroniumbromide hydrobromide     

Identification of CD8+ T cell epitopes within lytic antigens of human herpes virus 8

The human herpesvirus 8 (HHV-8) is a gamma herpesvirus with oncogenic potential which establishes a chronic infection that is normally controlled by the immune system of healthy individuals. In particular, CTL responses seem to play a key role in control of the infection. In this study, we characterized epitope-specific CTL responses in healthy HHV-8-seropositive individuals against four HHV-8 lytic Ags: open reading frames (ORF) 26, 70, K3, and K5. We found that the majority of subjects responded to at least one HHV-8 lytic Ag-derived epitope, and some of these epitopes represented dominant targets, suggesting that they could be relevant targets of CTL-mediated immunity in vivo, and may be involved in host control of HHV-8. Specifically, we identified three CTL epitopes from ORF 26, which are presented by HLA-A2, six CTL epitopes from ORF 70 presented by HLA-A2 (three epitopes), -A24 (two epitopes), and -B7 (one epitope), three CTL epitopes from ORF K3 presented by HLA-A2 (two epitopes) and -B7 (one epitope), and one HLA-A2 presented epitope derived from ORF K5. The identified epitopes may be regarded as useful tools for understanding the role of CTL responses to lytic Ags in individuals affected by HHV-8-associated disorders, and for the development of immunotherapies for the treatment/prevention of HHV-8-associated malignancies.     

An MSI tumor specific frameshift mutation in a coding microsatellite of MSH3 encodes for HLA-A0201-restricted CD8+ cytotoxic T cell epitopes

Background

Microsatellite instability (MSI) resulting from inactivation of the DNA mismatch repair system (MMR) characterizes a highly immunological subtype of colorectal carcinomas. Those tumors express multiple frameshift-mutated proteins which present a unique pool of tumor-specific antigens. The DNA MMR protein MSH3 is frequently mutated in MSI⁺ colorectal tumors, thus making it an attractive candidate for T cell-based immunotherapies.

Methodology/Principal Findings

FSP-specific CD8⁺ T cells were generated from a healthy donor using reverse immunology. Those T cells specifically recognized T2 cells sensitized with the respective peptides. Specific recognition and killing of MSI⁺ colorectal carcinoma cells harbouring the mutated reading frame was observed. The results obtained with T cell bulk cultures could be reproduced with T cell clones obtained from the same cultures. Blocking experiments (using antibodies and cold target inhibition) confirmed peptide as well as HLA-A0201-specificity.

Conclusions

We identified two novel HLA-A0201-restricted cytotoxic T cell epitopes derived from a (-1) frameshift mutation of a coding A(8) tract within the MSH3 gene. These were ³⁸⁶-FLLALWECSL (FSP18) and ³⁸⁷-LLALWECSL (FSP19) as well as ⁴⁰³-IVSRTLLLV (FSP23) and ⁴⁰²-LIVSRTLLLV (FSP31), respectively. These results suggest that MSH3(-1) represents another promising MSI⁺-induced target antigen. By identifying two distinct epitopes within MSH3(-1), the sustained immunogenicity of the frameshift mutated sequence was confirmed. Our data therefore encourage further exploitation of MSH3 as a piece for peptide-based vaccines either for therapeutic or –even more important– preventive purposes.     

Immunomodulation mediated through Leishmania donovani protein disulfide isomerase by eliciting CD8+ T-cell in cured visceral leishmaniasis subjects and identification of its possible HLA class-1 restricted T-cell epitopes

Protein disulphide isomerase (PDI) is one of the key enzymes essential for the survival of Leishmania donovani in the host. Our study suggested that PDI is associated with the generation of Th1-type of cellular responses in treated Visceral leishmaniasis (VL) subjects. The stimulation of Peripheral blood mononuclear cells (PBMCs) with recombinant Protein Disulphide Isomerase upregulated the reactive oxygen species generation, Nitric oxide release, IL12 and IFN-γ production indicating its pivotal role in protective immune response. Further, a pre-stimulation of PBMCs with Protein disulphide isomerase induced a strong IFN-γ response through CD8+ T cells in treated VL subjects. These findings also supported through the evidence that this antigen was processed and presented by major histocompatibility complex class I (MHC-1) dependent pathway and had an immunoprophylactic potential which can induce CD8+ T cell protective immune response in MHC class I dependent manner against VL. To find out the possible epitopes that might be responsible for CD8+ T cell specific IFN-γ response, computational approach was adopted. Six novel promiscuous epitopes were predicted to be highly immunogenic and can be presented by 32 different HLA allele to CD8+ T cells. Further investigation will explore more about their immunological relevance and usefulness as vaccine candidates.     

Phenotypical and functional analysis of memory and effector human CD8 T cells specific for mycobacterial antigens

Mycobacterium tuberculosis infects one-third of the global population and claims two million lives every year. Because memory CD8 T cells exhibit a high heterogeneity in terms of phenotype and functional characteristic, we investigated the frequency, phenotype, and functional properties of Ag85A epitope-specific HLA-A*0201 CD8 T cells in children affected by tuberculosis (TB) before and 4 mo after chemotherapy and healthy contact children. Using Ag85A peptide/HLA-A*0201 pentamer, we found a low frequency of blood peptide-specific CD8 T cells in tuberculous children before therapy, which consistently increased after therapy to levels detected in healthy contacts. Ex vivo analysis of the expression of CD45RA and CCR7 surface markers indicated a skewed representation of Ag85A epitope-specific CD8 T cells during active TB, with a predominance of T central memory cells and a decrease of terminally differentiated T cells, which was reversed after therapy. Accordingly, pentamer-specific CD8 T cells from tuberculous patients produced low levels of IFN-gamma and had low expression of perforin, which recovered after therapy. The finding of an elevated frequency of pentamer-specific CD8 T cells with T effector memory and terminally differentiated phenotypes in the cerebrospinal fluid of a child with tuberculous meningitis strongly indicates compartmentalization of such CD8 effectors at the site of disease. Our study represents the first characterization of Ag-specific memory and effector CD8 T cells during TB and may help to understand the type of immune response that vaccine candidates should stimulate to achieve protection.     

Identification and characterization of T cell-stimulating antigens from Leishmania by CD4 T cell expression cloning

Persistent immunity against Leishmania: infections in humans is mediated predominantly by CD4(+) T cells of the Th1 phenotype. Herein we report the expression cloning of eight Leishmania: Ags using parasite-specific T cell lines derived from an immune donor. The Ags identified by this technique include the flagellar proteins alpha- and beta-tubulin, histone H2b, ribosomal protein S4, malate dehydrogenase, and elongation factor 2, as well as two novel parasite proteins. None of these proteins have been previously reported as T cell-stimulating Ags from Leishmania: beta-tubulin-specific T cell clones generated against Leishmania: major amastigotes responded to Leishmania:-infected macrophages and dendritic cells. IFN-gamma enzyme-linked immunospot analysis demonstrated the presence of T cells specific for several of these Ags in PBMC from self-healing cutaneous leishmaniasis patients infected with either Leishmania: tropica or L. major. The responses elicited by Leishmania: histone H2b were particularly striking in terms of frequency of histone-specific T cells in PBMC (1 T cell of 6000 PBMC) as well as the percentage of responding donors (86%, 6 of 7). Ags identified by T cells from immune donors might constitute potential vaccine candidates for leishmaniasis.     

In vitro suppression of CD8+ T cell function by Friend virus-induced regulatory T cells

Regulatory T cell (Treg)-mediated suppression of CD8+ T cells has been implicated in the establishment and maintenance of chronic viral infections, but little is known about the mechanism of suppression. In this study an in vitro assay was developed to investigate the suppression of CD8+ T cells by Friend retrovirus (FV)-induced Tregs. CD4+CD25+ T cells isolated from mice chronically infected with the FV suppressed the development of effector function in naive CD8+ T cells without affecting their ability to proliferate or up-regulate activation markers. In vitro restimulation was not required for suppression by FV-induced Tregs, correlating with their high activation state in vivo. Suppression was mediated by direct T cell-T cell interactions and occurred in the absence of APCs. Furthermore, suppression occurred irrespective of the TCR specificity of the CD8+ T cells. Most interestingly, FV-induced Tregs were able to suppress the function of CD8+ effector T cells that had been physiologically activated during acute FV infection. The ability to suppress the effector function of activated CTLs is likely a requisite role for Tregs in limiting immunopathology by CD8+ T cells during antiviral immune responses. Such activity may also have adverse consequences by allowing viruses to establish and maintain chronic infections if suppression of antiviral immune responses occurs before virus eradication.     

Peptide-specific, allogeneic T cell response in vitro induced by a self-peptide binding to HLA-A2

The role of the bound peptide in alloreactive T-cell recognition is controversial, ranging from peptide-independent to peptide-specific recognition of alloreactive T-cells. The aim of this study is to find the evidence that there exist peptide/MHC complex (pMHC)-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a viral peptide (LMP2A426-434) or a self-peptide (Tyr369-377). The PBLs samples from 4 HLA-A2 positive (HLA-A2+ve) and 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The HLA-A2+ve PBL co-cultured with the LMP2A426-434pulsed T2 (T2/LMP) stands for the nominal T-cell response to a viral antigen, and the HLA-A2-ve PBLs co-cultured with the Tyr369-377 pulsed T2 (T2/Tyr) for alloreactive T-cell response to an allogeneic antigen.The specificity of the expanded CTLs after the LTMLC was detected by their specific cytotoxicity and binding ability to specific pMHC-tetramer. An HLA-A2 restricted, HIV peptide (Gag77-85) was included for control. The cultural bulk of HLA-A2+ve PBLs with the T2/LMP showed an elevated specific cytotoxicity against the T2/LMP compared to that against the T2/HIV (26.52%±3.72% vs 7.01%±0.87%, P＜0.001), and an increased frequency of binding to LMP-tetramer compared to that binding to HIV-tetramer (0.98%±0.33% vs 0.05%±0.01%, P=0.0014). The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the T2/Tyr than that against T2/HIV (28.07%±2.58% vs 6.87%±1.01%,P＜0.001), and a higher frequency of binding to the Tyr-tetramer than that binding to the HIV-tetramer (0.88%±0.3% vs 0.06%±0.03%, P=0.0018). Our results indicate that the LTMLC is able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs. A relatively large proportion of alloreactive CTLs should be pMHC-specific, i.e., the specificity of the alloreactive lines depends on both the bound peptide and the allotype of MHC. Our observations support the hypothesis that the cumularive effect of T cells specific to each peptide epitope could account for the strength and diversity of the alloresponse. The method using manipulated pMHC and the LTMLC to generate pMHC-specific, alloreactive CTLs is of potential importance for adoptive T-cell immunotherapy.     

Peptide-specific, allogeneic T cell response in vitro induced by a self-peptide binding to HLA-A2

The role of the bound peptide in alloreactive T-cell recognition is controversial, ranging from pep-tide-independent to peptide-specific recognition of alloreactive T-cells. The aim of this study is to find the evidence that there exist peptide/MHC complex (pMHC)-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a viral peptide (LMP2A426-434) or a self-peptide (Tyr369-377). The PBLs samples from 4 HLA-A2 positive (HLA-A2 ve) and 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The HLA-A2 ve PBL co-cultured with the LMP2A426-434 pulsed T2 (T2/LMP) stands for the nominal T-cell response to a viral antigen, and the HLA-A2-ve PBLs co-cultured with the Tyr369-377 pulsed T2 (T2/Tyr) for alloreactive T-cell response to an allogeneic antigen. The specificity of the expanded CTLs after the LTMLC was detected by their specific cytotoxicity and binding ability to specific pMHC-tetramer. An HLA-A2 restricted, HIV peptide (Gag77-85)was included for control. The cultural bulk of HLA-A2 ve PBLs with the T2/LMP showed an elevated specific cytotoxicity against the T2/LMP compared to that against the T2/HIV (26.52%±3.72% vs 7.01%±0.87%, P<0.001), and an increased frequency of binding to LMP-tetramer compared to that binding to HIV-tetramer (0.98%±0.33% vs 0.05%±0.01%, P=0.0014). The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the T2/Tyr than that against T2/HIV (28.07%±2.58% vs 6.87%±0.01 %, P<0.001), and a higher frequency of binding to the Tyr-tetramer than that binding to the HIV-tetramer (0.88%±0.3% vs 0.06%±0.03%, P=0.0018). Our results indicate that the LTMLC is able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs. A relatively large proportion of alloreactive CTLs should be pMHC-specific, i.e., the specificity of the alloreactive lines depends on both the bound peptide and the allotype of MHC. Our observations support the hypothesis that the cumulative effect of T cells specific to each peptide epitope could account for the strength and diversity of the alloresponse. The method using manipulated pMHC and the LTMLC to generate pMHC-specific, alloreactive CTLs is of potential importance for adoptive T-cell immunotherapy.     

The memory T cell response to West Nile virus in symptomatic humans following natural infection is not influenced by age and is dominated by a restricted set of CD8+ T cell epitopes

We examined the West Nile virus (WNV)-specific T cell response in a cohort of 52 patients with symptomatic WNV infections, including neuroinvasive and non-invasive disease. Although all virus proteins were shown to contain T cell epitopes, certain proteins, such as E, were more commonly targeted by the T cell response. Most patients exhibited reactivity toward 3-4 individual WNV peptides; however, several patients exhibited reactivity toward >10 individual peptides. The relative hierarchy of T cell reactivities in all patients showed a fixed pattern that was sustained throughout the 12-mo period of the current study. Surprisingly, we did not observe any relationship between age and either the breadth or magnitude of the T cell response following infection. We also did not observe a relationship between disease severity and either the breadth or magnitude of the T cell response. The T cell epitopes were distributed in a non-random fashion across the viral polyprotein and a limited number of epitopes appeared to dominate the CD8(+) T cell response within our cohort. These data provide important new insight into the T cell response against WNV in humans.     

Immuno-informatics based approaches to identify CD8+ T cell epitopes within the Leishmania donovani 3-ectonucleotidase in cured visceral leishmaniasis subjects

Leishmaniases are vector-borne diseases for which no vaccine exists. These diseases are caused by the Leishmania species complex. Activation of the CD8⁺ T cell is crucial for protection against intracellular pathogens, and peptide antigens are attractive strategies for the precise activation of CD8⁺ T in vaccine development against intracellular infections. The traditional approach to mine the epitopes is an arduous task. However, with the advent of immunoinformatics, in silico epitope prediction tools are available to expedite epitope identification. In this study, we employ different immunoinformatics tools to predict CD8⁺ T cell specific 9 mer epitopes presented by HLA-A*02 and HLA-B40 within the highly conserved 3′-ectonucleotidase of Leishmania donovani. We identify five promiscuous epitopes, which have no homologs in humans, theoretically cover 85% of the world's population and are highly conserved (100%) among Leishmania species. Presentation of selected peptides was confirmed by T2 cell line based HLA-stabilization assay, and three of them were found to be strong binders. The in vitro peptide stimulation of peripheral blood mononuclear cells (PBMC) from cured HLA-A02⁺ visceral leishmaniasis (VL) subjects produced significantly higher IFN-γ, IL-2 and IL-12 compared to no peptide control healthy subjects. Further, CD8⁺ cells from treated VL subjects produced significantly higher intracellular IFN-γ, lymphocyte proliferation and cytotoxic activity against selected peptides from the PBMCs of treated HLA-A02⁺ VL subjects. Thus, the CD8⁺ T cell specific epitopes shown in this study will speed up the development of polytope vaccines for leishmaniasis.     

Leishmania antigens are presented to CD8+ T cells by a transporter associated with antigen processing-independent pathway in vitro and in vivo

CD8+ T cells are generated in response to Leishmania major (Lm) or Toxoplasma gondii parasitic infections, indicating that exogenously delivered Ag can be processed for presentation by MHC class I molecules. We show that presentation of Lm nucleotidase (NT)-OVA is TAP independent in vivo and in vitro, and is inhibited by chloroquine, but not by proteasome inhibitors. In contrast, the presentation of T. gondii P30-OVA relies on the TAP/proteasome pathway. Presentation of OVA- or rNT-OVA-coated beads also bypassed TAP requirement above a certain Ag threshold. TAP was also dispensable for the presentation of wild-type Lm Ags to primed CD8+ T cells in vitro. Finally, in vivo priming of CD8+ T cells involved in acquired resistance to Lm was not compromised in TAP-deficient mice. Thus, Leishmania Ags appear to be confined to an intraphagosomal processing pathway that requires higher concentrations of Ags, suggesting that these parasites may have evolved strategies to impair the efficient endoplasmic reticulum-based, TAP-dependent cross-presentation pathway to avoid or delay CD8+ T cell priming.     

Dynamic antigen presentation patterns of Listeria monocytogenes-derived CD8 T cell epitopes in vivo

Little information exists regarding the presentation of antigenic peptides in infected tissues. In this study the in vivo presentation of four different CD8 T cell epitopes of Listeria monocytogenes was monitored. Peptide presentation was measured by a new, highly sensitive, ex vivo Ag presentation assay that was based on the testing of freshly isolated cells from infected spleens with peptide-specific CD8 T cell lines in an IFN-gamma-specific ELISPOT assay. Remarkably, the peptide presentation pattern of splenocytes and that of macrophages purified from spleens of L. monocytogenes-infected mice were different from those of in vitro infected macrophage-like cell lines. The in vivo Ag presentation pattern of splenocytes also exhibited dynamic changes during the first 48 h of infection. In vivo peptide presentation at later time points postinfection was biased toward immunodominant CD8 T cell epitopes, while at an early time point, 6 h postinfection, subdominant and dominant CD8 T cell epitopes were presented with similar strength. In summary, our studies show that Ag presentation during an infection is a highly dynamic process that only can be fully appreciated by the study of cells infected in their physiological environment.     

N-terminal trimer extension of nominal CD8 T cell epitopes is sufficient to promote cross-presentation to cognate CD8 T cells in vivo

Cross-priming is the process in which Ag-presenting dendritic cells (DCs) acquire, process, and present Ags scavenged from other cells, and use these cells to activate naive CD8 T cells. Cross-priming of cognate CD8 cells can result in either tolerance or immunity, depending upon the activation status of the Ag-presenting DC. Previous studies have shown that nominal peptide is inefficiently cross-presented and that proteins and large polypeptides that require proteasomal processing are the main source of naturally cross-presented Ags. In this study we show that N-terminal extension of nominal peptide by as few as three residues is sufficient to produce a substrate for TAP-dependent cross-presentation that is highly efficient in cross-priming murine CD8 T cells in vivo. On a molar basis, cross-priming with 3-mer-extended peptide is 20-fold more efficient than priming with intact protein. This method of peptide extension should prove of great value in facilitating in vivo studies of CD8 immunity and tolerance that rely on cross-presentation.     

Identification of HLA‐A*11:01‐restricted Mycobacterium tuberculosis CD8+ T cell epitopes

New vaccines are needed to combat Mycobacterium tuberculosis (MTB) infections. The currently employed Bacillus Calmette‐Guérin vaccine is becoming ineffective, due in part to the emergence of multidrug‐resistant tuberculosis (MDR‐TB) strains and the reduced immune capacity in cases of HIV coinfection. CD8⁺ T cells play an important role in the protective immunity against MTB infections, and the identification of immunogenic CD8⁺ T cell epitopes specific for MTB is essential for the design of peptide‐based vaccines. To identify CD8⁺ T cell epitopes of MTB proteins, we screened a set of 94 MTB antigens for HLA class I A*11:01‐binding motifs. HLA‐A*11:01 is one of the most prevalent HLA molecules in Southeast Asians, and definition of T cell epitopes it can restrict would provide significant coverage for the Asian population. Peptides that bound with high affinity to purified HLA molecules were subsequently evaluated in functional assays to detect interferon‐γ release and CD8⁺ T cell proliferation in active pulmonary TB patients. We identified six novel epitopes, each derived from a unique MTB antigen, which were recognized by CD8⁺ T cells from active pulmonary TB patients. In addition, a significant level of epitope‐specific T cells could be detected ex vivo in peripheral blood mononuclear cells from active TB patients by an HLA‐A*11:01 dextramer carrying the peptide Rv3130c_194‐204 (from the MTB triacylglycerol synthase Tgs1), which was the most frequently recognized epitope in our peptide library. In conclusion, this study identified six dominant CD8⁺ T cell epitopes that may be considered potential targets for subunit vaccines or diagnostic strategies against TB.     

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

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.     

Vaccines targeting tumor blood vessel antigens promote CD8(+) T cell-dependent tumor eradication or dormancy in HLA-A2 transgenic mice

We have recently shown that effective cytokine gene therapy of solid tumors in HLA-A2 transgenic (HHD) mice lacking murine MHC class I molecule expression results in the generation of HLA-A2-restricted CD8(+) T effector cells selectively recognizing tumor blood vessel-associated pericytes and/or vascular endothelial cells. Using an HHD model in which HLA-A2(neg) tumor (MC38 colon carcinoma or B16 melanoma) cells are not recognized by the CD8(+) T cell repertoire, we now show that vaccines on the basis of tumor-associated blood vessel Ags (TBVA) elicit protective Tc1-dependent immunity capable of mediating tumor regression or extending overall survival. Vaccine efficacy was not observed if (HLA-A2(neg)) wild-type C57BL/6 mice were instead used as recipient animals. In the HHD model, effective vaccination resulted in profound infiltration of tumor lesions by CD8(+) (but not CD4(+)) T cells, in a coordinate reduction of CD31(+) blood vessels in the tumor microenvironment, and in the "spreading" of CD8(+) T cell responses to alternate TBVA that were not intrinsic to the vaccine. Protective Tc1-mediated immunity was durable and directly recognized pericytes and/or vascular endothelial cells flow-sorted from tumor tissue but not from tumor-uninvolved normal kidneys harvested from these same animals. Strikingly, the depletion of CD8(+), but not CD4(+), T cells at late time points after effective therapy frequently resulted in the recurrence of disease at the site of the regressed primary lesion. This suggests that the vaccine-induced anti-TBVA T cell repertoire can mediate the clinically preferred outcomes of either effectively eradicating tumors or policing a state of (occult) tumor dormancy.     

Random screening of proteins for HLA-A*0201-binding nine-amino acid peptides is not sufficient for identifying CD8 T cell epitopes recognized in the context of HLA-A*0201

HLA-A2 is the most frequent HLA molecule in Caucasians with HLA-A*0201 representing the most frequent allele; it was also the first human HLA allele for which peptide binding prediction was developed. The Bioinformatics and Molecular Analysis Section of the National Institutes of Health (BIMAS) and the University of Tübingen (Syfpeithi) provide the most popular prediction algorithms of peptide/MHC interaction on the World Wide Web. To test these predictions, HLA-A*0201-binding nine-amino acid peptides were searched by both algorithms in 19 structural CMV proteins. According to Syfpeithi, the top 2% of predicted peptides should contain the naturally presented epitopes in 80% of predictions (www.syfpeithi.de). Because of the high number of predicted peptides, the analysis was limited to 10 randomly chosen proteins. The top 2% of peptides predicted by both algorithms were synthesized corresponding to 261 peptides in total. PBMC from 10 HLA-A*0201-positive and CMV-seropositive healthy blood donors were tested by ex vivo stimulation with all 261 peptides using crossover peptide pools. IFN-gamma production in T cells measured by CFC was used as readout. However, only one peptide was found to be stimulating in one single donor. As a result of this work, we report a potential new T cell target protein, one previously unknown CD8-T cell-stimulating peptide, and an extensive list of CMV-derived potentially strong HLA-A*0201-binding peptides that are not recognized by T cells of HLA-A*0201-positive CMV-seropositive donors. We conclude that MHC/peptide binding predictions are helpful for locating epitopes in known target proteins but not necessarily for screening epitopes in proteins not known to be T cell targets.