Selection, transmission, and reversion of an antigen-processing cytotoxic T-lymphocyte escape mutation in human immunodeficiency virus type 1 infection

Numerous studies now support that human immunodeficiency virus type 1 (HIV-1) evolution is influenced by immune selection pressure, with population studies showing an association between specific HLA alleles and mutations within defined cytotoxic T-lymphocyte epitopes. Here we combine sequence data and functional studies of CD8 T-cell responses to demonstrate that allele-specific immune pressures also select for mutations flanking CD8 epitopes that impair antigen processing. In persons expressing HLA-A3, we demonstrate consistent selection for a mutation in a C-terminal flanking residue of the normally immunodominant Gag KK9 epitope that prevents its processing and presentation, resulting in a rapid decline in the CD8 T-cell response. This single amino acid substitution also lies within a second HLA-A3-restricted epitope, with the mutation directly impairing recognition by CD8 T cells. Transmission of the mutation to subjects expressing HLA-A3 was shown to prevent the induction of normally immunodominant acute-phase responses to both epitopes. However, subsequent in vivo reversion of the mutation was coincident with delayed induction of new CD8 T-cell responses to both epitopes. These data demonstrate that mutations within the flanking region of an HIV-1 epitope can impair recognition by an established CD8 T-cell response and that transmission of these mutations alters the acute-phase CD8(+) T-cell response. Moreover, reversion of these mutations in the absence of the original immune pressure reveals the potential plasticity of immunologically selected evolutionary changes.     

In vivo hierarchy of immunodominant and subdominant HLA-A*0201-restricted T-cell epitopes of HBx antigen of hepatitis B virus

A polyepitopic CD8+ T-cell response is critical for the control of hepatitis B virus (HBV) infection. The HBV X protein (HBx) is a multifunctional protein that is important for the viral life cycle and for host-virus interactions. The aim of this study was to analyze the immunogenicity and dominance of various HLA-A*0201-restricted HBx-derived epitopes. For this purpose, we immunized HLA-A*0201-transgenic mice with HBx-derived peptides and DNA. This is a powerful model for studying the induction of HLA-A*0201-restricted immune responses in vivo, as these mice possess a cytotoxic T lymphocyte (CTL) repertoire representative of HLA-A2.1 individuals. We used cytotoxic tests and enzyme-linked immunosorbent spot (ELISPOT) assays to study the induction of specific cytotoxic and interferon (IFN)-gamma-secreting T cells. This allowed us to classify the HBx epitopes according to their T-cell activation capacity. After endogenous processing of the antigen synthesized in vivo after DNA-based immunization, we found that the HBx-specific T-cell response is targeted against one immunodominant epitope. Furthermore, following peptide immunization, we identified six additional novel subdominant T-cell epitopes. Inclusion of well-characterized epitopic sequences of HBx in a new vaccine for chronic HBV infections could help to broaden the T-cell response.     

Characterization of a Proteasome and TAP-independent Presentation of Intracellular Epitopes by HLA-B27 Molecules

Nascent HLA-class I molecules are stabilized by proteasome-derived peptides in the ER and the new complexes proceed to the cell surface through the post-ER vesicles. It has been shown, however, that less stable complexes can exchange peptides in the Trans Golgi Network (TGN). HLA-B27 are the most studied HLA-class I molecules due to their association with Ankylosing Spondylitis (AS). Chimeric proteins driven by TAT of HIV have been exploited by us to deliver viral epitopes, whose cross-presentation by the HLA-B27 molecules was proteasome and TAP-independent and not restricted to Antigen-Presenting Cells (APC). Here, using these chimeric proteins as epitope suppliers, we compared with each other and with the HLA-A2 molecules, the two HLA-B*2705 and B*2709 alleles differing at residue 116 (D116H) and differentially associated with AS. We found that the antigen presentation by the two HLA-B27 molecules was proteasome-, TAP-, and APC-independent whereas the presentation by the HLA-A2 molecules required proteasome, TAP and professional APC. Assuming that such difference could be due to the unpaired, highly reactive Cys-67 distinguishing the HLA-B27 molecules, C67S mutants in HLA-B*2705 and B*2709 and V67C mutant in HLA-A*0201 were also analyzed. The results showed that this mutation did not influence the HLA-A2-restricted antigen presentation while it drastically affected the HLA-B27-restricted presentation with, however, remarkable differences between B*2705 and B*2709. The data, together with the occurrence on the cell surface of unfolded molecules in the case of C67S-B*2705 mutant but not in that of C67S-B*2709 mutant, indicates that Cys-67 has a more critical role in stabilizing the B*2705 rather than the B*2709 complexes.     

Putative immunodominant human immunodeficiency virus-specific CD8(+) T-cell responses cannot be predicted by major histocompatibility complex class I haplotype

Recent studies of human immunodeficiency virus (HIV)-specific CD8(+) T cells have focused on responses to single, usually HLA-A2-restricted epitopes as surrogate measures of the overall response to HIV. However, the assumption that a response to one epitope is representative of the total response is unconfirmed. Here we assess epitope immunodominance and HIV-specific CD8(+) T-cell response complexity using cytokine flow cytometry to examine CD8(+) T-cell responses in 11 HLA-A2(+) HIV(+) individuals. Initial studies demonstrated that only 4 of 11 patients recognized the putative immunodominant HLA-A2-restricted p17 epitope SLYNTVATL, suggesting that the remaining subjects might lack significant HIV-specific CD8(+) T-cell responses. However, five of six SLYNTVATL nonresponders recognized other HIV epitopes, and two of four SLYNTVATL responders had greater responses to HIV peptides restricted by other class I alleles. In several individuals, no HLA-A2-restricted epitopes were recognized, but CD8(+) T-cell responses were detected to epitopes restricted by other HLA class I alleles. These data indicate that an individual's overall CD8(+) T-cell response to HIV is not adequately represented by the response to a single epitope and that individual major histocompatibility complex class I alleles do not predict an immunodominant response restricted by that allele. Accurate quantification of total HIV-specific CD8(+) T-cell responses will require assessment of the response to all possible epitopes.     

HLA-A2-restricted protection against lethal lymphocytic choriomeningitis

The consequences of human lymphocytic choriomeningitis virus (LCMV) infection can be severe, including aseptic meningitis in immunocompetent individuals, hydrocephalus or chorioretinitis in fetal infection, or a highly lethal outcome in immunosuppressed individuals. In murine models of LCMV infection, CD8(+) T cells play a primary role in providing protective immunity, and there is evidence that cellular immunity may also be important in related arenavirus infections in humans. For this reason, we sought to identify HLA-A2 supertype-restricted epitopes from the LCMV proteome and evaluate them as vaccine determinants in HLA transgenic mice. We identified four HLA-A*0201-restricted peptides-nucleoprotein NP(69-77), glycoprotein precursor GPC(10-18), GPC(447-455), and zinc-binding protein Z(49-58)-that displayed high-affinity binding (< or =275 nM) to HLA-A*0201, induced CD8(+) T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LCMV antigens in HLA-restricted human antigen presenting cells. One of the epitopes (GPC(447-455)), after peptide immunization of HLA-A*0201 mice, induced CD8(+) T cells capable of killing peptide-pulsed HLA-A*0201-restricted target cells in vivo and protected mice against lethal intracranial challenge with LCMV.     

HLA-A*7401-mediated control of HIV viremia is independent of its linkage disequilibrium with HLA-B*5703

The potential contribution of HLA-A alleles to viremic control in chronic HIV type 1 (HIV-1) infection has been relatively understudied compared with HLA-B. In these studies, we show that HLA-A*7401 is associated with favorable viremic control in extended southern African cohorts of >2100 C-clade-infected subjects. We present evidence that HLA-A*7401 operates an effect that is independent of HLA-B*5703, with which it is in linkage disequilibrium in some populations, to mediate lowered viremia. We describe a novel statistical approach to detecting additive effects between class I alleles in control of HIV-1 disease, highlighting improved viremic control in subjects with HLA-A*7401 combined with HLA-B*57. In common with HLA-B alleles that are associated with effective control of viremia, HLA-A*7401 presents highly targeted epitopes in several proteins, including Gag, Pol, Rev, and Nef, of which the Gag epitopes appear immunodominant. We identify eight novel putative HLA-A*7401-restricted epitopes, of which three have been defined to the optimal epitope. In common with HLA-B alleles linked with slow progression, viremic control through an HLA-A*7401-restricted response appears to be associated with the selection of escape mutants within Gag epitopes that reduce viral replicative capacity. These studies highlight the potentially important contribution of an HLA-A allele to immune control of HIV infection, which may have been concealed by a stronger effect mediated by an HLA-B allele with which it is in linkage disequilibrium. In addition, these studies identify a factor contributing to different HIV disease outcomes in individuals expressing HLA-B*5703.     

CD8 T cell responses to myelin oligodendrocyte glycoprotein-derived peptides in humanized HLA-A*0201-transgenic mice

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the CNS. Though originally believed to be CD4-mediated, additional immune effector mechanisms, including myelin-specific CD8(+) T cells, are now proposed to participate in the pathophysiology of MS. To study the immunologic and encephalitogenic behavior of HLA-A*0201-binding myelin-derived epitopes in vivo, we used a humanized HLA-A*0201-transgenic mouse model. Eight HLA-A*0201-binding peptides derived from myelin oligodendrocyte glycoprotein (MOG), an immunodominant myelin self-Ag, were identified in silico. After establishing their relative affinity for HLA-A*0201 and their capacity to form stable complexes with HLA-A*0201 in vitro, their immunological characteristics were studied in HLA-A*0201-transgenic mice. Five MOG peptides, which bound stably to HLA-A*0201 exhibited strong immunogenicity by inducing a sizeable MOG-specific HLA-A*0201-restricted CD8(+) T cell response in vivo. Of these five candidate epitopes, four were processed by MOG-transfected RMA target cells and two peptides proved immunodominant in vivo in response to a plasmid-encoding native full-length MOG. One of the immunodominant MOG peptides (MOG(181)) generated a cytotoxic CD8(+) T cell response able to aggravate CD4(+)-mediated EAE. Therefore, this detailed in vivo characterization provides a hierarchy of candidate epitopes for MOG-specific CD8(+) T cell responses in HLA-A*0201 MS patients identifying the encephalitogenic MOG(181) epitope as a primary candidate.     

Large-scale computational identification of HIV T-cell epitopes

Bioinformatics-driven T-cell epitope-identification methods can enhance vaccine target selection significantly. We evaluated three unrelated computational methods to screen Pol, Gag and Env sequences extracted from the Los Alamos HIV database for HLA-A*0201 and HLA-B*3501 T-cell epitope candidates. The hidden Markov model predicted 389 HLA-B*3501-restricted candidates from 374 HIV-1 and 97 HIV-2 sequences. The artificial neural network (ANN) model, and Bioinformatics and Molecular Analysis Section (BIMAS) quantitative matrix predictions for A*0201 yielded 1122 HIV-1 and 548 HIV-2 candidates. The overall sequence coverage of the predicted A*0201 T-cell epitopes was 2.7% (HIV-1)and 3.0% (HIV-2). HLA-B*3501-predicted epitopes covered 0.9% (HIV-1) and 1.4% (HIV-2) of the total sequence. Comparison of 890 ANN- and 397 BIMAS-derived HIV-1 A*0201- restricted epitope candidates showed that only 13-19% of the predicted and 26% of the experimentally confirmed T-cell epitopes were captured by both methods. Extrapolating these results, we estimated that at least 247 predicted HIV-1 epitopes are yet to be discovered as active A*0201-restricted T-cell epitopes. Adequate comparison and combined usage of various predictive bioinformatics methods, rather than uncritical use of any single prediction method, will enable cost-effective and efficient T-cell epitope screening.     

Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans

CD8(+) T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8(+) CTLs, epitopes for classical MHC class I-restricted CD8(+) T cells, which most likely represent a majority among CD8(+) T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K(b) transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4(+) T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8(+) T cells against Ag85B of M. tuberculosis in HLA-A2/K(b) transgenic mice and HLA-A*0201(+) humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8(+) CTLs were identified. These CD8(+) T cells produced IFN-gamma and TNF-alpha and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8(+) T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201(+) individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8(+) CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8(+) T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.     

Frequencies of ex vivo-activated human immunodeficiency virus type 1-specific gamma-interferon-producing CD8+ T cells in infected children correlate positively with plasma viral load

HIV-specific CD8+ T cells are critical in controlling human immunodeficiency virus (HIV) replication. We present the evaluation of a gamma-interferon (IFN-gamma)-based enzyme linked immunospot (ELISPOT) assay for the quantification of HIV-specific CD8+ T cells from HIV-infected children. We studied 20 HLA-A*0201-positive HIV-infected children. The IFN-gamma production in response to stimulation with two HLA-A*0201-restricted immunodominant CD8 epitopes (SLYNTVATL [SL9] in Gag and ILKEPVHGV [IV9] in Pol) was tested using the ELISPOT assay. The results were compared to labeling with the corresponding tetramers. Among the 20 children, 18 had detectable responses against the SL9 and/or the IV9 epitope using the ELISPOT assay (medians, 351 and 134 spot-forming cells/10(6) peripheral blood mononuclear cells, respectively). Comparison of results from the tetramer and ELISPOT assays suggests that only a fraction of HIV-specific CD8+ T cells were able to produce IFN-gamma. Most importantly, we found that the frequencies of IFN-gamma-producing CD8+ T cells were positively correlated with the viral load whereas the frequencies of tetramer-binding CD8+ T cells were not. The high sensitivity of the ELISPOT assay and the fact that this functional assay provided information different from that of tetramer labeling support its use for measurement of HIV-specific CD8+ T cells. In conclusion, our results show that the ex vivo-activated IFN-gamma-producing HIV-specific CD8+ T-cell subset is dependent upon continuous antigenic stimulation.     

A long peptide from MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination

CD4⁺ T cells contribute importantly to the antitumor T cell response, and thus, long peptides comprising CD4 and CD8 epitopes may be efficient cancer vaccines. We have previously identified an overexpressed antigen in melanoma, MELOE-1, presenting a CD8⁺ T cell epitope, MELOE-1_36–44, in the HLA-A*0201 context. A T cell repertoire against this epitope is present in HLA-A*0201+ healthy subjects and melanoma patients and the adjuvant injection of TIL containing MELOE-1 specific CD8⁺ T cells to melanoma patients was shown to be beneficial. In this study, we looked for CD4⁺ T cell epitopes in the vicinity of the HLA-A*0201 epitope. Stimulation of PBMC from healthy subjects with MELOE-1_26–46 revealed CD4 responses in multiple HLA contexts and by cloning responsive CD4⁺ T cells, we identified one HLA-DRβ1*1101-restricted and one HLA-DQβ1*0603-restricted epitope. We showed that the two epitopes could be efficiently presented to CD4⁺ T cells by MELOE-1-loaded dendritic cells but not by MELOE-1+ melanoma cell-lines. Finally, we showed that the long peptide MELOE-1_22–46, containing the two optimal class II epitopes and the HLA-A*0201 epitope, was efficiently processed by DC to stimulate CD4⁺ and CD8⁺ T cell responses in vitro, making it a potential candidate for melanoma vaccination.     

Escape from HLA-B*08-restricted CD8 T cells by hepatitis C virus is associated with fitness costs

The inherent sequence diversity of the hepatitis C virus (HCV) represents a major hurdle for the adaptive immune system to control viral replication. Mutational escape within targeted CD8 epitopes during acute HCV infection has been well documented and is one possible mechanism for T-cell failure. HLA-B*08 was recently identified as one HLA class I allele associated with spontaneous clearance of HCV replication. Selection of escape mutations in the immunodominant HLA-B*08-restricted epitope HSKKKCDEL(1395-1403) was observed during acute infection. However, little is known about the impact of escape mutations in this epitope on viral replication capacity. Their previously reported reversion back toward the consensus residue in patients who do not possess the B*08 allele suggests that the consensus sequence in this epitope is advantageous for viral replication in the absence of immune pressure. The aim of this study was to determine the impact of mutational escape from this immunodominant epitope on viral replication. We analyzed it with a patient cohort with chronic HCV genotype 1b infection and in a single-source outbreak (genotype 1b). Sequence changes in this highly conserved region are rare and selected almost exclusively in the presence of the HLA-B*08 allele. When tested in the subgenomic replicon (Con1), the observed mutations reduce viral replication compared with the prototype sequence. The results provide direct evidence that escape mutations in this epitope are associated with fitness costs and that the antiviral effect of HLA-B*08-restricted T cells is sufficiently strong to force the virus to adopt a relatively unfavorable sequence.     

The Magnitude and Specificity of Influenza A Virus-Specific Cytotoxic T-Lymphocyte Responses in Humans Is Related to HLA-A and -B Phenotype

The repertoire of human cytotoxic T-lymphocytes (CTL) in response to influenza A viruses has been shown to be directed towards multiple epitopes, with a dominant response to the HLA-A2-restricted M1(58-66) epitope. These studies, however, were performed with peripheral blood mononuclear cells (PBMC) of individuals selected randomly with respect to HLA phenotype or selected for the expression of one HLA allele without considering an influence of other HLA molecules. In addition, little information is available on the influence of HLA makeup on the overall CTL response against influenza viruses. Here, the influenza A virus-specific CTL response was investigated in groups of HLA-A and -B identical individuals. Between groups the individuals shared two or three of the four HLA-A and -B alleles. After in vitro stimulation of PBMC with influenza virus, the highest CTL activity was found in HLA-A2(+) donors. A similar pattern was observed for the precursor frequency of virus-specific CTL (CTLp) ex vivo, with a higher CTLp frequency in HLA-A2-positive donors than in HLA-A2-negative donors, which were unable to recognize the immunodominant M1(58-66) epitope. In addition, CTL activity and frequency of CTLp for the individual influenza virus epitopes were determined. The frequency of CTLp specific for the HLA-B8-restricted epitope NP(380-388) was threefold lower in HLA-B27-positive donors than in HLA-B27-negative donors. In addition, the frequency of CTLp specific for the HLA-A1-restricted epitope NP(44-52) was threefold higher in HLA-A1-, -A2-, -B8-, and -B35-positive donors than in other donors, which was confirmed by measuring the CTL activity in vitro. These findings indicate that the epitope specificity of the CTL response is related to the phenotype of the other HLA molecules. Furthermore, the magnitude of the influenza virus-specific CTL response seems dependent on the HLA-A and -B phenotypes.     

Hepatitis B virus splice-generated protein induces T-cell responses in HLA-transgenic mice and hepatitis B virus-infected patients

Hepatitis B virus splice-generated protein (HBSP), encoded by a spliced hepatitis B virus RNA, was recently identified in liver biopsy specimens from patients with chronic active hepatitis B. We investigated the possible generation of immunogenic peptides by the processing of this protein in vivo. We identified a panel of potential epitopes in HBSP by using predictive computational algorithms for peptide binding to HLA molecules. We used transgenic mice devoid of murine major histocompatibility complex (MHC) class I molecules and positive for human MHC class I molecules to characterize immune responses specific for HBSP. Two HLA-A2-restricted peptides and one immunodominant HLA-B7-restricted epitope were identified following the immunization of mice with DNA vectors encoding HBSP. Most importantly, a set of overlapping peptides covering the HBSP sequence induced significant HBSP-specific T-cell responses in peripheral blood mononuclear cells from patients with chronic hepatitis B. The response was multispecific, as several epitopes were recognized by CD8(+) and CD4(+) human T cells. This study provides the first evidence that this protein generated in vivo from an alternative reading frame of the hepatitis B virus genome activates T-cell responses in hepatitis B virus-infected patients. Given that hepatitis B is an immune response-mediated disease, the detection of T-cell responses directed against HBSP in patients with chronic hepatitis B suggests a potential role for this protein in liver disease progression.     

A novel cytosolic class I antigen-processing pathway for endoplasmic-reticulum-targeted proteins

Proteins bearing an endoplasmic reticulum (ER) leader are inserted into the ER followed by cleavage of the signal peptide. Major histocompatibility complex class I-restricted T-cell epitopes can be generated from these proteins by the proteasome after retrotranslocation into the cytosol. Here, we show that an HLA-A(*)0201-restricted epitope from prostate stem cell antigen contains the cleavage site of the ER signal peptidase. The resulting cleavage products fail to bind to HLA-A(*)0201 and are not recognized by T lymphocytes. As processing of prostate stem cell antigen by signal peptidase occurs immediately after co-translational insertion, the epitope must be processed from polypeptides that have never reached the ER. The processing of this epitope depends on the proteasome and the transporter associated with antigen processing and shows a novel pathway of class I processing that relies on the failure of ER-targeted proteins to reach their target compartment.     

Identification of five new HLA-B*3501-restricted epitopes derived from common melanoma-associated antigens, spontaneously recognized by tumor-infiltrating lymphocytes

We previously described HLA-B35-restricted melanoma tumor-infiltrating lymphocyte responses to frequently expressed melanoma-associated Ags: tyrosinase, Melan-A/MART-1, gp100, MAGE-A3/MAGE-A6, and NY-ESO-1. Using clones derived from these TIL, we identified in this study the corresponding epitopes. We show that five of these epitopes are new and that melanoma cells naturally present all the six epitopes. Interestingly, five of these epitopes correspond to or encompass melanoma-associated Ag epitopes presented in other HLA contexts, such as A2, A1, B51, and Cw3. In particular, the HLA-B35-restricted Melan-A epitope is mimicked by the peptide 26-35, already known as the most immunodominant melanoma epitope in the HLA-A*0201 context. Because this peptide lacked adequate anchor amino acid residues for efficient binding to HLA-B35, modified peptides were designed. Two of these analogues were found to induce higher PBL- and tumor-infiltrating lymphocyte-specific responses than the parental peptide, suggesting that they could be more immunogenic in HLA-B*3501 melanoma patients. These data have important implications for the formulation of polypeptide-based vaccines as well as for the monitoring of melanoma-specific CTL response in HLA-B*3501 melanoma patients.     

The majority of currently circulating human immunodeficiency virus type 1 clade B viruses fail to prime cytotoxic T-lymphocyte responses against an otherwise immunodominant HLA-A2-restricted epitope: implications for vaccine design

Human immunodeficiency virus type 1 (HIV-1) mutates to escape immune selection pressure, but there is little evidence of selection mediated through HLA-A2, the dominant class I allele in persons infected with clade B virus. Moreover, HLA-A2-restricted responses are largely absent in the acute phase of infection as the viral load is being reduced, suggesting that circulating viruses may lack immunodominant epitopes targeted through HLA-A2. Here we demonstrate an A2-restricted epitope within Vpr (Vpr59-67) that is targeted by acute-phase HIV-1-specific CD8+ T cells, but only in a subset of persons expressing HLA-A2. Individuals in the acute stage of infection with viruses containing the most common current sequence within this epitope (consensus sequence) were unable to mount epitope-specific T-cell responses, whereas subjects infected with the less frequent I60L variant all developed these responses. The I60L variant epitope was a stronger binder to HLA-A2 and was recognized by epitope-specific T cells at lower peptide concentrations than the consensus sequence epitope. These data demonstrate that HLA-A2 is capable of contributing to the acute-phase cytotoxic T-lymphocyte response in infected subjects, but that most currently circulating viruses lack a dominant immunogenic epitope presented by this allele, and suggest that immunodominant epitopes restricted by common HLA alleles may be lost as the epidemic matures.     

Hierarchy of alpha fetoprotein (AFP)-specific T cell responses in subjects with AFP-positive hepatocellular cancer

We identified a series of immunodominant and subdominant epitopes from alpha fetoprotein (AFP), restricted by HLA-A*0201, which are recognized by the human T cell repertoire. The four immunodominant epitopes have been tested for immunogenicity in vivo, in HLA-A*0201+AFP+ advanced stage hepatocellular cancer (HCC) patients, and have activated and expanded AFP-specific IFN-gamma-producing T cells in these patients, despite high serum levels of this self Ag. Here, we have examined the frequency, function, and avidity of the T cells specific for subdominant epitopes from AFP. We find that T cells specific for several of these epitopes are of similar or higher avidity than those specific for immunodominant epitopes. We then tested the peripheral blood of subjects ex vivo with different levels of serum AFP for the hierarchy of response to epitopes from this Ag and find that HCC patients have detectable frequencies of circulating IFN-gamma-producing AFP-specific CD8+ T cells to both immunodominant and subdominant epitopes. We find the immunodominant and subdominant peptide-specific T cells to be differentially expanded with different modes of Ag presentation. Whereas spontaneous and AFP protein-stimulated responses show evidence for immunodominance, AdVhAFP-transduced dendritic cell-stimulated responses were broader and not skewed. Importantly, these data identify subdominant epitopes from AFP that can activate high-avidity T cells, and that can be detected and expanded in HCC subjects. These subdominant epitope-specific T cells can also recognize tumor cells and may be important therapeutically.     

Naturally Processed Non-canonical HLA-A*02:01 Presented Peptides

Human leukocyte antigen (HLA) class I molecules generally present peptides (p) of 8 to 11 amino acids (aa) in length. Although an increasing number of examples with lengthy (>11 aa) peptides, presented mostly by HLA-B alleles, have been reported. Here we characterize HLA-A*02:01 restricted, in addition to the HLA-B*0702 and HLA-B*4402 restricted, lengthy peptides (>11 aa) arising from the B-cell ligandome. We analyzed a number of 15-mer peptides presented by HLA-A*02:01, and confirmed pHLA-I formation by HLA folding and thermal stability assays. Surprisingly the binding affinity and stability of the 15-mer epitopes in complex with HLA-A*02:01 were comparable with the values observed for canonical length (8 to 11 aa) HLA-A*02:01-restricted peptides. We solved the structures of two 15-mer epitopes in complex with HLA-A*02:01, within which the peptides adopted distinct super-bulged conformations. Moreover, we demonstrate that T-cells can recognize the 15-mer peptides in the context of HLA-A*02:01, indicating that these 15-mer peptides represent immunogenic ligands. Collectively, our data expand our understanding of longer epitopes in the context of HLA-I, highlighting that they are not limited to the HLA-B family, but can bind the ubiquitous HLA-A*02:01 molecule, and play an important role in T-cell immunity.