Genome-wide association studies (GWAS) have identified more than 220 loci associated with breast cancer susceptibility, yet identifying effector genes, their modes of action and prioritising therapeutic targets remains a significant challenge. To address this, we performed pooled CRISPR knockout and inhibition screens to identify genes at risk loci that influence cytotoxic T lymphocyte (CTL) killing of MCF7 breast cancer cells in co-culture. These screens uncovered 33 candidate modulating genes, of which we validated six by single gene editing in two cell lines. Deletion of IRF1, ATF7IP, and CASP8 conferred resistance to CTL killing, while disruption of CFLAR, CREBBP and PRMT7 enhanced sensitivity. Analysis of clinical data showed that PRMT7 expression is negatively correlated with CD8+ infiltration and survival in breast cancer patient cohorts. Pharmacological inhibition of PRMT7 sensitized breast cells to CTL killing in vitro, and Prmt7-deficient tumors exhibited reduced growth and increased CD8+ T cell infiltration in immunocompetent mice. Enhanced Prmt7-dependent tumor growth was not observed in immunodeficient mice, implicating Prmt7 in immune evasion. This study underscores the utility of CRISPR screens for high-throughput functional follow-up of GWAS findings and identifies PRMT7 inhibition as a promising therapeutic strategy.

BACKGROUND:Adoptive T-cell therapy targeting antigens expressed in glioblastoma has emerged as a potential therapeutic strategy to prevent or delay recurrence and prolong overall survival in this aggressive disease setting. Ephrin receptor A3 (EphA3), which is highly expressed in glioblastoma; in particular, on the tumor vasculature and brain cancer stem cells, is an ideal target for immune-based therapies. METHODS:We have designed an EphA3-targeted chimeric antigen receptor (CAR) using the single chain variable fragment of a novel monoclonal antibody, and assessed its therapeutic potential against EphA3-expressing patient-derived glioblastoma neurospheres, organoids and xenografted glioblastoma tumors in immunodeficient mice. RESULTS:In vitro expanded EphA3 CAR T cells from healthy individuals efficiently recognize and kill EphA3-positive glioblastoma cells in vitro. Furthermore, these effector cells demonstrated curative efficacy in an orthotopic xenograft model of glioblastoma. EphA3 CAR T cells were equally effective in targeting patient-derived neurospheres and infiltrate, disaggregate, and induce apoptosis in glioblastoma-derived organoids. CONCLUSIONS:This study provides compelling evidence supporting the therapeutic potential of EphA3 CAR T-cell therapy against glioblastoma by targeting EphA3 associated with brain cancer stem cells and the tumor vasculature. The ability to target patient-derived glioblastoma underscores the translational significance of this EphA3 CAR T-cell therapy in the pursuit of effective and targeted glioblastoma treatment strategies.

Genome-wide association studies have identified more than 220 loci associated with breast cancer susceptibility. A major challenge is now to identify the effector genes with plausible functions in the context of breast cancer risk. We have previously performed pooled CRISPR screens to identify target genes at risk loci that drive cancer hallmarks including proliferation or modulating DNA damage response. We now extend these screens to identify genes involved in response to cytotoxic T lymphocyte (CTL) killing. We performed knockout and inhibition screens to identify genes that affect the response of the MCF7 human breast cancer cell line to CTL killing in an in vitro co-culture system. We identified 33 candidate risk genes associated with resistance or sensitisation to T cell-mediated killing. Using single gene perturbation, we showed that deletion of candidate risk genes IRF1, ATF7IP, CCDC170 and CASP8 induced resistance, while ablation of CFLAR, CREBBP , and PRMT7 sensitized cells to CTL killing. We used reporter assays to show that the risk-associated alleles at rs736801 and rs3769821 reduced transactivation of the IRF1 and CASP8 promoters, respectively. We showed that pharmacological inhibition of PRMT7 rendered breast cells sensitive to CTL killing and PRMT7 levels were negatively correlated with CD8+ infiltration and patient survival in luminal A breast cancer patient cohorts. Our results demonstrate that phenotypic pooled CRISPR screens are a useful approach for high throughput functional follow-up of GWAS findings, identifying genes which alter immune responses to breast cancer which offer opportunities to enhance immunotherapy.### Competing Interest StatementThe authors have declared no competing interest.

Efforts are being made worldwide to understand the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic, including the impact of T cell immunity and cross-recognition with seasonal coronaviruses. Screening of SARS-CoV-2 peptide pools revealed that the nucleocapsid (N) protein induced an immunodominant response in HLA-B7+ COVID-19-recovered individuals that was also detectable in unexposed donors. A single N-encoded epitope that was highly conserved across circulating coronaviruses drove this immunodominant response. In vitro peptide stimulation and crystal structure analyses revealed T cell-mediated cross-reactivity toward circulating OC43 and HKU-1 betacoronaviruses but not 229E or NL63 alphacoronaviruses because of different peptide conformations. T cell receptor (TCR) sequencing indicated that cross-reactivity was driven by private TCR repertoires with a bias for TRBV27 and a long CDR3β loop. Our findings demonstrate the basis of selective T cell cross-reactivity for an immunodominant SARS-CoV-2 epitope and its homologs from seasonal coronaviruses, suggesting long-lasting protective immunity.

Significant efforts are being made worldwide to understand the immune response to SARS-CoV-2, responsible for the COVID-19 pandemic, including the role of preexisting T cell immunity. Understanding the mechanisms that promote crossrecognition by T cells induced by seasonal coronaviruses will be critical for future predictions on the role of pre-existing immunity in protection against severe disease. We demonstrate that the SARS-CoV-2 nucleocapsid (N) protein induces an immunodominant response in HLA-B7+ COVID-19-recovered individuals that is also readily detectable in unexposed donors. This immunodominant response is driven by a single N-encoded epitope that displays a high degree of conservation with the homologous region in circulating coronaviruses. We show that T cell-mediated crossreactivity can be detected towards the circulating OC43/HKU-1 coronaviruses, but not the 229E or NL63 coronaviruses, due to different peptide conformations. This cross-reactivity is driven by private T cell receptor repertoires with a bias for TRBV27 and a long CDR3b loop in unexposed and COVID-19-recovered individuals. Together, our findings demonstrate the basis of pre-existing immunity to a conserved and highly immunogenic SARS-CoV-2 epitope driven by cross-reactive memory T cells, suggesting long-lived protective immunity.

Mounting evidence indicates that infection with Epstein–Barr virus (EBV) has a major role in the pathogenesis of multiple sclerosis (MS). Defective elimination of EBV-infected B cells by CD8 + T cells might cause MS by allowing EBV-infected autoreactive B cells to accumulate in the brain. Here we undertake a comprehensive analysis of the T-cell response to EBV in MS, using flow cytometry and intracellular IFN-γ staining to measure T-cell responses to EBV-infected autologous lymphoblastoid cell lines and pools of human leukocyte antigen (HLA)-class-I-restricted peptides from EBV lytic or latent proteins and cytomegalovirus (CMV), in 95 patients and 56 EBV-seropositive healthy subjects. In 20 HLA-A2 + healthy subjects and 20 HLA-A2 + patients we also analysed CD8 + T cells specific for individual peptides, measured by binding to HLA-peptide complexes and production of IFN-γ, TNF-α and IL-2. We found a decreased CD8 + T-cell response to EBV lytic, but not CMV lytic, antigens at the onset of MS and at all subsequent disease stages. CD8 + T cells directed against EBV latent antigens were increased but had reduced cytokine polyfunctionality indicating T-cell exhaustion. During attacks the EBV-specific CD4 + and CD8 + T-cell populations expanded, with increased functionality of latent-specific CD8 + T cells. With increasing disease duration, EBV-specific CD4 + and CD8 + T cells progressively declined, consistent with T-cell exhaustion. The anti-EBNA1 IgG titre correlated inversely with the EBV-specific CD8 + T-cell frequency. We postulate that defective CD8 + T-cell control of EBV reactivation leads to an expanded population of latently infected cells, including autoreactive B cells.

Adoptive T cell therapy has emerged as a powerful strategy to treat human cancers especially haematological malignancies. Extension of these therapies to solid cancers remains a significant challenge especially in the context of defining immunological correlates of clinical responses. Here we describe results from a clinical study investigating autologous Epstein-Barr virus (EBV)-specific T cells generated using a novel AdE1-LMPpoly vector to treat patients with nasopharyngeal carcinoma (NPC) either pre-emptively in at-risk patients with no or minimal residual disease (N/MRD) or therapeutically in patients with active recurrent/metastatic disease (ARMD). Tolerability, safety and efficacy, including progression-free survival (PFS) and overall survival (OS), were evaluated following adoptive T-cell immunotherapy. Twenty-nine patients, including 20 with ARMD and nine with N/MRD, successfully completed T-cell therapy. After a median follow-up of 18.5 months, the median PFS was 5.5 months (95% CI 2.1 to 9.0 months) and the median OS was 38.1 months (95% CI 17.2 months to not reached). Post-immunotherapy analyses revealed that disease stabilization in ARMD patients was significantly associated with the functional and phenotypic composition of in vitro-expanded T cell immunotherapy. These included a higher proportion of effector CD8+ T-cells and an increased number of EBV-specific T-cells with broader antigen specificity. These observations indicate that adoptive immunotherapy with AdE1-LMPpoly-expanded T cells stabilizes relapsed, refractory NPC without significant toxicity. Promising clinical outcomes in N/MRD patients further suggest a potential role for this approach as a consolidation treatment following first-line chemotherapy.

T cell cross-reactivity underpins the molecular mimicry hypothesis in which microbial peptides sharing structural features with host peptides stimulate T cells that cross-react with self-peptides, thereby initiating and/or perpetuating autoimmune disease. EBV represents a potentially important factor in the pathogenesis of several T cell-mediated autoimmune disorders, with molecular mimicry a likely mechanism. In this study, we describe a human self-peptide (DELEIKAY) that is a homolog of a highly immunogenic EBV T cell epitope (SELEIKRY) presented by HLA-B*18:01. This self-peptide was shown to bind stably to HLA-B*18:01, and peptide elution/mass spectrometric studies showed it is naturally presented by this HLA molecule on the surface of human cells. A significant proportion of CD8(+) T cells raised from some healthy individuals against this EBV epitope cross-reacted with the self-peptide. A diverse array of TCRs was expressed by the cross-reactive T cells, with variable functional avidity for the self-peptide, including some T cells that appeared to avoid autoreactivity by a narrow margin, with only 10-fold more of the self-peptide required for equivalent activation as compared with the EBV peptide. Structural studies revealed that the self-peptide-HLA-B*18:01 complex is a structural mimic of the EBV peptide-HLA-B*18:01 complex, and that the strong antiviral T cell response is primarily dependent on the alanine/arginine mismatch at position 7. To our knowledge, this is the first report confirming the natural presentation of a self-peptide cross-recognized in the context of self-HLA by EBV-reactive CD8(+) T cells. These results illustrate how aberrant immune responses and immunopathological diseases could be generated by EBV infection.

20 Polymorphism in the human leukocyte antigen (HLA) loci ensures that the CD8 + T cell 21 response to viruses is directed against a diverse range of antigenic epitopes, thereby 22 minimizing the impact of virus escape mutation across the population. The BZLF1 antigen of 23 Epstein-Barr virus is an immunodominant target for CD8 + T cells but the response has only 24 been characterized in the context of a limited number of HLA molecules due to incomplete 25 epitope mapping. We have now greatly expanded the number of defined CD8 + T cell epitopes 26 from BZLF1, allowing the response to be evaluated in a much larger proportion of the 27 population. Some regions of the antigen fail to be recognized by CD8 + T cells while others 28 include clusters of overlapping epitopes presented by different HLA molecules. These highly 29 immunogenic regions of BZLF1 include polymorphic sequences, such that up to four 30 overlapping epitopes are impacted by a single amino acid variation common in different 31 regions of the world. This focusing of the immune response to limited regions of the viral 32 protein could be due to sequence similarity to human proteins creating “immune blind spots” 33 through self-tolerance. This study significantly enhances the understanding of the immune 34 response to BZLF1, and the precisely mapped T cell epitopes may be directly exploited in 35 vaccine development and adoptive immunotherapy. 36 37 on July 1, 2017 by gest http/jvi.asm .rg/ D ow nladed fom

ABSTRACT Polymorphism in the human leukocyte antigen (HLA) loci ensures that the CD8+ T cell response to viruses is directed against a diverse range of antigenic epitopes, thereby minimizing the impact of virus escape mutation across the population. The BZLF1 antigen of Epstein-Barr virus is an immunodominant target for CD8+ T cells, but the response has been characterized only in the context of a limited number of HLA molecules due to incomplete epitope mapping. We have now greatly expanded the number of defined CD8+ T cell epitopes from BZLF1, allowing the response to be evaluated in a much larger proportion of the population. Some regions of the antigen fail to be recognized by CD8+ T cells, while others include clusters of overlapping epitopes presented by different HLA molecules. These highly immunogenic regions of BZLF1 include polymorphic sequences, such that up to four overlapping epitopes are impacted by a single amino acid variation common in different regions of the world. This focusing of the immune response to limited regions of the viral protein could be due to sequence similarity to human proteins creating “immune blind spots” through self-tolerance. This study significantly enhances the understanding of the immune response to BZLF1, and the precisely mapped T cell epitopes may be directly exploited in vaccine development and adoptive immunotherapy. IMPORTANCE Epstein-Barr virus (EBV) is an important human pathogen, associated with several malignancies, including nasopharyngeal carcinoma and Hodgkin lymphoma. T lymphocytes are critical for virus control, and clinical trials aimed at manipulating this arm of the immune system have demonstrated efficacy in treating these EBV-associated diseases. These trials have utilized information on the precise location of viral epitopes for T cell recognition, for either measuring or enhancing responses. In this study, we have characterized the T cell response to the highly immunogenic BZLF1 antigen of EBV by greatly expanding the number of defined T cell epitopes. An unusual clustering of epitopes was identified, highlighting a small region of BZLF1 that is targeted by the immune response of a high proportion of the world's population. This focusing of the immune response could be utilized in developing vaccines/therapies with wide coverage, or it could potentially be exploited by the virus to escape the immune response.

Exposure to naturally occurring variants of herpesviruses in clinical settings can have a dramatic impact on anti-viral immunity. Here we have evaluated the molecular imprint of variant peptide-MHC complexes on the T-cell repertoire during human cytomegalovirus (CMV) infection and demonstrate that primary co-infection with genetic variants of CMV was coincident with development of strain-specific T-cell immunity followed by emergence of cross-reactive virus-specific T-cells. Cross-reactive CMV-specific T cells exhibited a highly conserved public T cell repertoire, while T cells directed towards specific genetic variants displayed oligoclonal repertoires, unique to each individual. T cell recognition foot–print and pMHC-I structural analyses revealed that the cross-reactive T cells accommodate alterations in the pMHC complex with a broader foot-print focussing on the core of the peptide epitope. These findings provide novel molecular insight into how infection with naturally occurring genetic variants of persistent human herpesviruses imprints on the evolution of the anti-viral T-cell repertoire.

BACKGROUND:Among the environmental factors associated with multiple sclerosis (MS) causation, some of the strongest associations are with Epstein-Barr virus (EBV), and to a lesser extent human herpesvirus 6 (HHV6). Associations with clinical course are less conclusive, however.METHODS:We evaluated serum anti-EBV-EA-R IgG and anti-HHV6 IgM, and EBV and HHV6 viral load (VL) for their associations with relapse, disability, and progression in disability in a prospective cohort of 198 participants with clinically definite MS.RESULTS:Anti-EBV-EA-R IgG was detected in 81.8% of cases at study entry, and titers remained essentially unchanged during the study. Anti-HHV6 IgM was detected in only one participant, and EBV-VL (29%) and HHV6-VL (1.8%) were detected in a minority of samples, and where detected levels were low. Our previously demonstrated association between anti-HHV6 IgG and relapse hazard was not affected by adjustment for parameters of reactivation. We found no evidence that any of the viral markers were associated with disability or progression in disability. In relation to relapse, only EBV-VL was positively associated, although this was strongly influenced by a single individual.CONCLUSION:Using a prospective cohort design, we found no convincing evidence that reactivation parameters of EBV or HHV6 were associated with subsequent MS relapse hazard or progression in disability, confirming previous findings, and indicating that herpesvirus reactivation is not an important driver of relapse or disability in this established MS population.

T cells play a central role in immunity as both regulators and effectors of immune function, following recognition of antigenic peptides presented by human leucocyte antigens (HLA) via the αβ T cell receptor (TCR). As such, the genes that encode the TCR have long been considered candidates for disease association. The human TCR is assembled from a total of 174 gene segments on chromosomes 7 and 14. Each α-chain is encoded by a variable (TRAV), a joining and a constant gene, while each β-chain is encoded by a variable (TRBV), a diversity, a joining and a constant gene (Davis & Bjorkman, 1988). Single nucleotide polymorphism (SNP) studies have reported considerable polymorphism in the TRAV and TRBV gene segments (Mackelprang et al, 2002). A total of 51 SNPs in the TRA locus and 72 SNPs in the TRB locus were found to result in amino acid changes (Mackelprang et al, 2002). Although the functional consequences of this sequence variation remain largely unknown, several studies have shown that allelic polymorphism in the TCR genes can influence antigen recognition (Gras et al, 2010). Furthermore, particular TCR loci have been associated with increased susceptibility to common autoimmune diseases such as multiple sclerosis (Watson et al, 2011) and narcolepsy (Hallmayer et al, 2009). A single missense SNP differentiates two TRBV9 alleles, with TRBV9*01 encoding a neutral glutamine and TRBV9*02 encoding a basic histidine at position 55 (IMGT® nomenclature), which is a framework (FR) residue lying directly adjacent to the complementarity determining region 2 (CDR2) loop of the β chain (Brzezinski et al, 2005). We have previously shown that this residue can contact peptide-laden HLA (pHLA) and can therefore influence antigen recognition (Gras et al, 2010). To determine if this polymorphism could influence the global composition of the T cell repertoire, lymphocyte cDNA from ten healthy, HLA-mismatched individuals who were heterozygous for the TCR SNP (Gras et al, 2010) was examined using a pyrosequencing technique (Wang & Elbein, 2007) to detect imbalanced expression of one TRBV9 allele over the other. Surprisingly, TRBV9*01 was clearly more frequently utilized than TRBV9*02 in the peripheral repertoire of the majority of donors (P = 0·004; Fig 1A). To determine if this bias was limited to a subset of T cells restricted by HLA class I or II molecules, we examined CD8+- and CD4+-sorted T cell populations using the same methodology. TRBV9*01-expressing T cells dominated in both the CD4+ (P < 0·0001; Fig 1B) and CD8+ (P = 0·004; Fig 1C) compartments, indicating that this general bias towards TRBV9*01 usage was not due to preferential thymic/peripheral selection by a particular pHLA complex. To investigate if the general bias towards TRBV9*01 usage is related to preferential peripheral selection by exposure to environmental antigens, we examined T cell populations isolated from umbilical cord blood (UCB). Heterozygous individuals were again identified, and analysis of allele usage in RNA isolated from these samples revealed significant bias towards TRBV9*01 expression in whole lymphocytes (P < 0·001; Fig 1A), CD4+ (P = 0·005; Fig 1B), and CD8+ (P = 0·056; Fig 1C) T cell populations, suggesting that the bias is introduced during lymphopoiesis. Note that healthy adult blood was obtained from donors at the Queensland Institute of Medical Research Berghofer Medical Research Institute, Australia, and UCB lymphocytes were obtained from The Anthony Nolan Trust, UK. This study was approved by ethics committees at each institution, and written informed consent was obtained according to the Declaration of Helsinki. We next analysed SNPs in the TCR α-chain gene segment TRAV14, which encodes for proline and glutamine (TRAV14*01/03) or glutamine and glutamic acid (TRAV14*02/04) at positions 30 (CDR1) and 61 (CDR2), respectively. Ten healthy HLA-mismatched adults who were heterozygous for these alleles were examined for evidence of imbalanced usage of the alleles. Interestingly, the TRAV14*01/03 alleles were used significantly more frequently than TRAV14*02/04 in unsorted lymphocytes (P = 0·004; Fig 2A). Furthermore, allele usage in sorted CD4+ T cell populations also showed a significant preference for the TRAV14*01/*03 alleles (P = 0·014; Fig 2B). Although imbalanced allele usage was a feature of the CD8+ subset in most of the donors, general bias towards TRAV14*01/*03 was not significant (Fig 2C). Fifteen UCB samples were also identified that were heterozygous for these allelic variants and again, significant bias towards TRAV14*01/03 usage was evident in unsorted lymphocytes (P = 0·033; Fig 2A). Five of these samples were also sorted into CD4+ or CD8+ subsets, and general bias towards TRAV14*01/03 was approaching significance for CD4+ cells (Fig 2B; P = 0·057) but not CD8+ cells (Fig 2C). Although we cannot rule out the possibility that unidentified cis-acting regulatory SNPs explain these observations, no polymorphisms have been identified within the promoter regions or recombination signal sequences for either TRBV9 or TRAV14 (Mackelprang et al, 2002). A more likely explanation is that missense SNPs at functionally important regions of the TCR can directly regulate variable gene usage in the T cell repertoire. The TRBV9 and TRAV14 SNPs are positioned at TCR regions that can contact pHLA (Gras et al, 2012), and may therefore influence positive/negative selection during thymocyte development and/or antigen recognition by mature T cells. Data from UCB indicate that the imbalanced allele usage is not dependent on exposure to environmental antigens and is therefore more likely to be introduced during thymocyte development. The SNPs appear to impact on thymic selection mediated by a wide range of HLA molecules, including both class I and II, because the imbalanced TRV allele usage was observed in both CD4+ and CD8+ cells from unrelated individuals. It is notable that the less favoured alleles, TRBV9*02 and TRAV14*02/04, encode for charged amino acids at the polymorphic positions, which are more likely to be destabilizing within protein complexes than the neutral amino acids encoded by the more frequently utilized alleles (TRBV9*01 and TRAV14*01/*03) (Tsai et al, 1997; Wang & Moult, 2001). Therefore, the avidity threshold required for thymic positive selection may be attained with a wider range of self-peptide-HLA complexes for TRBV9*01- and TRAV14*01/*03-expressing TCRs compared to the less ‘biochemically flexible’ allelic variants. Irrespective of the mechanisms involved, this report has clearly shown that nonsynonomous polymorphism in the coding regions of the human TCR loci can shape variable gene usage in the global T cell repertoire, and could therefore control thymic selection of particular clonotypes, including clonotypes protective and predictive of disease. The authors thank the Anthony Nolan Trust, UK for providing UCB used in this study. This work was supported by the National Health and Medical Research Council (NHMRC) of Australia (Grant No. APP1011498), and the Australian Research Council (ARC). SG is supported by an ARC Future Fellowship, JR by an NHMRC Australia Fellowship, JJM by an NHMRC Career Development Fellowship, and SRB by an NHMRC Principal Research Fellowship. RMB and SRB designed the study and wrote the manuscript; RMB, JMB, TE, and MAN conducted various experimental studies; and JR, JJM, and SG provided critical intellectual input and contributed to writing the manuscript. The authors report no conflict of interests.

Class I HLAs generally present peptides of 8-10 aa in length, although it is unclear whether peptide length preferences are affected by HLA polymorphism. In this study, we investigated the CD8(+) T cell response to the BZLF1 Ag of EBV, which includes overlapping sequences of different size that nevertheless conform to the binding motif of the large and abundant HLA-B*44 supertype. Whereas HLA-B*18:01(+) individuals responded strongly and exclusively to the octamer peptide (SELEIKRY180)-S-173, HLA-B*44:03(+) individuals responded to the atypically large dodecamer peptide (EECDSELEIKRY180)-E-169, which encompasses the octamer peptide. Moreover, the octamer peptide bound more stably to HLA-B*18: 01 than did the dodecamer peptide, whereas, conversely, HLA-B*44:03 bound only the longer peptide. Furthermore, crystal structures of these viral peptide-HLA complexes showed that the Ag-binding cleft of HLA-B*18:01 was more ideally suited to bind shorter peptides, whereas HLA-B*44: 03 exhibited characteristics that favored the presentation of longer peptides. Mass spectrometric identification of > 1000 naturally presented ligands revealed that HLA-B*18:01 was more biased toward presenting shorter peptides than was HLA-B*44:03. Collectively, these data highlight a mechanism through which polymorphism within an HLA class I supertype can diversify determinant selection and immune responses by varying peptide length preferences.

The TCR plays a critical role in recognizing intracellular pathogens and initiating pathways leading to the destruction of infected cells by the immune system. Although genetic variability is known to greatly impact on the human immune system and the outcome of infection, the influence of sequence variation leading to the inactivation or deletion of TCR gene segments is unknown. To investigate this issue, we examined the CD8(+) T cell response to an HLA-B7-restricted epitope ((265)RPHERNGFTVL(275)) from the pp65 Ag of human CMV that was highly biased and frequently dominated by a public TCR β-chain encoded by the variable gene segment TRBV4-3. Approximately 40% of humans lack T cells expressing TRBV4-3 because of a 21.5-kb insertion/deletion polymorphism, but these individuals remain responsive to this epitope, using a diverse T cell repertoire characterized by private TCR usage. Although most residues within the bulged 11-mer peptide were accessible for TCR contact, the public and private TCRs showed distinct patterns of sensitivity to amino acid substitution at different positions within the peptide, thereby suggesting that the repertoire diversity generated in the absence of the dominant public TRBV4-3(+) TCR could lead to better protection from viral escape mutation. Thus, variation in the size of the TRBV repertoire clearly contributes toward interindividual variability in immune responses and is presumably maintained in many ethnic groups to enhance the diversity of Ag-specific T cell responses.

In comparison to human leukocyte antigen (HLA) polymorphism, the impact of allelic sequence variation within T cell receptor (TCR) loci is much less understood. Particular TCR loci have been associated with autoimmunity, but the molecular basis for this phenomenon is undefined. We examined the T cell response to an HLA-B*3501–restricted epitope (HPVGEADYFEY) from Epstein-Barr virus (EBV), which is frequently dominated by a TRBV9*01+ public TCR (TK3). However, the common allelic variant TRBV9*02, which differs by a single amino acid near the CDR2β loop (Gln55→His55), was never used in this response. The structure of the TK3 TCR, its allelic variant, and a nonnaturally occurring mutant (Gln55→Ala55) in complex with HLA-B*3501HPVGEADYFEY revealed that the Gln55→His55 polymorphism affected the charge complementarity at the TCR–peptide-MHC interface, resulting in reduced functional recognition of the cognate and naturally occurring variants of this EBV peptide. Thus, polymorphism in the TCR loci may contribute toward variability in immune responses and the outcome of infection.

The major ligands presented by MHC class I molecules after natural antigen processing are peptides of eight to ten residues in length, and it is widely accepted that the binding preferences of MHC class I molecules play a dominant role in dictating this classic feature of antigen presentation. In this report, we have reassessed the peptide size specificity of class I human leukocyte antigens (HLAs). By lengthening previously defined T cell epitopes by central amino acid insertion, we demonstrate that the peptide length specificity of some common HLA class I alleles (HLA-B*3501, B*0702 and A*2402) is very broad, and includes peptides of up to 25 residues. These data suggest that the length limitation of naturally processed MHC class I-associated peptides is primarily controlled by peptide availability after antigen processing rather than the binding specificity of MHC class I molecules. Furthermore, the findings provide an explanation for recent reports highlighting that epitopes of >10 amino acids play a minor but significant role in virus-specific immune surveillance by CD8(+) T cells.