CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope cross-react with selective seasonal coronaviruses.

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 CDR3ß 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.

Erratum: Defective T-cell control of Epstein-Barr virus infection in multiple sclerosis.

[This corrects the article DOI: 10.1038/cti.2016.87.].

Defective T-cell control of Epstein-Barr virus infection in multiple sclerosis.

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.

T Cell Cross-Reactivity between a Highly Immunogenic EBV Epitope and a Self-Peptide Naturally Presented by HLA-B*18:01+ Cells.

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.

T cell epitope clustering in the highly immunogenic BZLF1 antigen of Epstein-Barr virus.

UNLABELLED: 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.

Molecular imprint of exposure to naturally occurring genetic variants of human cytomegalovirus on the T cell repertoire.

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.

HLA peptide length preferences control CD8+ T cell responses.

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 (173)SELEIKRY(180), HLA-B*44:03(+) individuals responded to the atypically large dodecamer peptide (169)EECDSELEIKRY(180), 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 impact of a large and frequent deletion in the human TCR ß locus on antiviral immunity.

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 ß-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.

Allelic polymorphism in the T cell receptor and its impact on immune 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 CDR2beta 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*3501(HPVGEADYFEY) 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 peptide length specificity of some HLA class I alleles is very broad and includes peptides of up to 25 amino acids in length.

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.

Widespread sequence variation in Epstein-Barr virus nuclear antigen 1 influences the antiviral T cell response.

Epstein-Barr virus (EBV) nuclear antigen (EBNA) 1 is perhaps the most widely studied EBV protein, because of its critical role in maintaining the EBV episome and its expression in all EBV-associated malignancies. Much of this research has focused exclusively on the EBV wild-type (wt) strain (B95-8). Sequence analysis of the gene encoding for EBNA1 in EBV isolates from 43 Caucasians has now revealed considerable EBNA1 sequence divergence from the EBV wt strain in the majority of isolates from this population group. Importantly, T cell recognition of an endogenously processed HLA-B8 - binding EBNA1 epitope was greatly influenced by this sequence polymorphism.

Preferential binding of unusually long peptides to MHC class I and its influence on the selection of target peptides for T cell recognition.

A classic feature of antigen presentation for CD8+ T cell recognition is that MHC class I molecules generally present peptides of 8-10 amino acids in length. However, recent studies have demonstrated that peptides of >10 residues play a significant role in immune surveillance by T cells restricted by some HLA class I alleles. In the present study, we describe several examples of unusually long viral peptides of 11 or 12 residues, recognized by CTLs in the context of HLA-B35. Interestingly, all these immunogenic peptides completely encompass shorter canonical length sequences that conform to the HLA-B35 binding motif, but which fail to stimulate detectable T cell responses. The mechanism for this phenomenon appears to involve the preferential binding to HLA-B35 of the atypically long CD8+ T cell target peptides over the overlapping canonical length sequences. These data suggest that the peptide length specificity of some HLA class I alleles is broad, allowing peptides of >10 residues to sometimes dominate over canonical length class I ligands as targets for T cell recognition.

Predictable alphabeta T-cell receptor selection toward an HLA-B*3501-restricted human cytomegalovirus epitope.

Human cytomegalovirus (HCMV) elicits a very large burden on the immune system, with approximately one in ten T cells being reserved solely to manage this infection. However, information on the clonotypic composition of these vast T-cell populations is limited. In this study, we sequenced 116 T-cell receptor (TcR) alpha/beta-chains specific for the highly immunogenic HLA-B*3501-resticted epitope IPSINVHHY from the pp65 antigen. Interestingly, T cells recovered from all donors bore an identical or near-identical TRBV28/TRBJ1-4/TRAV17/TRAJ33 TcR. The ability to predict the responding alphabeta TcR repertoire before viral infection should prove a powerful tool for basic and clinical immunology.

The impact of HLA-B micropolymorphism outside primary peptide anchor pockets on the CTL response to CMV.

The factors controlling epitope selection in the T cell response to persistent viruses are not fully understood, and we have examined this issue in the context of four HLA-B*35-binding peptides from the pp65 antigen of human cytomegalovirus, two of which are previously undescribed. Striking differences in the hierarchy of immunodominance between these four epitopes were observed in healthy virus carriers expressing HLA-B*3501 versus B*3508, two HLA-B allotypes that differ by a single amino acid at position 156 (HLA-B*3501, (156)Leucine; HLA-B*3508, (156)Arginine) that projects from the alpha2 helix into the centre of the peptide-binding groove. While HLA-B*3501(+) individuals responded most strongly to the (123)IPSINVHHY(131) and (366)HPTFTSQY(373) epitopes, HLA-B*3508(+) individuals responded preferentially to (103)CPSQEPMSIYVY(114) and (188)FPTKDVAL(195). By comparing peptide-MHC association and disassociation rates with peptide immunogenicity, it was clear that dissociation rates correlate more closely with the hierarchy of immunodominance among the four pp65 peptides. These findings demonstrate that MHC micropolymorphism at positions outside the primary anchor residue binding pockets can have a major impact on determinant selection in antiviral T cell responses. Such influences may provide the evolutionary pressure that maintains closely related MHC molecules in diverse human populations.

TCR alpha genes direct MHC restriction in the potent human T cell response to a class I-bound viral epitope.

The underlying generic properties of alphabeta TCRs that control MHC restriction remain largely unresolved. To investigate MHC restriction, we have examined the CTL response to a viral epitope that binds promiscuously to two human leukocyte Ags (HLAs) that differ by a single amino acid at position 156. Individuals expressing either HLA-B*3501 (156Leucine) or HLA-B*3508 (156Arginine) showed a potent CTL response to the 407HPVGEADYFEY417 epitope from EBV. Interestingly, the response was characterized by highly restricted TCR beta-chain usage in both HLA-B*3501+ and HLA-B*3508+ individuals; however, this conserved TRBV9+ beta-chain was associated with distinct TCR alpha-chains depending upon the HLA-B*35 allele expressed by the virus-exposed host. Functional assays confirmed that TCR alpha-chain usage determined the HLA restriction of the CTLs. Structural studies revealed significant differences in the mobility of the peptide when bound to HLA-B*3501 or HLA-B*3508. In HLA-B*3501, the bulged section of the peptide was disordered, whereas in HLA-B*3508 the bulged epitope adopted an ordered conformation. Collectively, these data demonstrate not only that mobile MHC-bound peptides can be highly immunogenic but can also stimulate an extremely biased TCR repertoire. In addition, TCR alpha-chain usage is shown to play a critical role in controlling MHC restriction between closely related allomorphs.

The immunogenicity of a viral cytotoxic T cell epitope is controlled by its MHC-bound conformation.

Thousands of potentially antigenic peptides are encoded by an infecting pathogen; however, only a small proportion induce measurable CD8(+) T cell responses. To investigate the factors that control peptide immunogenicity, we have examined the cytotoxic T lymphocyte (CTL) response to a previously undefined epitope ((77)APQPAPENAY(86)) from the BZLF1 protein of Epstein-Barr virus (EBV). This peptide binds well to two human histocompatibility leukocyte antigen (HLA) allotypes, HLA-B*3501 and HLA-B*3508, which differ by a single amino acid at position 156 ((156)Leucine vs. (156)Arginine, respectively). Surprisingly, only individuals expressing HLA-B*3508 show evidence of a CTL response to the (77)APQPAPENAY(86) epitope even though EBV-infected cells expressing HLA-B*3501 process and present similar amounts of peptide for CTL recognition, suggesting that factors other than peptide presentation levels are influencing immunogenicity. Functional and structural analysis revealed marked conformational differences in the peptide, when bound to each HLA-B35 allotype, that are dictated by the polymorphic HLA residue 156 and that directly affected T cell receptor recognition. These data indicate that the immunogenicity of an antigenic peptide is influenced not only by how well the peptide binds to major histocompatibility complex (MHC) molecules but also by its bound conformation. It also illustrates a novel mechanism through which MHC polymorphism can further diversify the immune response to infecting pathogens.

CTL recognition of a bulged viral peptide involves biased TCR selection.

MHC class I molecules generally present peptides of 8-10 aa long, forming an extended coil in the HLA cleft. Although longer peptides can also bind to class I molecules, they tend to bulge from the cleft and it is not known whether the TCR repertoire has sufficient plasticity to recognize these determinants during the antiviral CTL response. In this study, we show that unrelated individuals infected with EBV generate a significant CTL response directed toward an HLA-B*3501-restricted, 11-mer epitope from the BZLF1 Ag. The 11-mer determinant adopts a highly bulged conformation with seven of the peptide side chains being solvent-exposed and available for TCR interaction. Such a complex potentially creates a structural challenge for TCR corecognition of both HLA-B*3501 and the peptide Ag. Surprisingly, unrelated B*3501 donors recognizing the 11-mer use identical or closely related alphabeta TCR sequences that share particular CDR3 motifs. Within the small number of dominant CTL clonotypes observed, each has discrete fine specificity for the exposed side chain residues of the peptide. The data show that bulged viral peptides are indeed immunogenic but suggest that the highly constrained TCR repertoire reflects a limit to TCR diversity when responding to some unusual MHC peptide ligands.

T cell receptor recognition of a 'super-bulged' major histocompatibility complex class I-bound peptide.

Unusually long major histocompatibility complex (MHC) class I-restricted epitopes are important in immunity, but their 'bulged' conformation represents a potential obstacle to alphabeta T cell receptor (TCR)-MHC class I docking. To elucidate how such recognition is achieved while still preserving MHC restriction, we have determined here the structure of a TCR in complex with HLA-B(*)3508 presenting a peptide 13 amino acids in length. This complex was atypical of TCR-peptide-MHC class I interactions, being dominated at the interface by peptide-mediated interactions. The TCR assumed two distinct orientations, swiveling on top of the centrally bulged, rigid peptide such that only limited contacts were made with MHC class I. Although the TCR-peptide recognition resembled an antibody-antigen interaction, the TCR-MHC class I contacts defined a minimal 'generic footprint' of MHC-restriction. Thus our findings simultaneously demonstrate the considerable adaptability of the TCR and the 'shape' of MHC restriction.

Contemporaneous fluctuations in T cell responses to persistent herpes virus infections.

The classical paradigm for T cell dynamics suggests that the resolution of a primary acute virus infection is followed by the generation of a long-lived pool of memory T cells that is thought to be highly stable. Very limited alteration in this repertoire is expected until the immune system is re-challenged by reactivation of latent viruses or by cross-reactive pathogens. Contradicting this view, we show here that the T cell repertoire specific for two different latent herpes viruses in the peripheral blood displayed significant contemporaneous co-fluctuations of virus-specific CD8(+) T cells. The coordinated responses to two different viruses suggest that the fluctuations within the T cell repertoire may be driven by sub-clinical viral reactivation or a more generalized 'bystander' effect. The later contention was supported by the observation that, while absolute number of CD3(+) T cells and their subsets and also the cell surface phenotype of antigen-specific T cells remained relatively constant, a loss of CD62L expression in the total CD8(+) T cell population was coincident with the expansion of tetramer-positive virus-specific T cells. This study demonstrates that the dynamic process of T cell expansion and contractions in persistent viral infections is not limited to the acute phase of infection, but also continues during the latent phase of infection.