T cell allorecognition via molecular mimicry.

T cells often alloreact with foreign human leukocyte antigens (HLA). Here we showed the LC13 T cell receptor (TCR), selected for recognition on self-HLA-B( *)0801 bound to a viral peptide, alloreacts with B44 allotypes (HLA-B( *)4402 and HLA-B( *)4405) bound to two different allopeptides. Despite extensive polymorphism between HLA-B( *)0801, HLA-B( *)4402, and HLA-B( *)4405 and the disparate sequences of the viral and allopeptides, the LC13 TCR engaged these peptide-HLA (pHLA) complexes identically, accommodating mimicry of the viral peptide by the allopeptide. The viral and allopeptides adopted similar conformations only after TCR ligation, revealing an induced-fit mechanism of molecular mimicry. The LC13 T cells did not alloreact against HLA-B( *)4403, and the single residue polymorphism between HLA-B( *)4402 and HLA-B( *)4403 affected the plasticity of the allopeptide, revealing that molecular mimicry was associated with TCR specificity. Accordingly, molecular mimicry that is HLA and peptide dependent is a mechanism for human T cell alloreactivity between disparate cognate and allogeneic pHLA complexes.

Immune self-reactivity triggered by drug-modified HLA-peptide repertoire.

Human leukocyte antigens (HLAs) are highly polymorphic proteins that initiate immunity by presenting pathogen-derived peptides to T cells. HLA polymorphisms mostly map to the antigen-binding cleft, thereby diversifying the repertoire of self-derived and pathogen-derived peptide antigens selected by different HLA allotypes. A growing number of immunologically based drug reactions, including abacavir hypersensitivity syndrome (AHS) and carbamazepine-induced Stevens-Johnson syndrome (SJS), are associated with specific HLA alleles. However, little is known about the underlying mechanisms of these associations, including AHS, a prototypical HLA-associated drug reaction occurring exclusively in individuals with the common histocompatibility allele HLA-B*57:01, and with a relative risk of more than 1,000 (refs 6, 7). We show that unmodified abacavir binds non-covalently to HLA-B*57:01, lying across the bottom of the antigen-binding cleft and reaching into the F-pocket, where a carboxy-terminal tryptophan typically anchors peptides bound to HLA-B*57:01. Abacavir binds with exquisite specificity to HLA-B*57:01, changing the shape and chemistry of the antigen-binding cleft, thereby altering the repertoire of endogenous peptides that can bind HLA-B*57:01. In this way, abacavir guides the selection of new endogenous peptides, inducing a marked alteration in 'immunological self'. The resultant peptide-centric 'altered self' activates abacavir-specific T-cells, thereby driving polyclonal CD8 T-cell activation and a systemic reaction manifesting as AHS. We also show that carbamazepine, a widely used anti-epileptic drug associated with hypersensitivity reactions in HLA-B*15:02 individuals, binds to this allotype, producing alterations in the repertoire of presented self peptides. Our findings simultaneously highlight the importance of HLA polymorphism in the evolution of pharmacogenomics and provide a general mechanism for some of the growing number of HLA-linked hypersensitivities that involve small-molecule drugs.

Lack of Heterologous Cross-reactivity toward HLA-A*02:01 Restricted Viral Epitopes Is Underpinned by Distinct αßT Cell Receptor Signatures.

αßT cell receptor (TCR) genetic diversity is outnumbered by the quantity of pathogenic epitopes to be recognized. To provide efficient protective anti-viral immunity, a single TCR ideally needs to cross-react with a multitude of pathogenic epitopes. However, the frequency, extent, and mechanisms of TCR cross-reactivity remain unclear, with conflicting results on anti-viral T cell cross-reactivity observed in humans. Namely, both the presence and lack of T cell cross-reactivity have been reported with HLA-A*02:01-restricted epitopes from the Epstein-Barr and influenza viruses (BMLF-1 and M158, respectively) or with the hepatitis C and influenza viruses (NS31073 and NA231, respectively). Given the high sequence similarity of these paired viral epitopes (56 and 88%, respectively), the ubiquitous nature of the three viruses, and the high frequency of the HLA-A*02:01 allele, we selected these epitopes to establish the extent of T cell cross-reactivity. We combined ex vivo and in vitro functional assays, single-cell αßTCR repertoire sequencing, and structural analysis of these four epitopes in complex with HLA-A*02:01 to determine whether they could lead to heterologous T cell cross-reactivity. Our data show that sequence similarity does not translate to structural mimicry of the paired epitopes in complexes with HLA-A*02:01, resulting in induction of distinct αßTCR repertoires. The differences in epitope architecture might be an obstacle for TCR recognition, explaining the lack of T cell cross-reactivity observed. In conclusion, sequence similarity does not necessarily result in structural mimicry, and despite the need for cross-reactivity, antigen-specific TCR repertoires can remain highly specific.

Human leukocyte antigen class I-restricted activation of CD8+ T cells provides the immunogenetic basis of a systemic drug hypersensitivity.

The basis for strong immunogenetic associations between particular human leukocyte antigen (HLA) class I allotypes and inflammatory conditions like Behçet's disease (HLA-B51) and ankylosing spondylitis (HLA-B27) remain mysterious. Recently, however, even stronger HLA associations are reported in drug hypersensitivities to the reverse-transcriptase inhibitor abacavir (HLA-B57), the gout prophylactic allopurinol (HLA-B58), and the antiepileptic carbamazepine (HLA-B*1502), providing a defined disease trigger and suggesting a general mechanism for these associations. We show that systemic reactions to abacavir were driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells. Recognition of abacavir required the transporter associated with antigen presentation and tapasin, was fixation sensitive, and was uniquely restricted by HLA-B*5701 and not closely related HLA allotypes with polymorphisms in the antigen-binding cleft. Hence, the strong association of HLA-B*5701 with abacavir hypersensitivity reflects specificity through creation of a unique ligand as well as HLA-restricted antigen presentation, suggesting a basis for the strong HLA class I-association with certain inflammatory disorders.

An extensive antigenic footprint underpins immunodominant TCR adaptability against a hypervariable viral determinant.

Mutations in T cell epitopes are implicated in hepatitis C virus (HCV) persistence and can impinge on vaccine development. We recently demonstrated a narrow bias in the human TCR repertoire targeted at an immunodominant, but highly mutable, HLA-B*0801-restricted epitope ((1395)HSKKKCDEL(1403) [HSK]). To investigate if the narrow TCR repertoire facilitates CTL escape, structural and biophysical studies were undertaken, alongside comprehensive functional analysis of T cells targeted at the natural variants of HLA-B*0801-HSK in different HCV genotypes and quasispecies. Interestingly, within the TCR-HLA-B*0801-HSK complex, the TCR contacts all available surface-exposed residues of the HSK determinant. This broad epitope coverage facilitates cross-genotypic reactivity and recognition of common mutations reported in HCV quasispecies, albeit to a varying degree. Certain mutations did abrogate T cell reactivity; however, natural variants comprising these mutations are reportedly rare and transient in nature, presumably due to fitness costs. Overall, despite a narrow bias, the TCR accommodated frequent mutations by acting like a blanket over the hypervariable epitope, thereby providing effective viral immunity. Our findings simultaneously advance the understanding of anti-HCV immunity and indicate the potential for cross-genotype HCV vaccines.

Drug hypersensitivity and human leukocyte antigens of the major histocompatibility complex.

The human leukocyte antigen (HLA) genes are the most polymorphic in the human genome and are critical in regulating specific immunity, hence their historical discovery as "immune response" genes. HLA allotypes are also implicated in unwanted immune reactions, including drug hypersensitivity syndrome, in which small therapeutic drugs interact with antigenic peptides to drive T cell responses restricted by host HLA. Abacavir, allo-purinol, and carbamazepine are three commonly used drugs that cause a T cell-mediated hypersensitivity that is HLA linked, with each drug exhibiting striking specificity for presentation by defined HLA allotypes. Recent findings have begun to unearth the mechanistic basis for these HLA associations, and here we review recent advances in the field of HLA-associated drug hypersensitivities.

The design and proof of concept for a CD8(+) T cell-based vaccine inducing cross-subtype protection against influenza A virus.

In this study, we examined the reactivity of human peripheral blood mononuclear cells to a panel of influenza A virus (IAV) CD8(+) T-cell epitopes that are recognised by the major human leukocyte antigen (HLA) groups represented in the human population. We examined the level of recognition in a sample of the human population and the potential coverage that could be achieved if these were incorporated into a T-cell epitope-based vaccine. We then designed a candidate influenza vaccine that incorporated three of the examined HLA-A2-restricted influenza epitopes into Pam2Cys-based lipopeptides. These lipopeptides do not require the addition of an adjuvant and can be delivered directly to the respiratory mucosa enabling the generation of local memory cell populations that are crucial for clearance of influenza. Intranasal administration of a mixture of three lipopeptides to HLA-A2 transgenic HHD mice elicited multiple CD8(+) T-cell specificities in the spleen and lung that closely mimicked the response generated following natural infection with influenza. These CD8(+) T cells were associated with viral reduction following H3N1 influenza virus challenge for as long as 3 months after lipopeptide administration. In addition, lipopeptides containing IAV-targeting epitopes conferred substantial benefit against death following infection with a virulent H1N1 strain. Because CD8(+) T cell epitopes are often derived from highly conserved regions of influenza viruses, such vaccines need not be reformulated annually and unlike current antibody-inducing vaccines could provide cross-protective immunity against newly emerging pandemic viruses.

Nucleoprotein of influenza A virus is a major target of immunodominant CD8+ T-cell responses.

Influenza A virus causes annual epidemics and sporadic pandemics, resulting in significant morbidity and mortality worldwide. Vaccines are currently available; however, they induce a non-strain-cross protective humoral immune response directed against the rapidly mutating surface glycoproteins, and thus need to be updated annually. As T cells are directed against more conserved internal influenza proteins, a T-cell-based vaccine has the potential to induce long-lasting and cross-strain protective CD8(+) T-cell immunity, and in that way minimize the severity of influenza infection. However, to rationally design such vaccines, we need to identify immunogenic T-cell regions within the most antigenic viral proteins. In this study, we have used a systematic approach to identify immunodominant peptides in HLA-A2-negative donors. A broad range of CD8(+) T-cell responses were observed and 6/7 donors had an immunodominant response against the relatively conserved internal nucleoprotein (NP). Dissecting the minimal epitope regions within the immunogenic NP led to the identification of six novel immunodominant epitopes, which include a 12-mer and an 8-mer peptides. The majority of immunodominant epitopes was clustered within the carboxyl terminal 2/3 of the NP protein and were highly conserved. We also subjected NP to three common computer algorithms for epitope prediction and found that most of the novel epitopes would not have been predicted. Our study emphasizes the importance of using a systematic approach to identify immunodominant CD8(+) T-cell responses and suggests that the epitope-rich regions within NP present a promising target for the T-cell-mediated multi-strain influenza vaccine.

Antigen-driven patterns of TCR bias are shared across diverse outcomes of human hepatitis C virus infection.

Hepatitis C virus (HCV) infection causes significant morbidity and mortality worldwide. T cells play a central role in HCV clearance; however, there is currently little understanding of whether the disease outcome in HCV infection is influenced by the choice of TCR repertoire. TCR repertoires used against two immunodominant HCV determinants--the highly polymorphic, HLA-B*0801 restricted (1395)HSKKKCDEL(1403) (HSK) and the comparatively conserved, HLA-A*0101-restricted, (1435)ATDALMTGY(1443) (ATD)--were analyzed in clearly defined cohorts of HLA-matched, HCV-infected individuals with persistent infection and HCV clearance. In comparison with ATD, TCR repertoire selected against HSK was more narrowly focused, supporting reports of mutational escape in this epitope, in persistent HCV infection. Notwithstanding the Ag-driven divergence, T cell repertoire selection against either Ag was comparable in subjects with diverse disease outcomes. Biased T cell repertoires were observed early in infection and were evident not only in persistently infected individuals but also in subjects with HCV clearance, suggesting that these are not exclusively characteristic of viral persistence. Comprehensive clonal analysis of Ag-specific T cells revealed widespread use of public TCRs displaying a high degree of predictability in TRBV/TRBJ gene usage, CDR3 length, and amino acid composition. These public TCRs were observed against both ATD and HSK and were shared across diverse disease outcomes. Collectively, these observations indicate that repertoire diversity rather than particular Vß segments are better associated with HCV persistence/clearance in humans. Notably, many of the anti-HCV TCRs switched TRBV and TRBJ genes around a conserved, N nucleotide-encoded CDR3 core, revealing TCR sequence mosaicism as a potential host mechanism to combat this highly variant virus.

CD1d-lipid-antigen recognition by the semi-invariant NKT T-cell receptor.

The CD1 family is a large cluster of non-polymorphic, major histocompatibility complex (MHC) class-I-like molecules that bind distinct lipid-based antigens that are recognized by T cells. The most studied group of T cells that interact with lipid antigens are natural killer T (NKT) cells, which characteristically express a semi-invariant T-cell receptor (NKT TCR) that specifically recognizes the CD1 family member, CD1d. NKT-cell-mediated recognition of the CD1d-antigen complex has been implicated in microbial immunity, tumour immunity, autoimmunity and allergy. Here we describe the structure of a human NKT TCR in complex with CD1d bound to the potent NKT-cell agonist alpha-galactosylceramide, the archetypal CD1d-restricted glycolipid. In contrast to T-cell receptor-peptide-antigen-MHC complexes, the NKT TCR docked parallel to, and at the extreme end of the CD1d-binding cleft, which enables a lock-and-key type interaction with the lipid antigen. The structure provides a basis for the interaction between the highly conserved NKT TCR alpha-chain and the CD1d-antigen complex that is typified in innate immunity, and also indicates how variability of the NKT TCR beta-chain can impact on recognition of other CD1d-antigen complexes. These findings provide direct insight into how a T-cell receptor recognizes a lipid-antigen-presenting molecule of the immune system.

A structural basis for selection and cross-species reactivity of the semi-invariant NKT cell receptor in CD1d/glycolipid recognition.

Little is known regarding the basis for selection of the semi-invariant alphabeta T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d-glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3beta composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR alpha- and semi-invariant beta-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved "hot spot" that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3beta loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR-CD1d-glycolipid interaction in which the invariant CDR3alpha loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant alphabeta TCR and the unique antigen specificity of NKT cells.

Kinetics of Abacavir-Induced Remodelling of the Major Histocompatibility Complex Class I Peptide Repertoire.

Abacavir hypersensitivity syndrome can occur in individuals expressing the HLA-B*57:01 major histocompatibility complex class I allotype when utilising the drug abacavir as a part of their anti-retroviral regimen. The drug is known to bind within the HLA-B*57:01 antigen binding cleft, leading to the selection of novel self-peptide ligands, thus provoking life-threatening immune responses. However, the sub-cellular location of abacavir binding and the mechanics of altered peptide selection are not well understood. Here, we probed the impact of abacavir on the assembly of HLA-B*57:01 peptide complexes. We show that whilst abacavir had minimal impact on the maturation or average stability of HLA-B*57:01 molecules, abacavir was able to differentially enhance the formation, selectively decrease the dissociation, and alter tapasin loading dependency of certain HLA-B*57:01-peptide complexes. Our data reveals a spectrum of abacavir mediated effects on the immunopeptidome which reconciles the heterogeneous functional T cell data reported in the literature.

Natural HLA class I polymorphism controls the pathway of antigen presentation and susceptibility to viral evasion.

HLA class I polymorphism creates diversity in epitope specificity and T cell repertoire. We show that HLA polymorphism also controls the choice of Ag presentation pathway. A single amino acid polymorphism that distinguishes HLA-B*4402 (Asp116) from B*4405 (Tyr116) permits B*4405 to constitutively acquire peptides without any detectable incorporation into the transporter associated with Ag presentation (TAP)-associated peptide loading complex even under conditions of extreme peptide starvation. This mode of peptide capture is less susceptible to viral interference than the conventional loading pathway used by HLA-B*4402 that involves assembly of class I molecules within the peptide loading complex. Thus, B*4402 and B*4405 are at opposite extremes of a natural spectrum in HLA class I dependence on the PLC for Ag presentation. These findings unveil a new layer of MHC polymorphism that affects the generic pathway of Ag loading, revealing an unsuspected evolutionary trade-off in selection for optimal HLA class I loading versus effective pathogen evasion.

Optimization of LMP-specific CTL expansion for potential adoptive immunotherapy in NPC patients.

Nasopharyngeal carcinoma (NPC) is Epstein-Barr virus (EBV) positive in all undifferentiated cases, expressing the latency II phenotype of latent membrane proteins (LMPs) 1 and 2, in addition to EBV nuclear antigen (EBNA) 1. Several studies have attempted to treat NPC with EBV-specific cytotoxic T lymphocyte (CTL) with a partial response. To improve this therapy, there is a need to expand CTL targeted to the latency II antigens of EBV, rather than the immunodominant EBV nuclear antigens 3-6 peptides typically expanded by lymphoblastoid cells. In order to maximize the expansion of LMP-specific CTL in vitro for use in adoptive immunotherapy of nasopharyngeal carcinoma patients, we used lymphoblastoid cell lines coated with synthetic peptides corresponding to CTL determinants from the LMP proteins. We investigated several issues pertaining to the expansion of an immunologically weak CTL response, including peptide and interleukin-2 concentration, and screening assays for selecting the optimal peptide for use in expansion of LMP-specific CTL. Although screening of ex vivo peripheral blood mononuclear cells did not prove to be useful in the selection of an LMP peptide for use in CTL cultures, the peptide and interleukin-2 concentrations were critical for the maximum expansion of CTL. Therefore, it is imperative that stimulation protocols are optimized for the expansion of LMP-specific CTL.

TCD8 response in diverse outcomes of recurrent exposure to hepatitis C virus.

To analyse the immune correlates in a setting of recurrent exposure to hepatitis C virus (HCV), we studied T(CD8) responses in injecting drug users (IDUs) with different disease outcomes. Ex vivo HCV-specific T(CD8) responses assessed by interferon-gamma (IFNgamma) enzyme-linked immunospot (ELISPOT) were comparable in human lymphocyte antigen (HLA)-matched IDUs with spontaneous HCV clearance or persistent infection. A detailed characterization of these T(CD8) cells in age and HLA-matched IDUs demonstrated that HCV clearance and protection from reinfection correlated with HCV-specific T(CD8) cells that could proliferate in vitro, possessed cytotoxic potential and produced IFNgamma and tumour-necrosis factor-alpha, rather than with the circulating frequency of responding T(CD8) cells determined ex vivo. While validating the importance of multifunctional T(CD8) in mediating protection in IDUs with recurrent exposure to HCV our findings highlight that the magnitude and/or breadth of HCV-specific T(CD8) determined in ex vivo ELISPOT may not be the sole determinant of protection especially in a setting of recurrent exposure.

Phase I trial of a CD8+ T-cell peptide epitope-based vaccine for infectious mononucleosis.

A single blind, randomized, placebo-controlled, single-center phase I clinical trial of a CD8(+) T-cell peptide epitope vaccine against infectious mononucleosis was conducted with 14 HLA B*0801-positive, Epstein-Barr virus (EBV)-seronegative adults. The vaccine comprised the HLA B*0801-restricted peptide epitope FLRGRAYGL and tetanus toxoid formulated in a water-in-oil adjuvant, Montanide ISA 720. FLRGRAYGL-specific responses were detected in 8/9 peptide-vaccine recipients and 0/4 placebo vaccine recipients by gamma interferon enzyme-linked immunospot assay and/or limiting-dilution analysis. The same T-cell receptor Vbeta CDR3 sequence that is found in FLRGRAYGL-specific T cells from most EBV-seropositive individuals could also be detected in the peripheral blood of vaccine recipients. The vaccine was well tolerated, with the main side effect being mild to moderate injection site reactions. After a 2- to 12-year follow-up, 1/2 placebo vaccinees who acquired EBV developed infectious mononucleosis, whereas 4/4 vaccinees who acquired EBV after completing peptide vaccination seroconverted asymptomatically. Single-epitope vaccination did not predispose individuals to disease, nor did it significantly influence development of a normal repertoire of EBV-specific CD8(+) T-cell responses following seroconversion.

High incidence of hepatitis C virus reinfection in a cohort of injecting drug users.

UNLABELLED: An estimated 170 million people worldwide carry the hepatitis C virus (HCV), and in more developed countries the prevalence and incidence of HCV is particularly high among injecting drug users (IDUs). Spontaneous clearance of HCV infection and reinfection is well recognized but the level of protection against further infection conferred by HCV infection and clearance remains uncertain. We conducted a prospective study of HCV infection in IDUs recruited in Melbourne, Australia, using a much shorter median testing interval than in previous studies. Incidences of naive infection and reinfection were calculated by the person-year method and Cox proportional hazards regression used to adjust for covariates. A significantly higher HCV incidence rate was measured in previously infected IDUs (46.8% per year) compared with HCV-naive IDUs (15.5% per year). The hazard ratio for previously infected IDUs compared to HCV-naive IDUs, after adjustment for time-dependent covariates, was 2.54 (95% confidence interval, 1.11-5.78, P > |z| < 0.05). Viral persistence after reinfection appeared similar to that following naive infection. CONCLUSION: Our data suggest that HCV infection in IDUs is more likely following prior infection and clearance than in HCV-naive individuals, implying no increased immunity against further infection. This result has important implications for the future development of an HCV vaccine.

Fiber-modified recombinant adenoviral constructs encoding hepatitis C virus proteins induce potent HCV-specific T cell response.

Hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTLs) play an important role in HCV clearance. The frequency of HCV-specific T(CD8) in peripheral blood of HCV-infected donors is very low and HCV cannot be cultivated for reinfection of antigen presenting cells, making it difficult to detect T(CD8) of broad HCV specificities from peripheral blood mononuclear cells (PBMCs). We have developed a recombinant adenoviral system that efficiently reactivates and expands HCV-specific CTLs from PBMCs of HCV-infected donors. Replication-incompetent adenoviruses expressing individual HCV proteins (core and NS3) were produced and PBMCs from HCV-infected donors were transduced with these recombinant adeno-HCV constructs to stimulate HCV-specific CTL populations. T cells expanded from adeno-HCV stimulated cultures were potent producers of HCV-specific IFN-gamma and TNF-alpha and efficiently lysed target cells pulsed with HCV peptides. These constructs could stimulate T(CD8) directed towards multiple HCV peptides while preserving the determinant hierarchy. This approach therefore overcomes some of the shortcomings of the selective expansion of CTLs with peptide-based vaccine strategies. These findings provide an effective approach for the expansion of HCV-specific CTLs from PBMCs of HCV-infected patients and have potential for immunotherapeutic/vaccine development.

Consecutive infections and clearances of different hepatitis C virus genotypes in an injecting drug user.

BACKGROUND: The hepatitis C virus (HCV) causes significant morbidity and mortality worldwide, and is highly prevalent among injecting drug users (IDUs). Whether initial HCV infection and clearance provides protection from reinfection has not been established, but is an important question for vaccine development. OBJECTIVE: To elucidate an unusual history of HCV infection and clearance in an IDU. STUDY DESIGN: The subject was interviewed and gave blood samples at approximately three-month intervals; all samples were tested for anti-HCV and HCV RNA, genotyped if RNA detected, and checked for mixed genotypes; phylogenetic analysis performed on the subject's and injecting partners' core HCV sequences. RESULTS: We observed consecutive infections with HCV genotypes 3a, 1a and 6l, and intervening clearances, in a young IDU over 449 days. Genotypes 1a and 6l were probably acquired from the subject's injecting partners, who had genetically related infections. CONCLUSION: This case illustrates (1) the ease with which IDUs can acquire HCV, (2) that prior HCV infection does not protect against reinfection with heterologous strains, and (3) that IDUs can clear consecutive HCV infections. Our subject's history of HCV infection and clearance offers hope for vaccine development, yet demonstrates that HCV vaccines must have cross-genotypic effectiveness.

Broad CD8+ T cell cross-recognition of distinct influenza A strains in humans.

Newly-emerged and vaccine-mismatched influenza A viruses (IAVs) result in a rapid global spread of the virus due to minimal antibody-mediated immunity. In that case, established CD8+ T-cells can reduce disease severity. However, as mutations occur sporadically within immunogenic IAV-derived T-cell peptides, understanding of T-cell receptor (TCRαß) cross-reactivity towards IAV variants is needed for a vaccine design. Here, we investigate TCRαß cross-strain recognition across IAV variants within two immunodominant human IAV-specific CD8+ T-cell epitopes, HLA-B*37:01-restricted NP338-346 (B37-NP338) and HLA-A*01:01-restricted NP44-52 (A1-NP44). We find high abundance of cross-reactive TCRαß clonotypes recognizing distinct IAV variants. Structures of the wild-type and variant peptides revealed preserved conformation of the bound peptides. Structures of a cross-reactive TCR-HLA-B37-NP338 complex suggest that the conserved conformation of the variants underpins TCR cross-reactivity. Overall, cross-reactive CD8+ T-cell responses, underpinned by conserved epitope structure, facilitates recognition of distinct IAV variants, thus CD8+ T-cell-targeted vaccines could provide protection across different IAV strains.