Flexibility in T-cell receptor ligand repertoires depends on MHC and T-cell receptor clonotype.

T-cell receptors (TCR) recognize peptides complexed to self-major histocompatibility complex (MHC) molecules. Recognition of peptide/MHC ligands by the TCR is highly peptide specific. However, certain TCRs can also recognize sequence-related and -unrelated ('mimicry') epitopes presented by homologous MHC molecules. Using two human, human leucocyte antigen-DR1 (HLA-DR1)-restricted T-cell clones specific for HA p307-319, we identified several diverse combinations of peptide-MHC complexes that are functionally equivalent in their ability to trigger T-cell stimulation. These findings demonstrate that a single TCR can productively interact with different peptides complexed to self- as well as non-self-MHC molecules. This extended reactivity is human leucocyte antigen (HLA) allele and TCR clonotype dependent, as the peptide repertoire recognized depends on the presenting HLA-DR molecule and varies among different TCRs that both recognize the HA p307-319/DR1 complex. Importantly, certain peptide analogues can completely change the HLA-restriction pattern of the TCR: T-cell recognition of the wild-type peptide that was absent in the context of a non-self HLA-DR molecule, was restored by complementing substitutions in altered peptide ligands, that could not be presented by the original restriction element. This mechanism may play an important role in allorecognition.     

Steric recognition of T-cell receptor contact residues is required to map mutant epitopes by immunoinformatical programmes

MHC class I-restricted CD8 T-lymphocyte epitopes comprise anchor motifs, T-cell receptor (TCR) contact residues and the peptide backbone. Serial variant epitopes with substitution of amino acids at either anchor motifs or TCR contact residues have been synthesized for specific interferon-γ responses to clarify the TCR recognition mechanism as well as to assess the epitope prediction capacity of immunoinformatical programmes. CD8 T lymphocytes recognise the steric configuration of functional groups at the TCR contact side chain with a parallel observation that peptide backbones of various epitopes adapt to the conserved conformation upon binding to the same MHC class I molecule. Variant epitopes with amino acid substitutions at the TCR contact site are not recognised by specific CD8 T lymphocytes without compromising their binding capacity to MHC class I molecules, which demonstrates two discrete antigen presentation events for the binding of peptides to MHC class I molecules and for TCR recognition. The predicted outcome of immunoinformatical programmes is not consistent with the results of epitope identification by laboratory experiments in the absence of information on the interaction with TCR contact residues. Immunoinformatical programmes based on the binding affinity to MHC class I molecules are not sufficient for the accurate prediction of CD8 T-lymphocyte epitopes. The predictive capacity is further improved to distinguish mutant epitopes from the non-mutated epitopes if the peptide-TCR interface is integrated into the computing simulation programme.     

Role of polymorphic residues of human leucocyte antigen-DR molecules on the binding of human immunodeficiency virus peptides.

A study was made of the binding properties of 96 human immunodeficiency virus peptides to human leucocyte antigen (HLA)-DR1 and HLA-DR103 molecules, which differ by three amino acids at positions 67, 70 and 71 in the beta chains. The affinity of the peptides was characterized by their inhibitory concentrations in competitive binding assays which displace half of the labelled influenza haemagglutinin peptide HA306-318 (IC50). Among the high-affinity peptides (IC50 < or = 1 microM), seven bound to DR1, three to DR103 and five equally well to both alleles (promiscuous peptides). Thirty-two other peptides showed medium or low affinity for DR molecules. The role of polymorphic residues was analysed using six mutated DR molecules, intermediates between DR1 and DR103 and differing by one or two substitutions at positions 67, 70 or 71. We reached the same conclusions when using DR1-specific or DR103-specific peptides: modification of residue 70 had no effect on peptide affinity, but single substitution at positions 67 or 71 decreased the allele specificity of the peptides while double substitution at 67 and 71 completely reversed the peptide specificity. In functional assays, DR-binding peptides are able to outcompete specific T-cell proliferation. Furthermore, modification at position 67 or 70 significantly affects the T-cell response and mutation at position 71 abolishes completely the T-cell proliferation. Thus, the polymorphic positions 67 and 71 contributed to the peptide binding with direct effects on T-cell receptor (TCR) recognition while position 70 seems to be mostly engaged in TCR interactions. Furthermore, our results suggest that polymorphic residues may select allele-specific peptides and also influence the conformation of promiscuous peptides.     

Characterization of MHC- and TCR-binding residues of the myelin oligodendrocyte glycoprotein 38-51 peptide

Myelin oligodendrocyte glycoprotein (MOG) is a major experimental autoimmune encephalomyelitis (EAE) antigen in H-2b mice and a potential autoantigen in multiple sclerosis. How well MOG peptides bind to MHC and how TCR recognize the peptide/MHC complex have important implications for thymic selection as well as T cell activation in the periphery. In this study, we have characterized amino acids in the MOG(38-51) peptide important for peptide binding to I-Ab, and for TCR recognition of the peptide/MHC complex. We found that the amino acids R41, F44, R46 and V47 constituted the major TCR contact residues, as alanine substitution at these positions abrogated T cell responses without decreasing their binding affinity to I-Ab. In addition, G38 and W39 were found to be minor TCR contact residues. Finally, substituting tyrosine for alanine at position 40 decreased binding to I-Ab by approximately 50% and prevented induction of T cell responses in C57BL/6J mice upon immunization. Thus, Y40 is the dominant MHC-binding residue of the MOG(38-51) peptide and most likely occupies the p1 pocket of I-Ab. Our results could be useful to design peptides with altered agretopes and epitopes of the MOG(38-51) peptide to study their therapeutic potential in the EAE model.     

Residue 67 in the DR β1*0101 and DR β1*0103 chains strongly influences antigen presentation and DR-peptide molecular complex conformation

Abstract: Two closely-related molecules, DR(α,β*0101) and DR(α,β*0103), whose β chains only differ by three amino acids at positions 67, 70, and 71, and six intermediate molecules obtained by site-directed mutagenesis were used to ascertain the respective roles of the three polymorphic residues. Substitutions at positions 70 (D→Q), 71 (E→R) and 67 (I or L→F) strongly affected HA 306–318-specific T-cell recognition. The consequences of the substitution of residue 67 by a phenylalanine depended on the modified HLA-DR molecule. Although this substitution completely inhibited peptide-specific DR1-restricted T-cell recognition, its manifestations on the DR103-restricted T-cell response were variable (abolishing proliferation of some cell lines and not others), no matter what the peptide presented was (HA 306–319 or HIV P25 peptides). We also observed that inhibition of the proliferation of an alloreactive anti-DR103 T-cell clone, caused by a substitution at position 70, was completely canceled by substitution of residue 67 by a phenylalanine. The observations based on functional experiments, thus, suggest that residue 67 plays an important role in determining conformation of the peptide presented to the T cells. Molecular modeling was used to predict changes induced by amino acid substitutions and highly supports functional data. Substitution of residue 67 by a phenylalanine could have repercussions on the structure of HLA-DR molecule/peptide complexes and affect T-cell recognition.     

Distinct effects of altered allopeptide analogs on a human self-restricted T cell clone

Alloreactive T cells involved in indirect recognition play a key role in initiating and sustaining graft rejection. One of the most promising approaches to achieve specific immunosuppression of indirect allorecognition resides in the use of chemically modified allopeptides. In order to design and test such peptide analogs, we have defined the dominant immunogenic peptide of the HLA-DRB1^*0101 antigen recognized by DRB1^*1101 responders. Next we engineered structural variants of this peptide (DRB1^*0101/residues 22-35), carrying single amino acid substitutions at postulated MHC and TCR contact residues. These analogs were tested for: (i) binding affinity to recombinant HLA-DRB1^*1101 protein (rDR11), and (ii) stimulatory activity exerted on a human anti-DR1/22-35 self-restricted T cell clone. The binding affinity of the analogs carrying non-homologous substitutions at putative anchor positions (24V/E and 29R/A) was significantly decreased, while little or no effect was observed in either peptide-binding or T cell proliferation assays for conserved substitution (24V/Y and 29R/K). This indicates that positions 24 and 29 are primarily involved in contacting the HLA-DR11 molecule. In contrast, single amino acid substitutions at positions 25 through 28 strongly affected the proliferative response of the clone, even when binding affinity to rDR11 was not altered. This finding suggests that positions 25 through 28 are TCR contact residues. Two peptide analogs (26L/I and 27L/V) displayed a higher stimulatory activity than the wild-type peptide and induced high-zone tolerance. Two other peptides (25R/A and 28E/Q), while binding to rDR11, did not exhibit any stimulatory activity and blocked the presentation and recognition of the wild-type peptide. Our data underscore the therapeutic potential of allopeptide analogs, as well as their value in dissecting the fine antigenic structure of a peptide determinant.     

Effects of localized HLA class II beta chain polymorphism on binding of antigenic peptide and stimulation of T cells.

The relationship between HLA-DR1 polymorphism and recognition of antigen by T cells was investigated. Two allelic variants of HLA-DR1, which differ by amino acid substitution at positions 85 and 86 of the beta chain, were characterized for the effect of substitution on recognition of foreign antigen by DR1-restricted T cells. Substitution of alanine and valine for valine and glycine residues at positions 85 and 86 of the DR1 beta chain resulted in deficient T-cell stimulation as demonstrated by the requirement for higher concentrations of antigen to induce maximal levels of T-cell proliferation, induction of lower levels of proliferation at optimal antigen concentrations, and slower kinetics of formation of stimulatory peptide-DR1 complexes. Direct binding studies employing both biotinylated and radioiodinated forms of antigenic peptide demonstrated quantitatively lower levels of peptide bound to substituted DR1 molecules and low levels of site-specific binding as assessed by competitive inhibition analyses. The effect of MHC class II polymorphism on peptide-binding affinity as opposed to induction of appropriate peptide conformation and the impact of polymorphism at DR1 beta chain positions 85 and 86 on allorecognition of HLA-DR1 are discussed.     

Recognition of allo-peptide is governed by novel anchor imposition and limited variations in TCR contact residues

Immune specificity of a T cell is determined by the TCR contact residues exposed on the antigenic peptide/MHC complex. Naturally processed, biallelic epitopes from H7 minor histocompatibility (mH) antigen vary in position 7 (p7) from aspartic acid (D) to a glutamic acid (E), which differ by an additional methylene (-CH(2)) in the side chain. Here, we show that this variation generates a strong anti-H7a or anti-H7b cytotoxic T cell responses. Further, the H7 allelic peptides use p6 asparagine as their central anchor residue and amino acid variations in either the canonical p5 or the predicted p6 anchor positions in the antigenic epitope were detrimental for TCR recognition. In addition, introduction of any other amino acids, except asparagine, in the polymorphic p7 significantly abolished the ability of anti-H7b TCR recognition. This demonstrates that only an asparagine with an amine group as a side chain instead of a charged oxygen radical could effectively stimulate the anti-H7b specific T cells. Our findings provide evidence that mH antigen-specific TCRs are highly stringent in recognizing their cognate epitopes.     

Contemplating immunopeptidomes to better predict them

The identification of T-cell epitopes is key for a complete molecular understanding of immune recognition mechanisms in infectious diseases, autoimmunity and cancer. T-cell epitopes further provide targets for personalized vaccines and T-cell therapy, with several therapeutic applications in cancer immunotherapy and elsewhere. T-cell epitopes consist of short peptides displayed on Major Histocompatibility Complex (MHC) molecules. The recent advances in mass spectrometry (MS) based technologies to profile the ensemble of peptides displayed on MHC molecules – the so-called immunopeptidome – had a major impact on our understanding of antigen presentation and MHC ligands. On the one hand, these techniques enabled researchers to directly identify hundreds of thousands of peptides presented on MHC molecules, including some that elicited T-cell recognition. On the other hand, the data collected in these experiments revealed fundamental properties of antigen presentation pathways and significantly improved our ability to predict naturally presented MHC ligands and T-cell epitopes across the wide spectrum of MHC alleles found in human and other organisms. Here we review recent computational developments to analyze experimentally determined immunopeptidomes and harness these data to improve our understanding of antigen presentation and MHC binding specificities, as well as our ability to predict MHC ligands. We further discuss the strengths and limitations of the latest approaches to move beyond predictions of antigen presentation and tackle the challenges of predicting TCR recognition and immunogenicity.     

Serial triggering of TCRs: a basis for the sensitivity and specificity of antigen recognition

Sensitivity of antigen detection by B cells correlates with high-affinity binding. This paradigm does not appear to hold for the T-cell receptor (TCR), which is able to bind its ligand — peptide in the context of major histocompatibility complex (MHC) — with low affinity. Here, Salvatore Valitutti and Antonio Lanzavecchia propose that the efficiency of T-cell antigen recognition is dependent upon optimal kinetics of the TCR—peptide—MHC interaction, allowing serial engagements and triggering of many TCRs by a few peptide—MHC complexes.     

Antigen-specific immunomodulation for type 1 diabetes by novel recombinant antibodies directed against diabetes-associates auto-reactive T cell epitope

The trimolecular complex composed of autoreactive T-cell receptor, MHC class II, and an autoantigenic peptide plays a central role in the activation of pathogenic Islet-specific CD4+ T cells in type 1 diabetes (T1D). We isolated and characterized novel antibodies against autoreactive T-cell epitopes associated with T1D. Our antibodies mimic the specificity of the T-cell receptor (TCR), while binding MHC class II/peptide complexes in an autoantigen peptide specific, MHC-restricted manner. The isolated TCR-like antibodies were directed against the minimal T-cell epitope GAD-555–567 in the context of the HLA-DR4-diabetic-associated molecule. A representative high-affinity TCR-like antibody clone (G3H8) enabled the detection of intra- and extra-cellular DR4/GAD-555–567 complexes in antigen presenting cells. I561M single mutation at the central position (P5) of the GAD-555–567 peptide abolished the binding of G3H8 to the DR4/GAD complex, demonstrating its high fine TCR-like specificity. The G3H8 TCR-like antibody significantly inhibited GAD-555–567 specific, DR4 restricted T-cell response in vitro and in vivo in HLA-DR4 transgenic mice. Our findings constitute a proof-of-concept for the utility of TCR-like antibodies as antigen-specific immunomodulation agents for regulating pathogenic T-cells and suggest that TCR-like antibodies targeting autoreactive MHC class II epitopes are valuable research tools that enable studies related to antigen presentation as well as novel therapeutic agents that may be used to modulate autoimmune disorders such as T1D.     

The effect of a single amino acid substitution within the V3 loop of HIV-1 gp120 on HLA-DR1-restricted CD4 T-cell recognition.

Viral variation has been proposed to play a role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1) infection, and is an important consideration in vaccine design. During the course of an infection, isolates with sequence changes in CD8 T-cell and B-cell epitopes arise. To determine whether sequence variation within the V3 loop of HIV-1 gp120 affects HLA-DR beta 1*0101-restricted CD4 T-cell recognition, we have generated CD4 T-cell clones (TLC) specific to gp120 V3 loop peptides. Four HLA-DR beta 1*0101-restricted groups of TLC were defined by distinct patterns of responses to a panel of peptides, consistent with a highly diverse T-cell repertoire recognizing the 30 amino acid stretch (296-326) of the gp120 V3 loop. Nevertheless, a single residue change at position 311 was found to abolish the recognition of two of the four groups of TLC. This was not due to an effect of the residue at 311 on binding to major histocompatibility complex (MHC), because: (1) irrespective of the residue at 311, peptides competed well with the influenza haemagglutinin peptide 307-319 for binding to cell-bound DR1; and (2) R311-specific TLC were also HLA DR beta 1*0101 restricted. Instead, the substitution of arginine for serine at position 311 blocked the interaction of the peptide with the T-cell receptor. Thus, despite the diversity of the T-cell response to the V3 loop of HIV-1, a single amino acid change can have a considerable influence on the responding T-cell population. As residue 311 is one of the most variable of the V3 loop residues, these results suggest that CD4 recognition can also exert pressure on viral variation consistent with a role for these cells in antiviral immunity.     

Allogeneic core amino acids of an immunodominant allopeptide are important for MHC binding and TCR recognition

The indirect alloimmune response seems to be restricted to a few dominant major histocompatibility complex (MHC)-derived peptides responsible for T-cell activation in allograft rejection. The molecular mechanisms of indirect T-cell activation have been studied using peptide analogues derived from the dominant allopeptide in vitro, whereas the in vivo effects of peptide analogues have not been well characterized yet. In the present study, we generated allochimeric peptide analogues by replacing the three allogeneic amino acids 5L, 9L, and 10T in the sequence of the dominant MHC class I allopeptide P1. These allochimeric peptide analogues were used to define the allogeneic amino acids critical for the MHC binding and TCR recognition. We found that position 5 (5L) of the dominant allopeptide acts as an MHC-binding residue, while the other two allogeneic positions, 9 and 10, are important for the T-cell receptor (TCR) recognition. A peptide containing the MHC-binding residue 5L, as the only different amino acid between donor (RT1.A(u)) and recipient (RT1.A(l)) sequences, did not induce proliferation of lymph node cells primed with the dominant peptide and prevented dominant peptide-induced acceleration of allograft rejection. Identification of MHC and TCR contact residues should facilitate the development of antigen-specific therapies to inhibit or regulate the indirect alloimmune response.     

Inhibitory effects on HLA-DR1-specific T-cell activation by influenza virus haemagglutinin-derived peptides

Collagen (CII) 263-272 peptide, an autoantigen in rheumatoid arthritis, is a specific human leukocyte antigen (HLA)-DR1/4-binding peptide recognized by T-cell receptors (TCR). The affinity of influenza virus haemagglutinin (HA) 306-318 peptide for the antigen-binding groove of HLA-DR1/4 molecules is higher than that of CII263-272. The HLA-DR1/4-binding residues of HA306-318 are located in the region 308-317. Altered HA308-317 peptides with substitutions of TCR-contact residues may inhibit HLA-DR1/4-specific T-cell activation by blocking the antigen-binding site of HLA-DR1/4 molecules. To evaluate the role of altered HA308-317 peptides in HLA-DR1-restricted T-cell activation, we synthesized three altered HA308-317 peptides. The specific binding of altered HA308-317 peptides to HLA-DR1 molecules was examined using flow cytometry. Effects of altered HA308-317 peptides on HLA-DR1-specific T-cell hybridoma were studied by measuring T-cell proliferation and surface expression of CD69 or CD25. The results showed that altered HA308-317 peptides were able to bind to HLA-DR1 molecules and competed with CII263-272 or wildtype HA308-317 peptide. Compared with wildtype CII263-272 or HA308-317, altered HA308-317 peptides did not stimulate significant T-cell proliferation and CD69 or CD25 expression. Furthermore, the altered HA308-317 peptides inhibited HLA-DR1-specific T-cell activation induced by CII263-272 or wildtype HA308-317 peptide, which may suggest an effective therapeutic strategy in inhibition of HLA-DR1-specific T-cell responses in autoimmunity.     

Permissive recognition of a mycobacterial T-cell epitope: localization of overlapping epitope core sequences recognized in association with multiple major histocompatibility complex class II I-A molecules.

Most T-cell epitopes are recognized in the context of a single or limited number of major histocompatibility complex (MHC) class II molecules. We have shown previously, however, that the immunodominant p61-80 epitope from the Mycobacterium tuberculosis 19,000 MW protein is recognized in a genetically permissive manner. In this study, permissive recognition of p61-80 was analysed in three murine MHC haplotypes (H-2b,d and k) with respect to: (i) T-cell-epitope core structure; (ii) I-A/I-E class II MHC restriction; and (iii) the identification of critical amino acid residues within the core region. Overlapping epitope core sequences composed of 6 to 8 amino acids were identified for each of the three H-2 haplotypes by T-cell epitope scanning (PEPSCAN) using peptide-specific T-cell lines. The epitope core sequences recognized by peptide and 19,000 MW protein-specific T cells were similar. In all three haplotypes, responses to p61-80 were restricted by class II MHC I-A molecules. To identify residues within the epitope core critically required for recognition, single substitution (alanine or leucine) analogue peptides were tested for their capacity to stimulate p61-80-specific T-cell hybridomas. A heterogeneous pattern of reactivity was observed, even among individual hybridomas derived from the same H-2 haplotype. Although every core residue could be defined as critical for at least one hybridoma, only one critical substitution (74Val-->Ala) was common to all hybridomas. The identification and structural analysis of genetically permissive epitopes of mycobacteria may be a useful strategy for the rational design of peptide-based vaccines for tuberculosis.     

Structural basis for ineffective T‐cell responses to MHC anchor residue‐improved “heteroclitic” peptides

MHC anchor residue‐modified “heteroclitic” peptides have been used in many cancer vaccine trials and often induce greater immune responses than the wild‐type peptide. The best‐studied system to date is the decamer MART‐1/Melan‐A_26–35 peptide, EAAGIGILTV, where the natural alanine at position 2 has been modified to leucine to improve human leukocyte antigen (HLA)‐A*0201 anchoring. The resulting EL AGIGILTV peptide has been used in many studies. We recently showed that T cells primed with the EL AGIGILTV peptide can fail to recognize the natural tumor‐expressed peptide efficiently, thereby providing a potential molecular reason for why clinical trials of this peptide have been unsuccessful. Here, we solved the structure of a TCR in complex with HLA‐A*0201‐EAAGIGILTV peptide and compared it with its heteroclitic counterpart , HLA‐A*0201‐EL AGIGILTV. The data demonstrate that a suboptimal anchor residue at position 2 enables the TCR to “pull” the peptide away from the MHC binding groove, facilitating extra contacts with both the peptide and MHC surface. These data explain how a TCR can distinguish between two epitopes that differ by only a single MHC anchor residue and demonstrate how weak MHC anchoring can enable an induced‐fit interaction with the TCR. Our findings constitute a novel demonstration of the extreme sensitivity of the TCR to minor alterations in peptide conformation.     

Structure of an autoimmune T cell receptor complexed with class II peptide-MHC: insights into MHC bias and antigen specificity

T cell receptor crossreactivity with different peptide ligands and biased recognition of MHC are coupled features of antigen recognition that are necessary for the T cell's diverse functional repertoire. In the crystal structure between an autoreactive, EAE T cell clone 172.10 and myelin basic protein (1-11) presented by class II MHC I-Au, recognition of the MHC is dominated by the Vbeta domain of the TCR, which interacts with the MHC alpha chain in a manner suggestive of a germline-encoded TCR/MHC "anchor point." Strikingly, there are few specific contacts between the TCR CDR3 loops and the MBP peptide. We also find that over 1,000,000 different peptides derived from combinatorial libraries can activate 172.10, yet the TCR strongly prefers the native MBP contact residues. We suggest that while TCR scanning of pMHC may be degenerate due to the TCR germline bias for MHC, recognition of structurally distinct agonist peptides is not indicative of TCR promiscuity, but rather highly specific alternative solutions to TCR engagement.     

Human Th1 cell lines recognize the Mycobacterium tuberculosis ESAT-6 antigen and its peptides in association with frequently expressed HLA class II molecules

We have used a synthetic-peptide approach to map epitope regions of the Mycobacterium tuberculosis ESAT-6 antigen recognized by human T cells in relation to major histocompatibility complex (MHC) restriction. ESAT-6-specific CD4+ T-cell lines were established by stimulating peripheral blood mononuclear cells from 25 HLA-DR-typed tuberculosis patients with complete antigen in vitro. The established T-cell lines were then screened for proliferation and interferon-gamma (IFN-gamma) secretion in response to eight overlapping 20-mer peptides covering the ESAT-6 sequence. The response of the T-cell lines to ESAT-6 and peptides from a human leucocyte antigen (HLA)-heterogeneous group of donors suggested the presence of multiple epitopes and promiscuous recognition of the antigen. Analysis of antigen and peptide recognition in the presence of anti-HLA class I and class II antibodies suggested that the T-cell lines recognized ESAT-6 in association with HLA-DR and -DQ molecules. Furthermore, testing of selected T-cell lines with ESAT-6 and the peptides in the presence of autologous and allogeneic HLA-DR- and -DQ-typed antigen-presenting cells identified HLA-DR2, -DR52 and -DQ2 amongst the HLA molecules involved in the presentation of ESAT-6 and its peptides to human Th1 cells. In addition, the T-cell lines were cytotoxic for monocytes and macrophages pulsed with ESAT-6 and peptides. In conclusion, the recognition of ESAT-6 by IFN-gamma-secreting and cytotoxic CD4+ T cells in association with frequently expressed HLA class II molecules supports the application of this antigen to either specific diagnosis or subunit vaccine design.     

Conserved T-cell receptor class II major histocompatibility complex contact detected in a T-lymphocyte population.

T-cell receptor (TCR) interacts with an antigenic peptide deeply buried in the major histocompatibility complex (MHC) molecule. How class II MHC is contacted by TCR during antigen recognition remains largely elusive. Here we used a panel of I-Ek mutants to identify two I-Ek residues that were frequently contacted by TCR among a large pool of T cells specific for the same antigen. The restricted TCR interaction with I-Ek was independent of the antigen peptides. We also identified a dominant heteroclitic residue on I-Ek, beta81H, in which mutation led to increased recognition of antigens in individual T-cell clones. Moreover, both the conserved TCR-I-Ek interaction and the heteroclitic TCR-I-Ek recognition were detected in T lymphocytes freshly isolated from mice primed with the specific antigens. The identical TCR-I-Ek interaction in a heterogeneous T-cell population suggested the dominance of invariant TCR-class II MHC interaction.