Analysis of the Conservation of T Cell Receptor Alpha and Beta Chain Variable Regions Gene in pp65 Peptide-Specific HLA-A＊0201-Restricted CD8^＋ T Cells

Many viral epitope specific T cell receptors （TCRs） in MHC-matched individuals have been demonstrated to involve conserved amino acid motifs in β chain complementarity-determining region 3 （CDR3）. However, it is not sure whether the conserved motifs can also be found in TCR β chain. In previous studies, we developed a modified method to enlarge the percentage of cytomegalovirus （CMV） pp65 peptide-specific CD8^＋ T cells in PBMC by continuous peptide stimulation in vitro, which provides sufficient number of specific T cells for detection. In this study, we further analyzed the restrictive usage of TCR Vα and Vβ gene families and investigated the CDR3 gene sequence of pp65 peptide-specific CD8β T cells. Analysis of CDR3 spectratypes suggested a restricted usage of TCR α chain AV8, AV12, AV21, AV31 families and TCR βchain BV3, BV14, BV21, BV23, BVll families in donor CD8^＋ T cells stimulated by pp65 peptide. The sequences of these T cells involved similar sequence （TX） G （X） A in CDR3 region of TCR α chain and L （XT） G （X） A in TCR β chain.     

Regulatory T cells play a role in T-cell receptor CDR2 peptide regulation of experimental autoimmune encephalomyelitis

Eliciting T-cell receptor (TCR) -specific responsiveness has been known to provide an effective autoregulatory mechanism for limiting inflammation mediated by T effector cells. Our previous use of TCR peptides derived from the CDR3 regions of a pathogenic TCR effectively reversed ongoing experimental autoimmune encephalomyelitis (EAE) in a humanized TCR transgenic model. In this study, we use the TCR BV8S2 CDR2 peptide in the non-transgenic C57BL/6 EAE model to down-regulate the heterogeneous TCR BV8S2(+) MOG-35-55-specific pathogenic T-cell population and demonstrate successful treatment of EAE after disease onset. Suppression of disease was associated with reduced MOG-35-55-specific and non-specific T-cell production of interleukin-17a and interferon-γ in the central nervous system, as well as reduced numbers of CD4(+) and Foxp3(+) T cells in the central nervous system. With the use of Foxp3-GFP and Foxp3 conditional knockout mice, we demonstrate that the TCR CDR2 peptide treatment effect is dependent on the presence of Foxp3(+) regulatory T cells and that regulatory T cell numbers are significantly expanded in the periphery of treated mice. Hence, TCR CDR2 peptide therapy is effective in regulating heterogeneous, pathogenic T-cell populations through the activity of the Foxp3(+) regulatory T cell population.     

Contrasting contributions of complementarity-determining region 2 and hypervariable region 4 of rat BV8S2+ (Vbeta8.2) TCR to the recognition of myelin basic protein and different types of bacterial superantigens

In experimental autoimmune encephalomyelitis (EAE) of LEW rats, BV8S2(+) (V(beta)8.2) T cells dominate the RT1B(l)-restricted response to guinea pig myelin basic protein (gpMBP), and respond to the superantigens (SAg) Staphylococcus enterotoxin C1 (SEC1), Mycoplasma arthritidis SAg (MAS) and Yersinia pseudotuberculosis mitogen (YPM). T cells expressing the closely related BV8S4 differ from BV8S2 T cells in their response to gpMBP, and the SAg SEC1 and MAS, but not in their response to YPM. The functional differences between BV8S2 and BV8S4, which vary in complementarity-determining/hypervariable region 4 (CDR4/HV4) and CDR2, were analyzed by cloning and mutating a TCR with features typical for gpMBP-specific BV8S2(+) TCR. The wild-type BV8S2 receptor and the BV8S4-like CDR2 + 4beta double mutant of BV8S2 showed the same differences in ligand specificity as polyclonal BV8S2(+) and BV8S4(+) lymphocyte populations. The CDR2beta mutant lost its reactivity for SEC1 and gpMBP(68-88), but the CDR4/HV4beta mutation abolished only activation by SEC1. Thus, CDR2 and HV4 contribute not only differently to recognition of peptide antigens, but also to recognition of different types of bacterial SAg.     

识别猪SLA的特异性人T细胞系TCR BV基因取用格局和CDR3结构多样性分析

不同T细胞克隆TCR分子的序列不同 ,所识别的抗原特异性也不同。其中第三互补决定区 (CDR3)变异最大 ,是TCR主要的抗原结合部位。本文采用荧光标记半定量PCR技术 ,用DNA测序仪作程序分析 ,了解猪细胞抗原致敏前后的人T细胞群和 5个T细胞系 2 4个TCRBV基因家族取用格局 ,并以TCRα链C区的基因片断作为内参对取用情况作定量估计。发现首次抗原致敏后培养 2周的T细胞除了BV2 4、BV8和BV10未能检测出 ,其它BV基因都有不同程度的取用。然而 ,5个细胞系的TCRBV基因呈现十分有限的取用格局 ,其中两个CD4+ T细胞系都取用BV12和BV14;3个CD8+ T细胞系中都优势取用BV1,有两个还取用BV19。CD4+ T细胞系和CD8+ T细胞系之间TCRBV无交叉取用 ,提示两类细胞识别的抗原表位存在差异。进一步用变性凝胶扫描分析上述T细胞系取用TCRBV中的CDR3的多样性 ,发现未经抗原致敏的T细胞BV的CDR3结构为多峰型且呈正态分布 ,表明涉及多种结构不同的细胞克隆 ;而抗原特异性T细胞系CDR3除了一个CD8+ T细胞系BV1有两个主峰外其它无例外地都显示单峰或者仅一个主峰 ,这从另一个角度证明建系T细胞的单克隆性。     

Composition and variation analysis of the TCR β-chain CDR3 repertoire in systemic lupus erythematosus using high-throughput sequencing

The ability of T lymphocytes to mount an immune response against a diverse array of pathogens is primarily conveyed by the amino acid (aa) sequence of the hypervariable complementarity-determining region 3 (CDR3) segments of the T cell receptor (TCR). In this study, we used a combination of multiplex-PCR, Illumina sequencing and IMGT/HighV-QUEST for a standardized analysis of the characteristics and polymorphisms of the T-cell receptor BV complementarity-determining region 3 (TCR BV CDR3) gene in peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy donors (NC). We found the distributions of CDR3, VD indel, and DJ indel lengths to be comparable between the SLE and NC groups. The degree of clonal expansion in the SLE group was significantly greater than in the NC group, and the expression levels of 10 TRβV segments and 6 TRβJ segments were also significantly different in the SLE group. Regarding public T cell responses, 3CDR3 DNA sequences and 4 aa sequences were shared by all SLE patients and may serve as biomarkers for SLE disease risk, diagnosis and/or prognosis.     

High‐sequence diversity and structural conservation in the human T‐cell receptor β junctional region during thymic development

The T‐cell repertoire depends on intrathymic genetic rearrangement events in the T‐cell receptor (TCR) locus, followed by positive and negative selection. The repertoire thus generated is highly diverse, but recent data indicate that the recombination of gene segments is less stochastic than previously suggested. Very little is known of the junctional complementarity determining region 3 (CDR3), which is to a large degree not germline encoded. We have analyzed the development of the human TCR β CDR3 repertoire, from the nonselected CD4⁺CD8⁺CD3^? cells up to the fully selected CD4⁺CD8^? thymocytes. In addition to spectratyping, a fraction of the CDR3 repertoire was sequenced and a structural in silico analysis of the CDR3 loop characteristics performed. Our data show that the thymic TCR repertoire is extremely diverse, and the effect of the selection events can be detected as a measurable loss of polyclonality in the CDR3 loop. However, the main physicochemical features of the CDR3 loop were found already at the nonselected repertoire and showed no progressive changes during the selection. Thus, the main structural characteristics of the CDR3 loop were already determined by the recombination process and not significantly affected by the extensive thymocyte death associated with selection in the thymus.     

Identification of oligoclonal CD4 T cells in diffuse large B cell lymphomas

Human B cell lymphomas often contain CD4 T cells. Here we show that, in diffuse large B cell lymphomas (DLCL), such T cells are oligoclonal. The CDR3 lengths and nucleotide sequences of oligoclonal TCRBV of CD4 T cells in an original and relapsed lymphoma from one patient were compared. Three BV23 sequences were identical (12/17 and 16/16 clones in primary and relapsed lymphomas, respectively), but were absent in CD4 T cells from another patient's DLCL. Two of the repetitive BV23 sequences were found in peripheral blood CD4 T cells (5/17 clones); gamma-irradiated DLCL from this patient stimulated syngeneic BV23 response in CD4 cells (92% of BV23 had the same CDR3 length). Skew in TCRBV representation was observed in CD4 T cells from all the DLCL. One DLCL, with overrepresentation of BV13S1 in CD4 cells, stimulated the same TCR in CD4 cells from three unrelated individuals. These findings support the conclusion that there is clonal selection of CD4 T cells in DLCL.     

Analysis of the interindividual conservation of T cell receptor alpha- and beta-chain variable regions gene in the peripheral blood of patients with systemic lupus erythematosus

The aim of this study was to find conserved motifs in specific T cell receptor (TCR) alpha- and beta-chains, and to analyse the association between complementarity determining region 3 (CDR3) spectratype and systemic lupus erythematosus (SLE) activity. TCR alpha-and beta-chain CDR3 spectratypes were analysed in 20 SLE patients. The CDR3 spectratypes of three patients were monitored over time, and the CDR3 regions of clonally expanded T cells were sequenced. CDR3 spectratype analysis showed prominent usage of TCR AV8, AV14, AV23, AV30, AV31, BV2, BV8, BV11, BV14, BV16, BV19 and BV24 families in SLE patients. The CDR3 spectratype showed dynamic change correlating with SLE activity. The sequence of the CDR3 region in clonally expanded T cells suggested a conserved GGX amino acid motif in both alpha- and beta-chains. The Ja34 and Jb2s1 region genes were found in high frequency. Both TCR Valpha and Vbeta gene usage is highly restricted in SLE, suggesting that the TCRs recognize a limited number of antigenic epitopes. The conserved motifs and limited use of joining region genes may indicate the recognition of similar antigenic epitopes in multiple individuals.     

Therapeutic vaccination with a trivalent T-cell receptor (TCR) peptide vaccine restores deficient FoxP3 expression and TCR recognition in subjects with multiple sclerosis

Therapeutic vaccination using T-cell receptor (TCR) peptides from V genes commonly expressed by potentially pathogenic T cells remains an approach of interest for treatment of multiple sclerosis (MS) and other autoimmune diseases. We developed a trivalent TCR vaccine containing complementarity determining region (CDR) 2 peptides from BV5S2, BV6S5 and BV13S1 emulsified in incomplete Freund''s adjuvant that reliably induced high frequencies of TCR-specific T cells. To evaluate induction of regulatory T-cell subtypes, immunological and clinical parameters were followed in 23 treatment-naïve subjects with relapsing-remitting or progressive MS who received 12 monthly injections of the trivalent peptide vaccine over 1 year in an open-label study design. Prior to vaccination, subjects had reduced expression of forkhead box (Fox) P3 message and protein, and reduced recognition of the expressed TCR repertoire by TCR-reactive cells compared with healthy control donors. After three or four injections, most vaccinated MS subjects developed high frequencies of circulating interleukin (IL)-10-secreting T cells specific for the injected TCR peptides and significantly enhanced expression of FoxP3 by regulatory T cells present in both ‘native’ CD4⁺ CD25⁺ and ‘inducible’ CD4⁺ CD25⁻ peripheral blood mononuclear cells (PBMC). At the end of the trial, PBMC from vaccinated MS subjects retained or further increased FoxP3 expression levels, exhibited significantly enhanced recognition of the TCR V gene repertoire apparently generated by perturbation of the TCR network, and significantly suppressed neuroantigen but not recall antigen responses. These findings demonstrate that therapeutic vaccination using only three commonly expressed BV gene determinants can induce an expanded immunoregulatory network in vivo that may optimally control complex autoreactive responses that characterize the inflammatory phase of MS.     

T淋巴瘤EL-4细胞双受体表达研究

目的:检测T淋巴瘤EL-4细胞T细胞受体(T cell receptor,TCR)的表达情况,为进一步研究T细胞等位基因排斥机制奠定基础。方法:以小鼠脾细胞作为阳性对照,分别提取脾细胞和EL-4细胞mRNA,逆转录为cDNA,RT-PCR扩增24个TCR BV家族CDR3区,结合基因扫描(GeneScan)和基因测序技术,对有表达的TCR BV家族进行CDR3谱型和序列分析。结果:小鼠脾细胞24 BV家族均有表达,各TCR BV家族CDR3谱型均呈高斯分布(Gaussian distribution),表明24 BV家族均为多克隆性。EL-4细胞被同时检测到TCR BV10和BV12家族表达,CDR3谱型均呈单峰,两个家族的RT-PCR产物经测序鉴定证实确属TCR序列,且均为框内编码。结论:EL-4细胞存在两套框内重排的TCRβ链,表明其TCRβ链可能存在等位基因排斥"缺陷"现象。本研究为探索T细胞等位基因排斥机制奠定了基础。     

Prevention and treatment of experimental autoimmune encephalomyelitis with clonotypic CDR3 peptides: CD4+ FoxP3+ T‐regulatory cells suppress interleukin‐2‐dependent expansion of myelin basic protein‐specific T cells

T‐cell receptor (TCR)‐derived peptides are recognized by the immune system and are capable of modulating autoimmune responses. Using the myelin basic protein (MBP) TCR 1501 transgenic mouse model, we demonstrated that TCR CDR3 peptides from the transgenic TCR can provide a protective effect when therapy is initiated before the induction of experimental autoimmune encephalomyelitis (EAE). More importantly, TCR CDR3 peptide therapy can ameliorate the disease when administered after EAE onset. Concurrent with the therapeutic effects, we observed reduced T‐cell proliferation and reduced interleukin‐2 (IL‐2) levels in response to stimulation with MBP‐85‐99 peptide in splenocyte cultures from mice receiving TCR CDR3 peptides compared with that of control mice. Moreover, we found that Foxp3⁺ CD4 T cells from mice protected with TCR CDR3 peptide are preferentially expanded in the presence of IL‐2. This is supportive of a proposed mechanism where Foxp3⁺ T‐regulatory cells induced by therapy with MBP‐85‐99 TCR CDR3 peptides limit expansion and the encephalitogenic activity of MBP‐85‐99‐specific T cells by regulating the levels of secreted IL‐2.     

Identification of conserved T cell receptor CDR3 residues contacting known exposed peptide side chains from a major histocompatibility complex class I-bound determinant

We have analyzed the T cell receptor (TCR) repertoire found in the major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response to the protein ovalbumin (OVA). Despite skewing towards the expression of Vβ5.2⁺ TCR by OVA-specific CTL from C57BL/6 mice, we found a relatively high degree of diversity in V(D)J usage in both TCR α- and β-chains. Closer examination showed that the majority of these sequences encoded negatively and positively charged residues at their respective TCR α- and β-chain VJ or VDJ junctions. These junctions form the third complementarity-determining regions (CDR3) of the TCR polypeptides involved in the direct interaction with the class I-bound peptide. Crystallographic analyses of K^b-peptide complexes predict that the major determinant from OVA, peptide OVA_257–264 (SIINFEKL), contains two exposed charged side chains which can contact the TCR. These are the negatively charged glutamic acid at determinant position 6 (P6) and the positively charged lysine at P7. To examine whether the TCR α-chain makes contact with P7 lysine, we established a single chain TCR transgenic C57BL/6 mouse line where all T cells express a TCR β-chain derived from the Vβ5.2⁺ clone B3. OVA-specific T cells derived from in vivo primed transgenic mice preferentially expressed TCR α-chains that also contained negatively charged junctional residues despite some further variation in Vα and Jα sequences. Stimulation of naive TCR β-chain transgenic T cells with a P7 substitution peptide analogue induced a T cell response that was no longer cross-reactive with the wild-type OVA_257–264 determinant, sugesting that the TCR α-chain from the T cell clone B3 can determine the specificity for this residue. Consequently, these results reveal the existence of conserved residues in the CDR3 of TCR α- and β-chains specific for OVA_257–264 and identify their possible orientation over the peptide-class I complex.     

Degeneracy of T cell receptor recognition of an influenza virus hemagglutinin epitope restricted by HLA-DQ and -DR class II molecules

The T cell receptor (TcR) recognizes antigens in the form of short peptide fragments bound to major histocompatibility (MHC) molecules. TcR have an immunoglobulin (Ig)-like structure and, in an analogous manner to antigen recognition by Ig, the third complementarity determining regions (CDR3) of the TcR are believed to provide the primary contact with the peptide lying in the MHC groove. CDR1 and CDR2 are thought to contact the presenting MHC molecule. We have analyzed seven human CD4⁺ T cell clones that recognize a conserved peptide epitope (residues 255–270) within the influenza virus hemagglutinin (H3) HA1 subunit. Two T cell clones recognized the peptide in the context of HLA-DRB1*1001 and HLA-DQB1* 0602/DQA1*0102, respectively, and shared V_α, V_β and J_β gene segments. Only the junctional regions encoding the CDR3 regions of the two TcR chains were different. This suggests that the CDR3 regions of these TcR interact with the MHC class II molecule. Six of the T cell clones were restricted by the HLA-DRB1*1001. Two of these T cell clones expressed Vβ9.1 and three expressed Vβ13 gene segments; the remaining clone expressed Vβ7.2, a close homologue of V_β9.1. A diverse selection of V_α and J gene segments contributed to the junctional heterogeneity of the TcR, indicating a diversity of sequence combinations recognizing the epitope. Nevertheless, five out of six T cell clones bore a motif in the V_α CDR3 loop consisting of adjacent acidic and polar amino acid residues, eight residues from the carboxyl end of each CDR3.     

Subtle changes in TCRα CDR1 profoundly increase the sensitivity of CD4 T cells

Changes in the peptide and MHC molecules have been extensively examined for how they alter T cell activation, but many fewer studies have examined the TCR. Structural studies of how TCR differences alter T cell specificity have focused on broad variation in the CDR3 loops. However, changes in the CDR1 and 2 loops can also alter TCR recognition of pMHC. In this study we focus on two mutations in the CDR1α loop of the TCR that increased the affinity of a TCR for agonist Hb(64-76)/I-E^k by increasing the on-rate of the reaction. These same mutations also conferred broader recognition of altered peptide ligands. TCR transgenic mice expressing the CDR1α mutations had altered thymic selection, as most of the T cells were negatively selected compared to T cells expressing the wildtype TCR. The few T cells that escaped negative selection and were found in the periphery were rendered anergic, thereby avoiding autoimmunity. T cells with the CDR1α mutations were completely deleted in the presence of Hb(64-76) as an endogenous peptide. Interestingly, the wildtype T cells were not eliminated, identifying a threshold affinity for negative selection where a 3-fold increase in affinity is the difference between incomplete and complete deletion. Overall, these studies highlight how small changes in the TCR can increase the affinity of TCR:pMHC but with the consequences of skewing selection and producing an unresponsive T cell.     

The structural dynamics and energetics of an immunodominant T cell receptor are programmed by its Vbeta domain

Immunodominant and public T cell receptor (TCR) usage is relatively common in many viral diseases yet surprising in the context of the large naive TCR repertoire. We examined the highly conserved Vbeta17:Valpha10.2 JM22 T cell response to the influenza matrix peptide (58-66)-HLA-A*0201 (HLA-A2-flu) through extensive kinetic, thermodynamic, and structural analyses. We found several conformational adjustments that accompany JM22-HLA-A2-flu binding and identified a binding "hotspot" within the Vbeta domain of the TCR. Within this hotspot, key germline-encoded CDR1 and CDR2 loop residues and a crucial but commonly coded residue in the hypervariable region of CDR3 provide the basis for the substantial bias in the selection of the germline-encoded Vbeta17 domain. The chances of having a substantial number of T cells in the naive repertoire that have HLA-A2-flu-specific Vbeta17 receptors may consequently be relatively high, thus explaining the immunodominant usage of this clonotype.     

Human TCR as antigen: homologies and potentially cross-reactive HLA-DR2-restricted epitopes within the AV and BV CDR2 loops

The major function of the T-cell receptor is to confer antigen specificity to T cells. However, nascent TCR proteins that are not assembled into functional heterodimers may be processed and displayed with self MHC molecules on the T-cell surface, and are thought to be the genesis of autoregulatory T cells that can limit inflammatory responses through T-T network interactions. In previous work, we and others have exploited this natural regulatory system using TCR peptides to amplify regulatory T cells that potentially can treat human autoimmune diseases such as multiple sclerosis (MS) and arthritis. The development of this approach is limited by the diversity of human TCR V gene sequences, and by lack of knowledge of exactly which regions of the V gene proteins are immunogenic in association with various MHC alleles. To identify similar amino acid sequences within and among human V gene families that might have immunologic cross reactivity, we aligned 74 known AV and 109 known BV protein sequences into homologous groups using the ClustalX program. Moreover, with a focus on CDR2 peptides that have previously been used to induce regulatory T cells in clinical trials, we established homologous peptide groups, and then identified the optimal amino acid motifs for binding to two alleles, HLA-DRB1*1501 and DRB5*0101, that have been associated with susceptibility to MS. From this analysis, > 75% of AV and BV CDR2 sequences were predicted to bind with at least moderate avidity to each of the DR2 alleles, thus enhancing the likelihood that they could be antigenic. Further ordering of putative TCR contact residues revealed a different set of homology groupings, including many intrafamily sequence matches and some interfamily matches that might allow immunological cross reactivity. Particularly striking were DRB1*1501-restricted IH-S and IY-S motifs shared by BV11, BV12, and BV13 and BV3, BV12, BV13, and BV17 family members, respectively, and DRB5*0101-restricted RL-H and RL-Y motifs shared by BV11, BV12, and BV13 and BV13 and BV17 family members, respectively. This analysis may be useful in designing an array of clinically useful homologous peptides with optimal MHC binding properties and highly cross-reactive TCR binding motifs.     

An example of immunodominance: engagement of synonymous TCR by invariant CDR3 beta

The structural basis of the T cell response against immunodominant tetanus toxin (TT)-derived peptides was investigated using TT-specific T cell clones raised from a DRB1*0301 homozygous donor. Three peptides forming T cell epitopes were identified, including one, TT(1272-1284), that stimulated four different TT-specific T cell clones. TCR sequence analysis revealed that these synonymous TCR shared only arginine at the third position of the CDR3 beta loop. This prominent residue may form a salt bridge with a corresponding aspartate at the relative position 8 (P8) of the antigenic peptide TT(1272-1284) as suggested from amino acid replacement analysis. A similar scenario was observed for a second TT epitope, TT(279-296), and its corresponding TCR. These examples show that immunodominance may result from a single strong amino acid interaction between TCR CDR3 beta loops here in contact with the C-terminus of the antigenic peptide. Such a dominant interaction could compensate for weaker contacts between other residues of the TCR and the antigenic peptide, and would allow the recognition of a single peptide-MHC complex by a broader synonymous TCR repertoire and could thus contribute to its immunodominance.     

Functional characterization of a T-cell receptor BV6+ T-cell clone derived from a leprosy lesion

Human infection with Mycobacterium leprae, an intracellular bacterium, presents as a clinical and immunological spectrum; thus leprosy provides an opportunity to investigate mechanisms of T-cell responsiveness to a microbial pathogen. Analysis of the T-cell receptor (TCR) repertoire in leprosy lesions revealed that TCR BV6(+) T cells containing a conserved CDR3 motif are over-represented in lesions from patients with the localized form of the disease. Here, we derived a T-cell clone from a leprosy lesion that expressed TCR BV6 and the conserved CDR3 sequence L-S-G. This T-cell clone produced a T helper type 1 cytokine pattern, directly lysed M. leprae-pulsed antigen-presenting cells by the granule exocytosis pathway, and expressed the antimicrobial protein granulysin. BV6(+) T cells may therefore functionally contribute to the cell-mediated immune response against M. leprae.     

Engagement of a T cell receptor by major histocompatibility complex irrespective of peptide

T cell receptors (TCR) identify target cells presenting a ligand consisting of a major histocompatibility complex molecule (MHC) and an antigenic peptide. A considerable amount of evidence indicates that the TCR contacts both the peptide and the MHC components of the ligand. In fully differentiated T cells the interaction between the peptide and the TCR makes the critical contribution to eliciting a cellular response. However, during the positive selection of thymocytes the contribution of peptide relative to MHC is less well established. Indeed it has been suggested that the critical interaction for positive selection is between the TCR and the MHC molecule and that peptides can be viewed as either allowing or obstructing this contact. This predicts that a given TCR is capable of engaging multiple MHC/peptide complexes. In this study a system is described which detects simply engagement of the TCR by MHC/peptide complexes rather than the functional outcome of such interactions. Using this approach the extent to which peptides can influence contacts between the TCR and the MHC molecule has been examined. The results show that the TCR does in fact engage a wide range of ligands in an MHC-restricted but largely peptide-independent manner, suggesting that only a few peptides are able to prevent the TCR from contacting the MHC molecule.