Reciprocal expression in CD4 or CD8 subsets of different members of the V alpha 11 gene family correlates with sequence polymorphism

Previous staining studies with TCR V alpha 11-specific mAbs showed that V alpha 11.1/11.2 (AV11S1 and S2) expression was selectively favored in the CD4+ peripheral T cell population. As this phenomenon was essentially independent of the MHC haplotype, it was suggested that AV11S1 and S2 TCRs exert a preference for recognition of class II MHC molecules. The V alpha segment of the TCR alpha-chain is suggested to have a primary role in shaping the T cell repertoire due to selection for class I or II molecules acting through the complementarity determining regions (CDR) 1 alpha and CDR2 alpha residues. We have analyzed the repertoire of V alpha 11 family members expressed in C57BL/6 mice and have identified a new member of this family; AV11S8. We show that, whereas AV11S1 and S2 are more frequent in CD4+ cells, AV11S3 and S8 are more frequent in CD8+ cells. The sequences in the CDR1 alpha and CDR2 alpha correlate with differential expression in CD4+ or CD8+ cells, a phenomenon that is also observed in BALB/c mice. With no apparent restriction in TCR J alpha usage or CDR3 alpha length in C57BL/6, these findings support the idea of V alpha-dependent T cell repertoire selection through preferential recognition of MHC class I or class II molecules.     

Amino acids specifying MHC class preference in TCR V alpha 2 regions.

Some TCR variable regions are preferentially expressed in CD4+ or CD8+ T cells, reflecting a predilection for interacting with MHC class II or class I molecules. The molecular basis for MHC class bias has been studied previously, in particular for V alpha 3 family members, pointing to a dominant role for two amino acid positions in complementary-determining regions (CDRs) 1 and 2. We have evaluated the generality of these findings by examining the MHC class bias of V alpha 2 family members, an attractive system because it shows more variability within the CDR1 and -2, exhibits variation in the framework regions, and includes a member for which the crystal structure has been determined. We find that preferential recognition of MHC class I or II molecules does not always depend on residues at the same positions of CDR1 and -2; rules for one family may be reversed in another. Instead, there are multiple influences exerted by various CDR1/2 positions as well as the CDR3s of both the TCR alpha- and TCR beta-chains.     

An MHC interaction site maps to the amino-terminal half of the T cell receptor alpha chain variable domain.

We have used cloned T cell receptor (TCR) genes from closely related CD4 T cell lines to probe the interaction of the TCR with several specific major histocompatibility complex (MHC) class II ligands. Complementarity determining region 3 (CDR3) equivalents of both alpha and beta TCR chains are required for antigen-MHC recognition. Our data provide novel information about the rotational orientation of TCR-MHC contacts in that exchange of the amino terminal portion of the TCR alpha chain containing the putative CDR1 and CDR2 regions results in both gain and loss of MHC class II specificity by the resulting receptor. These two TCRs differ primarily in recognition of polymorphisms in the second hypervariable region of the MHC class II alpha chain. These results document the involvement of CDR1 and/or CDR2 of the TCR alpha chain in MHC recognition and suggest a rotational orientation of this TCR to its MHC ligand.     

A new twist in TCR diversity revealed by a forbidden alphabeta TCR

We report crystal structures of a negatively selected T cell receptor (TCR) that recognizes two I-A^u-restricted myelin basic protein peptides and one of its peptide/major histocompatibility complex (pMHC) ligands. Unusual complementarity-determining region (CDR) structural features revealed by our analyses identify a previously unrecognized mechanism by which the highly variable CDR3 regions define ligand specificity. In addition to the pMHC contact residues contributed by CDR3, the CDR3 residues buried deep within the Vα/Vβ interface exert indirect effects on recognition by influencing the Vα/Vβ interdomain angle. This phenomenon represents an additional mechanism for increasing the potential diversity of the TCR repertoire. Both the direct and indirect effects exerted by CDR residues can impact global TCR/MHC docking. Analysis of the available TCR structures in light of these results highlights the significance of the Vα/Vβ interdomain angle in determining specificity and indicates that TCR/pMHC interface features do not distinguish autoimmune from non-autoimmune class II-restricted TCRs.     

Immunobiological analysis of TCR single-chain transgenic mice reveals new possibilities for interaction between CDR3alpha and an antigenic peptide bound to MHC class I

The interaction between TCRs and peptides presented by MHC molecules determines the specificity of the T cell-mediated immune response. To elucidate the biologically important structural features of this interaction, we generated TCR beta-chain transgenic mice using a TCR derived from a T cell clone specific for the immunodominant peptide of vesicular stomatitis virus (RGYVYQGL, VSV8) presented by H-2K(b). We immunized these mice with VSV8 or analogs substituted at TCR contact residues (positions 1, 4, and 6) and analyzed the CDR3alpha sequences of the elicited T cells. In VSV8-specific CTLs, we observed a highly conserved residue at position 93 of CDR3alpha and preferred Jalpha usage, indicating that multiple residues of CDR3alpha are critical for recognition of the peptide. Certain substitutions at peptide position 4 induced changes at position 93 and in Jalpha usage, suggesting a potential interaction between CDR3alpha and position 4. Cross-reactivity data revealed the foremost importance of the Jalpha region in determining Ag specificity. Surprisingly, substitution at position 6 of VSV8 to a negatively charged residue induced a change at position 93 of CDR3alpha to a positively charged residue, suggesting that CDR3alpha may interact with position 6 in certain circumstances. Analogous interactions between the TCR alpha-chain and residues in the C-terminal half of the peptide have not yet been revealed by the limited number of TCR/peptide-MHC crystal structures reported to date. The transgenic mouse approach allows hundreds of TCR/peptide-MHC interactions to be examined comparatively easily, thus permitting a wide-ranging analysis of the possibilities for Ag recognition in vivo.     

T cell receptor gene segment utilization by HLA-DR1-alloreactive T cell clones.

Transplantation of histoincompatible tissues leads to allograft rejection, which involves recognition of allogeneic MHC molecules by Ag-specific receptors expressed on T cells. The interaction of these molecules is highly specific yet poorly understood. We have investigated the relationship between TCR gene utilization and allo-MHC restriction patterns by using a one-way polymerase chain reaction to amplify the alpha- and beta-chain mRNA from a panel of 10 HLA-DR1-alloreactive T lymphocyte clones. Two previously unreported V alpha and five J alpha gene sequences were obtained. Although a few V alpha, V beta, and J alpha genes were utilized more than once, no correlation between TCR gene usage and DR1 alloreactivity was identified. At the sequence level, the presumed TCR alpha- and beta-chain CDR1 and CDR2 regions displayed limited diversity, whereas the CDR3 or junctional sequences were highly variable. Although most TCR probably interact with subtly different surface features of the DR1 alloantigen, we predict that TCR with similar CDR1 and CDR2 sequences would contact essentially identical regions of the DR1 molecule. The lack of sequence conservation in the junctional regions suggests that different endogenous peptides also may be recognized. Thus, alloreactive T cells may recognize not only allogeneic MHC molecules but perhaps also bound endogenous peptides.     

Plasticity in the Contribution of T Cell Receptor Variable Region Residues to Binding of Peptide–HLA-A2 Complexes

One hypothesis accounting for major histocompatibility complex (MHC) restriction by T cell receptors (TCRs) holds that there are several evolutionary conserved residues in TCR variable regions that contact MHC. While this “germline codon” hypothesis is supported by various lines of evidence, it has been difficult to test. The difficulty stems in part from the fact that TCRs exhibit low affinities for pep/MHC, thus limiting the range of binding energies that can be assigned to these key interactions using mutational analyses. To measure the magnitude of binding energies involved, here we used high-affinity TCRs engineered by mutagenesis of CDR3. The TCRs included a high-affinity, MART-1/HLA-A2-specific single-chain TCR and two other high-affinity TCRs that all contain the same Vα region and recognize the same MHC allele (HLA-A2), with different peptides and Vβ regions. Mutational analysis of residues in CDR1 and CDR2 of the three Vα2 regions showed the importance of the key germline codon residue Y51. However, two other proposed key residues showed significant differences among the TCRs in their relative contributions to binding. With the use of single-position, yeast-display libraries in two of the key residues, MART-1/HLA-A2 selections also revealed strong preferences for wild-type germline codon residues, but several alternative residues could also accommodate binding and, hence, MHC restriction. Thus, although a single residue (Y51) could account for a proportion of the energy associated with positive selection (i.e., MHC restriction), there is significant plasticity in requirements for particular side chains in CDR1 and CDR2 and in their relative binding contributions among different TCRs.     

Differential sensitivity to mutations in a single peptide by two TCRs having identical beta-chains and closely related alpha-chains

The TCR on CD4 T cells binds to and recognizes MHC class II:antigenic peptide complexes through molecular contacts with the peptide amino acid residues that face up and out of the peptide-binding groove. This interaction primarily involves the complementarity-determining regions (CDR) of the TCR alpha- and ss-chains contacting up to five residues of the peptide. We have used two TCRs that recognize the same antigenic peptide and have identical Vss8.2 chains, but differ in all three CDR of their related Valpha2 chains, to examine the fine specificity of the TCR:peptide contacts that lead to activation. By generating a peptide library containing all 20 aa residues in the five potential TCR contact sites, we were able to demonstrate that the two similar TCRs responded differentially when agonist, nonagonist, and antagonist peptide functions were examined. Dual substituted peptides containing an agonist residue at the N terminus, which interacts with CDR2alpha, and an antagonist residue at the C terminus, which interacts with the CDR3ss, were used to show that the nature of the overall signal through the TCR is determined by a combination of the type of signal received through both the TCR alpha- and ss-chains.     

Random length assortment of human and mouse T cell receptor for antigen alpha and beta chain CDR3.

In view of the recently determined three-dimensional structures of complexes formed by the T cell receptor for antigen (TCR), the processed peptide and the MHC class I molecule, it is expected that the combined configuration formed by the third complementarity determining regions (CDR3) of TCR alpha and beta chains will be very restricted in size and shape due to the limited length variations of the processed peptides. Thus, the combined TCR alpha and beta chain CDR3 lengths should have a fairly narrow distribution. This feature can be due to the selective association of long alpha chain CDR3 with short beta chain CDR3 and vice versa or due to random assortment of alpha and beta chain CDR3 of even narrower length distribution. Based on existing translated amino acid sequence data, it has been found that the latter mechanism is responsible.     

Modeling of the TCR-MHC-peptide complex

The methodology for generating a homology model of the T1 TCR-PbCS-K(d) class I major histocompatibility complex (MHC) class I complex is presented. The resulting model provides a qualitative explanation of the effect of over 50 different mutations in the region of the complementarity determining region (CDR) loops of the T cell receptor (TCR), the peptide and the MHC's alpha(1)/alpha(2) helices. The peptide is modified by an azido benzoic acid photoreactive group, which is part of the epitope recognized by the TCR. The construction of the model makes use of closely related homologs (the A6 TCR-Tax-HLA A2 complex, the 2C TCR, the 14.3.d TCR Vbeta chain, the 1934.4 TCR Valpha chain, and the H-2 K(b)-ovalbumine peptide), ab initio sampling of CDR loops conformations and experimental data to select from the set of possibilities. The model shows a complex arrangement of the CDR3alpha, CDR1beta, CDR2beta and CDR3beta loops that leads to the highly specific recognition of the photoreactive group. The protocol can be applied systematically to a series of related sequences, permitting the analysis at the structural level of the large TCR repertoire specific for a given peptide-MHC complex.     

High-affinity, peptide-specific T cell receptors can be generated by mutations in CDR1, CDR2 or CDR3

The third complementarity-determining regions (CDR3s) of antibodies and T cell receptors (TCRs) have been shown to play a major role in antigen binding and specificity. Consistent with this notion, we demonstrated previously that high-affinity, peptide-specific TCRs could be generated in vitro by mutations in the CDR3alpha region of the 2C TCR. In contrast, it has been argued that CDR1 and CDR2 are involved to a greater extent than CDR3s in the process of MHC restriction, due to their engagement of MHC helices. Based on this premise, we initiated the present study to explore whether higher affinity TCRs generated through mutations in these CDRs or other regions would lead to significant reductions in peptide specificity (i.e. the result of greater binding energy gained through interactions with major histocompatibility complex (MHC) helices). Yeast-display technology and flow sorting were used to select high-affinity TCRs from libraries of CDR mutants or random mutants. High-affinity TCRs with mutations in the first residue of the Valpha, CDR1, CDR2, or CDR3 were isolated. Unexpectedly, every TCR mutant, including those in CDR1 and CDR2, retained remarkable peptide specificity. Molecular modeling of various mutants suggested that such exquisite specificity may be due to: (1) enhanced electrostatic interactions with key peptide or MHC residues; or (2) stabilization of CDRs in specific conformations. The results indicate that the TCR is positioned so that virtually every CDR can contribute to the antigen-specificity of a T cell. The conserved diagonal docking of TCRs could thus orient each CDR loop to sense the peptide directly or indirectly through peptide-induced effects on the MHC.     

Amino acid residues in the T cell receptor CDR3 determine the antigenic reactivity patterns of insulin-reactive hybridomas

We examined TCR gene usage in a panel of beef insulin/I-Ad-restricted T cell hybrids obtained from BALB/c mice. These hybrids demonstrated several distinct patterns of reactivity defined by their ability to respond to species variants of insulin. Correlation of TCR-alpha and -beta-gene usage with these patterns of reactivity demonstrated that TCR gene usage was restricted within Ag reactivity groups. In particular, V-J junctional regions (CDR3 equivalent) were restricted with conserved junctional amino acid motifs present in both TCR-alpha- and -beta-chains. Comparison of TCR gene usage in hybrids expressing identical V alpha and V beta gene segments but demonstrating different patterns of reactivity revealed that changes in either J alpha and/or J beta gene segment usage could alter antigenic reactivity. Indeed, single or limited amino acid differences within the CDR3 region were sufficient to markedly alter fine specificity. These data demonstrate the critical role for CDR3 in determining antigenic reactivity in beef insulin-reactive hybrids and are compatible with the current model of TCR/peptide/MHC interaction.     

Functional analysis of the TCR binding domain of toxic shock syndrome toxin-1 predicts further diversity in MHC class II/superantigen/TCR ternary complexes

Superantigens (SAGs) aberrantly alter immune system function through simultaneous interaction with lateral surfaces of MHC class II molecules on APCs and with particular variable regions of the TCR beta-chain (Vbeta). To further define the interface between the bacterial SAG toxic shock syndrome toxin-1 (TSST-1) and the TCR, we performed alanine scanning mutagenesis within the putative TCR binding region of TSST-1 along the central alpha helix adjacent to the N-terminal alpha helix and the beta7-beta9 loop as well as with two universally conserved SAG residues (Leu(137) and Tyr(144) in TSST-1). Mutants were analyzed for multiple functional activities, and various residues appeared to play minor or insignificant roles in the TCR interaction. The locations of six residues (Gly(16), Trp(116), Glu(132), His(135), Gln(136), and Gln(139)), each individually critical for functional activity as well as direct interaction with the human TCR Vbeta2.1-chain, indicate that the interface occurs in a novel region of the SAG molecule. Based on these data, a model of the MHC/TSST-1/TCR ternary complex predicts similarities seen with other characterized SAGs, although the CDR3 loop of Vbeta2.1 is probably involved in direct SAG-TCR molecular interactions, possibly contributing to the TCR Vbeta specificity of TSST-1.     

Production,characterization, and immunogenicity of a soluble rat single chain T cell receptor specific for an encephalitogenic peptide

The encephalitogenic rat T cell clone C14 recognizes the myelin basic protein 69-89 peptide in the context of the RT1B major histocompatibility complex (MHC) class II molecule. Modeling of the C14 TCR molecule indicated that previously identified CDR3 motifs are likely to be central to interaction with MHC class II-presented peptide. Here we report the cloning and expression of C14-derived single chain TCR (scTCR) molecules in an Escherichia coli expression system. The recombinant molecule consists of the Valpha2 domain connected to the Vbeta8.2 domain via a 15-residue linker. Soluble C14 scTCR was purified using conventional chromatography techniques and refolded by a rapid dilution procedure. C14 scTCR was able to bind soluble rat MHC class II molecules bearing covalently coupled Gp-BP-(69-89) peptide, as analyzed using surface plasmon resonance. Immune recognition of the C14 scTCR protein as an antigen revealed that limited regions of the TCR may be more likely to induce responsiveness.     

Distinct CDR3 conformations in TCRs determine the level of cross-reactivity for diverse antigens, but not the docking orientation

T cells are known to cross-react with diverse peptide MHC Ags through their alphabeta TCR. To explore the basis of such cross-reactivity, we examined the 2C TCR that recognizes two structurally distinct ligands, SIY-K(b) and alloantigen QL9-L(d). In this study we characterized the cross-reactivity of several high-affinity 2C TCR variants that contained mutations only in the CDR3alpha loop. Two of the TCR lost their ability to cross-react with the reciprocal ligand (SIY-K(b)), whereas another TCR (m67) maintained reactivity with both ligands. Crystal structures of four of the TCRs in complex with QL9-L(d) showed that CDR1, CDR2, and CDR3beta conformations and docking orientations were remarkably similar. Although the CDR3alpha loop of TCR m67 conferred a 2000-fold higher affinity for SIY-K(b), the TCR maintained the same docking angle on QL9-L(d) as the 2C TCR. Thus, CDR3alpha dictated the affinity and level of cross-reactivity, yet it did so without affecting the conserved docking orientation.     

The three-dimensional structure of a T-cell antigen receptor V alpha V beta heterodimer reveals a novel arrangement of the V beta domain.

The crystal structure of a mouse T-cell antigen receptor (TCR) Fv fragment complexed to the Fab fragment of a specific anti-clonotypic antibody has been determined to 2.6 A resolution. The polypeptide backbone of the TCR V alpha domain is very similar to those of other crystallographically determined V alphas, whereas the V beta structure is so far unique among TCR V beta domains in that it displays a switch of the c" strand from the inner to the outer beta-sheet. The beta chain variable region of this TCR antigen-binding site is characterized by a rather elongated third complementarity-determining region (CDR3beta) that packs tightly against the CDR3 loop of the alpha chain, without leaving any intervening hydrophobic pocket. Thus, the conformation of the CDR loops with the highest potential diversity distinguishes the structure of this TCR antigen-binding site from those for which crystallographic data are available. On the basis of all these results, we infer that a significant conformational change of the CDR3beta loop found in our TCR is required for binding to its cognate peptide-MHC ligand.     

Thymocyte maturation: selection for in-frame TCR alpha-chain rearrangement is followed by selection for shorter TCR beta-chain complementarity-determining region 3

Thymocyte maturation consists of a number of stages, the goal of which is the production of functioning T cells that respond to foreign antigenic peptides using their clonotypic receptors. Selection of a productively rearranged TCR beta-chain is the first stage in the process and occurs at the double-negative to double-positive (DP) transition. Later maturation stages are based on changes in markers such as CD5, CD69, or IL-7R. A stage in which a-chains are selected has also been identified using beta-chain transgenic mice. Here we identify two additional selection stages in human thymocytes based on characteristics of the TCR. alpha selection is measured directly by identification of in-frame rearrangements and is associated with the appearance of CD3 on the DP thymocyte surface. The next stage has not yet been described and involves selection of thymocytes that express shorter TCR beta-chain complementarity-determining region 3 (CDR3). This stage is associated with the acquisition of high levels of CDR3 by DP cells and the transition to SP thymocytes. The extent of CDR3 length selection observed is a function of the TCR V and J genes. We propose that CDR3 length selection is based on recognition of the MHC. Thus, there exist limitations on the allowable length of that portion of the TCR most intimately in contact with MHC and peptide. This may be a physical representation of positive selection.     

Hapten addition to an MHC class I-binding peptide causes substantial adjustments of the TCR structure of the responding CD8(+) T cells

T cell responses against hapten-modified peptides play an important role in the pathogenesis of certain diseases, including contact dermatitis and allergy. However, the structural features of TCRs recognizing bulky, potentially mobile hapten groups remain poorly defined. To analyze the structural basis of TCR recognition of defined hapten-modified peptides, the immunodominant octapeptide derived from vesicular stomatitis virus nucleoprotein (VSV8) was modified with a trinitrophenyl (TNP) group at the primary TCR contact residues (position 4 or 6) and used for immunization of mice carrying either the TCR alpha- or beta-chain of a VSV8 (unmodified)/H-2K(b)-specific CTL clone as a transgene. Such mice allow independent analysis of one TCR chain by maintaining the other fixed. The TCR V gene usage of the responding T cell population was specifically altered depending upon the presence of the TNP group and its position on the peptide. The CDR3 sequences of the TNP-modified peptide-specific TCRs showed a preferential J region usage in both the CDR3alpha and beta loops, indicating that the J regions of both CDR3s are critical for recognition of TNP-modified peptides. In contrast to our previous observations showing the prime importance of CDR3beta residues encoded by D-segment or N-addition nucleotides for recognition of position 6 of unmodified VSV8, our studies of TNP-modified peptides demonstrate the importance of the Jbeta region, while the Jalpha region was crucial for recognizing both TNP-modified and unmodified peptides. These data suggest that different structural strategies are utilized by the CDR3alpha and beta loops to allow interaction with a haptenated peptide.     

Antigen Specificity of �æ� T Cells Depends Primarily on the Flanking Sequences of CDR3��

The structural basis that determines the specificity of γδ T cell receptor (TCR) recognition remains undefined. Our previous data show that the complementary determining region of human TCRδ (CDR3δ) is critical to ligand binding. Here we used linear and configurational approaches to examine the roles of V, N-D-N, or J regions in CDR3δ-mediated antigen recognition. Surprisingly, we found that the binding activities of CDR3δ from different γδ TCRs to their target tissues and ligands depend on the conserved flanking sequences (V and J) but not as much on the D region of CDR3δ fragment. We further defined the key residues in the V and J regions of CDR3δ fragments, including the cysteine residue in the V fragment and the leucine residue in the J fragment that determine their ligand binding specificity. Our results demonstrate that TCRδ primarily uses conserved flanking regions to bind ligands. This finding may provide an explanation for the limited number of γδ TCR ligands that have as yet been identified.Extensive studies suggest that γδ T cells play important roles in host defense against microbial infections, monitoring of tumorigenesis, immunoregulation, and development of autoimmunity (1–3). However, little is known about the structural basis of antigenic recognition by γδ T cell receptor (TCR)³ because of the limited identified specific ligands for γδ TCR and the lack of structural information revealing how γδ TCR might interact with such ligands.The crystallographic structure of a murine γδ TCR in complex with major histocompatibility complex class (MHC) Ib T22 (4, 5) showed that the CDR loops οf γδ TCR, predominantly germline-encoded residues of the complementary determining region of human TCRδ (CDR3δ), are in direct contact with T22, suggesting that the primary sequence of CDR3 in γδ TCR, especially CDR3δ, serves as a key determinant for the specificity of antigen recognition. Our recent finding that CDR3δ peptide mimics human γδ TCR binding to tumor cells and tissues is consistent with the role of CDR3δ in γδ TCR recognition (6).Based on this finding, we used synthesized CDR3δ peptide as a probe to screen putative protein ligands in tumor protein extracts by affinity chromatography analysis. With this novel strategy, we have successfully identified seven tumor-related epitopes, two hepatitis B virus (HBV) infection-related antigenic epitopes, and two self proteins including heat shock protein (HSP) 60 and human mutS homolog 2 (hMSH2) that are recognized by human γδ TCR (7). These results further support that the primary sequence of CDR3δ in γδ TCR determine the specificity of antigen binding.CDR3δ is composed of fragments derived from V, N-D-N, and J gene segments. The flanking sequences composed of V and J fragment is conserved while N-D-N region is diverse. The diversity of γδ TCRs is supposedly higher than that of TCRαβ due to the link of D gene fragment and the insertion of nucleotide acids (8). However, the number of identified antigenic ligands recognized by γδ TCR remains very limited. It has been demonstrated that γδ TCR recognizes some protein antigens and small phosphate or amine-containing compounds, including nonclassical MHC class I molecule T22 and T10 in mice (9), UL-16-binding protein (ULBP) (10) and mitochondrial F1-ATPase in humans (11). Nevertheless, important questions regarding γδ TCR recognition remain to be addressed. For example, given the seemingly high diversity of γδ TCR, why have only limited antigenic ligands been identified? What are the contributions of individual fragments of CDR3δ to antigen recognition? In αβ TCR, a single mutation in D gene fragment (12) abolishes its antigenic recognition, whereas the contribution of the different fragments in γδ TCR recognition remain unknown. Answers to these questions will shed important insights to antigen recognition of γδ T cells.In this study, we investigated the contribution of individual fragments of CDR3δ in antigen recognition. We mutated V, N-D-N, or J fragments of a Vδ2 TCR CDR3 sequence (OT3) in peptide and engineered γδ TCR. We found that the conserved flanking regions of CDR3δ play a critical role in antigenic binding to OEC cells/tissues or hMSH2 protein, a new ligand for γδTCR we found recently (7). Furthermore, we have identified the cysteine residue in V fragment and the leucine residue in J fragment as critical residues in the binding activity of γδ TCR. These results demonstrate that TCRδ chain uses the conserved flanking regions to recognize their antigens, suggesting that ligands for γδ ΤCR may also be conserved and limited in number.