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 V beta 5.2+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 alpha- and beta-chains. Closer examination showed that the majority of these sequences encoded negatively and positively charged residues at their respective TCR alpha- and beta-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 Kb-peptide complexes predict that the major determinant from OVA, peptide OVA257-264 (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 alpha-chain makes contact with P7 lysine, we established a single chain TCR transgenic C57BL/6 mouse line where all T cells express a TCR beta-chain derived from the V beta 5.2+ clone B3. OVA-specific T cells derived from in vivo primed transgenic mice preferentially expressed TCR alpha-chains that also contained negatively charged junctional residues despite some further variation in V alpha and J alpha sequences. Stimulation of naive TCR beta-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 OVA257-264 determinant, suggesting that the TCR alpha-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 alpha- and beta-chains specific for OVA257-264 and identify their possible orientation over the peptide-class I complex.

Ovalbumin-specific, Kb-restricted T cells recognize the minimal fully active synthetic peptide ovalbumin (OVA)257-264. This sequence coincides with the eight residue, allele-specific peptide binding motif previously predicted from direct sequencing of naturally occurring Kb-associated peptides (Falk, K., Rotzscke, O., Stevanovic, S., Jung, G., and Rammensee, H.-G., Nature 351:290, 1990). T cell recognition of a panel of analogs with single residue substitutions between the two putative Kb anchor residues at OVA261 and OVA264 suggested that at least one residue, Glu at position 262, is involved in TCR interaction. OVA-specific cytotoxic T lymphocytes (CTL) derived from TCR beta-chain transgenic mice, where the beta-chain originates from an OVA-specific, Kb-restricted CTL B3, showed that differences in TCR alpha-chain pairing determined the specificity for OVA residue 262. These data support the notion that residue 262 of the OVA T cell determinant, corresponding to position 6 within the Kb-binding motif, represents a contact site for TCR. This residue interacts directly with the TCR alpha-chain or with a site on the TCR beta-chain whose conformation is affected by TCR alpha-chain pairing.

Summary' Many antigen-specific T cell responses show profound V-region biases in one or both chains of the TCR aP heterodimer. We have examined how changes in residues within an MHC-bound peptide can influence V-region selection. Single-chain TCR transgenic mice were derived using a p-chain specific for the K'^-restricted peptide from ovalbumin, OVA257_264-Transgenic T cells were stimulated in vitro using OVA257_2(,4 or a single residue variant having a lysine to aspartic acid substitution at determinant position 7 (7D). Recent crystallographic analyses have shown that this variant residue is involved in direct contact with the TCR. Sequence analysis of the T cell populations showed that the majority OVA257_264-specific T cells used ValO-positive TCR while the majority of the 7D-specific TCR expressed the Va4 element. In both peptide responses we found that some cell lines appeared to be made up of more than one clone expressing the same Va sequence but having limited variation at the V-J junction. These results show that the TCR cotitact residues within the target peptide can influence V-region bias and suggest that the first and second hypervariable regions of the TCR are directly or indirectly involved in determining peptide specificity.