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 maj or histocompatibility complex class I-restricted cytotoxic T lymphocyt e (CTL) response to the protein ovalbumin (OVA). Despite skewing towar ds the expression of Vbeta5.2+ TCR by OVA-specific CTL from C57BL/6 mi ce, we found a relatively high degree of diversity in V(D)J usage in b oth TCR alpha- and beta-chains. Closer examination showed that the maj ority of these sequences encoded negatively and positively charged res idues at their respective TCR alpha- and beta-chain VJ or VDJ junction s. 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 OVA257- 264 (SIINFEKL), contains two exposed charged side chains which can con tact the TCR. These are the negatively charged glutamic acid at determ inant position 6 (P6) and the positively charged lysine at P7. To exam ine whether the TCR alpha-chain makes contact with P7 lysine, we estab lished a single chain TCR transgenic C57BL/6 mouse line where all T ce lls express a TCR beta-chain derived from the Vbeta5.2+ clone B3. OVA- specific T cells derived from in vivo primed transgenic mice preferent ially expressed TCR alpha-chains that also contained negatively charge d junctional residues despite some further variation in Va and Jalpha 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, s ugesting that the TCR alpha-chain from the T cell clone B3 can determi ne the specificity for this residue. Consequently, these results revea l the existence of conserved residues in the CDR3 of TCR alpha- and be ta-chains specific for OVA257-264 and identify their possible orientat ion over the peptide-class I complex.