ROLE OF NON-ANCHOR RESIDUES OF D-B-RESTRICTED PEPTIDES IN CLASS-I BINDING AND TCR TRIGGERING

To understand better, the role of non-anchor residues of class I restr icted T cell epitopes in class I binding and TCR stimulation, a panel of peptides was synthesized in which each of the non-anchor positions of the D-b-restricted influenza peptide, ASNENMETM, was changed to eac h of the 20 natural amino acids (AAs). The relative affinity of all th e peptides for D-b was determined and their ability to stimulate anti- ASNENMETM cytotoxic T cell hybridomas was also assessed. The results i llustrated that for D-b binding, the AAs with the most solvent exposur e had the smallest effect on binding. Changes at other positions affec ted binding to different degrees. Results for the recognition by the T cell hybridomas indicated that a peptide-MHC complex represents a mul titude of epitopes, as each hybridoma recognized a different subset of peptides. Most changes in the highly solvent-exposed residues negativ ely affected recognition by all hybridomas while changes in other posi tions affected each hybridoma differently, independent of the directio n of the side chain of the AA at that position. Furthermore, the use o f saturating concentrations of low and high binding peptides showed th at, as long as the class I-peptide complex is formed, the T-cell recep tor does not differentiate between high and low binding peptides. This indicates that, although the stability of the class I-peptide complex is highly dependent on peptide affinity, the class I MHC conformation induced by low affinity peptides does not necessarily differ signific antly from that induced by high affinity peptides. The results of pept ide-class I recognition by one ASNENMETM-specific hybridoma was used t o construct a peptide that differed from ASNENMETM at four of the nine residues, yet stimulated the hybridoma to a level comparable to ASNEN METM. In addition, peptides bearing the canonical D-b-binding motif bu t unable to bind to the class I molecule with high affinity could be m ade to bind D-b, by changing unfavorable AAs to favorable ones at appr opriate positions. The extended motif determined was used to identify more accurately the peptides derived from Coxsakie b3 virus that would bind D-b. It was also shown that some of the canonical characteristic s of the peptide motif could be obviated and still obtain high affinit y binding, provided optimal AAs were present at secondary anchor posit ions. (C) 1997 Elsevier Science Ltd.