USE OF GLOBAL AMINO-ACID REPLACEMENTS TO DEFINE THE REQUIREMENTS FOR MHC BINDING AND T-CELL RECOGNITION OF MOTH CYTOCHROME-C-(93-103)

Substitution with all naturally occurring L-amino acids at each of 11 residues of the IE(k)-restricted month cytochrome c (93-103) epitope h as allowed us to analyze the requirements for MHC binding and T cell r ecognition to a level of definition not previously possible. Substitut ions at only three positions systematically affect MHC binding and thr ee others appear to be the major TCR contacts. Interestingly, changing residues involved in MHC binding can ablate T cell recognition withou t altering MHC association. Additionally, residue identity at two posi tions that do not appear critical for MHC binding, nor to be involved in specific T cell contact, nonetheless dramatically affect T cell res ponses. This suggests that peptides differing only slightly in sequenc e can have significantly altered conformations within the class II MHC binding groove. We have also developed a simple scoring program that uses the binding data to quantitate how well a given peptide fits the MCC motif. All strongly immunogenic IE(k)-restricted epitopes score hi ghly (greater than or equal to 0.70, where 1.0 is perfect concordance) , and only 3% of all potential nonameric peptides in the two main prot ein sequence databases have scores greater than 0.70. This indicates t hat the global amino acid replacement approach using a single peptide is an efficient means of deriving binding motifs for a given class II MHC molecule, and should aid in the identification of novel T cell epi topes.