The importance of pairwise interactions between peptide residues in the delineation of TCR specificity

A minimal, nonamer epitope (TEMEKEGKI) from the reverse transcriptase protein of HIV-1, restricted by H-2Kk, was identified and the function of individual residues determined. Besides classical anchor residues at positions 2 and 9, methionine at position 3 was identified as an important MHC anchor and improved binding of a different (malarial) nonamer epitope to H-2Kk, albeit while also abolishing CTL recognition. Lysine at position 5 was replaceable by alanine for CTL raised against wild-type peptide but abolished recognition for CTL raised against the variant 5ALA peptide, indicating a unidirectional cross-reactivity. Interestingly, one CTL line raised against the 5ALA substituted peptide was permissive for a double substitution at positions 5 and 6, in which lysine was permissive at position 5 only if the adjacent glutamic acid was replaced by alanine. Extensive analysis revealed three distinct patterns of responses with peptides doubly substituted in this region: recognition of both single substitutions but not the double substitution, recognition of only one single substitution but also the double substitution, or recognition of both single substitutions and the double substitution. A second complementary substitution can therefore restore function lost through a first substitution. Thus, no residue acts independently of its neighbors, and pairs of substitutions may give results not predictable from the effects of each taken singly. This finding may have bearing on viral infections (such as HIV), in which the accumulation of two mutations in the epitope may lead to the reengagement of memory CTL previously silenced by the initial mutation.