Elongated peptides, not the predicted nonapeptide stimulate a major histocompatibility complex class I‐restricted cytotoxic T lymphocyte clone with specificity for a bacterial heat shock protein

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Abstract
The peptides recognized by an H‐2Db‐restricted CD8 cytotoxic T lymphocyte (CTL) clone which is specific for the 60‐kDa mycobacterial heat shock protein (hsp) and cross‐reacts with stressed host cells were characterized. None of the nonapeptides from hsp60 conforming to the H‐2Db binding motif were able to sensitize target cells for lysis by this CTL clone. Sequence analysis of the stimulatory fraction from a trypsin digest of hsp60, together with synthetic peptide studies, defined a cluster of overlapping epitopes. Carboxy‐terminal extension by at least one amino acid of the nonamer predicted to bind best to H‐2Db was essential for CTL recognition. Two such elongated peptides, a 10‐mer and a 12‐mer stimulated the clone at similarly low concentrations in the 100 pM range. We assume that these two peptides comply best with the natural epitope. In contrast, the 11‐mer was inactive. The stimulatory 10‐mer bound to H‐2Db with an efficacy similar to that of the nonapeptide corresponding to the H‐2Db motif, as revealed by peptide induced major histocompatibility complex (MHC) surface expression on RMA‐S cells and competitive blocking of epitope recognition by the nonamer. Binding of these carboxy‐terminally extended peptides to the MHC groove can be explained by anchoring through the amino acid residue Asn in position 5 of the peptide and by intrusion of the hydrophobic carboxy‐terminal Ala (10‐mer) or Leu (12‐mer), but not Gly (11‐mer), into the hydrophobic pocket of the H‐2Db cleft. Because the carboxy‐terminal part is thus larger than predicted this region of the peptide may arch up from the binding groove. We assume that recognition of steric components of the MHC/peptide complex broaden the range of epitope specificity for a single T cell receptor. This flexibility not only promotes recognition of several overlapping peptides from a single antigen, but may also increase the chance of cross‐reaction with similar peptides from unrelated proteins, including autoantigens. Consistent with this latter assumption, the T cell clone cross‐recognizes mycobacterial hsp60 and stressed host cells.