CHARACTERIZATION OF PEPTIDE BINDING TO THE MURINE MHC CLASS-I H-2K(K)MOLECULE - SEQUENCING OF THE BOUND PEPTIDES AND DIRECT BINDING OF SYNTHETIC PEPTIDES TO ISOLATED CLASS-I MOLECULES

Peptides that are bound by the murine class I MHC molecule H-2K(k) hav e been isolated and sequenced. The initial step in the fractionation w as affinity column isolation of the peptide-class I complex from eithe r RDM-4 or x5563 tumor cell lines. Acid denaturation of the complex fo llowed by HPLC fractionation of the peptides allowed us to sequence in dividual peptides, as well as pools of peptides. To date, a total of 1 0 sequences have been characterized, and all were 8 mers. The sequence s were variable except for glutamic acid in the second position (P2) a nd isoleucine in the eighth (P8), which were highly conserved. To furt her study peptide binding to H-2K(k), a competitive binding assay cons isting of the immobilized histocompatibility protein and a biotinylate d self-peptide for signal generation was developed. A complete set of single-alanine variants for this one self-peptide was tested in the as say, demonstrating that substitution at P2 and P8 markedly decreased t he affinity for the class I molecule; alanine at position 3 had an int ermediate effect on binding. A comparison of the identified self-pepti des for binding to H-2K(k) showed that they differed in affinity by mo re than one order of magnitude. influenza virus nucleoprotein peptide, SDY EGR LI, associated with the plate-bound class I molecule, and the resulting MHC-peptide complex could trigger TNF release by influenza- primed CTLs. This result demonstrated the functional activity of the p latebound H-2K(k)-peptide complex.