RECOGNITION BY HLA-A2-RESTRICTED CYTOTOXIC T-LYMPHOCYTES OF ENDOGENOUSLY GENERATED AND EXOGENOUSLY PROVIDED SYNTHETIC PEPTIDE ANALOGS OF THE INFLUENZA-A VIRUS MATRIX PROTEIN

Experiments were carried out to determine whether complexes between MH C class I molecules and synthetic peptides are representative of those formed under more physiologically relevant conditions, with peptides derived intracellularly from processed antigens. Lysis of cells sensit ized with exogenously provided and endogenously generated peptide anal ogues of the optimal nonameric peptide 58-66 (GILGFVFTL; derived from the influenza virus matrix protein) was compared. Endogenous loading w as accomplished by expressing minigene DNA coding for alanine-substitu ted analogues of peptide 58-66 in HLA-A2-positive cells. Susceptibilit y to lysis by HLA-A2-restricted, peptide-specific cytotoxic lymphocyte s was compared with lysis of cells sensitized with the same synthetic peptides. Although results were quite comparable, differences were obs erved. The endogenously presented analogues 58-66L60A, G61A, T65A, and L66A were recognized more efficiently than the corresponding exogenou sly presented analogues. This difference in recognition was most strik ing for peptide 58-66G61A. These results indicate the need for caution in using synthetic peptides in defining peptide binding motifs. Addit ional experiments with endogenously expressed analogues of 58-66 with substitutions other than alanine were carried out to define the intera ction between this peptide and HLA-A2. Results are compatible with the interpretation that residues 58, 59, and 60 interact with pockets A, B, and D, respectively, in the HLA-A2 binding groove and that these in teractions contribute to peptide binding.