DIRECT ANALYSIS OF PEPTIDE BINDING TO CELL-ASSOCIATED MHC CLASS-I MOLECULES

Exogenously added synthetic peptides can mimic endogenously produced antigenic peptides recognized on target cells by MHC class I-restricted cytolytic T lymphocytes. While it is assumed that exogenous peptides associate with class I molecules on the target cell surface, direct binding of peptides to cell-associated class I molecules has been difficult to demonstrate. Using a newly developed binding assay based on photoaffinity labeling, we have investigated the interaction of two antigenic peptides, known to be recognized in the context of H-2K(d) or H-2D(b), respectively, with 20 distinct class I alleles on living cells. None of the class I alleles tested, with the exception of H-2K(d) or H-2D(b), bound either of the peptides, thus demonstrating the exquisite specificity of peptide binding to class I molecules. Moreover, peptide binding to cell-associated H-2K(d) was drastically reduced when metabolic energy, de novo protein synthesis or protein egress from the endoplasmic reticulum was inhibited. It is thus likely that exogenously added peptides do not associate with the bulk of class I molecules expressed at the cell surface, but rather bind to short-lived molecules devoid of endogenous peptides.