Tumors express peptide antigens capable of being recognized by tumor-specific cytotoxic T lymphocytes (CTL)

SummaryT lymphocytes recognize antigens consisting ofpeptides presented by class I and II major histocompatibility complex (MHC) molecules. The peptides identified so far have been predictable from the amino acid sequences of proteins. We have identified the natural peptide target of a CTL clone that recognizes the tyrosinase gene product on melanoma cells. The peptide results from posttranslational conversion ofasparagine to aspartic acid. This change is of central importance for peptide recognition by melanoma-specific T cells, but has no impact on peptide binding to the MHC molecule. This posttranslational modification has not been previously described for any MHC-associated peptide and represents the first demonstration of posttranslational modification of a naturally processed class I-associated peptide. This observation is relevant to the identification and prediction of potential peptide antigens. The most likely mechanism for production of this peptide leads to the suggestion that antigenic peptides can be derived from proteins that are translated into the endoplasmic reticulum.C lass I molecules of MHC bind to peptides derived from intracellular pathogens or from proteins expressed in tumor cells, and present them on the cell surface to the host immune system (1-3). Identification of the specific peptides that constitute T cell epitopes has been difficult without prior knowledge of the source protein. However, peptides recognized by human melanoma--specific T cells have recently been identified from five proteins using two alternative strategies. One approach has been to generate genomic or cDNA libraries from tumor cells followed by transfection of progressively smaller subsets of these molecular clones into cells that express the appropriate MHC molecule, but not the tumor-specific epitope (4--14). Molecular clones that encode T cell epitopes are identified by their ability to reconstitute tumor-specific T cell recognition of the transfected cells. The exact T cell epitope is then identified by a combination of molecular subcloningThe contributions of the first two authors were equivalent and their order should be considered arbitrary. and the use of synthetic peptides based on the predicted amino acid sequence. This approach led to the identification of antigens encoded by genes whose expression is specific for tumors, such as MAGE and of other antigens related to melanocyte differentiation such as tyrosinase (4, 13). In the second approach, naturally occurring peptides associated with MHC molecules on the tumor cells are directly extracted, fractionated by HPLC, and used to reconstitute recognition by melanoma-specific CTL of a nonmelanoma cell expressing appropriate MHC molecules (15). The peptide epitope within a reconstituting peptide fraction is identified and sequenced by tandem mass spectrometry (16,17). Using this approach, a peptide, YLEPG-PVTA, from the protein Pmel-17/gp100, was identified as an epitope for HLA-A2.1-restricted, melanoma-specific CTL from multiple individuals (18).

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Polymorphism of MHC class Ia molecules severely constrains vaccine development against intracellular pathogens. Antigen presentation by MHC class Ib molecules, which are generally conserved between different individuals, may circumvent this obstacle. Herein, we use tandem mass spectrometry to identify a Listeria monocytogenes pentapeptide antigen that is presented to T lymphocytes by the H2-M3 MHC class Ib molecule. The peptide contains N-formyl methionine at the N terminus and exclusively hydrophobic amino acids. Mice of the H-2 d, H-2 b,and H-2 k haplotypes respond to this peptide upon infection with Listeria monocytogenes. Identification of antigens presented by MHC class Ib molecules is feasible and may provide opportunities for relatively unrestricted vaccine development.