A MAGE-A1 HLA-A*0201 epitope identified by mass spectrometry

Peptides presented by HLA-A*0201 molecules on the surface of the human brea st carcinoma cell line KS24.22 after IFN-gamma induction were analyzed by t he "Predict-Calibrate-Detect" approach, which combines epitope prediction a nd high-performance liquid chromatography mass spectrometry. One of the pre dicted epitopes, MAGE-A1(278-286) (KVLEYVIKV), mas found to be presented by HLA-A*0201, with an estimated copy number of 18 molecules/cell. HLA-A*0201 transgenic mice (HHD mice) were used to generate CTL lines that stained po sitive with an HLA-A*0201 tetramer folded around the KVLEYVIKV peptide and killed peptide-loaded mouse target cells expressing HLA-A*0201. IFN-gamma - treated or -nontreated HLA-A*0201 expressing HeLa cells transiently transfe cted with a plasmid expressing the IMAGE-AI gene stimulated in vitro cytoki ne production by the CTL lines, Moreover, IFN-gamma -treated KS24.22 cells, but not IFN-gamma -treated HLA-A*0201(+) MAGE-A1(-) cells or IFN-gamma -tr eated HLA-A*0201(-) MAGE-A1(+) cells, were killed by these CTLs, Thus, the combination of HLA epitope prediction, peptide analysis, and immunological methods is a powerful approach for the identification of tumor-associated e pitopes.