Naturally processed and concealed HLA-A2.1-restricted epitopes from tumor-associated antigen tyrosinase-related protein-2

In this study, a computer-assisted reverse immunology approach was utilized in order to identify potentially antigenic peptides derived from the differentiation antigen TRP-2, a melanosomal protein frequently expressed in melanoma. Among the seven peptides complying with HLA-A2.1-binding motifs, two induced specific CD8⁺ cytotoxic T lymphocytes. HLA-A2.1⁺ melanoma cells expressing TRP-2 were lysed by clones specific for TRP-2<INF>360–368</INF> (TLDSQVMSL) peptide, thus identifying it as a naturally processed epitope. Other T-cell clones directed against TRP-2<INF>476–484</INF> (VMGTLVALV) were unable to lyse HLA-matched TRP-2⁺ cell lines. The role of intracellular proteolytic processing in the generation of this epitope was investigated by transfecting mini-genes encoding the TRP-2<INF>476–484</INF> peptide alone or carrying N- or C-terminal extensions. Specific T-cell clones recognized target cells expressing the cytotoxic T-lymphocyte (CTL)-defined epitope or its C-terminally extended precursor, but failed to recognize cells expressing the N-terminally extended TRP-2<INF>476–484</INF> peptide, suggesting the presence of a negative processing signal (NPS). Regarding C-terminus-flanking regions, mutational analysis indicates that the GLY485 residue plays a key role in the processing of the TRP-2<INF>476–484</INF> epitope. Interestingly, proteasome inhibitors preventing the generation of the MART-1/Melan-A<INF>27–35</INF> immunodominant melanoma tumor-associated antigen (TAA) promoted detectable presentation of TRP-2<INF>476–484</INF> epitope in HLA-A2.1⁺ and TRP-2⁺ tumor lines, as witnessed by cytokine release by specific T-cell clones. Int. J. Cancer 87:241–246, 2000. © 2000 Wiley-Liss, Inc.