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 diffe rentiation antigen TRP-2, a melanosomal protein frequently expressed in mel anoma, Among the seven peptides complying with HLA-A2.1-binding motifs, two induced specific CD8(+) cytotoxic T lymphocytes, HLA-A2.1(+) melanoma cell s expressing TRP-1 were lysed by clones specific for TRP-2(360-368) (TLDSQV MSL) peptide, thus identifying it as a naturally processed epitope. Other T -cell clones directed against TRP-2(476-484) (VMGTLVALV) were unable to lys e HLA-matched TRP-2(+) cell lines. The role of intracellular proteolytic pr ocessing in the generation of this epitope was investigated by transfecting minigenes encoding the TRP-2(476-484) peptide alone or carrying N- or C-te rminal extensions, Specific T-cell clones recognized target cells expressin g the cytotoxic T-lymphocyte (CTL)-defined epitope or its C-terminally exte nded precursor, but failed to recognize cells expressing the N-terminally e xtended TRP-2(476-484) peptide, suggesting the presence of a negative proce ssing signal (NPS). Regarding C-terminus-flanking regions, mutational analy sis indicates that the GLY485 residue plays a key role in the processing of the TRP-2(476-484) epitope. Interestingly, proteasome inhibitors preventin g the generation of the MART-I/Melan-A(27-35) immunodominant melanoma tumor -associated antigen (TAA) promoted detectable presentation of TRP-2(476-484 ) 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. (C) 2 000 Wiley-Liss, Inc.