A MAGE-C2 antigenic peptide processed by the immunoproteasome is recognized by cytolytic T cells isolated from a melanoma patient after successful immunotherapy.

We have pursued our analysis of a melanoma patient who showed almost complete tumor regression following vaccination with MAGE-A1 and MAGE-A3 antigens. We previously described high frequencies of tumor-specific CTL precursors in blood samples collected after but also before vaccination. A set of CTL clones were derived that recognized antigens different from those of the vaccine. Two of these antigens were peptides encoded by another MAGE gene, MAGE-C2. Here we describe the antigen recognized by another tumor-specific CTL clone. It proved to be a third antigenic peptide encoded by gene MAGE-C2, ASSTLYLVF. It is presented by HLA-B57 molecules and proteasome-dependent. Tumor cells exposed to interferon-gamma (IFN-γ) were better recognized by the anti-MAGE-C2(42-50) CTL clone. This mainly resulted from a better processing of the peptide by the immunoproteasome as compared to the standard proteasome. Mass spectrometric analyses showed that the latter destroyed the antigenic peptide by cleaving between two internal hydrophobic residues. Despite its higher "chymotryptic-like" (posthydrophobic) activity, the immunoproteasome did not cleave at this position, in line with the suggestion that hydrophobic residues immediately downstream from a cleavage site impair cleavage by the immunoproteasome. We previously reported that one of the other MAGE-C2 peptides recognized by CTL from this patient was also better processed by the immunoproteasome. Together, these results support the notion that the tumor regression of this patient was mediated by an antitumor response shaped by IFN-γ and dominated by CTL directed against peptides that are better produced by the immunoproteasome, such as the MAGE-C2 peptides.