MHC molecules present protein-derived peptides to T lymphocytes. By developing TiO2-based microcentrifugation columns, we identified the first phosphorylated MHC class I ligands from tumor tissue (renal cell carcinoma) and, by comparison to healthy renal tissue, found one Brf1-derived ligand as potentially tumor-associated. We further discovered the first natural phosphorylated MHC class II ligands. They revealed several novel phosphorylation sites of significant transmembrane receptors, such as Frizzled 6, CXCR4 and CD20.

Viral peptides are presented by HLA class I on infected cells to activate CD8+ T cells. Several immunogenic peptides have been identified indirectly by epitope prediction and screening of T cell responses to poxviral vectors, including modified vaccinia virus Ankara (MVA) currently being tested as recombinant or smallpox vaccines. However, for the development of optimal vaccination and immunomonitoring strategies, it is essential to characterize the actual viral HLA ligand repertoire of infected cells. We used an innovative approach to identify naturally processed MVA HLA ligands by differential HPLC-coupled mass spectrometry. We describe 12 viral peptides presented by HLA-A*0201 and 3 by HLA-B*0702. All HLA-A*0201 ligands participated in the memory response of MVA-immune donors, and several were immunogenic in Dryvax vaccinees. Eight epitopes were novel. Viral HLA ligand presentation and viral protein abundance did not correlate. All ligands were expressed early during the viral life cycle, and a pool of three of these mediated stronger protection against a lethal challenge in mice as compared with late epitopes. This highlights the reliability of the comparative mass spectrometry-based technique to identify relevant viral CD8+ T cell epitopes for optimizing the monitoring of protective immune responses and the development of effective peptide-based vaccines.