Peroxynitrite affects MHC I peptide repertoire presented on tumor cells and impedes the efficacy of antitumor cytotoxic T-lymphocytes

Abstract

Abstract Despite the advancements in enhancing antigen-specific T cell responses in cancer their clinical benefit remains limited. Recent studies demonstrated that tumor microenvironment factors are able to dampen in vivo anti-tumor T cell response. Previously, we found that myeloid cells produce large amounts of peroxynitrite (PNT) in the tumor. The pretreatment with PNT blocked binding of exogeneous peptides by tumor MHC I and decreased tumor killing by cytotoxic T lymphocytes (CTL). We hypothesized that PNT affects tumor MHC I peptide repertoire, resulting in inability of CTL to recognize and eliminate tumor cells. To test this hypothesis, we treated tumor cells with PNT and analyzed MHC I peptides by LC-MS/MS. We found that PNT dramatically affected the repertoire of tumor MHC I peptides. We identified two groups of peptides: “stable” peptides that were retained on MHC class I after PNT treatment and “non-stable” peptides that were significantly reduced after treatment. The characterization of peptides had revealed similar binding capacity for both “stable” and “non-stable” peptides. However, the dissociation rate of “non-stable” peptides from peptide-MHC I complexes was significantly higher. We found that PNT treatment impaired proteasomal activity in tumor cells. This may skew MHC I repertoire towards more “stable” peptides. To examine biological relevance of PNT, we generated CTLs specific to representative “stable” and “non-stable” peptides. We confirmed that PNT pretreatment of tumor cells decreased the killing efficacy of CTLs specific to “non-stable”, but not to “stable” peptides. Overall, our findings demonstrate a novel PNT-mediated mechanism of tumor cell resistance to CTLs that could be targeted for cancer immunotherapy.