Abstract 6615: Peroxynitrite mediates immune escape of tumor cells from cytotoxic T cells in situ
Authors: Tcyganov Evgenii N. , Gabrilovich Dmitry I. 
Published: September 2020

Abstract
Despite improved ability to generate strong tumor-specific T cell responses, clinical benefits remain relatively moderate. Only a small proportion of predicted neoepitopes was shown to induce specific cytotoxic T cells (CTLs) or serve as an efficient target for T cell-based therapy. We suggest here that low efficacy of CTLs could be at least partially explained by the fact that tumor microenvironment (TME) modulates antigen presentation and epitope profile expressed by tumor. As a result, tumor escapes recognition by CTLs. TME is enriched in peroxynitrite (PNT), a potent oxidant. It is mostly produced by infiltrating myeloid cells, but also by some tumors (as melanoma). We hypothesized that PNT is able to affect antigen presentation on tumor cells and facilitates tumor immune escape.
To check this hypothesis, we treated murine EG7 tumor cells with PNT, isolated MHC I peptides and compared their expression on treated versus non-treated cells using SILAC mass spectrometry. We found that PNT treatment significantly decreased expression of a portion of MHC I peptides presented by tumor cells, whereas other peptides remained relatively intact. We found that PNT-sensitive and resistant peptides demonstrated similar binding capacity to MHC I. However, the off-rate of PNT-sensitive peptides was significantly higher. PNT strongly affected proteasomal activity of tumor cells suggesting the mechanism for under-representation of MHC I peptides with high off-rate level. To investigate the biological role of this effect, we generated CTLs specific to a pool of PNT-sensitive or resistant peptides. In vitro we observed that both types of CTLs effectively killed tumor cells. Pre-treatment of tumor cells with PNT significantly reduced killing only by CTLs specific to PNT-sensitive peptides. By ELISPOT we showed that most of antitumor-specific CTL responses in mice with different tumors were induced against PNT-resistant, but not PNT-sensitive species. Moreover, the adoptive transfer of CTLs specific to PNT-resistant peptides combined with immune checkpoint inhibition (ICI) was able to induce tumor rejection in 70% of mice, whereas similar setting for CTLs specific to PNT-sensitive peptides didn't affect tumor growth. PNT inhibition combined with ICI significantly delayed the tumor growth in mice. In addition, we showed that PNT had similar effect on MHC I peptide profile for human melanoma cells. Finally, we measured nitrotyrosine (NT) level (marker of PNT presence) in tumor tissues resected from melanoma patients before the start of ICI therapy. We retrospectively correlated NT levels with patient ability to respond to ICI therapy. We found that high NT level was a predictor of poor prognosis and patient un-responsiveness to ICI.
Overall, our study demonstrates PNT ability to affect antigen presentation in tumor site. In addition, we suggest PNT as a promising target for ICI-combined therapy as well as a biomarker for resistance to ICI.
Citation Format: Evgenii N. Tcyganov, Dmitry I. Gabrilovich. Peroxynitrite mediates immune escape of tumor cells from cytotoxic T cells in situ [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6615.