Vormehr and Reinhard et al. used a matrix of 2474 peptides representing all point mutations in CT26 murine colon carcinoma to screen for neoantigen-specific CD8+ T cells following treatment with a TLR7 agonist, MHCII neoepitope vaccination, or anti-PD-L1. From the neoepitopes, only mutated Smc3-specific CD8+ T cells were detected, which failed to lyse CT26 cells or control tumor growth, and engineered Smc3-TCR T cells did not recognize CT26 cells. Analysis of MHCI-bound peptides revealed variable and low expression of Smc3-MHCI complexes on CT26 cells, indicating that the Smc3 neoantigen may elicit specific yet non-functional CD8+ T cells.
Cancer-associated mutations, mostly single nucleotide variations, can act as neoepitopes and prime targets for effective anti-cancer T-cell immunity. T cells recognizing cancer mutations are critical for the clinical activity of immune checkpoint blockade (ICB) and they are potent vaccine antigens. High frequencies of mutation-specific T cells are rarely spontaneously induced. Hence, therapies that broaden the tumor specific T-cell response are of interest. Here, we analyzed neoepitope-specific CD8+ T-cell responses mounted either spontaneously or after immunotherapy regimens, which induce local tumor inflammation and cell death, in mice bearing tumors of the widely used colon carcinoma cell line CT26. A comprehensive immune reactivity screening of 2474 peptides covering 628 transcribed CT26 point mutations was conducted. All tested treatment regimens were found to induce a single significant CD8+ T-cell response against a non-synonymous D733A point mutation in the Smc3 gene. Surprisingly, even though Smc3 D733A turned out to be the immune-dominant neoepitope in CT26 tumor bearing mice, neither T cells specific for this neoepitope nor their T cell receptors (TCRs) were able to recognize or lyse tumor cells. Moreover, vaccination with the D733A neoepitope did not result in anti-tumoral activity despite induction of specific T cells. This is to our knowledge the first report that neoepitope specific CD8+ T cells primed by tumor-released antigen exposure in vivo can be functionally irrelevant.