Abstract A059: Breaking tolerance in autochthonous tumors

Abstract Persistent antigen exposure drives T cell dysfunction, promoting immune evasion and cancer progression. Although immune therapies have shown efficacy, many patients fail to respond, reflecting limitations observed in traditional transplanted tumor mouse models. We established an autochthonous tumor model (TTC mice) in which the SV40 large T antigen (Tag) oncogene, fused to firefly luciferase for tumor visualization, is induced in tyrosinase-expressing cells via doxycycline (dox) from birth. Within four months of dox exposure, TTC mice develop Tag-specific tolerance, indicating dysfunctional T cell activity, and from six months on, they typically form autochthonous tumors. Tolerance was broken by a sequential regimen of dox withdrawal to reduce antigen exposure, whole-body irradiation (5 Gray), and vaccination with Tag-expressing tumor cells. This approach restored the function of tumor-reactive CD8+ T cells, which otherwise displayed exhausted or progenitor-exhausted phenotypes. When dysfunctional CD8+ T cells were transferred into Rag2-/- mice that were subsequently immunized with Tag-expressing tumor cells, they expanded but failed to reject tumors. TCR sequencing revealed Tag-specific T cells that were undetectable by peptide-MHC tetramer staining due to TCR downregulation, e.g., reduced expression of TCR complex genes (Cd3, Zap70, Cd8) in CD8+ T cells. Depletion of CD4+ or CD25+ T cells from the spleen of tumor-bearing TTC mice, prior to transfer, restored Tag-specific CD8+ T cell function and enabled tumor rejection, suggesting that regulatory T cells (Tregs) suppress tumor-reactive CD8+ T cells. Single-cell sequencing performed on dysfunctional TTC T cells, compared with T cells from the CD4-depleted cohort, revealed upregulation of inhibitory receptor Lag-3, on both CD8+ T cells and Tregs, supporting the notion that Tregs actively inhibit CD8+ T cell function. Currently, we are analyzing whether Lag-3 blockade can induce tumor rejection in combination with vaccination of Tag+ cells. Together, these findings indicate that tumor-reactive CD8+ T cells are maintained in a dysfunctional state by TCR downregulation and by Tregs, whose removal restores tumor immunity. Citation Format: Lydia Dyck, Kathleen Anders, Mai Huong To, Miha Milek, Elmehdi Belbaraka, Maria Teresa. Norcia, Benedikt Obermayer, Dieter Beule, Inmaculada Martinez-Reyes, Thomas Blankenstein. Breaking tolerance in autochthonous tumors [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A059.