ROLE OF IL-10 PRODUCING EOMES+ TR1-LIKE CELLS IN MELANOMA

The regulatory mechanisms that prevent the adaptive immune system’s ability to eradicate tumors are crucial for the development of effective cancer immunotherapies. In particular, the immunosuppressive functions of FOXP3+ T regulatory cells (Tregs) and EOMES+ type 1 regulatory T (Tr1) cells, which produce the anti-inflammatory cytokine IL-10, have been identified as key modulators of anti-tumor immunity. IL-10 plays a dual role in cancer, functioning as a checkpoint receptor agonist during cytotoxic T cell priming and potentially enhancing established CD8+ T cell responses. We demonstrated that EOMES+ Tr1-like cells are not only enriched and clonally expanded in human tumors, but they also suppress CD8+ T cell activity. Elevated levels of an EOMES+ Tr1-like gene signature correlate with poor patient survival and predict responses to anti-PD1 immunotherapy in melanoma patients. Moreover, in vitro studies have demonstrated that human EOMES+ Tr1-like cells exhibit a reduced capacity to suppress antigen- experienced CD8+ T cells in the presence of anti-PD1 antibodies. In this study, we employed the C57Bl/6 mouse model of B16-OVA melanoma to investigate the molecular mechanisms by which EOMES+ Tr1-like cells modulate anti-tumor T cell responses in vivo. Our findings reveal that EOMES+ Tr1 cells producing IL-10 are significantly enriched in tumors and contribute to promote tumor growth by suppressing cytotoxic T cell activity. Thus, adoptive transfer experiments with IL-10-deficient FOXP3-CD4+ T cells confirmed the critical role 7 of Tr1-derived IL-10 in promoting tumor progression and impairing anti-tumor immunity. Furthermore, therapeutic treatment with anti-PD-L1 antibodies reduced T cell IL-10 production and inhibited tumor growth, a phenomenon that was absent in the absence of Tr1-derived IL-10. Using MHC tetramer staining, we showed that a subset of tumor-infiltrating EOMES+ Tr1-like cells were specific for the neoantigen Ovalbumin (OVA). Single-cell RNA sequencing of purified OVA- specific CD4+ T cells revealed two major clusters: FOXP3+ Tregs and FOXP3- CD4+T-cells containing a Tr1-like population expressing granzyme K (GzmK). Performing a Differential Gene Expression (DEG) analysis on Tr1 cells, we observed no significant differences between OVA− and OVA+ cells. However, we identified differentially expressed genes between untreated and anti-PD-L1-treated mice, suggesting a heightened stress response and increased exhaustion in intratumoral Tr1 cells following treatment. In melanoma patients, Tr1 cells were significantly enriched within tumors, exhibiting a high proliferation rate. Circulating EOMES+ Tr1 cells in immune checkpoint blockade (ICB) treated patients showed an increased IFNg production, as well as for CD4+ T helper cells and CD8+ T cells, while Tregs have a significant reduction in IL-10 production, suggesting an enhancement of cytotoxic differentiation following treatment. When stimulated with melanoma-associated antigens, they produced both IFN-γ and IL-10, particularly in response to MelanA, while healthy donors did not respond to any tumor antigens. Moreover, circulating Tr1 cells from patients harbouring V600E mutation in the BRAF gene respond to the mutated peptide, suggesting the neo antigen specificity of these cells. Notably, melanoma patients treated with ICB exhibited a diminished capacity for IL-10 production in circulating tumor antigen-specific Tr1 cells, mirroring the effects observed in intratumoral CD4+ T cells in the mouse model, while CD4+ CTL and CD8+ T cells showed upregulation of IFNg response to tumor antigens in ICB treated patients. In summary, these data suggest a pivotal role of IL-10-producing EOMES+ Tr1 cells in modulating anti-tumor immunity. This study also provides the first evidence that a fraction of circulating Tr1 cells in melanoma patients are tumor antigen specific. Our findings suggest that immune checkpoint blockade therapy may be particularly effective when Tr1 cells are abundant in the tumor microenvironment, providing new insights for optimizing immunotherapeutic strategies.