Colorectal cancer-infiltrating bacteria promote the proliferation of cytotoxic lymphocyte populations endowed with antitumor properties

Colorectal cancer (CRC) is the second-leading cause of cancer deaths worldwide. During CRC carcinogenesis alteration of the gut epithelial barrier allows the translocation of commensal bacteria from the gut to the tumor tissue and their interaction with infiltrating immune cells. Infiltration by cytotoxic T lymphocytes is associated with improved survival, but their potential interplay with tumor-infiltrating bacteria has not been fully investigated.We previously identified a panel of bacteria whose abundance in CRC tissues correlates with the extent of lymphocytic infiltration. In this work, we have investigated the capacity of these bacteria to induce lymphocyte-mediated immune responses.Peripheral blood mononuclear cells from healthy donors or patients were cultured with cryopreserved bacteria of interest and assessed for proliferation based on CFSE dilution. Proliferating cells mostly consisted of CD4-CD8- double negative (DN) T cells expressing gammadeltaTCRs, and in particular the Vdelta2 chain. CD4+ and CD8+ TCRalphaß+ T cells were also detected among proliferating cells. Following activation, DN Vdelta2+ T cells released IFNg, TNFalpha, perforin, and granzymes suggesting a cytotoxic potential. Their concomitant expression of NKG2D led us to investigate whether they could mediate tumor-cell killing against CRC cell lines. In contrast to CD4+ and CD8+, bacteria-reactive DN T cells showed MHC-independent tumor cell killing.Analysis performed by large-scale flow cytometry and single-cell RNA sequencing of primary CRC samples revealed the presence of DN TCRgammadelta+ T cells among tumor-infiltrating lymphocytes (TILs). Interestingly, their phenotypes and transcriptomic profiles were found to partially overlap with those of bacteria-reactive DN T cells from peripheral blood. The reactivity of DN TILs to CRC-associated bacteria is currently under investigation.Bacteria-mediated activation of CRC-infiltrating DN T cells may favor a double-sided targeting of the tumor, contributing to an effective antitumor immune response.