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Background Despite improved melanoma outlook following the introduction of anti-PD1 therapy, 50% of patients bear treatment resistant tumors. Though the drivers of resistance remain largely unidentified, recent studies have correlated the composition of the gut microbiome with response to anti-PD1 therapy in melanoma. 1–3 However, the mechanisms behind these correlations remain. Previously, we demonstrated that Helicobacter hepaticus (Hhep) colonization drives CD4+ T cell dependent anti-tumor immunity in a murine model of colorectal cancer.4 We have since learned that Hhep colonization combined with anti-PD1 drives anti-tumor immunity in a PD1-resistent murine melanoma, providing a model to mechanistically unravel how the gut microbiome synergizes with anti-PD1 to promote immunotherapy response in distant tumors. We hypothesized that translocation of Hhep to the tumor fosters an anti-tumor microenvironment via Hhep-specific T cell polarization in the presence of anti-PD1.Methods We used intradermal B16-F10 melanoma in mice colonized with Hhep via oral gavage and treated with systemic anti-PD1 blockade. We monitored tumor growth and survival as well as the infiltration and function of transferred tumor- and Hhep-specific TCR transgenic T cells through flow cytometry, immunofluorescence, single cell RNA sequencing, and TCR sequencing. We also visualized Hhep within the colon and tumor with fluorescent in situ hybridization (FISH) and Microbiota-inclusive Passive Clearing Technique (MiPACT).Results Strikingly, we found that Hhep colonization significantly decreased tumor burden when combined with anti-PD1. Hhep was only present in tumors of mice treated with both Hhep and anti-PD1. Dual treatment resulted in a significant increase in Hhep-specific CD4+ TH1 cells intratumorally in contrast to Hhep colonization alone which resulted in lower infiltration of Hhep-specific TFH cells, demonstrating a shift in not only quantity but also function. Despite no significant difference in infiltration of tumor specific CD8+ T cells, cells isolated from dual treated mice expressed fewer inhibitory receptors. Interestingly, when B16-F10 tumors were injected subcutaneously rather than intradermally, Hhep and anti-PD1 mediated decreases in tumor burden were lost and Hhep was no longer visible intratumorally, demonstrating a tissue specific component to this anti-tumor mechanism. Further experiments suggest that PD1 blockade itself increases the seeding of beneficial intratumoral bacteria, like Hhep. Conclusions Overall, we have found that combined Hhep and PD1 treatment decreased tumor burden, altered the intratumoral Hhep-specific T cell population, and promoted translocation of bacteria to the tumor. Better understanding of the crosstalk between checkpoint inhibitors and the microbial translocation has the potential to drive microbe-targeted therapeutic intervention for treatment resistant tumors in the clinic.References Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT, Daillère R, Fluckiger A, Messaoudene M, Rauber C, Roberti MP, et al. Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science. 2018;359:91–97.Davar D, Dzutsev AK, McCulloch JA, Rodrigues RR, Chauvin J-M, Morrison RM, Deblasio RN, Menna C, Ding Q, Pagliano O, et al. Fecal microbiota transplant overcomes resistance to anti-PD-1 therapy in melanoma patients. Science. 2021;371:595–602.Gopalakrishnan V, Spencer CN, Nezi L, Reuben A, Andrews MC, Karpinets TV, Prieto PA, Vicente D, Hoffman K, Wei SC, et al. Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients. Science. 2018;359:97–103.Overacre-Delgoffe AE, Bumgarner HJ, Cillo AR, Burr AHP, Tometich JT, Bhattacharjee A, Bruno TC, Vignali DAA, Hand TW. Microbiota-specific T follicular helper cells drive tertiary lymphoid structures and anti-tumor immunity against colorectal cancer. Immunity. 2021;54:2812–2824.