1259 Going the distance: <i>Helicobacter hepaticus</i> colonization drives immunotherapy response in melanoma

<h3>Background</h3> While melanoma outlook has improved with the introduction of anti-PD1 immunotherapy, 50% of patients are treatment resistant with largely unknown barriers to response. Recent studies correlated the composition of the gut microbiome with immunotherapy response in melanoma; however, the mechanism behind these correlations remains unclear and the beneficial bacteria identified are remarkably dissimilar.^1–3 We previously demonstrated that <i>Helicobacter hepaticus</i> (<i>Hhep</i>) colonization drives CD4⁺ T cell dependent anti-tumor immunity in a murine model of colorectal cancer, but whether <i>Hhep</i> supports anti-tumor immunity at distant tumor sites was unknown.⁴ We hypothesize that <i>Hhep</i> drives anti-tumor immunity at distant sites through bacteria-specific T cell polarization in the presence of anti-PD1. <h3>Methods</h3> At both intradermal and subcutaneous sites, we used B16 melanoma (anti-PD1 resistant) and MC38 colon adenocarcinoma (anti-PD1 sensitive) to determine whether microbiome modulation through <i>Hhep</i> colonization could synergize with anti-PD1 blockade. We monitored tumor growth and survival as well as the infiltration and function of transferred tumor- and <i>Hhep</i>-specific TCR transgenic T cells through flow cytometry and immunofluorescence. We also visualized <i>Hhep</i> within the colon and tumor with fluorescent <i>in situ</i> hybridization. <h3>Results</h3> Unlike our colorectal cancer model, <i>Hhep</i> colonization without anti-PD1 therapy increased tumor burden in our B16 intradermal model. However, when <i>Hhep</i> colonization was combined with anti-PD1, we observed decreased tumor growth. <i>Hhep</i>-specific CD4⁺ T cells were found intratumorally, regardless of anti-PD1 treatment. However, <i>Hhep</i> only colonized the tumor in mice that received anti-PD1. This combination of <i>Hhep</i> and anti-PD1 blockade shifted both <i>Hhep</i>-specific and endogenous intratumoral CD4⁺ T cells from T_FH to T_H1 phenotype, indicated by loss of Bcl6 expression and increased Tbet expression. Additionally, tumor-specific CD8⁺ T cells were less exhausted as shown through reduced inhibitory receptor expression. However, when either MC38 or B16 models were moved from the dermal to subcutaneous layer of skin, the effects seen by both <i>Hhep</i> alone and <i>Hhep</i> with anti-PD1 intradermally were lost, suggesting that the tissue location of the tumor is critical for bacteria-driven anti-tumor effects. <h3>Conclusions</h3> Here, we show that addition of a single bacterial species confers anti-tumor immunity in a distant, refractory tumor model. Ultimately, these findings provide novel insight into the mechanism by which microbiome modulation can boost immunotherapy response at distant tumor sites – a phenomenon currently recognized but previously mechanistically undefined. Taken together, this study highlights the promise of microbiome-driven immunotherapeutic targets in refractory tumors and illuminates the contributions of the tissue microenvironment in modulating this response. <h3>References</h3> Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT, Daillère R, Fluckiger A, Messaoudene M, Rauber C, Roberti MP, <i>et al</i>. Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors.<i> Science</i> 2018;<b>359</b>:91–97. 10.1126/science.aan3706. Davar D, Dzutsev AK, McCulloch JA, Rodrigues RR, Chauvin J-M, Morrison RM, Deblasio RN, Menna C, Ding Q, Pagliano O, <i>et al</i>. Fecal microbiota transplant overcomes resistance to anti-PD-1 therapy in melanoma patients. <i>Science</i> 2021;<b>371</b>:595–602. 10.1126/science.abf3363. Gopalakrishnan V, Spencer CN, Nezi L, Reuben A, Andrews MC, Karpinets TV, Prieto PA, Vicente D, Hoffman K, Wei SC, <i>et al</i>. Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients. <i>Science</i> 2018;<b>359</b>:97–103. 10.1126/science.aan4236. 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. <i>Immunity</i> 2021;<b>54</b>:2812–2824.e4. 10.1016/j.immuni.2021.11.003.