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Background PD-1 immunotherapy revolutionized cancer treatment since its approval in 2014 but still fails in the majority of metastatic melanoma patients, emphasizing the need to further understand its impact on the immune system. 1 Recently, combination immunotherapy, treatment with multiple checkpoint inhibitors, or immunotherapy paired with chemotherapy, has shown efficacy across cancer types2 but choosing the most beneficial treatment is challenging. A crucial component in directing each patient towards therapy with the highest chance of success is to identify biomarkers predictive of failure of specific therapeutic protocols. Our recent work identified the anti-apoptotic protein ICOS as a mediator of Treg persistence in melanoma during PD-1 immunotherapy.3 Based on these findings, we hypothesize that high expression of ICOS on Tregs in melanoma patients before αPD-1 treatment correlates with resistance to immunotherapy.Methods We utilized an immunogenic melanoma model (D4M-S) to understand the mechanisms behind Treg accumulation during immunotherapy. Using flow cytometry and multiphoton intravital microscopy (MP-IVM) of transgenic mouse models, we quantified tumor immune cell numbers and function during treatment with αPD-1. We extended our findings to humans through analysis of melanoma patient RNA-seq and immunofluorescence data. Additionally, we profiled T cells in blood and lymph node aspirates using flow cytometry from stage III-IV melanoma patients before they began immunotherapy to correlate with responsiveness to PD-1 blockade.Results We found that αPD-1 leads to tumor Treg accumulation in patients and D4M-S tumors. MP-IVM revealed that αPD-1 does not enhance tumor Treg activation and that PD-1 deficient tumor Tregs accumulated during αPD-1. CD8 + T cells responded to αPD-1 with enhanced production of IL-2, a cytokine that improved survival of tumor Tregs through their expression of ICOS (figure 1). To evaluate ICOS as a biomarker of therapy failure, our ongoing study will enroll 20 therapy naive patients. We will immunophenotype routine blood samples and, if patients are stage III, lymph node aspirates taken before the start of immunotherapy (figure 2). As the study progresses, we will correlate ICOS expression with clinical response to immunotherapy. Our preliminary results from this clinical study will be discussed at SITC.Conclusions Our findings indicate that indirect mechanisms are the primary driver of Treg accumulation in D4M-S tumors treated with αPD-1, which is substantially different from what was previously published. 4 5 Our data suggest that Treg-expressed ICOS can be a marker of poor responsiveness to PD-1 blockade, and our ongoing clinical study will test this theory.Acknowledgements This study was funded by Melanoma Research Alliance young investigator award # 929155 to F.M., and pilot grants from the Chao Family Comprehensive Cancer Center (University of California Irvine). S.G. is supported by NIH T32 AI177324 and an NSF-GRFP fellowship 2235784.References Wolchok JD, et al. Final, 10-year outcomes with nivolumab plus ipilimumab in advanced melanoma. New England Journal of Medicine 2025;392:11–22.Butterfield LH, Najjar YG. Immunotherapy combination approaches: mechanisms, biomarkers and clinical observations. Nature Reviews Immunology 2024;24: 399–416.Geels SN, et al. Interruption of the intratumor CD8+ T cell:Treg crosstalk improves the efficacy of PD-1 immunotherapy. Cancer Cell 2024;42:1051–1066.e7.Kamada T, et al. PD-1+ regulatory T cells amplified by PD-1 blockade promote hyperprogression of cancer. Proc Natl Acad Sci U S A. 2019;116: 9999–10008.Kumagai S, et al. The PD-1 expression balance between effector and regulatory T cells predicts the clinical efficacy of PD-1 blockade therapies. Nat Immunol. 2020;21:1346–1358.Ethics Approval This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California Irvine; approval number AUP22-092 and with a pending IRB.Abstract 455 Figure 1Interruption of the intratumor CD8+ T cell:Treg crosstalk through ICOSL blockade improves the efficacy of PD-1 immunotherapy. Data summary describing mechanism of intratumor support to Tregs through CD8 IL-2 production during PD-1 blockadeAbstract 455 Figure 2Evaluating ICOS expression as a biomarker for PD-1 therapy success in melanoma patients. Timeline of samples to correlate ICOS expression pretherapy to immunotherapy resistance