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Background Checkpoint blockade immunotherapies (CBIs) transformed cancer treatment, yet benefit only one-third of patients. 1 2 The continuous exposure to natural microbiota modulates anti-tumoral immune responses, with specific bacteria acting as adjuvants for CBIs.3–6 However, the underlying mechanisms remain poorly defined. Specific pathogen-free (SPF) lab mice are inadequate for addressing this question due to limited microbiota diversity and immature immune systems.7 Instead, we utilized C57BL/6 mice with wild-derived microbiota (Wildling mice), which possess immune-experienced, fully mature immune systems.8 Studies using various mouse models with wild-derived microbiota show improved tumor control.9 10 We hypothesized that natural immunity adjuvates CBIs by modulating the activation dynamics of intratumoral CD8+ T cells.Methods To quantify TCR-mediated CD8 + T cell activation dynamics, we implanted poorly immunogenic D4M melanoma cells into SPF or Wildling E8IcrexRosa26LSL-Salsa6f mice, which express the genetically-encoded calcium signaling reporter Salsa6f11 in CD8+ T cells, and performed multiphoton intravital microscopy.12 13 Two mice per group were imaged before and 12h post anti-PD-1 injection. To further assess T cell function, we performed flow cytometry analysis of key functional markers expressed by tumor-associated CD8+ T cells in wild-type SPF and Wildling mice treated with control or anti-PD-1 antibodies, in two independent experiments. Finally, we explored the clinical advantage of the Wildling mice’s natural microbiota compared to SPF mice for tumor growth control and anti-PD-1 response across four independent experiments.Results Intravital imaging of the tumor environment and flow cytometry revealed a statistically significant 9.6-fold increase in tumor-infiltrating CD8 + T cell density in the Wildling mice compared to SPF mice, along with their enhanced TCR activation following anti-PD-1 treatment. Increased intratumor CD8+ T cell activation in Wildling mice correlated with significantly higher IFNγ and Granzyme B expression following anti-PD-1 treatment even in the terminally differentiated Tim3+ CD8+ T cells, which remained unresponsive to PD-1 blockade in SPF mice. Importantly, Wildling mice controlled the growth of poorly immunogenic melanoma better than SPF mice after anti-PD-1 immunotherapy.Conclusions Our findings show that immune experience enhances CD8 + activation to improve immunotherapy-mediated antitumor immune responses. To elucidate the underlying mechanisms, we are currently examining how continuous exposure to diverse microbiota affects memory CD8+ T cell pools, their epigenetic reprogramming, and CD8+ T cell recruitment/reactivation at tumor sites. Understanding these mechanisms will pave the way for innovative strategies to prime patients to CBI responsiveness and improve tumor control.References Larkin J, Chiarion-Sileni V, Gonzalez R, Grob J-J, Rutkowski P, Lao CD, Cowey CL, Schadendorf D, Wagstaff J, Dummer R, et al. Five-year survival with combined nivolumab and ipilimumab in advanced melanoma. New England Journal of Medicine. 2019;381(16):1535–1546.Wolchok JD, Chiarion-Sileni V, Rutkowski P, Cowey CL, Schadendorf D, Wagstaff J, Queirolo P, Dummer R, Butler MO, Hill AG, et al. Final, 10-year outcomes with nivolumab plus ipilimumab in advanced melanoma. New England Journal of Medicine. 2025;392(1):11–22.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(6529):595–602.Park EM, Chelvanambi M, Bhutiani N, Kroemer G, Zitvogel L, Wargo JA. 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