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Background Gliomas remain one of the most treatment-refractory cancers, with immunosuppressive tumor microenvironments that limit the efficacy of immune checkpoint blockade (ICB). Adoptive TIL therapy may overcome this limitation, but its success depends on robust ex vivo expansion and in vivo persistence of tumor-reactive T cells. We demonstrate that dual ICB with anti-PD-1 and anti-CTLA-4 primes the tumor immune microenvironment to enhance TIL recovery, functionality, and therapeutic efficacy upon reinfusion.Methods C57BL/6 mice were implanted intracranially with 5×10 4 GL261 or SB28 cells (n=16/model). Mice received intraperitoneal PBS or 200 µg each of anti-PD-1 and anti-CTLA-4 on Days 1 and 4. On Day 15, tumors were dissociated, dead cells removed, and CD45+ TILs isolated. Flow cytometry assessed expression of tumor reactive T cell markers. TILs were co-cultured with irradiated splenocytes (1:100), anti-CD3, and high-dose IL-2 (1000 IU/mL) for 14 days. IFN-γ levels were quantified in supernatants by ELISA upon TIL coculture with autologous tumor cells. Expanded TILs were reinfused into tumor-bearing mice (Day -10 inoculation) across six treatment arms. Survival scores were tracked post-reinfusion.Results Dual ICB preconditioning increased TIL recovery (GL261: 3.7×10 6 vs. 0.9×106 cells/tumor, p<0.001). No significant difference was found in tumor reactive T cell subpopulations with prior ICB exposure. IFN-γ secretion was markedly elevated in ICB-primed TIL cultures (p<0.0001), confirming functional activation upon tumor reexposure. Kaplan-Meier analysis revealed that ICB- TIL therapy extended survival in both GL261 and SB28 models. While control and vehicle TIL groups showed median survival between 20-22 days across models, ICB TIL notably prolonged survival, with the GL261 mice exhibiting a median survival beyond the study’s observation period (30+ days), and the SB28 mice reaching approximately 25 days, highlighting a synergistic effect.Conclusions Our data demonstrates that dual checkpoint blockade enhances the quantity and quality of tumor-infiltrating lymphocytes, but also translates into durable responses in aggressive murine glioma models. Dual ICB enhances both the yield and functionality of glioma-derived TILs and significantly improves survival upon adoptive reinfusion. These findings validate our hypothesis and support clinical translation of ICB-primed TIL therapy as a next-generation immunotherapeutic strategy for glioma.