GITR activation potentiates anti-tumor immunity of tumor-infiltrating lymphocytes expanded from glioblastoma by rescuing exhaustion

Abstract

Autologous tumor-infiltrating lymphocyte (TIL) therapy holds transformative potential for solid tumors, yet its efficacy in glioblastoma remains limited by T cell exhaustion and immunosuppression. In the current study, we optimized an effective and reliable method for in vitro expansion of TILs from glioblastoma lesions and assessed their tumor-killing capacity both in vitro and in vivo. Single-cell RNA sequencing (scRNA-seq) of expanded TILs uncovered their heterogeneity and identified a cytotoxic tissue-resident memory (TRM) CD8⁺ TIL subset with a unique exhaustion signature. Notably, the co-stimulatory factor GITR (encoded by TNFRSF18) is highly expressed not only on immunosuppressive regulatory T (Treg) cells but also on exhausted CD8⁺ TILs. GITR agonism via αGITR antibody achieved dual effects: it directly enhanced CD8⁺ TIL activation while simultaneously abrogating Treg-mediated immunosuppression. This dual-action mechanism synergized with αPD-1 therapy to amplify TIL reactivation, significantly enhancing tumor control in vivo. Mechanistically, GITR activation potentiated anti-tumor responses by promoting immunological synapse (IS) formation and function in TILs via the NF-κB/KALRN signaling axis. Our findings established GITR as a crucial regulator of CD8⁺ TIL anti-tumor immunity, positioning GITR targeting as a novel strategy to improve TIL therapy for glioblastoma, with promising implications for clinical application.