Phenotypic Characterization and Cytotoxic Profiling of Tumor-infiltrating Lymphocytes (TILs) in Pediatric Gliomas

Introduction: Pediatric gliomas are the leading cause of cancer-related mortality in children and young adults, characterized by significant heterogeneity and a lack of effective therapeutic options. Cellular therapies, such as tumor-infiltrating lymphocyte (TIL) transfer, have shown promise in other cancers, such as in melanoma. In glioma, ex vivo expansion of TILs followed by reinfusion has the potential to overcome the diverse tumor antigen landscape, offering an advantage over approaches like cancer vaccines or TCR-T/CAR-T therapies. Notably, TIL transfer relies on the rational that T cells found in the tumor microenvironment can recognize and thereby eliminate tumor cells. However, we have limited information regarding tumor-reactivity of TILs in pediatric gliomas, thus hindering development of TIL transfer therapies for these tumors.

Methods: We have optimized a TIL expansion protocol (REP) and assessed TIL reactivity in pediatric gliomas. Tumor reactivity and functionality of TILs were assessed by IFN-γ secretion, tumor cell death, and the expression of markers of tumor-antigen experienced T cell phenotype via flow cytometry for established markers of T cell activation and memory (4-1BB, PD1, CD103, and CD39), isolated from human glioma samples (n=16; 8 LGG, 8 HGG) and from murine glioma models (SB28, GL261, and SB de novo).

Results: Our findings demonstrate that TILs from pediatric glioma patients express markers associated with tumor antigen reactivity and can be successfully expanded ex vivo using REP, providing proof-of-concept for TIL expansion and a pathway for clinical translation. Moreover, we show that TILs, when compared to naïve or tumor-bearing splenocytes, exhibit preferential cytotoxicity against autologous tumor cells. These suggest the presence of tumor-specific TILs in pediatric gliomas.

Conclusion : This approach holds promise not only for enhancing anti-tumor immune responses but also for overcoming the immunosuppressive tumor microenvironment that often limits the efficacy of conventional therapies. Incorporating TIL-based therapies into glioma treatment could provide a long-term strategy for improving survival outcomes in pediatric glioma patients.