Karolina Woroniecka, Pakawat Chongsathidkiet, Kristen Rhodin, Hanna Kemeny, Cosette Dechant, S. Harrison Farber, Aladine A. Elsamadicy, Xiuyu Cui, Shohei Koyama, Christina Jackson, Landon J. Hansen, Tanner M. Johanns, Luis Sanchez-Perez, Vidyalakshmi Chandramohan, Yen-Rei Andrea Yu, Darell D. Bigner, Amber Giles, Patrick Healy, Glenn Dranoff, Kent J. Weinhold, Gavin P. Dunn, Peter E. Fecci

T cell dysfunction is a hallmark of GBM. While anergy and tolerance have been well characterized, T cell exhaustion remains relatively unexplored. Exhaustion, characterized in part by the upregulation of multiple immune checkpoints, is a known contributor to failures amidst immune checkpoint blockade, a strategy that has lacked success thus far in GBM. This study is among the first to examine, and credential as bona fide, exhaustion among T cells infiltrating human and murine GBM. Tumor-infiltrating and peripheral blood lymphocytes (TIL, PBL) were isolated from patients with GBM. Levels of exhaustion-associated inhibitory receptors and post-stimulation levels of the cytokines IFN-g, TNF-a, and IL-2 were assessed by flow cytometry. T cell receptor (TCR) Vβ chain expansion was also assessed in TIL and PBL. Similar analysis was extended to TIL isolated from intracranial and subcutaneous immunocompetent murine models of glioma, breast, lung, and melanoma cancers. Our data reveal that GBM elicits a particularly severe T cell exhaustion signature among infiltrating T cells characterized by: 1) prominent upregulation of multiple immune checkpoints; 2) stereotyped T cell transcriptional programs matching classical virus-induced exhaustion; and 3) notable T cell hypo-responsiveness in tumor-specific T cells. Exhaustion signatures differ predictably with tumor identity, but remain stable across manipulated tumor locations. Distinct cancers possess similarly distinct mechanisms for exhausting T cells. The poor TIL function and severe exhaustion observed in GBM highlights the need to better understand this tumor-imposed mode of T cell dysfunction in order to formulate effective immunotherapeutic strategies targeting GBM.