Glioblastoma (GBM) is the most common primary malignant brain tumor and remains universally lethal. Median survival is persistently less than two years, despite modest improvements to standard of care. Immunotherapies, though FDA-approved in other solid tumors, are hampered in GBM by the tumor’s marked heterogeneity and immunosuppressive influences. Propagating multiple modes of T-cell dysfunction facilitates GBM’s ability to escape immunotherapeutic targeting [1, 2]. T-cell anergy [3] and tolerance [4, 5] are well-characterized in GBM patients, while other contributing manners of T-cell dysfunction, including sequestration [6], senescence, and exhaustion [7], are beginning to be further explored [8]. We uncovered recently that T-cell exhaustion likely makes substantial contributions to dysfunction among T-cells that successfully arrive at GBM, and may provide direct mechanistic limitations to the efficacy of checkpoint blockade [7]. Exhaustion is a hypo-responsive T-cell state resulting from chronic antigenic exposure under sub-optimal conditions. It was initially described amidst chronic viral infection (such as with chronic lymphocytic choriomeningitis virus (LCMV)) but is increasingly appreciated in cancer. Exhaustion represents a specific transcriptional program that is often characterized by up-regulation of the various co-inhibitory receptors constituting immune checkpoints. Blocking the classical immune checkpoints, PD-1 and CTLA-4, to rejuvenate T-cell function is an FDA-approved strategy in many cancers, yet a phase III clinical trial of PD-1 blockade demonstrated limited efficacy against GBM. Recent work has shown that one mode of resistance to PD-1 blockade involves the emergence of alternative immune checkpoints/exhaustion markers on T-cells, such as TIM-3 and LAG-3 [9]. We therefore sought to determine the prevalence of exhaustion and these alternative immune checkpoints among T-cells in GBM.

We found that GBM does indeed elicit a severe exhaustion signature amidst T-cells [7]. T-cells infiltrating human GBM tumors (TIL) were found to express multiple immune checkpoints, including PD-1, TIM-3, LAG-3, TIGIT, and CD39. TIL were likewise less able to secrete the cytokines IFN-γ, IL-2, or TNF-α than T-cells isolated from patient or control blood. Among patient samples, triple-positive PD-1+TIM-3+LAG-3+ CD8+ T-cells were least functional compared to PD-1 negative and PD-1 single-positive cells, showing that mounting expression of the alternative immune checkpoints TIM-3 and LAG-