Immune checkpoint inhibitor-induced reinvigoration of tumor-infiltrating CD8+ T cells is determined by their differentiation status in glioblastoma

Purpose: Immune checkpoint inhibitors (ICI) are used for the treatment of various cancers, but clinical trials of antiprogrammed cell death protein 1 (PD-1) with patients with recurrent glioblastoma (GBM) have failed to show clinical benefits. In this study, we examined the differentiation status of CD8⁺ tumor-infiltrating lymphocytes (TIL) from patients with primary GBM and their reinvigoration by ICIs to understand the nature of T-cell exhaustion in GBM. Experimental Design: We isolated TILs from 98 patients with newly diagnosed GBM and examined the expression of immune checkpoint receptors and T-cell transcription factors using flow cytometry. TILs were ex vivo stimulated with anti-CD3 in the presence of anti-PD-1 and/or anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) and their proliferation assessed. Results:CD8⁺ TILs had significantly increased expression of immune checkpoint receptors, including PD-1 and CTLA-4, compared with peripheral blood CD8⁺ T cells. Among CD8+ TILs, PD-1⁺ cells exhibited more terminally differentiated phenotypes (i.e., Eomes^hiT-bet^lo) than PD-1^- cells. These data were confirmed by analyzing NY-ESO-1_157- specific CD8⁺ TILs. Evaluating the proliferation of CD8⁺ TILs after ex vivo stimulation with anti-CD3 and anti-PD-1, we found that proliferation inversely correlated with the percentage of Eomes^hiT-bet^lo cells among PD-1+CD8+ TILs. When anti- CTLA-4 was used in combination with anti-PD-1, an additional increase in CD8⁺ TIL proliferation was observed in patients with low percentages of Eomes^hiT-bet^lo CD8+ TILs, who responded well to anti-PD-1 in ex vivo assays, but not in patients with high percentages of Eomes^hiT-bet^lo CD8+ TILs, who did not respond to anti-PD-1. Conclusions: In primary GBM, the differentiation status of CD8⁺ TILs determines their reinvigoration ability upon ICI treatment.