Co-blockade of TGFβ and PD-1 reinvigorates glioblastoma-infiltrating CD8+ T cells that characteristically upregulate TGFβR expression

Abstract Purpose: Clinical trials have shown limited efficacy of anti-programmed cell death protein 1 (PD-1) treatment for glioblastoma (GBM). In this study, we examined the expression of TGFbRI in GBM-infiltrating CD8+ T cells and the characteristics of TGFbRI+CD8+ T cells. We examined the ex vivo effects of co-blockade of PD-1 and TGFb on the functions of GBM-infiltrating CD8+ T cells. Experimental Design: Using flow cytometry, we examined the phenotypes of tumor-infiltrating CD8+ T cells from newly diagnosed GBM patients. We performed single-cell RNA/TCR-sequencing to characterize the tumor-infiltrating TGFbRI+CD8+ T cells. We also examined the effects of co-blockade of PD-1 and TGFb on the functions of tumor-infiltrating CD8+ T cells in ex vivo assays. Results: GBM-infiltrating CD8+ T cells expressed significantly increased levels of TGFβRI compared to peripheral blood CD8+ T cells. Among tumor-infiltrating CD8+ T cells, TGFbRI+CD8+ T cells exhibited increased expression of immune checkpoint inhibitory receptors, and tumor antigen-specific cells were enriched in TGFbRI+CD8+ T cells. Single-cell profiling revealed that tumor-infiltrating TGFBR1+CD8+ T cells demonstrated more clonal expansion and upregulation of TCR signaling genes compared to TGFBR1-CD8+ T cells. In vitro, anti-CD3 stimulation upregulated TGFbRI expression on CD8+ T cells. GBM patients with a high frequency of TGFbRI+CD8+ T cells presented with increased TGFb signaling intensity. Importantly, combined blockade of PD-1 and TGFb significantly enhanced the functions of tumor-infiltrating CD8+ T cells ex vivo. Conclusions: Our findings provide a basis for further investigation of co-blockade of PD-1 and TGFβ for the treatment of patients with GBM.