Granzyme F: Exhaustion Marker and Modulator of Chimeric Antigen Receptor T Cell–Mediated Cytotoxicity

Abstract Granzymes are a family of proteases used by CD8 T cells to mediate cytotoxicity and other less-defined activities. The substrate and mechanism of action of many granzymes are unknown, although they diverge among the family members. In this study, we show that mouse CD8+ tumor-infiltrating lymphocytes (TILs) express a unique array of granzymes relative to CD8 T cells outside the tumor microenvironment in multiple tumor models. Granzyme F was one of the most highly upregulated genes in TILs and was exclusively detected in PD1/TIM3 double-positive CD8 TILs. To determine the function of granzyme F and to improve the cytotoxic response to leukemia, we constructed chimeric Ag receptor T cells to overexpress a single granzyme, granzyme F or the better-characterized granzyme A or B. Using these doubly recombinant T cells, we demonstrated that granzyme F expression improved T cell–mediated cytotoxicity against target leukemia cells and induced a form of cell death other than chimeric Ag receptor T cells expressing only endogenous granzymes or exogenous granzyme A or B. However, increasing expression of granzyme F also had a detrimental impact on the viability of the host T cells, decreasing their persistence in circulation in vivo. These results suggest a unique role for granzyme F as a marker of terminally differentiated CD8 T cells with increased cytotoxicity, but also increased self-directed cytotoxicity, suggesting a potential mechanism for the end of the terminal exhaustion pathway.