Armored human CAR T_reg cells with PD1 promoter-driven IL-10 have enhanced suppressive function

Regulatory T cell (T_reg cell) therapy has been transformed through the use of chimeric antigen receptors (CARs). We previously found that human T_reg cells minimally produce IL-10 and have a limited capacity to control innate immunity compared to type 1 regulatory T cells (T_r1 cells). To create "hybrid" CAR T_reg cells with T_r1 cell-like properties, we examined whether the PDCD1 locus could be exploited to endow T_reg cells with CAR-regulated IL-10 expression. CRISPR-mediated PD1 deletion increased CAR T_reg cell activation, and knock-in of IL10 under control of the PD1 promoter resulted in CAR-induced IL-10 secretion. IL10 knock-in improved CAR T_reg cell function, as determined by increased suppression of dendritic cells and alloantigen- and islet autoantigen-specific T cells. In vivo, IL10 knock-in CAR T_reg cells were stable, safe, and suppressed dendritic cells and xenogeneic graft-versus-host disease. CRISPR-mediated engineering to simultaneously remove an inhibitory signal and enhance a suppressive mechanism is a previously unexplored approach to improve CAR T_reg cell potency.