Fourth generation CAR Tregs with PDCD1-driven IL-10 have enhanced suppressive function

The potency of regulatory T cell (Treg) therapy has been transformed through use of chimeric antigen receptors (CAR). However, to date, CAR Treg therapy has not achieved long-lasting tolerance in mouse models, suggesting that additional engineering is required to unlock the full potential of these cells. We previously found that human Tregs produce minimal amounts of IL-10 and have a limited capacity to control innate immunity in comparison to type I regulatory (Tr1) cells. Seeking to create "hybrid" CAR Tregs that were engineered with Tr1-like properties, we examined whether the PDCD1 locus could be exploited to endow Tregs with the ability to secrete high levels of IL-10 in a CAR-regulated manner. CRISPR-mediated PD1-deletion increased the activation potential of CAR Tregs without compromising in vivo stability. Knock-in of IL10 under control of the PD1 promoter facilitated CAR-mediated secretion of IL-10 in large quantities, and improved CAR Treg function, as determined by significant inhibition of dendritic cell antigen presentation and enhanced suppression of alloantigen- and islet autoantigen-specific T cells. Overall, CRISPR-mediated engineering to simultaneously remove an inhibitory signal and enhance suppressive mechanisms is a new approach to enhance the therapeutic potency of CAR Tregs.