Combinatorial genetic engineering strategy for immune protection of stem cell-derived beta cells by chimeric antigen receptor regulatory T cells

Regenerative medicine is a rapidly expanding field harnessing human pluripotent stem cell (hPSC)-derived cells and tissues to treat many diseases, including type 1 diabetes. However, graft immune protection remains a key challenge. Chimeric antigen receptor (CAR) technology confers new specificities to effector T cells and immunosuppressive regulatory T cells (Tregs). One challenge in CAR design is identifying target molecules unique to the cells of interest. Here, we employ combinatorial genetic engineering to confer CAR-Treg-mediated localized immune protection to stem cell-derived cells. We engineered hPSCs to express truncated epidermal growth factor receptor (EGFRt), a biologically inert and generalizable target for CAR-Treg homing and activation, and generated CAR-Tregs recognizing EGFRt. Strikingly, CAR-Tregs suppressed innate and adaptive immune responses in vitro and prevented EGFRt-hPSC-derived pancreatic beta-like cell (sBC [stem cell-derived beta cell]) graft immune destruction in vivo. Collectively, we provide proof of concept that hPSCs and Tregs can be co-engineered to protect hPSC-derived cells from immune rejection upon transplantation. [Display omitted] •Gene editing of human pluripotent stem cells (hPSCs) to express unique bait protein•Regulatory T cell engineering with bait-specific chimeric antigen receptor (CAR-Treg)•CAR-Tregs are suppressive in vitro when activated by bait-expressing cells•CAR-Tregs protect bait-expressing hPSC-derived beta cells from immune destruction in vivo Immune protection of human pluripotent stem cell (hPSC)-derived grafts remains a key hurdle in regenerative medicine. Barra and Robino et al. engineered hPSCs to express an inert bait protein and bait-specific immunosuppressive chimeric antigen receptor regulatory T cells (CAR-Tregs), accomplishing localized hPSC-derived beta cell immune protection in humanized mice.