Human OX40L-CAR-T_regs target activated antigen-presenting cells and control T cell alloreactivity

Regulatory T cells (T_regs) make major contributions to immune homeostasis. Because T_reg dysfunction can lead to both allo- and autoimmunity, there is interest in correcting these disorders through T_reg adoptive transfer. Two of the central challenges in clinically deploying T_reg cellular therapies are ensuring phenotypic stability and maximizing potency. Here, we describe an approach to address both issues through the creation of OX40 ligand (OX40L)-specific chimeric antigen receptor (CAR)-T_regs under the control of a synthetic forkhead box P3 (FOXP3) promoter. The creation of these CAR-T_regs enabled selective T_reg stimulation by engagement of OX40L, a key activation antigen in alloimmunity, including both graft-versus-host disease and solid organ transplant rejection, and autoimmunity, including rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus. We demonstrated that OX40L-CAR-T_regs were robustly activated in the presence of OX40L-expressing cells, leading to up-regulation of T_reg suppressive proteins without induction of proinflammatory cytokine production. Compared with control T_regs, OX40L-CAR-T_regs more potently suppressed alloreactive T cell proliferation in vitro and were directly inhibitory toward activated monocyte-derived dendritic cells (DCs). We identified trogocytosis as one of the central mechanisms by which these CAR-T_regs effectively decrease extracellular display of OX40L, resulting in decreased DC stimulatory capacity. OX40L-CAR-T_regs demonstrated an enhanced ability to control xenogeneic graft-versus-host disease compared with control T_regs without abolishing the graft-versus-leukemia effect. These results suggest that OX40L-CAR-T_regs may have wide applicability as a potent cellular therapy to control both allo- and autoimmune diseases.