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.