Xiangni Wu ^1,2 Pin-I Chen ¹ Robert L. Whitener ³ Matthew MacDougall ⁴ Vy M. N. Coykendall ³ Hao Yan ¹ Yong Bin Kim ⁵ William Harper ¹ Shiva Pathak ¹ Bettina Iliopoulou ¹ Allison Hestor ¹ Diane C. Saunders ⁶ Erick Spears ⁶ Jean Sévigny ⁷ David M. Maahs ⁸ Marina Basina ⁹ Seth A. Sharp ¹⁰ Anna L. Gloyn ¹⁰ Alvin C. Powers ⁶ Seung K. Kim ³ Kent P. Jensen ¹ Everett H. Meyer ^1*

• ¹ Division of Blood and Marrow Transplantation & Cellular Therapy, Department of Medicine, School of Medicine, Stanford University, Stanford, California, United States
• ² Department of Internal Medicine, School of Medicine, University of Missouri–Kansas City, Kansas City, Kansas, United States
• ³ Department of Developmental Biology, School of Medicine, Stanford University, Stanford, California, United States
• ⁴ Department of Pediatrics, School of Medicine, Stanford University, Palo Alto, California, United States
• ⁵ Department of Chemical Engineering, School of Engineering, Stanford University, Stanford, California, United States
• ⁶ Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, United States
• ⁷ Centre de recherche sur le cancer de l’Université Laval, Centre de recherche du CHU de Québec, Université Laval, Québec, Quebec, Canada
• ⁸ Division of Endocrinology and Diabetes, Department of Pediatrics, School of Medicine, Stanford University, Palo Alto, California, United States
• ⁹ School of Medicine, Stanford University, Stanford, California, United States
• ¹⁰ Departments of Genetics, School of Medicine, Stanford University, Stanford, California, United States

Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases, including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs, most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets, may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes, a human beta cell line and human islet beta cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet beta cells. In exploring subsets and mechanisms that may explain this pattern, we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39 low/-Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39- CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly, beta cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased beta cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet beta cells in the setting of CAR immunotherapy. In summary, introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction.