Project Summary / Abstract This application is in response to a specific request for proposals to develop immune cell engineering towards the treatment of type 1 diabetes (T1D). We propose to evaluate genetic engineering approaches in immune T regulatory cells (Treg) by integrating chimeric antigen receptor (CAR) proteins that are engineered with an external targeting domain (scFv) and internal stimulatory domain. This approach has revolutionized cancer cell therapy with heightened specificity and effectiveness of T cell action. We and others have found evidence that CAR Treg can help mediate immune protection of islets and may even act upon islets themselves to reduce stress and cell death. We propose to (a) determine how enhanced targeting and activation of Treg to human islets might improve islet function and local immune modulation to protect islets, (b) evaluate a method for enhanced islet targeting through the development and testing of a dual-targeting CAR system that exploits downstream T cell receptor signaling molecules that have not been previously evaluated and (c) determine how enhanced targeting and activation of Treg to human monocytes might result in more immunoregulatory monocytes that could help to alter response to islet autoantigens and prevent immune destruction. One very important component of our proposal is that we suspect that CAR Treg from patients with T1D may not function as well as CAR Treg from normal individuals. In fact, there is no data about this available. We think that some CAR Treg from some T1D patients might be more cytotoxic, less effective or more inflammatory and we propose to evaluate if this is true statistically and also to develop an approach to introduce and overexpress a set of genes known to be important for Treg function as a way of making sure that all Treg in all cases will exert effects that are wanted. We postulate that developing these Treg methods will produce novel clinical strategies to prevent T1D in high risk patients and to suppress autoimmunity and preserve β-cell mass in patients with recent-onset T1D.