PROJECT SUMMARY Mesenchymal stromal cells (MSCs) have regenerative, immunosuppressive properties, but using MSCs to treat autoimmunity has resulted in mixed clinical results at best. Lackluster therapeutic efficacy has been attributed to insufficient trafficking to the tissue of interest and suboptimal immunosuppression. Our preliminary data indicate that using lentiviruses with specific enhancers, we can successfully engineer MSCs to express chimeric antigen receptors (CARs) and become CAR-MSC. We also have demonstrated that incorporating specifically designed CARs targeting gut integrins into MSCs results in their enhanced immunosuppression and trafficking to the colon in preclinical models. Engineering MSCs maintain their stemness and CAR-MSCs are safe in canine models. This led to our central hypothesis that engineering MSCs with a CAR enhances their efficacy in graft versus host disease (GVHD) and autoimmune inflammatory bowel disease (IBD) and colitis. In this project, we will leverage our available tools of viral vectors, CRISPR constructs, mouse models, and established biobanks to develop CAR-MSCs that demonstrate enhanced therapeutic efficacy over unmodified MSCs in the treatment of GVHD. In Aim 1 of this proposal, we will determine the mechanisms by which CAR-MSCs exert their enhanced immunosuppression. In Aim 2 of this proposal, we will study cellular trafficking in preclinical models. In Aim 3 of this proposal, we will optimize signaling of CAR-MSCs. At the conclusion of this project, we will have developed a new, translatable cellular therapeutic strategy to treat GVHD and IBD by engineering CAR-MSCs to have improved targeting and immunosuppression over unmodified MSCs. This platform can be applicable in different autoimmune diseases.