Project Summary/Abstract Hematopoietic stem cell transplantation (HSCT) has become a powerful treatment for a myriad of disorders. Patients diagnosed with blood disorders such as leukemia or aplastic anemia that are treated with HSCT have seen highly improved outcomes, and in some of these cases HSCT is the only curative option. However, donors and recipients of these transplants are commonly not HLA identical, resulting in an immune response due to interactions between the recipient’s alloreactive memory T cells that recognize “non-self” antigens on the donor cells. The most common method of inducing tolerance to the donor cells and tissue is by lifelong administration of immunosuppressive drugs that come with the risk of opportunistic infections and toxicities. The overarching goal of this project is to develop the necessary genetic tools to restore immune tolerance without the need for life-long immunosuppression. Our approach to achieving this is to propose a split CAR T cell system that uses off-the-shelf antibody adapter molecules to specifically deplete the recipient’s memory T cells that are reactive against the donor, while preserving other T cell subsets to maintain a robust immune system. This system has the ability to switch targets without reengineering the T cells and respond to multiple antigens to target only alloreactive T-cells and enhance the establishment of mixed chimerism and transplantation tolerance. If successful, the findings of this research project will demonstrate the feasibility of using CAR T cell therapy for the induction of immune tolerance that can be applied beyond HSCT to the treatment of many autoimmune conditions including type 1 diabetes, rheumatoid arthritis, vitiligo and a myriad of disorders that originate from autoreactive T cell dysfunction.