Project Summary/Abstract The balance between immune tolerance and activation lies at the heart of most pathologies. Immunological tolerance refers to the set of processes that prevent immune activation against non-pathogenic antigens. The key distinguishing feature of tolerance compared to other forms of immunosuppression is that it operates to inhibit reactivity only to specific antigens and does not render the host immunosuppressed with respect to unrelated pathogens. Immune homeostasis relies upon precise tuning of this tolerance-activation axis, and disruption of this balance results in autoimmunity or malignancy. To date, nearly all our treatments for autoimmune disease result in some form of nonspecific immunosuppression. Conversely, while immunotherapies represent the greatest paradigm shift in oncology in decades, they largely act to induce broad immune activation in a nonspecific manner that often results in adverse events, including autoimmune pathologies. In solid organ transplantation, our inability to induce complete graft tolerance often requires the use of lifelong immunosuppressants. Thus, despite over a half-century of research into immunological tolerance, there remains a pressing need to develop therapies capable of controlling antigen-specific immunological tolerance for a wide range of diseases and clinical settings. In this proposal, we seek to develop a new class of modular immunotherapies that couple intrinsic T cell biology with synthetic biology and genome engineering to reeducate endogenous tolerance programs in the host. We hypothesize that by activating or disrupting aberrant tolerance programs, we can treat autoimmune disease and metastatic cancer. Lymph nodes are the anatomical hubs of peripheral tolerance induction, a feature that is coopted by malignancies as they metastasize throughout the host. Unlike conventional engineered cell therapies whose mechanism of action relies upon cytolysis of pathogenic cells, including tumors or autoreactive lymphocytes, the engineered cell therapies that we propose instead function by trafficking to lymph nodes to alter endogenous tolerance induction resulting in resolution of autoimmunity or treatment of metastatic cancer. To achieve these goals, we will develop a T cell therapy that specifically homes to lymph nodes by coopting intrinsic naïve T cell homing machinery. In the context of metastatic cancer, we will augment this approach with the inclusion of chimeric antigen receptors (CARs) that break immune tolerance and induce activation upon ligation of the CARs following trafficking to LNs. In the context of autoimmunity, our therapies will induce tolerance following recognition of autoantigen presentation by antigen presenting cells in lymph nodes. Thus, at the conclusion of this project, we will deliver a new class of modular immunotherapies that harnesses the endogenous immune response in an antigen-specific manner. This approach has the potential to deliver cures to patie...