Project Summary/Abstract Type 1 diabetes (T1D) is an autoimmune disease leading to pancreatic beta cell destruction and lifelong dependence on exogenous insulin injections. Immunomodulatory agents that aim to reverse beta cell autoimmunity have been shown to delay T1D diagnosis but cannot halt the decline of C-peptide levels that reflect insulin production. Autologous cell therapy (ACT) is an investigational therapy that aims to restore the immune set point back to tolerance by infusion of ex vivo expanded regulatory T cells (Tregs). Tregs are responsible for downregulating the immune response and their absence in animal models of diabetes leads to accelerated disease progression. Early clinical trials of ACT, however, show that transfused Tregs persist for years in patients but were ineffective in preventing C-peptide decline. This project proposes to generate islet specific T follicular regulatory (Tfr) cells from Tregs through gene editing to improve their utility as ACT for T1D. Tfr cells are a specialized subset of Tregs that act in the germinal centers of lymph nodes where mature B cells are activated by T follicular helper (Tfh) cells. Early B cell activation is an important step in T1D prognosis as the detection of class-switched islet autoantibodies in the pre-diabetic phase predicts onset of disease. Therefore, creating Tfr-like cells represents a potential avenue of T1D prevention through suppression of Tfh-mediated activation. The following Specific Aims outline the objectives for utilizing genome targeting to produce Tfr-like cells as potential cellular therapies of T1D. In Aim 1, islet reactive Tregs will be produced through non-viral genome editing to knock in islet reactive TCR in the TCR locus. Engineered Tregs will be tested in vitro for islet antigen reactivity and tested in vivo through adoptive transfer into NOD mice to determine its effects on diabetes incidence. In Aim 2, the Tfr cell characteristic chemokine receptor CXCR5 will be knocked in the Rosa26 locus of islet reactive Tregs and tested for responsivity to CXCL13. Engineered CXCR5-positive islet reactive Tregs will be transferred into NOD mice to assess diabetes incidence and Treg trafficking to the pancreas and pancreatic lymph nodes. The proposed training will take place at the University of Florida Diabetes Institute under the guidance of Dr. Todd Brusko and Dr. Michael Haller. The training plan will provide the applicant with research design and technical skills in autoimmunity, cell engineering, and diabetes models as well as professional skills in teaching and scientific writing to facilitate growth as an independent investigator.