Dynamic FOXP3 Expression in the Development of Regulatory T cells from Induced Pluripotent Stem Cells - PROJECT SUMMARY Over 20 million individuals in the United States live with an autoimmune disease, resulting in significant disability and mortality. The increasing prevalence of autoimmune diseases raises the stakes to develop new therapies. Harnessing Tregs as a cellular therapy is a promising and attractive treatment approach for autoimmunity. However, Tregs represent a rare cell subset and can exhibit unstable suppressive function due to the unstable expression of master transcription factor FOXP3. We propose a novel approach to study the role of endogenous and exogenous FOXP3 expression in thymic-like Treg development from a self-renewing source: induced pluripotent stem cells (iPSC). Pilot data demonstrate the ability of artificial thymic organoids (ATOs) – developed in the Crooks’s Lab – to support the differentiation of CD4+FOXP3+ T cells from iPSC (henceforth “iPSC-Tregs”). In Aim 1, Dr. Yiu will test methods to enhance endogenous FOXP3 expression in iPSC and compare iPSC-Tregs to Tregs isolated from the thymus (tTregs) and peripheral blood (pTregs) based on their immunophenotype, transcriptional pattern, hypomethylation, and suppressive function. Because constitutive FOXP3 expression blocks T cell development, in Aim 2, Dr. Yiu will link exogenous FOXP3 transgene expression to endogenous genes that are only expressed at mature T cell stages, via CRISPR/Cas9. In Aim 3, Dr. Yiu will transduce iPSC with exogenous T cell receptors (TCRs) isolated from either a non-Treg or Treg cell, to determine the effect of TCR expression and identity on Treg fate and antigen-specific suppressive function. This proposal is a five-year research career development plan for the Principal Investigator, Dr. Gloria Yiu, M.D., Ph.D., a physician-scientist and Assistant Professor of Medicine in Rheumatology at the University of California, Los Angeles (UCLA). During the award period, Dr. Yiu will be mentored by Dr. Gay Crooks, M.D., an expert in stem and T cell development, distinguished physician-scientist, and accomplished mentor with a productive track record of mentoring physician-scientists. Dr. Yiu will also obtain Treg expertise from co-mentor Dr. Maureen Su, M.D., a leading physician-scientist at the forefront of mouse and human autoimmunity. The goal of this proposal is to build upon Dr. Yiu’s rigorous background in cellular immunology and gain new training in gene editing, advanced transcriptomic and proteomic techniques, and bioinformatics. This will be accomplished through high- yield didactic coursework and guidance from advisory committee members, who were carefully selected for their relevant scientific expertise and impressive records of mentorship. UCLA’s Department of Medicine and Division of Rheumatology will provide the resources, support, and infrastructure to assist Dr. Yiu in achieving the aims described in this proposal and her long-term goal of developing into an independent physician-scientist studying Treg development for the treatment of autoimmunity.
