PROJECT SUMMARY/ABSTRACT Regulatory and effector T cells serve distinct immunological roles that contribute to immune homeostasis. Whereas effector T cells have the functional capacity to protect the host from infectious threats and cancer, regulatory T cells (Tregs) are suppressive in nature and prevent runaway effector T cell-mediated inflammation and autoimmunity. Although these are distinct cell lineages determined primarily during thymic selection, it is now understood that the two can interconvert – T cells can assume regulatory phenotypes in the periphery, and Tregs may lose suppressive capacity to become pro-inflammatory “ex-regulatory” T cells (exTregs). Because exTregs can subvert traditional regulatory T cells to exacerbate inflammation and contribute to pathologic autoimmune progression, there is great interest in understanding how and under what conditions exTregs develop. In preliminary work from our lab, we found that single cell RNA sequencing reveals extensive phenotypic heterogeneity of primary Tregs and exTregs from mouse gut, lungs, and lymph nodes. I propose using single cell genomics approaches to fully define the gene regulatory programs that govern exTreg development and polarization from destabilized Tregs. In my first aim, I will deeply analyze the single cell RNA sequencing data set to define the heterogeneity of exTreg phenotypes in distinct tissue microenvironments from which they were isolated. For my second aim, I will leverage my sponsors’ expertise in high-throughput genetic perturbation of primary mouse Tregs to perform a forward genetic screen to map the gene regulatory programs that maintain Treg identity and destabilize Tregs to exTregs induced by pro-inflammatory cytokines IL-4 or IL-6. My sponsor Alex Marson has extensive expertise in the genetics of immune cell function and autoimmunity, and in genetic engineering of primary T cells using CRISPR-Cas9. My co-sponsor Chun (Jimmie) Ye is a computational immunologist and human geneticist whose laboratory uses innovative technologies like single cell RNA sequencing to molecularly define processes of immune cell development and function. Additionally, I have secured scientific and career mentorship from renowned immunologists Dr. Jeffrey Bluestone (characterized exTregs in autoimmune inflammation) and Dr. Qizhi Tang (studies immune tolerance and autoimmunity). Concurrently, I am completing a longitudinal clinical preceptorship in pediatric immunology with Dr. Alice Chan, director of the UCSF Benioff Children’s Hospital Pediatric Immune Dysregulation Clinic and expert in genetic diagnosis of pediatric immune disorders. Overall, the proposed work will further our understanding of exTregs development and their role in autoimmune pathogenesis and progression. Moreover, this fellowship support will foster my training in immunology and computational biology, supporting me on my path through combined MD-PhD training towards a career as an academic pediatric immunologist...