Coordinating Central Tolerance: Defining Distinct Roles for Nr4a1 and Nr4a3 in Thymocyte Selection

About This Grant

PROJECT SUMMARY/ABSTRACT Dr. Nielsen’s long-term goal is to understand molecular mechanisms that drive autoimmune diseases to improve their diagnosis and treatment. This five-year research and training plan will launch her independent career studying thymic T cell tolerance. To select a functional, self-tolerant T cell repertoire, developing thymocytes test their vast, random array of T cell receptors (TCRs) at key checkpoints to 1) ensure MHC restriction, 2) “tune” their capacity to distinguish self from foreign stimuli, and 3) “prune” self-reactive TCRs via clonal deletion or diversion to a regulatory T cell (Treg) fate. Nr4a transcription factors – whose expression scales with self-reactivity – collectively translate the TCR signals that enforce clonal deletion, Treg diversion, and aspects of TCR “tuning.” However, distinct protein expression dynamics for family members NR4A1 and NR4A3 suggest a “division of labor” in thymic selection which has yet to be unmasked. This proposal’s central hypotheses are: 1) NR4A1’s low threshold for induction and rapid decay allow it sense and tune TCR signaling during positive selection, 2) collective NR4A “dosage” determines deletion and Treg diversion, and NR4A3’s high threshold for induction and sustained expression enforce death and diversion of select self-reactive clonotypes in the medulla. Using biochemical assays of TCR signaling, Aim 1 characterizes how TCR tuning and NR4As operate in human thymocytes and – using genetic mouse models – determines the roles of NR4A1 and NR4A3 in TCR tuning. Aim 2 leverages high-resolution TCR sequencing and unbiased auto-antibody profiling (PhIP-seq) in to identify how the NR4As uniquely and cooperatively shape the thymic TCR repertoire. Aim 3 employs single-cell transcriptomics (CITE-seq) combined with a TCR signaling reporter to decode the transcriptional networks by which the NR4As link TCR signaling to tolerogenic outcomes during thymic selection. Completion of this project will provide unprecedent molecular insights into TCR signaling mechanisms and the formation of a tolerant TCR repertoire, illuminating fundamental principles of autoimmunity and informing strategies for T cell-based immunotherapies. Dr. Nielsen will perform this research at the University of California, San Francisco, a world-renowned institution for immunology. She has assembled a strong mentorship and collaborative team: Dr. Julie Zikherman (primary mentor, lymphocyte signaling), Dr. Mark Anderson (co-mentor, thymic tolerance), Dr. Ellen Robey (T cell selection), Dr. Mike Waterfield (gene regulation), Dr. Jimmie Ye (single-cell genomic technologies), and Dr. Wan-Lin Lo (University of Utah, TCR signaling in human cells). Through hands-on training, formal coursework, and engagement with the global immunology community, she will develop essential lab management skills and master advanced immunologic and genomic techniques, positioning herself as an independent investigator driving cutting-edge research in immune tolerance.