CD4 T cells express T cell receptors (TCRs) that recognize peptides loaded on major histocompatibility complex II molecules (p:MHCII). While CD4 T cell recognition of foreign-peptides mediates immunity, recognition of self-peptides may promote autoimmunity. Therefore, self-reactive CD4 T cells must be tolerized. Self tolerance is not always complete, as evidenced by the tens of millions of people with type I diabetes, multiple sclerosis, or other autoimmune diseases. A key to understanding autoimmunity is to study the cells that mediate disease and the means by which they are normally regulated. By studying these mechanisms we may learn how specific breaches in tolerance occur, enhancing our ability to design therapies that target relevant pathways rather than broadly trying to resolve inflammation. Unfortunately, it has not been feasible to track rare, self-reactive T cells with known specificity in normal individuals. Therefore, much of our knowledge comes from study of transgenic mice that express model self-antigens and corresponding TCRs. This work has identified possible modes of tolerance including clonal deletion, regulatory T cell (Treg) induction, Treg-mediated suppression, anergy, and peripheral deletion. However, it is now clear that TCR transgenic systems are frequently unphysiological, generating an imperfect understanding of how tolerance pathways interact. For example, elevated clonal frequencies diminish the efficiency of Treg production. Further, self-p:MHCII-reactive CD4 T cells may be tolerized by multiple mechanisms, but a single clone within a larger repertoire may only be susceptible to one. The objective of this proposal is to determine if and how these pathways contribute to tolerance in normal individuals by studying how endogenous polyclonal self-reactive CD4 T cells are tolerized to a single self-p:MHCII ligand. The central hypothesis is that different modes of tolerance regulate distinct self-reactive clones within the polyclonal repertoir according to their TCR affinities. The specific aims of this proposal are to: 1) Determine how different mechanisms promote polyclonal CD4 T cell tolerance to a self-p:MHCII ligand and 2) Identify the role of TCR affinity in determining the mode of tolerance to a self-p:MHCII ligand. These studies will use innovative p:MHCII technologies, cutting edge single-cell techniques (RNA analyses, single cell transfers, TCR affinity measurements), and in vivo studies to comprehensively determine how tolerance is enforced to a single self-p:MHCII ligand within a polyclonal repertoire. Our results will provide important insights into how tolerance is normally established, an essential first step towards developing therapeutic avenues for patients with autoimmunity.