Naturally occurring Foxp3+ regulatory T (Treg) cells play an important role in the maintenance of immune homeostasis and prevention of autoimmunity. Although it is commonly thought that self-reactivity is important for Treg cell selection, the quantitative parameters that govern this process has not been reported. In this proposal, we will analyze the role of TCR self-reactivity in thymic Treg cell differentiation as wel as negative selection. In Aim 1, we will study these thymic tolerance mechanisms using a novel panel of TCRs with broad reactivity to ovalbumin peptide (OVA323-339). If self-reactivity is the primary driving force for thymic Treg cell development as suggested by our preliminary data, then the antigens presented in the thymus will determine the Treg cell repertoire. In Aim 2, we will address whether the thymic APC subsets serve non-redundant capacities in Treg cell generation and negative selection. Finally, we will assess in Aim 3 whether the level of self-reactivity that triggers thymic tolerance mechanisms also triggers peripheral Treg cell generation. We will also assess the degree of self-reactivity needed for Treg cells to prevent autoimmunity, as well as for effector cells to cause pathology. Understanding the mechanisms that govern Treg cell development may lead to the development of therapies which utilize Treg cell-mediated therapies for the treatment of autoimmune disease.