CD4+CD25+ regulatory T (Treg) cells express the forkhead transcription factor Foxp3 and are essential for the maintenance of dominant immunological tolerance to the 'extended self, which includes self proteins and the commensal microbial flora. Global failure of T reg cells due to mutations in Foxp3 results in a lethal syndrome of systemic autoimmunity, Iymphoproliferation, and allergic dysregulation, underscoring the essential contribution of T reg cells to the control of immune responses. There are two types of Treg cells, the so called 'natural'T reg (nT reg) cells that arise as a specific lineage in the thymus, and 'induced'T reg (iTreg) cells that are generated from conventional T cells in the periphery. The broad, long- term objective of this project is to elucidate the fundamental mechanisms underpinning Treg cell maturation and function. The specific aims of this proposal are intended to identify the individual contributions of iT reg and nTreg cells to tolerance and to dissect the mechanisms responsible for functional distinctions between the two cell types. The proposed studies will test the hypothesis that complete tolerance requires both iTreg and nT reg cells. We postulate that each cell type serves a non-redundant and synergistic role, mandated by distinct TCR repertoires, variations in gene expression patterns, and divergent functional attributes. In the proposed experiments, we will use thymic organ cultures to establish the TCR affinity requirements that control nT reg selection in the thymus. We will employ incisive genetic tools to functionally define the iTreg and nT reg cell populations used to rescue Foxp3 deficient mice and mice with inflammatory bowel disease by adoptive transfer immunotherapy. These investigations will provide fundamental and preclinical insights into mechanisms governing the maintenance of dominant tolerance by Treg cells, as well as the dysregulated immune responses and autoimmunity unleashed by Treg cell deficiency.