Despite marked improvements in understanding the immune system, tolerance is still not reliably achieved. Recent studies have shown that intercellular regulation is required to modulate immune responses and induce or maintain tolerance. In particular, regulatory T cells (Treg) have emerged as major elements of the apparatus that controls immunity and tolerance. Among the regulatory T cells that have been described are anergic CD4+CD25+ Treg. These cells express the Foxp3 transcription factor, which is to date the most specific marker for cells that possess the Treg suppressive function. Foxp3 may be a "master switch" for the transcriptional program for Treg function. Despite progress in the field, many issues and characteristics remain incompletely defined or unexplored. For example, how are Foxp3 and the Treg program induced and maintained? What is the function of Foxp3 and the gene set induced down stream? What is the function of Treg that express all or part of the Foxp3 program? How do Treg modify innate and adaptive immunity? Our preliminary results show that TGFbeta enhances survival of Treg which are already committed to express the Foxp3 program. Importantly, TGFbeta induces de novo Foxp3 expression and Treg function in uncommitted naive cells, and Foxp3 induction is blocked by CD28 co-stimulation in an IL-4 dependent fashion. Committed or induced Treg inhibit innate immune responses by pancreatic islets, thereby enhancing islet engraftment and cure of diabetes. Based on these findings, we hypothesize that TGFbeta is a key regulator of the signaling pathways that initiate and maintain Foxp3 expression and function of Treg. To investigate this hypothesis we propose the following Aims: 1.) Determine how TGFbeta generates Treg. We will investigate the signals important for induction and inhibition of Foxp3 expression. We will investigate the cellular functions of TGFbeta driven Treg that express Foxp3. We will determine the plasticity of the Foxp3/Treg program in individual T cells. 2.) Determine how Foxp3 generates Treg. We will investigate the function of cells in which Foxp3 expression is driven by retroviral gene transfer. We will determine the effects of forced Foxp3 expression on T cell function and the plasticity of that function. We will investigate the signals and molecules that permit or inhibit Foxp3 function. 3.) Determine how Treg function. TGFbeta or Foxp3 driven Treg will be evaluated for their ability to migrate, undergo homeostatic proliferation, and transition to a memory phenotype. In the autoimmune model of colitis, the ability of these cells to regulate the homeostatic proliferation and autoimmune function of effector T cells will be determined, with attention to specific effector molecules, including IL-10 and TGFbeta, and costimulatory molecules. The mechanism of Treg suppression of the innate immune response of pancreatic islets will be determined in vitro and in vivo, analyzing effector molecules in Treg-islet interactions.