A small subset of T lymphocytes expresses FoxP3 and is called as regulatory T cells or Tregs. Transplantation of various organs is often required to treat many human diseases or disease-related complications. However, transplanted tissues need to survive rejection by host T lymphocytes. Host-derived donor-specific Tregs offer a natural way of suppressing rejection of transplanted organs without side effects or off targets associated with immunosuppressive drugs. Tregs are found at very low frequency and animal models have shown that large numbers of Tregs are required to achieve clinical efficacy;therefore elucidation of the mechanisms underlying the expansion of Tregs will facilitate the therapeutic application of this specific subset of T lymphocytes for induction of transplantation tolerance. We have found that sustained and clustered CD3 and CD28 signaling induces apoptosis of effector T cells and allows expansion of Tregs in less than 2 weeks. Unsorted human CD4 T cells (initially 6% FoxP3+ cells) cultured for 12 days under this condition were highly enriched for Tregs (>75% cells Foxp3+ cells at end of culture). Therefore, this procedure has great promise and potential for clinical application of Tregs. The proposed study will investigate in detail the expansion of donor reactive Tregs from mice and humans for induction of transplantation tolerance and elucidate the mechanism of selective Treg expansion in this system. Aim 1 will test the hypothesis that Treg specific transcription factor FoxP3 actively suppresses apoptosis in this system, allowing selective proliferation and expansion of Tregs. Aim 2 will employ MHC-peptide tetramers for induction of TCR ligation to test the hypothesis that sustained and clustered signaling by immobilized MHC-peptide tetramers and anti-CD28 selectively expand alloantigen specific functional Tregs from mice and humans. The outcome of these studies will greatly facilitate clinical application of Tregs to improve transplantation tolerance. PUBLIC HEALTH RELEVANCE: Regulatory T cells offer a natural way of inducing longevity of transplanted organs to recipients without side effects. Using a novel system, proposed study aims to expand alloreactive Tregs to facilitate the long term acceptance of transplanted tissue to mismatch recipients.