Initiation and maintenance of peripheral T cell tolerance relies on a complex set of events dependent on the responding T cell subpopulation, the antigen and the antigen presenting cells (APC), expression of co- stimulatory molecules and cytokines, and the genes expressed during the early signalling cascade following exposure to antigen. The overall goals of this Tolerance Program Project are to dissect the mechanisms which lead to peripheral T cell tolerance by examining the molecular, biochemical, and immunobiological consequences of T cell interactions with antigen presenting cells. To achieve these general goals, four complementary projects will be undertaken: 1. To study the early molecular events following T cell activation to determine the genetic elements that control the functional outcome of T cell receptor (TCR)/ligation. The molecular basis of T cell tolerance will be studied by analyzing the transcriptional regulation of the IL-2 gene following T cell activation and tolerance induction. In addition, novel genes, including a newly discovered bcl-2-like gene (bcl-x), will be studied for its role in T cell inactivation and antigen-induced cell death (apoptosis). 2. To dissect the role of cell surface adhesion molecules in regulating T cell tolerance. Functional activation of T cells depends not only on the interaction of the TCR with its ligand but, in addition, the signals transduced by so called co-stimulatory molecules present on T cells that bind to ligands expressed on APC. The role of one such co- stimulatory molecule/ligand interaction, CD28/B7, in the induction of peripheral anergy will be examined both in vitro and in vivo using a combination of mAbs, soluble receptors, and genetically-deficient mice. Other cell surface molecules such as CTLA4, HSA, and ICAM-1 will also be studied to determine their role in peripheral tolerance induction and maintenance. These studies will include analyses of both clonal, naive, and in vivo-derived T cells. 3. To determine the mechanism of T cell tolerance induction in distinct T cell subsets using different APC. A variety of T cell subsets exist. Activation of these individual subsets are dictated by the nature of the antigens recognized, characteristics of the APC, and cytokine milieu. Only the Th1 of T cells have been studied, in depth, with regard to anergy induction. Other T cell subsets including: Th2 cells; CD8+ IL-2 producing cells; and CD8+ IL-4 producing cells will be examined for tolerance induction using T cell clones derived against selected nominal antigens, and subsets will be compared for differences in expression of genetic and biochemical components that regulate T cell activation. 4. The in vivo basis for peripheral T cell tolerance will be examined using transplantation and transgenic mouse models. Individual antigen-presenting cells derived from epithelial tissue and pancreatic islets will be manipulated using gene therapy and transgenic mouse technology in order to express selective co-stimulatory molecules in these cell types. These animals will be examined in vivo for the initiation and maintenance of tolerance. T cells derived from these animals will be examined molecularly and biochemically to compare in vivo tolerized T cells inactivated in vitro. Members of this Program Project grant bring together in a unique set of reagents, model systems, and insights into the mechanisms of T cell anergy and tolerance. Together, these projects should provide information on the mechanism of peripheral T cell tolerance and help to develop new therapeutic modalities treating autoimmunity and graft rejection.