PrOJeCt 3 Regulation of immune responses is critical in health and disease. Nowhere is this clearer than following allogeneic hematopoietic cell transplantation (HCT) where induction of effective immune responses eliminates residual disease in the graft vs tumor (GVT) effect, whereas an aberrant immune reaction towards the host can result in life threatening graft vs host disease (GVHD). We and others have found that CD4+CD25+FoxP3+ regulatory T cells (Treg) are capable of controlling GVHD yet allow for GVT effects and promote more effective immune reconstitution. In this proposal we will build upon extensive progress in the initial funding period to study critical biological characteristics of the mechanisms by which Treg control GVHD reactions. We have developed novel imaging technology utilizing bioluminescence (BLl) that allows us to serially and non-invasively study the trafficking and survival of distinct cell populations including Treg. Our hypothesis is that Treg traffic to critical nodal sites where they interact with antigen presenting cells (APCs) and through this interaction down modulate the proliferation of effector CD4+ and CD8+ alloreactive T cells. In this proposal we will study the role of the interaction between Treg and CD11C+ APCs by selectively depleting this latter population from recipient animals. We will evaluate the role of critical molecules expressed by Treg in this process and explore the importance of interaction with APCs and expression of cell surface receptors and enzymes in immune regulation mediated by Treg. Using BLl and novel transgenic mouse lines developed in our laboratories we will explore mechanisms utilized by Treg including trafficking to critical sites, proliferation and control of alloreactive T cell expansion. We will also utilize data generated in the initial term of this grant to explore novel approaches to expanding Treg both in vitro and in vivo. In particular we have demonstrated the Treg utilize different signaling pathways upon activation than conventional CD4+ T cells and we will explore approaches to the expansion of Treg both in living animals and in cultures. We expect that these studies will significantly increase our basic understanding of the biology and mechanisms of Treg in the settings of HCT and organ transplantation.