Graft versus host disease (GvHD) is the major limitation to expanding allogeneic stem cell transplantation for the treatment of patients with hematological cancers and stem cell disorders. Acute GvHD (aGvHD) is due to the recognition of minor and major MHC disparities between the donor and host that lead to a cascade of immunological events resulting in the inflammatory state characteristic of aGvHD. Chronic GvHD (cGvHD) is a more protean manifestation of immune reactivity between donor and host, which can lead to extensive fibrotic changes in tissues, immune dysfunction and death. The pathophysiology of cGvHD is less understood compared to aGvHD partly due to the lack of informative animal models. Over the past 10 years, investigators have identified a number of new T cell lineages characterized by the activity of specific transcription factors that mediate much of the function in those cells. Of these, one of the most scrutinized has been natural regulatory T cells that express constitutively CD4 and CD25 and the transcription factor Foxp3. Treg cells can inhibit the proliferation and effector function of T and B cells in the peripheral compartment and are critical in maintaining tolerance to self-antigens. Little is known regarding how Treg cells function in vivo. Our group demonstrated that Treg cells with increased expression of L-selectin functioned best to prevent GvHD. This suggested that Treg cells function by blocking T cell activation in lymphoid tissue. However, we also published that Treg cells are required to migrate to the liver and lung to prevent GvHD occurring at those sites. How Tregs function in lymphoid tissue or parechymal organs is not clear and is the focus of this proposal. In specific aim 1, we will focus on studies to understand how Treg cells used to prevent or treat GvHD block Teff cell function. We will use extremely novel intravital imaging of Tregs, effector T cells and antigen presenting cells in tissues such as the liver and skin to determine mechanistically how Treg interact with Teff and APCs to prevent or treat GvHD. We will investigate whether treatment of ongoing GvHD is best done using Treg cells targeted to GvHD-involved organs. An understanding of how Treg cells work is critical to inform clinical trials using Treg cells to treat or prevent aGvHD. In specific aim 2, we will focus on a very novel finding from our laboratory that Treg cells, which do not migrate into secondary lymphoid tissue when transplanted with allogeneic bone marrow and alloreactive T cells, cause cGvHD manifested by fibrotic changes in the skin, eye and liver. This suggests that cGvHD may be due to impaired Treg function in the peripheral compartment leading to aberrant activation of donor T and B cells against host antigens that should be seen as [unreadable]self.[unreadable] We will investigate the mechanism by which CCR7 -/- Treg cells given with allogeneic bone marrow and T cells mediate cGvHD focusing on the role of donor and host T and B cells in this process. This work has the potential to alter our understanding of the mechanism by which cGvHD occurs and could offer new treatment avenues for its therapy.