Project Summary Acute kidney injury (AKI) is associated with high mortality in native kidneys, decreased allograft survival in transplants, and very high health care cost. Among the most common etiologies of AKI in both native and transplanted kidneys is ischemia-reperfusion injury (IRI). There is currently no specific therapy for AKI and the recovery process is incompletely understood. A major unknown is precise roles of each T cell type. This is important as some T cells are involved in causing AKI, whereas others are involved in preventing tissue damage and improving repair. Our team is focusing on characterizing and understanding the role of double negative (DN) T cells in healthy kidney and during AKI. DN T cells are one of the least understood T cell types because of their paucity in peripheral lymphoid organs and thus have been relatively ignored. However, we recently discovered DNT cells as a large constituent of kidney αβT cells (hereafter referred to as KDNT cells). Significance of KDNT cells is underscored by their proven suppressive functions in vitro and immunoregulatory protective function during experimental AKI. This renewal application is based on strong recently published and unpublished preliminary data showing that KDNT cells are divided into two (PD-1+ and NK1.1+) subsets in murine and human kidney. Murine PD-1+ DNT cells are actively dividing in the steady state that is accompanied by a proliferative burst in response to experimental IRI by mechanisms that are not restricted by known classical or non-classical MHC I and II molecules. The non-dividing NK1.1+ subset is regulated by 2m-dependent non-classical MHC class I in mice. The major goals of this renewal application are to investigate the regulatory role of PD-1 molecule, relationship of KDNT cells and renal tubular epithelial cells (RTEC) in health and AKI, and translational of our murine findings to humans using human kidney samples and from discarded deceased donors. Our specific Aims: Test the hypothesis that PD-1/PD-L system regulates homeostasis and effector function of kidney DN T cells in health and AKI. 2) Test the hypothesis that kidney DN and TREC regulate each other via a bidirectional loop during health and AKI. 3) Test the hypothesis that human KDNT cells are suppresser cells that regulate homeostasis of RTEC during health and AKI. We have all the key critical elements to achieve these goals that include an in vitro KDNT/RTEC culture system, unique access to human kidney samples pre and post ischemia (from nephrectomies) and discarded deceased donor kidneys, and a synergistic team of investigators with an established collaborative track record and complementary expertise needed for successful outcome.