Each pancreatic islet from each subject is represented by one symbol, with red symbols representing islets containing T cell infiltration. cell PD-L1 expression was dependent on T cell infiltration, as cells from Rag1-deficient mice lacked PD-L1. Using Rag1-deficient NOD mouse islets, we determined that IFN- promotes cell PD-L1 expression. We performed analogous experiments using human OTS186935 samples, and found a significant increase in cell OTS186935 PD-L1 expression in type 1 diabetic samples compared to type 2 diabetic, autoantibody positive, and non-diabetic samples. Among type 1 diabetic samples, cell PD-L1 expression correlated with insulitis. experiments with human islets from non-diabetic individuals showed that IFN- promoted cell PD-L1 expression. These results suggest that insulin-producing cells respond to pancreatic Nedd4l inflammation and IFN- production by upregulating PD-L1 expression to limit self-reactive T cells. Introduction The inhibitory receptor Programmed Death-1 (PD-1) and its ligands Programmed Death Ligand (PD-L) 1 and 2 are critical regulators of immune cell function and autoimmunity1C7. Genetic deficiency of in C57BL/6 and BALB/c mice leads to spontaneous lupus-like disease or autoimmune cardiomyopathy, respectively5,7, while non-obese diabetic (NOD) mice lacking either PD-1 or PD-L1 developed accelerated type 1 diabetes (T1D)4,6. Antibody blockade experiments suggest that PD-1:PD-L1 interactions, but not PD-1:PD-L2, are necessary for the maintenance of tolerance in the NOD model of T1D8C14. Several lines of evidence also suggest that the PD-1:PD-L1 pathway plays a role in maintaining islet tolerance in humans as recent onset patients with T1D have elevated gene expression levels of (PD-L1)?in whole-blood RNA analysis15. Additionally, single nucleotide polymorphisms in the or genes have been associated with T1D16C18. Finally, adverse events such as rapid autoimmunity including T1D can develop following checkpoint blockade in cancer patients19,20, further suggesting a role for this inhibitory pathway in autoimmunity. PD-1 is rapidly expressed on the surface of T cells following activation, to diminish their proliferation and effector function upon ligand binding21. Many cells throughout the body can express PD-L1 including both hematopoietic and non-hematopoietic cells22. PD-L1 is constitutively expressed on resting T cells, B cells, dendritic cells, and macrophages, and is further upregulated upon cellular activation or in response to cytokines1,23C25. Previous work suggests that PD-1:PD-L1 interactions within the pancreas may limit autoimmune diabetes6,8,26. Despite this body of knowledge, the timing, location, and specific cellular interactions that are regulated by PD-1:PD-L1 in T1D remain unclear. While previous reports have shown intra-islet PD-L1 expression OTS186935 on infiltrating mononuclear cells6,27, and suggest a role for non-hematopoietic PD-L1 expression to limit diabetes, it is unclear if cells themselves express PD-L1 and how this expression is regulated during diabetes progression. Additionally, enforcing PD-L1 expression on cells under the insulin promoter has shown conflicting results, as NOD mice were protected from disease28 while diabetes-resistant mice were rendered susceptible with insulin promoter-driven PD-L1 expression29. In this study, we measured islet cell PD-L1 expression and regulation during diabetes pathogenesis. The goals of this study were to improve upon previous strategies for flow cytometric analysis of individual, insulin-positive, live cells, and determine the specific regulators, location, and timing of PD-L1 expression in both mouse and human cells. We utilized multicolor flow cytometry and epifluorescent microscopy to measure PD-L1 expression on islet cells during spontaneous diabetes in NOD mice, and found that PD-L1 expression increased as mice approach diabetes onset, and was associated with islet infiltration. We also investigated the effect of cytokines on PD-L1 expression. The promoter contains two interferon regulatory factor-1 (IRF-1) binding sites, and previous work has shown that type 1 and type 2 interferons (IFN) induce PD-L1 expression on T cells, B cells, endothelial cells, epithelial cells, and tumor cells1,22. We found that IFN- and to a lesser extent, IFN-, promoted increased frequency of PD-L1+ cells, and increased expression on a per cell basis. Similar to our findings in mice, within human pancreas we found that increased PD-L1 expression correlated with increased inflammatory T cell infiltration in pancreatic lesions. Interestingly, we observed a minor increase in PD-L1 staining in autoantibody positive patients in the absence of overt autoimmune diabetes and found that Th1-associated cytokine IFN- modulated PD-L1 expression on isolated human islets. Taken together, this OTS186935 work illustrates that both mouse and human islet cells express PD-L1 in response to the same inflammatory cues, which may help delay islet destruction, but is ultimately insufficient to prevent cell death. Results PD-L1 expression on islet cells We first performed a time course analysis of Programmed Death Ligand 1 (PD-L1) expression on islet cells from the pancreas of NOD mice during type 1 diabetes. In NOD mice, cells were identified as side and forward.