ABSTRACT Type 1 diabetes (T1D) is a major autoimmune disease that poses significant problems to afflicted individuals, the development of effective therapeutic interventions, and public health initiatives at large. Initiated and perpetuated by a complex interplay of genetic and environmental risk factors, insulin-secreting pancreatic beta- cells are progressively destroyed by aberrant immune responses leading to elevated blood glucose levels as well as serious disturbances of protein, fat and carbohydrate metabolism. Currently, no cure or effective prevention is available, and despite insulin treatment, serious long-term complications are frequent. Much of the progress in T1D research attained over the past four decades comes from the study of animal models yet despite many insights generated, a genuine appreciation of the human disease requires better knowledge about pathological processes unfolding directly in the human pancreas. The pursuit of this goal, however, faces two major challenges: the difficulty to obtain suitable tissues from pre/diabetic donors; and the fact that pertinent pathological alterations are often mild and distributed in a variegated fashion across the pancreas. Thus, information obtainable by traditional analysis of individual pancreatic tissue sections is limited. In the present proposal, which constitutes a direct continuation of our previously NIH-funded work on pancreatic histopathology, we will address these challenges by leveraging the resources of the Network of Pancreatic Organ Donors with Diabetes (nPOD) tissue repository together with the recent development or adaptation of two novel, highly multiplexed tissue staining technologies, and with the combined expertise and long-standing interactions of the von Herrath and Homann laboratories. This particular constellation of resources, technologies and experience allows us to tackle two critical knowledge gaps in specific: the detailed in situ phenotypes, functionalities, and interactions of major immune and endocrine cell populations in the pre/diabetic pancreas; and the contribution of specific cytokine signatures to as revealed in these cells to the hyperexpression of MHC-I, a histopathognomonic feature of T1D. Accordingly, we have developed a research plan that employs high-dimensional multiplexing strategies to interrogate the precise phenotype, activation status, contextual microanatomical localization and distribution of CD8+ and CD4+T cells, antigen presenting cells and B cells, and endocrine cells across the pre/diabetic human pancreas (Aim 1), and that will correlate major cytokine expression patterns (type 1 interferons, IFN?, TNF?) and functional signatures with their cellular sources and targets as potential causes for MHC-I hyperexpression (Aim 2). Moreover, by conducting the work with tissue specimens from the same donors using two different multiplexing technologies in two different laboratories, we seek to achieve robustness and reproducibility of experimental readouts. Altogether, we propose that an integrated analysis of up to 30 phenotypic, functional and topological parameters will define spatiotemporal pathogenetic landmarks that will permit a partial reconstruction of the highly dynamic in situ autoimmune processes operative in T1D disease. Mapping the histopathological landscape of the pre/diabetic human pancreas in exquisite detail not only is important for our conception of disease pathogenesis but ultimately may inform the development of novel or improved prevention strategies and treatment modalities.