PROJECT SUMMARY/ABSTRACT Type 1 Diabetes (T1D) is an autoimmune disease caused by aberrant T-cell mediated targeted destruction of insulin-producing beta cells in the pancreas, resulting in loss of blood glucose regulation, with increased long- term risks of vascular and neuropathic comorbidities. Despite the fact that T1D is one of the most studied organ- specific autoimmune diseases, the various strategies aimed at intervention, prevention, or reversal of this disease have failed to succeed due to incomplete knowledge about the precise mechanisms of their action, as only peripheral assessments of systemic impacts (e.g., circulating cytokine changes, C-peptide levels) are feasible. This lack of mechanistic understanding of these interventions, as well as substantial time and cost of clinical trials, is a profound obstacle in improving therapeutic outcomes. To address these significant knowledge gaps, there is a substantial clinical need to develop human-based ex vivo systems capable of intimately studying the interplay of islets and immune cells, as well as the contribution of environmental factors on immune cell activation, homing, and cytotoxicity. The primary hypothesis of this proposal is that the development of an islet- immune platform has the potential to provide unique insight into T1D, with investigation of activation pathways and screening of interventional approaches. Thus, the objective of this proposal is to engineer, validate, and utilize a unique in vitro 3-D platform for the interrogation of human T1D immunopathogenesis by converging innovative cells with biomaterials, in situ imaging, and microphysiological systems (MPS). Aim 1 will seek to establish and validate this 3D biomaterial-based co-culture platform. To validate the system, a tiered approach, building from single antigen murine model cells to human T1D-antigen cells, will be employed. Once validated, Aim 2 will translate this platform to study human-centric T1D-relevant pathways and interventions. Finally, Aim 3 will seek to integrate spatial and fluidic features by translating the 3D material to an established microphysiological system (MPS) platform, which will permit the study of T cell migration from a fluidic microenvironment to the beta cell niche. Results from this proposal should provide a validated and enabling tool for the study of human T1D-relevant pathophysiology, interventions, and therapeutics. While the proposed field of application for this platform is T1D, other autoimmune diseases can benefit from this engineered benchtop platofrm, as they share homologous hallmarks of immune cell dysregulation.