ABSTRACT The destruction of β cells in Type 1 Diabetes (T1D) is mediated by islet-reactive cytotoxic CD8+ T cells that infiltrate the pancreatic islet. Extracellular Vesicles (EVs) are bilayer membrane structures of diameters 30 – 1000 nm released into the blood by cells throughout the body, at concentrations on the order of 1010 per ml. Their molecular content of proteins, dsDNA oligomers, microRNAs (miRNAs), mRNAs, and other analytes, may play multiple functional roles via EV trafficking, and may also provide a diagnostic report back on the disease site or tissue of origin. As such, EVs can serve as potential sources of T1D biomarkers, perhaps even providing insights into the genetic and functional characteristics of the pancreatic islet microenvironment. We propose a program pursuing T1D biomarkers from islet-derived vesicles that couples our cutting-edge, microfluidic nanoparticle enrichment technology to a suite of multiparameter analytical methods. In AIM 1, we utilize a distinct set of bulk and single-EV (sEV) transcriptomic and proteomic methods to comprehensively investigate the molecular contents of islet cells from healthy donors and individuals with T1D, and EVs derived from those cultured cells. This will yield a set of proteins and RNAs that can uniquely identify EVs from β cells, as well as insights on the heterogeneity of the islet EVs. Here we also begin to resolve the molecular signatures that are unique to T1D pathogenesis. The study of islet-infiltrating CD8+ T cells is often impeded by the limited access to donor islet tissues. The fact that islet-infiltrating CD8+ T cells shed EVs into the blood stream provides a unique opportunity for monitoring the activities of the islet-infiltrating T cells with circulating EVs. In AIM 2, we propose a nanoparticle technology to capture T-cell receptor (TCR) bearing EVs in circulation. We plan to characterize these EVs from healthy and T1D sera to determine the EV populations that arise from pancreatic islet infiltrating CD8+ T cells. Monitoring these EV populations can reveal key local events in the pancreas such as T cell activation and cytotoxicity. As we anticipate a low blood abundance of islet-derived EVs, in AIM 3, we provide a microfluidics-enabled solution to enrich EVs derived from pancreatic β cells and infiltrating CD8+ T cells from human sera. This integrated sample processing workflow allows automatic isolation of islet-derived EVs from limited volumes of serum, with minimal hands on time. Analysis of banked serum specimens will yield a set of candidate biomarkers associated with T1D development, which will be validated and refined with an independent cohort. Importantly, the proposed study will not only provide EV- based T1D biomarkers, but also generate datasets that are of high value to the design and development of antigen-specific immunotherapies for T1D. Our multidisciplinary team is comprised of scientists and clinicians with expertise in EV biology, T1D pathogenesis,...