Type 1 diabetes (T1D) results from an autoimmune disorder characterized by T-cell-mediated destruction of pancreatic beta cells. While the role of auto-reactive T cells in targeting beta cells has been well defined, the pathological mechanisms operating in beta cells remains poorly understood. To investigate beta cell stress responses during disease progression but before diabetes onset, we performed single-cell RNA-seq (scRNA-seq) on islet cells from 8 week and 14 nonobese diabetic(NOD) mice. This analysis revealed two transcriptionally-distinct sub-populations of beta cells in the 14 week beta cells, which had hallmarks of stress response senescence and the senescence-associated secretory phenotype (SASP). The SASP consists of proinflammatory cytokines, chemokines, extracellular matrix factors, metalloproteases and growth factors secreted from senescent cells that regulate the tissue microenvironment and provide signals for immune surveillance and senescent cell clearance. As the SASP has been previously implicated in the pathogenesis of a variety of age-related diseases, we hypothesized that accumulation of SASP beta cells could be a contributing factor to disease progression in T1D. Consistent with our hypothesis, further studies revealed that targeted elimination of SASP beta cells with small molecule inhibitors prevented T1D in the NOD mouse model.To determine whether beta cells undergo senescence and SASP during the natural history of T1D in humans, we have already performed two studies on a cohort of nPOD specimens from similarly age nondiabetic, autoantibody-positive and T1D donors. Xgal staining of cryosections was used to detect the activity of senescence-associated βgalactosidase (SA-βgal), a sensitive and widely used biomarker for senescent cells in vivo. This analysis showed dramatically elevated SA-βgal activity in the islets of T1D donors as compared with nondiabetic and autoantibody-positive donors, consistent with a higher number of senescent beta cells. We also performed immunohistochemistry for senescence (CDKN1A, CDKN2A, Ser139 phospho-H2A.X) and SASP (IL-6, IL-8, MMP9, SERPINE1) markers. While nondiabetic donors generally had no expression of these markers in beta cells, autoantibody-positive and T1D donors had significantly increased levels. These data extend and build upon our findings in the NOD mouse model and suggest involvement of beta cell SASP inthe progression of human T1D. In order to test the hypothesis that beta cell SASP plays a role in T1D pathogenesis in humans, as well as to investigate whether hallmarks of SASP could provide novel biomarkers for disease progression, the following experiments requiring nPOD specimens are planned:1) Evaluate exosome RNA biomarkers in nPOD donor serum/plasma. We will evaluate candidate exosome small noncoding RNA biomarkers identified from small RNA-seq analysis of SASP islet exosome RNA human serum from nondiabetic, autoantibody-positive and T1D donors; and 2) Evaluate therapy for targeted elimination of human SASP beta cells using nPOD donor islets. Using our current small molecule therapy for clearance of SASP beta cells in NOD mice and islets, we will perform similar studies on nPOD donor islets in culture to provide proof-of-concept of this therapy for ablating human SASP beta cells.