Recent evidence put forth by our group and others suggests type 1 diabetes (T1D) pathogenesis involves a combination of immune, islet, and acinar pancreas defects. Hence, there is a need to understand each of these facets in concert, linking islet and acinar cell function with precise cellular composition of the insulitis lesion, throughout T1D progression. We hypothesize that intrinsic β-cell stress triggers innate inflammation resulting in immune trafficking and localized islet and acinar pathology, culminating in immune-mediated destruction of β- cells. To address this hypothesis, Project 1 will utilize high parameter imaging to provide deep profiling of immune and pancreas phenotypes including across projects (SH2B3 [collaboration with Project 2]; HLA class- II, CD226, and SIRPG [collaboration with Project 3]), and address how immune infiltration affects islet and acinar cell composition and function. In Aim 1, we propose to perform MACSima ultra high-content imaging to assess in situ expression of 70 immune and pancreas cell markers, including vascular and lymphatic annotation with signatures of inflammation, extravasation/trafficking, (collaboration with Project 2) or immune cell residency in donors with well-characterized insulitis. We will compare inflamed vs non-inflamed islets, within and across donors, to determine how insulitis correlates with, islet and acinar cell phenotypes. Aim 2 further proposes single cell multi-omics analysis involving spectral flow cytometry as well as scRNA-Seq with antibody-based CITE-Seq and paired T cell receptor sequencing (TCR-Seq) for linked transcriptomic, proteomic, and repertoire information on CD45+ and CD45- cell fractions isolated from freshly acquired organ donor pancreas tissues. The transcriptomic markers affected in slices with and without insulitis will be correlated to changes in cell status. Finally, in Aim 3 we propose to assay islet and acinar tissue function using our novel pancreas slice culture platform. Specifically, we will apply hypothesized diabetogenic conditions (type 1 or 2 interferons, glucotoxicity) to pancreas slices followed by T cell “avatars” expressing islet antigen-specific TCRs (collaboration with Project 3) to evaluate the impact of this “investigator-initiated insulitis” on pancreas secretory function and cellular signaling in situ. We will quantitate real time live cellular imaging of Ca2+ signalling activity within islet and acinar tissue areas from T1D, AAb+, and control organ donor pancreata in response to established endocrine (glucose, arginine, KCl) and exocrine stimuli (carbachol). Through intermittent sampling of the perifusate, we will also test (pro)hormone (proinsulin, insulin, glucagon) and enzyme (lipase, trypsin) secretion. We will then correlate these functional data with molecular features via MACSima imaging and scRNA-Seq/CITE-Seq/TCR-Seq, facilitating direct correlation of phenotype and function. We expect to identify altered molecular pathways and ...