Co-registration of Cell Organization, Phenotype and Function in the Human Pancreas During Type 1 Diabetes

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. In addition to autoimmunity and β-cell death, it has
become clear that T1D is characterized by a whole-organ pathology with reduced pancreas size, reduced
exocrine enzyme levels in serum, and altered α- and β-cell function, including impaired insulin processing, even
in the islet autoantibody positive (AAb+) pre-T1D condition. Hence, there is a need to understand each of these
facets in concert, linking cellular phenotype and function, together with studies of the human pancreas tissue
microenvironment, throughout T1D progression. We hypothesize that alterations to β-cell status and its
surrounding environment are key determinants of impaired β-cell function, exocrine function, and infiltration
(insulitis). We propose to assay islet and acinar tissue function using our novel pancreas slice culture platform
(Aim 1a) to test (pro)hormone (proinsulin, insulin, glucagon) and enzyme (lipase, trypsinogen) secretion from
T1D, AAb+, and control organ donor pancreata in response to established endocrine (glucose, arginine, KCl)
and exocrine stimuli (cholecystokinin, carbachol). We will correlate these functional data with molecular features
via scRNAseq (single cell RNA sequencing) with antibody-based CITEseq (Cellular Indexing of Transcriptomes
and Epitopes) and scATACseq (single cell assay for transposase-accessible chromatin sequencing); this, for the
purpose of cell identification together with transcriptomic and epigenomic analyses (Aims 1b). Pancreas slices
will also be subjected to these same stimulatory conditions for live cellular imaging of Ca2+ signalling activity
within islet and acinar tissue areas in real time (Aim 2a), then fixed and analyzed by imaging mass cytometry
(IMC). We will assess in situ expression of 120 immune and pancreas cell markers with cellular resolution (Aim
2b), followed by spatial and temporal analysis of IMC data to determine how islet, immune and acinar cell
phenotypes correlate with tissue and cellular function, using our histoCAT (histology topography cytometry
analysis toolbox). This will enable computational analysis with 3D reconstruction from serial sections (Aim 2c).
Finally, in Aim 3, human donor pancreas slices will be subjected to diabetogenic stimuli (inflammatory cytokines,
glucotoxicity) and interventions targeting β-cell stress [imatinib (tyrosine kinase inhibitor), MSL-7 (autophagy
enhancer), exenatide (GLP-1 receptor agonist)], and similarly evaluated by single cell and IMC profiling. With
over 14 years of experience in procurement of transplant-quality human pancreata through the Network for
Pancreatic Organ donors with Diabetes (nPOD), we are uniquely poised to perform the proposed studies. We
expect to identify altered molecular pathways and tissue features linking β-cell, whole-islet, and acinar cell
phenotypes with cellular function in AAb+ and T1D pancreata. We anticipate these same defects will arise in
slices subjected to diabetogenic stimuli, providing a platform to test known and novel candidates for targeted
intervention to reduce β-cell stress, restore islet and acinar cell function, as well as prevent disease progression.

Grant Number: 5R01DK131059-05
NIH Institute/Center: NIH
Principal Investigator: MARK ATKINSON