Objective
This proposal aims to investigate early histopathological changes in the pancreas of donors at risk for T1D development before the clinical onset of the disease. We will use pancreas tissue from organ donors at risk of developing T1D who are positive for single (sAAb+) and multiple (mAAb+) autoantibodies. Using 3D light sheet fluorescence (LSFM) imaging and 2D high-parameter PhenoCycler immunofluorescence imaging approaches, we aim to identify changes in islet distribution by size and endocrine cell composition, immune infiltration scores in endocrine and exocrine compartments, pancreatic islets that are more infiltrated, as well as proinsulin and prohormone convertase PC1/3 to insulin ratios.
Background Rationale
People who develop T1D often have disease-specific immune markers in their blood months to years before diagnosis. However, the changes that occur in the pancreas during the time of such markers, especially and including T1D-associated autoantibodies, remains limited. Studies from the nPOD consortium have, however, shown that insulitis is rare (~3%) in sAAb⁺ donors, increases to ~40-50% of mAAb⁺ donors, and is present in nearly 100% of new-onset T1D cases. These findings raise many critical questions, including: Why do only a subset of high-risk individuals develop insulitis?; Why, even among mAAb⁺ donors with insulitis, is it only observed in certain islets?; and What factors among mAAb+ islets render them more susceptible to insulitis vs. sAAb+? Hence, an improved understanding of the biological processes driving T1D progression, particularly during the earliest stages of the disease, would appear essential to guide optimal development of therapeutic strategies that preserve β-cells. Apart from insulitis presence, the proportion of infiltrated islets and T cell density changes significantly throughout the course of T1D, with marked increases seen across the spectrum from single AAb+ positive to mAAb+, and eventually new-onset disease cases. Recently submitted reports involving 2D and 3D imaging data supports a notion that insulin-positive small islets have been underappreciated in previous studies of T1D pathogenesis. Previous 2D histological studies have shown that some β cells persist in the pancreas of individuals with T1D, but they are often enriched for inefficiently processed insulin. Additionally, expression of the proinsulin-processing enzyme PC1/3 is reduced in T1D pancreata, consistent with accumulated proinsulin. However, whether hormone processing defects are a cause or consequence of T1D is not clear. By analyzing tissues from donors with different levels of T1D risk, we hope to find early warning signs of disease. The proposed work will utilize two tools capable of addressing current knowledge gaps: 3D light-sheet fluorescence microscopy (LSFM) and 2D spatial phenotyping via PhenoCycler multiplex imaging.
Description of Project
Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic islets are infiltrated by a variety of immune cells, leading to the targeted destruction of the insulin-producing β-cells. Pancreatic islets play a crucial role in regulating blood glucose through the secretion of endocrine hormones. The loss of insulin-secreting β-cells results in dysglycemia (i.e., abnormal glucose levels), which ultimately forms the basis for a diagnosis of overt disease. In T1D, β-cell loss is preceded by the development of autoantibodies that target β-cell antigens—such as glutamic acid decarboxylase (GADA), insulin (IAA), zinc transporter 8 (ZnT8A), and insulinoma-associated protein-2 (IA-2A). These autoantibodies typically appear months to years before clinical symptoms and provide valuable prognostic insights. This proposal aims to investigate early structural changes in the pancreas of non-diabetic individuals with varying levels of risk for T1D, as defined by the number of diabetes-related autoantibodies. Using advanced 2-dimensional (2D) and 3D imaging techniques, we will analyze pancreas tissue samples obtained from the Breakthrough T1D supported Network for Pancreatic Organ donors with Diabetes (nPOD) biobank. Specifically, we will conduct quantitative assessments of immune cell infiltration in the islets (termed insulitis) and the surrounding exocrine tissue, islet distribution by size and endocrine cell composition, and levels of proinsulin and prohormone convertase PC1/3, which are participants in insulin processing. Defects in insulin processing will be assessed in 2D view using a high-parameter PhenoCycler immunofluorescence imaging platform. I hypothesize that multiple autoantibodies positive donors—particularly those positive for IA-2A and exhibiting insulitis—will show reduced proportions of small insulin-positive glucagon-negative islets, increased frequencies of insulin- negative glucagon-positive cell clusters, disturbances in insulin processing, and elevated levels of T cells and antigen-presenting cells. If successful, this research may offer critical insights into the mechanisms underlying T1D pathogenesis, provide support for promoting earlier risk detection, and the development of strategies to prevent or delay the onset of the disease.
Anticipated Outcome
I anticipate that tissue from mAAb+ donors—particularly those positive for IA-2A and exhibiting insulitis—will show reduced proportions of small insulin-positive, glucagon-negative endocrine structures, increased frequencies of insulin-negative, glucagon-positive cell clusters, increased frequencies of T cells and antigen presenting cells and defects in insulin processing such as increased proinsulin and decreased levels of prohormone processing enzyme. I also expect to observe that specific types of islets—based on their size and hormone content—are more likely to be attacked by the immune system in the early stages of T1D. These findings have the potential to help determine early histopathological changes in T1D, identify who is truly at the most risk for disease development, and guide future therapies aimed at protecting insulin-producing cells before the disorder fully develops.
Relevance to T1D
Understanding how T1D begins—at the cellular level—can lead to earlier diagnosis and better prevention. By studying pancreas samples from people who are at risk but not yet diabetic, this research could help to predict who will develop the disease and how to optimally organize a strategy to halt the disorder’s progress before overt (i.e., symptomatic) disease occurs. Importantly, the vast majority of T1D treatment strategies focus on seeking prevention of disease or preservation of insulin production in those recently diagnosed with the disorder are largely immune centric. While the rationale for such an approach clearly exists, my proposed efforts will diverge from this norm through the investigation of additional pathomorphological levels using rare and highly unique tissues from donors who at risk for T1D progression, remarkably novel imaging techniques. If and when successful, these experiments should benefit the community by identifying early indicators of disease progression in multiple autoantibody-positive donors at risk for T1D, potentially informing the development of new treatments to prevent islet cell death and dysfunction.