One in Ten CD8+ Cells in the Pancreas of Living Individuals With Recent-Onset Type 1 Diabetes Recognizes the Preproinsulin Epitope PPI15-24

Abstract

In type 1 diabetes (T1D), a lifelong autoimmune disease, T cells infiltrate the islets and the exocrine pancreas in high numbers. CD8⁺ T cells are the main cell type found in the insulitic lesion, and CD8⁺ T cells reactive against β-cell antigens have been detected in peripheral blood and in the pancreas of patients with short- or long-term disease. In the Diabetes Virus Detection (DiViD) study, researchers collected pancreatic tissue, by pancreatic tail resection, from living patients with recent-onset T1D. These tissues have been extensively studied by the scientific community, but the autoreactive nature of the T-cell infiltrate has remained unexplored. Our objective was to determine the number and localization of these cells in pancreas samples obtained through the DiViD study. Here, we demonstrate the presence of high frequencies of CD8⁺ T cells reactive against a highly relevant epitope derived from the preproinsulin signal peptide in pancreatic tissue samples from these donors. We also show the heterogeneity of islet distribution and CD8⁺ T-cell infiltration. Our findings contribute to the current limited existing knowledge of T-cell reactivity in the pancreas of donors with recent-onset T1D and indicate that antigen-specific therapies directed toward preproinsulin could have high clinical impact.

Figure 1

Heterogeneity of islet distribution and CD8⁺ T-cell infiltration in the DiViD cases. Bar graphs and tables show: the mean number of islets per section (# islets/section) (A); CD8 T-cell density in the whole tissue section expressed as number of CD8⁺ cells/mm² (B); and mean number of CD8⁺ cells per islet (# CD8⁺ cells/islet) (C). Error bars represent the SD.

Figure 2

Representative images from immunofluorescence in situ tetramer staining of pancreas sections from DiViD-2. Staining was performed for the indicated tetramer (positive cells are marked with red crosses in whole-slide images), glucagon (white staining), and DAPI (blue staining). Consecutive sections are shown to illustrate the staining and quantification of antigen-specific cells reactive against PPI, CMV, EBV, FLU, EV, and a negative control without tetramer (no tetramer). Scale bars, 1,000 µm in whole-slide images. Magnified images show staining for tetramer (red) and CD8 (green). Yellow arrows indicate tetramer-positive cells. Scale bars, 25 µm for PPI; 20 µm for CMV.

Figure 3

Quantification of tetramer-positive cells in the entire tissue section. A–D: Bar graphs show the percentage of antigen (Ag)-specific CD8⁺ T cells for each tissue section and individual (DiViD-1, 2, 4, and 5). PPI, CMV, EBV, FLU, EV, and negative control without tetramer (no tetramer). E: The summary table shows the percentage of Ag-specific CD8⁺ cells for each antigen and patient.