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Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that has a strong HLA association, where a number of self-epitopes have been implicated in disease pathogenesis. Human pancreatic islet-infiltrating CD4 + T cell clones not only respond to proinsulin C-peptide (PI 40-54; GQVELGGGPGAGSLQ) but also cross-react with a hybrid insulin peptide (HIP; PI 40-47 -IAPP 74-80; GQVELGGG-NAVEVLK) presented by HLA-DQ8. How T cell receptors recognize self-peptide and cross-react to HIPs is unclear. We investigated the cross-reactivity of the CD4 + T cell clones reactive to native PI 40-54 epitope and multiple HIPs fused at the same N-terminus (PI 40-54 ) to the degradation products of two highly expressed pancreatic islet proteins, neuropeptide Y (NPY 68-74 ) and amyloid polypeptide (IAPP 23-29 and IAPP 74-80 ). We observed that five out of the seven selected SKW3 T cell lines expressing TCRs isolated from CD4 + T cells of people with T1D responded to multiple HIPs. Despite shared TRAV26-1-TRBV5-1 gene usage in some T cells, these clones cross-reacted to varying degrees with the PI 40-54 and HIP epitopes. Crystal structures of two TRAV26-1 + -TRBV5-1 + T cell receptors (TCRs) in complex with PI 40-54 and HIPs bound to HLA-DQ8 revealed that the two TCRs had distinct mechanisms responsible for their differential recognition of the PI 40-54 and HIP epitopes. Alanine scanning mutagenesis of the PI 40-54 and HIPs determined that the P2, P7, and P8 residues in these epitopes were key determinants of TCR specificity. Accordingly, we provide a molecular basis for cross-reactivity towards native insulin and HIP epitopes presented by HLA-DQ8., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Autoimmune diabetes is a disorder of immune homeostasis that leads to targeted insulin-secreting islet β cell destruction characterized by insulitis. Human amylin (hA) is an important neuroendocrine hormone co-secreted with insulin by pancreatic β cells. Here, we report hA immune-modulatory action through inducing regulatory T cells. We ex vivo-treated human peripheral blood mononuclear cells (hPBMCs) with hA for 24 h and counted CD4+Foxp3+ regulatory T cells (Treg) using flow cytometry. Diabetic status was monitored and splenic Treg were measured in non-obese diabetic (NOD) male mice. NOD mice were intraperitoneally injected once daily with hA (n = 25) or solvent for control (n = 25) for 7 months continuously. Spleen tissues were collected at the end of intervention and processed for flow cytometry and Western blot. We found a 2.9-fold (p < 0.05) increase of CD4+Foxp3+ Treg in hPBMCs treated with 10 nmol/L hA compared with negative control. Incidence of diabetes in hA-treated NOD mice decreased 44% (p = 0.045) in the 6th month and 57% (p = 0.0002) in the 7th month. Meanwhile, the hA treatment induced a 1.5-fold increase of CD4+Foxp3+ Treg from mouse splenocytes (p = 0.0013). Expression of transforming growth factor-β (TGF-β) and toll-like receptor-4 (TLR-4) were upregulated in hA-treated mice. Human amylin might protect against autoimmune diabetes via the induction of CD4+Foxp3+ Treg, which suggests a novel approach to improve autoimmune conditions.

Aims/hypothesis: Islet amyloid polypeptide (IAPP) is a beta cell hormone secreted together with insulin upon glucose stimulation. IAPP participates in normal glucose regulation, but IAPP is also known for its ability to misfold and form islet amyloid. Amyloid fibrils form through smaller cell toxic intermediates and deposited amyloid disrupts normal islet architecture. Even though IAPP and amyloid formation are much discussed in type 2 diabetes, our aim was to study the significance of IAPP in type 1 diabetes., Results: Plasma IAPP levels in children and adolescents with newly diagnosed type 1 diabetes (n = 224) were analysed and concentrations exceeding 100 pmol/L (127.2-888.7 pmol/L) were found in 11% (25/224). The IAPP increase did not correlate with C-peptide levels., Conclusions/interpretation: Plasma levels of IAPP and insulin deviate in a subpopulation of young with newly-diagnosed type 1 diabetes. The determined elevated levels of IAPP might increase the risk for IAPP misfolding and formation of cell toxic amyloid in beta cells. This finding add IAPP-aggregation to the list over putative pathological factors causing type 1 diabetes.

We previously reported a peptide KS20 from islet amyloid polypeptide (IAPP) to be the target Ag for a highly diabetogenic CD4 T cell clone BDC-5.2.9. To track IAPP-reactive T cells in NOD mice and determine how they contribute to the pathogenesis of type 1 diabetes, we designed a new I-Ag7 tetramer with high affinity for BDC-5.2.9 that contains the peptide KS20. We found that significant numbers of KS20 tetramer(+) CD4 T cells can be detected in the pancreas of prediabetic and diabetic NOD mice. To verify pathogenicity of IAPP-reactive cells, we sorted KS20 tetramer(+) cells and cloned them from uncloned T cell lines isolated from spleen and lymph nodes of diabetic mice. We isolated a new KS20-reactive Th1 CD4 T cell clone that rapidly transfers diabetes. Our results suggest that IAPP triggers a broad autoimmune response by CD4 T cells in NOD mice.

Objective: To investigate autoantigens in β-cells, we have used a panel of pathogenic T-cell clones that were derived from the NOD mouse. Our particular focus in this study was on the identification of the target antigen for the highly diabetogenic T-cell clone BDC-5.2.9., Research Design and Methods: To purify β-cell antigens, we applied sequential size exclusion chromatography and reverse-phase high-performance liquid chromatography to membrane preparations of β-cell tumors. The presence of antigen was monitored by measuring the interferon-γ production of BDC-5.2.9 in response to chromatographic fractions in the presence of NOD antigen-presenting cells. Peak antigenic fractions were analyzed by ion-trap mass spectrometry, and candidate proteins were further investigated through peptide analysis and, where possible, testing of islet tissue from gene knockout mice., Results: Mass-spectrometric analysis revealed the presence of islet amyloid polypeptide (IAPP) in antigen-containing fractions. Confirmation of IAPP as the antigen target was demonstrated by the inability of islets from IAPP-deficient mice to stimulate BDC-5.2.9 in vitro and in vivo and by the existence of an IAPP-derived peptide that strongly stimulates BCD-5.2.9., Conclusions: IAPP is the target antigen for the diabetogenic CD4 T-cell clone BDC-5.2.9.