Abstract

The mechanisms underlying the major histocompatibility complex class II (MHCII) type 1 diabetes (T1D) association remain incompletely understood. We have previously shown that thymocytes expressing the highly diabetogenic, I-Ag7-restricted 4.1-T-cell receptor (TCR) are MHCII-promiscuous, and that, in MHCII-heterozygous mice, they sequentially undergo positive and negative selection/Treg deviation by recognizing pro- and anti-diabetogenic MHCII molecules on cortical thymic epithelial cells and medullary hematopoietic antigen-presenting cells (APCs), respectively. Here, we use a novel autoantigen discovery approach to define the antigenic specificity of this TCR in the context of I-Ag7. This was done by screening the ability of random epitope–GS linker–I-chain fusion pools to form agonistic peptide–MHCII complexes on the surface of I- chain-transgenic artificial APCs. Pool deconvolution, I-Ag7-binding register-fixing, TCR contact residue mapping, and alanine scanning mutagenesis resulted in the identification of a 4.1-TCR recognition motif XL(G/A)XEXE(D/E)X that was shared by seven agonistic hybrid insulin peptides (HIPs) resulting from the fusion of several different chromogranin A and/or insulin C fragments, including post-translationally modified variants. These data validate a novel, highly sensitive MHCII-restricted epitope discovery approach for orphan TCRs and suggest thymic selection of autoantigen-promiscuous TCRs as a mechanism for the murine T1D–I-Ag7-association.

Highlights

  • Compelling experimental evidence have shown that type 1 diabetes (T1D) onset and progression require the activation and recruitment of autoreactive CD4+ T cells

  • Thymocytes expressing T-cell receptor (TCR) capable of recognizing peptide–major histocompatibility complexes displaying epitopes that are selectively expressed in peripheral tissues, such as post-translationally modified epitopes or hybrid insulin peptides (HIPs), have the highest likelihood to contribute to the initiation and/or progression of autoimmunity when appropriately recruited and activated [3,4,5]

  • We first sought to use this approach to ascertain the ability of the 4.1-TCR to recognize previously described T1Drelevant autoantigens [19, 20], including pro-insulin (INS), chromogranin A (ChrA), islet amyloid polypeptide (IAPP), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), glutamic acid decarboxylase 65 (GAD65), zinc transporter 8 (ZnT8), and islet tyrosine phosphatase 2 (I-A2)

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Summary

Introduction

Compelling experimental evidence have shown that type 1 diabetes (T1D) onset and progression require the activation and recruitment of autoreactive CD4+ T cells. These T cells orchestrate the activation of downstream effectors of diabetogenic autoimmunity, such as B cells and beta cell-cytotoxic CD8+ T cells [1]. In non-autoimmune-prone individuals, autoreactive T cells are either deleted (thymocyte negative selection) or programmed to become autoreactive regulatory T cells [2]. Both pathways require the recognition of cognate autoantigenic epitopes that are either expressed in thymic medullary epithelial cells or are delivered to the thymus by bone. Whereas alleles encoding DQb chains carrying Ala, Val, or Ser at position

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