Through analyses of β-chain variable regions in self-reactive T cell antigen receptors, Huseby and colleagues show that an index based on the interfacial hydrophobicity of amino acids at positions 6 and 7 of the CDR3β segment distinguishes self-reactive T cell antigen receptors. Studies of individual T cell antigen receptors (TCRs) have shed some light on structural features that underlie self-reactivity. However, the general rules that can be used to predict whether TCRs are self-reactive have not been fully elucidated. Here we found that the interfacial hydrophobicity of amino acids at positions 6 and 7 of the complementarity-determining region CDR3β robustly promoted the development of self-reactive TCRs. This property was found irrespective of the member of the β-chain variable region (Vβ) family present in the TCR or the length of the CDR3β. An index based on these findings distinguished Vβ2+, Vβ6+ and Vβ8.2+ regulatory T cells from conventional T cells and also distinguished CD4+ T cells selected by the major histocompatibility complex (MHC) class II molecule I-Ag7 (associated with the development of type 1 diabetes in NOD mice) from those selected by a non–autoimmunity-promoting MHC class II molecule I-Ab. Our results provide a means for distinguishing normal T cell repertoires versus autoimmunity-prone T cell repertoires.

Posttranslational modification (PTM) of islet autoantigens can cause lack of central tolerance in type 1 diabetes (T1D). Tissue transglutaminase (tTG), involved in PTM of gluten antigens in celiac disease, creates negatively charged peptides favored by T1D-predisposing HLA-DQ molecules, offering an attractive candidate modifying islet autoantigens in T1D. The highly predisposing HLA-DQ8cis/trans molecules share preferences for negatively charged peptides, as well as distinct peptide-binding characteristics that distinguish their peptide-binding repertoire. We screened islet autoantigens with the tTG substrate motif for candidate-modified epitopes binding to HLA-DQ8cis/trans and identified 31 candidate islet epitopes. Deamidation was confirmed for 28 peptides (90%). Two of these epitopes preferentially bound to HLA-DQ8cis and six to HLA-DQ8trans upon deamidation, whereas all other peptides bound equally to HLA-DQ8cis/trans. HLA-DQ8cis–restricted T cells from a new-onset T1D patient could only be generated against a deamidated proinsulin peptide, but cross-reacted with native proinsulin peptide upon restimulation. The rate of T-cell autoreactivity in recent-onset T1D patients extended from 42% to native insulin to 68% adding responses to modified proinsulin, versus 20% and 37% respectively, in healthy donors. Most patients responded by interferon-γ, whereas most healthy donors produced interleukin-10 only. Thus, T-cell autoreactivity exists to modified islet epitopes that differs in quality and quantity between patients and healthy donors.

Defects in T cell receptor (TCR) repertoire are proposed to predispose to autoimmunity. Here we show, by analyzing >2 × 10⁸TCRB sequences of circulating naive, central memory, regulatory and stem cell-like memory CD4⁺ T cell subsets from patients with type 1 diabetes and healthy donors, that patients have shorter TCRB complementarity-determining region 3s (CDR3), in all cell subsets, introduced by increased deletions/reduced insertions during VDJ rearrangement. High frequency of short CDR3s is also observed in unproductive TCRB sequences, which are not subjected to thymic culling, suggesting that the shorter CDR3s arise independently of positive/negative selection. Moreover, TCRB CDR3 clonotypes expressed by autoantigen-specific CD4⁺ T cells are shorter compared with anti-viral T cells, and with those from healthy donors. Thus, early events in thymic T cell development and repertoire generation are abnormal in type 1 diabetes, which suggest that short CDR3s increase the potential for self-recognition, conferring heightened risk of autoimmune disease.

HLA-DQ2/8 heterozygous individuals are at far greater risk for type 1 diabetes (T1D) development by expressing HLA-DQ8trans on antigen-presenting cells compared with HLA-DQ2 or -DQ8 homozygous individuals. Dendritic cells (DC) initiate and shape adaptive immune responses by presenting HLA-epitope complexes to naïve T cells. To dissect the role of HLA-DQ8trans in presenting natural islet epitopes, we analyzed the islet peptidome of HLA-DQ2, -DQ8, and -DQ2/8 by pulsing DC with preproinsulin (PPI), IA-2, and GAD65. Quality and quantity of islet epitopes presented by HLA-DQ2/8 differed from -DQ2 or -DQ8. We identified two PPI epitopes solely processed and presented by HLA-DQ2/8 DC: an HLA-DQ8trans–binding signal-sequence epitope previously identified as CD8 T-cell epitope and a second epitope that we previously identified as CD4 T-cell epitope with increased binding to HLA-DQ8trans upon posttranslational modification. IA-2 epitopes retrieved from HLA-DQ2/8 and -DQ8 DC bound to HLA-DQ8cis/trans. No GAD65 epitopes were eluted from HLA-DQ. T-cell responses were detected against the novel islet epitopes in blood from patients with T1D but scantly detected in healthy donor subjects. We report the first PPI and IA-2 natural epitopes presented by highest-risk HLA-DQ8trans. The selective processing and presentation of HLA-DQ8trans–binding islet epitopes provides insight in the mechanism of excessive genetic risk imposed by HLA-DQ2/8 heterozygosity and may assist immune monitoring of disease progression and therapeutic intervention as well as provide therapeutic targets for immunotherapy in subjects at risk for T1D.

Autoimmune diseases result from autoantigen-mediated activation of adaptive immunity; intriguingly, autoantigen-specific T cells are also present in healthy donors. An assessment of dynamic changes of this autoreactive repertoire in both health and disease is thus warranted. Here we investigate the physiological versus pathogenic autoreactive processes in the context of Type 1 diabetes (T1D) and one of its landmark autoantigens, glutamic acid decarboxylase 65 (GAD65). Using single cell gene expression profiling and tandem T cell receptor (TCR) sequencing, we find that GAD65-specific true naïve cells are present in both health and disease, with GAD65-specific effector and memory responses showing similar ratios in healthy donors and patients. Deeper assessment of phenotype and TCR repertoire uncover differential features in GAD65-specific TCRs, including lower clonal sizes of healthy donor-derived clonotypes in patients. We thus propose a model whereby physiological autoimmunity against GAD65 is needed during early life, and that alterations of these physiological autoimmune processes in predisposed individuals trigger overt Type 1 diabetes.