Studies of individual T cells receptors (TCRs) have shed some light on structural features that underlie self-reactivity. However, general rules that predict whether TCRs are self-reactive have not been fully elucidated. Analyses of thymocytes expressing all Vβ family members show that the interfacial hydrophobicity of amino acids at positions 6 and 7 of the CDR3β segment robustly promotes the development of self-reactive TCRs. An index based on these findings distinguishes Vβ2+, Vβ6+ and Vβ8.2+ regulatory T cells from conventional T cells, as well as T cells selected on a major histocompatibility complex (MHC) allele associated with mouse type-1 diabetes from those selected on a non-autoimmune promoting MHC. These results provide a means for distinguishing normal and autoimmune-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-g, 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 × 108 TCRB 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.

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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 HLADQ8trans-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 HLADQ8trans. 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.Type 1 diabetes (T1D) is an autoimmune disease characterized by autoreactive T-cell-mediated destruction of the insulin-producing pancreatic b-cells (1-4). The search for naturally processed and presented epitopes (NPPE) for high-risk HLA class II as a target for autoreactive CD4 T cells in T1D has been focus of attention over the years. Most attention was given to HLA-DR-binding epitopes (5,6), whereas HLA-DQ-binding epitopes deserve investigation, too. Indeed, subjects heterozygous for HLA-DQ2 and -DQ8 are endorsed with by far the highest risk for development of T1D, but what functional consequences explain this synergistically increased risk compared with a double dose of HLA-DQ2 or -DQ8 remain unclear. We previously revealed the unique peptide binding properties of HLA-DQ molecules composed of the products of DQA1*0201 (coding for the a-chain of DQ2) and DQB1*0302 (coding for the b-chain of DQ8), the so-called HLA-DQ8trans molecule (7-9). The islet epitopes presented by HLA-DQ8trans are largely unknown. Importantly, HLA-DQ8cis/trans-restricted CD4 T-cell clones have been isolated from human insulitis lesions, and T-cell autoreactivity was confirmed for several proinsulin peptides, underscoring the potential relevance of preproinsulin (PPI) peptides presented by HLA-DQ in diabetogenesis (10-13). We contend that knowledge of the HLA-DQ8trans islet peptidome provides insight in the mechanism by which HLA-DQ2/8 hete...

SummaryType 1 diabetes results from destruction of insulin-producing beta cells in pancreatic islets and is characterized by islet cell autoimmunity. Autoreactivity against non-beta cell-specific antigens has also been reported, including targeting of the calcium-binding protein S100β. In preclinical models, reactivity of this type is a key component of the early development of insulitis. To examine the nature of this response in type 1 diabetes, we identified naturally processed and presented peptide epitopes derived from S100β, determined their affinity for the human leucocyte antigen (HLA)-DRB1*04:01 molecule and studied T cell responses in patients, together with healthy donors. We found that S100β reactivity, characterized by interferon (IFN)-γ secretion, is a characteristic of type 1 diabetes of varying duration. Our results confirm S100β as a target of the cellular autoimmune response in type 1 diabetes with the identification of new peptide epitopes targeted during the development of the disease, and support the preclinical findings that autoreactivity against non-beta cell-specific autoantigens may have a role in type 1 diabetes pathogenesis.

Aims/hypothesis Antigen-specific therapy aims to modify inflammatory T cell responses in type 1 diabetes and restore immune tolerance. One strategy employs GAD65 conjugated to aluminium hydroxide (GAD-alum) to take advantage of the T helper (Th)2-biasing adjuvant properties of alum and thereby regulate pathological Th1 autoimmunity. We explored the cellular and molecular mechanism of GAD-alum action in the setting of a previously reported randomised placebo-controlled clinical trial conducted by Type 1 Diabetes TrialNet. Methods In the clinical trial conducted by Type 1 Diabetes TrialNet, participants were immunised with 20 μg GAD-alum (twice or three times) or alum alone and peripheral blood mononuclear cell samples were banked at baseline and post treatment. In the present study, GAD-specific T cell responses were measured in these samples and GAD-specific T cell lines and clones were generated, which were then further characterised. Results At day 91 post immunisation, we detected GAD-specific IL-13 + CD4 T cell responses significantly more frequently in participants immunised with GAD-alum (71% and 94% treated twice or three times, respectively) compared with those immunised with alum alone (38%; p = 0.003 and p = 0.0002, respectively) accompanied by high secreted levels of IL-13, IL-4 and IL-5, confirming a GAD-specific, GAD-alum-induced Th2 response. Of note, GAD-specific, IL-13 + CD4 T cells observed after immunisation co-secreted IFN-γ, displaying a bifunctional Th1/Th2 phenotype. Single-cell transcriptome analysis identified IL13 and IFNG expression in concert with the canonical Th2 and Th1 transcription factor genes GATA3 and TBX21, respectively. T cell receptor β-chain (TCRB) CDR3 regions of GAD-specific bifunctional T cells were identified in circulating naive and central memory CD4 T cell pools of non-immunised participants with newonset type 1 diabetes and healthy individuals, suggesting the potential for bifunctional responses to be generated de novo by GAD-alum immunisation or via expansion from an existing public repertoire. Conclusions/interpretation GAD-alum immunisation activates and propagates GAD-specific CD4 T cells with a distinctive bifunctional phenotype, the functional analysis of which might be important in understanding therapeutic responses.

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