GM-CSF producing autoreactive CD4+ T cells in type 1 diabetes.

The phenotype of autoreactive T cells in type 1 diabetes is described as Th1, Th17 and/or Th21, but is largely uncharacterized. We combined multi-parameter cytokine profiling and proliferation, and identified GM-CSF producing cells as a component of the response to beta cell autoantigens proinsulin and GAD65. Overall cytokine profiles of CD4+ T cell were not altered in type 1 diabetes. In contrast, patients with recent onset type 1 diabetes had increased frequencies of proinsulin-responsive CD4+CD45RA- T cells producing GM-CSF (p=0.002), IFNγ (p=0.004), IL-17A (p=0.008), IL-21 (p=0.011), and IL-22 (p=0.007), and GAD65-responsive CD4+CD45RA- T cells producing IL-21 (p=0.039). CD4+ T cells with a GM-CSF+IFNγ-IL-17A-IL-21-IL-22- phenotype were increased in patients for responses to both proinsulin (p=0.006) and GAD65 (p=0.037). GM-CSF producing T cells are a novel phenotype in the repertoire of T helper cells in type 1 diabetes and consolidate a Th1/Th17 pro-inflammatory pathogenesis in the disease.

Tetraspanin 7 autoantibodies in type 1 diabetes.

AIMS/HYPOTHESIS: Autoantibodies to pancreatic beta cell proteins are markers of asymptomatic type 1 diabetes. The aim was to determine whether autoantibodies to the beta cell protein tetraspanin 7 would improve the ability to identify autoimmunity against pancreatic beta cells. METHODS: Full length and external domain fragments of tetraspanin 7 were expressed as luciferase-tagged fusion proteins and used in immunoprecipitation assays to measure autoantibodies in samples from 363 patients with type 1 diabetes at onset of disease, 503 beta cell autoantibody negative first-degree relatives of patients, and 212 relatives with autoantibodies to insulin, glutamic acid decarboxylase, insulinoma antigen 2 or zinc transporter 8. RESULTS: Antibody binding was observed against the full length and external domains of tetraspanin 7, and was strongest against the full length protein. Autoantibodies that could be inhibited by untagged tetraspanin 7 were detected in 5 (1%) of 503 autoantibody negative relatives, 3 (3.2%) of 94 autoantibody negative patients, 95 (35.3%) of 269 autoantibody positive patients, 1 (1%) of 98 single autoantibody positive relatives and 25 (21.9%) of 114 multiple autoantibody positive relatives. Progression to diabetes did not differ between multiple autoantibody positive relatives with and without tetraspanin 7 autoantibodies. CONCLUSIONS/INTERPRETATION: Tetraspanin 7 is an autoantigen in type 1 diabetes. Tetraspanin 7 autoantibodies are a marker of type 1 diabetes, but provide minor additional value to existing autoantibodies in identifying beta cell autoimmunity.

Maternal Type 1 Diabetes Reduces Autoantigen-Responsive CD4+ T Cells in Offspring.

Autoimmunity against pancreatic ß-cell autoantigens is a characteristic of childhood type 1 diabetes (T1D). Autoimmunity usually appears in genetically susceptible children with the development of autoantibodies against (pro)insulin in early childhood. The offspring of mothers with T1D are protected from this process. The aim of this study was to determine whether the protection conferred by maternal T1D is associated with improved neonatal tolerance against (pro)insulin. Consistent with improved neonatal tolerance, the offspring of mothers with T1D had reduced cord blood CD4+ T-cell responses to proinsulin and insulin, a reduction in the inflammatory profile of their proinsulin-responsive CD4+ T cells, and improved regulation of CD4+ T cell responses to proinsulin at 9 months of age, as compared with offspring with a father or sibling with T1D. Maternal T1D was also associated with a modest reduction in CpG methylation of the INS gene in cord blood mononuclear cells from offspring with a susceptible INS genotype. Our findings support the concept that a maternal T1D environment improves neonatal immune tolerance against the autoantigen (pro)insulin.

Association of Dendritic Cell Signatures With Autoimmune Inflammation Revealed by Single-Cell Profiling.

OBJECTIVE: To identify single-cell transcriptional signatures of dendritic cells (DCs) that are associated with autoimmunity, and determine whether those DC signatures are correlated with the clinical heterogeneity of autoimmune disease. METHODS: Blood-derived DCs were single-cell sorted from the peripheral blood of patients with rheumatoid arthritis, systemic lupus erythematosus, or type 1 diabetes as well as healthy individuals. DCs were analyzed using single-cell gene expression assays, performed immediately after isolation or after in vitro stimulation of the cells. In addition, protein expression was measured using fluorescence-activated cell sorting. RESULTS: CD1c+ conventional DCs and plasmacytoid DCs from healthy individuals exhibited diverse transcriptional signatures, while the DC transcriptional signatures in patients with autoimmune disease were altered. In particular, distinct DC clusters, characterized by up-regulation of TAP1, IRF7, and IFNAR1, were abundant in patients with systemic autoimmune disease, whereas DCs from patients with type 1 diabetes had decreased expression of the regulatory genes PTPN6, TGFB, and TYROBP. The frequency of CD1c+ conventional DCs that expressed a systemic autoimmune profile directly correlated with the extent of disease activity in patients with rheumatoid arthritis (Spearman's r = 0.60, P = 0.03). CONCLUSION: DC transcriptional signatures are altered in patients with autoimmune disease and are associated with the level of disease activity, suggesting that immune cell transcriptional profiling could improve our ability to detect and understand the heterogeneity of these diseases, and could guide treatment choices in patients with a complex autoimmune disease.

CD4+ T cell activation, function, and metabolism are inhibited by low concentrations of DMSO.

Dimethyl sulfoxide (DMSO) is a polar organic solvent used in a wide range of biological applications. DMSO is routinely used as a cryoprotectant for long-term cell freezing as well as to dissolve peptides and drugs for immune cell functional assays. Here, human CD4+ T cell activation, cytokine production, proliferation, and metabolism were investigated after stimulation in the presence of 0.01% to 1%, DMSO, representing concentrations commonly used in vitro. Surface expression of the activation markers CD69, CD25 and CD154 after polyclonal activation of CD4+ T cells was inhibited by 0.25% or higher concentrations of DMSO. The frequencies of IL-21+, IL-4+, and IL-22+ CD4+ T cells, following polyclonal activation were variably inhibited by DMSO at concentrations ranging from 0.25% to 1%, whereas IFNγ+ cells were unaffected. CD4+ T cell proliferation after anti-CD3 or antigen stimulation was inhibited by 0.5% DMSO and abolished by 1% DMSO. After polyclonal stimulation, glucose uptake was inhibited in the presence of 1% DMSO, but only minor effects on CD4+ T cell respiration were observed. Consistent with the immune effects, the gene expression of early signaling and activation pathways were inhibited in CD4+ T cells in the presence of 1% DMSO. Our study revealed that DMSO at concentrations generally used for in vitro studies of T cells impacts multiple features of T cell function. Therefore, we urge care when adding DMSO-containing preparations to T cell cultures.

CD8+ T cells specific for the islet autoantigen IGRP are restricted in their T cell receptor chain usage.

CD8+ T cells directed against beta cell autoantigens are considered relevant for the pathogenesis of type 1 diabetes. Using single cell T cell receptor sequencing of CD8+ T cells specific for the IGRP265-273 epitope, we examined whether there was expansion of clonotypes and sharing of T cell receptor chains in autoreactive CD8+ T cell repertoires. HLA-A*0201 positive type 1 diabetes patients (n = 19) and controls (n = 18) were analysed. TCR α- and ß-chain sequences of 418 patient-derived IGRP265-273-multimer+ CD8+ T cells representing 48 clonotypes were obtained. Expanded populations of IGRP265-273-specific CD8+ T cells with dominant clonotypes that had TCR α-chains shared across patients were observed. The SGGSNYKLTF motif corresponding to TRAJ53 was contained in 384 (91.9%) cells, and in 20 (41.7%) patient-derived clonotypes. TRAJ53 together with TRAV29/DV5 was found in 15 (31.3%) clonotypes. Using next generation TCR α-chain sequencing, we found enrichment of one of these TCR α-chains in the memory CD8+ T cells of patients as compared to healthy controls. CD8+ T cell clones bearing the enriched motifs mediated antigen-specific target cell lysis. We provide the first evidence for restriction of T cell receptor motifs in the alpha chain of human CD8+ T cells with specificity to a beta cell antigen.