Differential expression of genes controlling lymphocyte differentiation and migration in two distinct endotypes of type 1 diabetes.

AIMS: Morphological studies of pancreas samples obtained from young people with recent-onset type 1 diabetes have revealed distinct patterns of immune cell infiltration of the pancreatic islets suggestive of two age-associated type 1 diabetes endotypes that differ by inflammatory responses and rates of disease progression. The objective of this study was to investigate whether these proposed disease endotypes are associated with pathological differences in immune cell activation and cytokine secretion by applying multiplexed gene expression analysis to pancreatic tissue from recent-onset type 1 diabetes cases. METHODS: RNA was extracted from samples of fixed, paraffin-embedded pancreas tissue from type 1 diabetes cases characterised by endotype and from controls without diabetes. Expression levels of 750 genes associated with autoimmune inflammation were determined by hybridisation to a panel of capture and reporter probes and these were counted as a measure of gene expression. Normalised counts were analysed for differences in expression between 29 type 1 diabetes cases and 7 controls without diabetes, and between the two type 1 diabetes endotypes. RESULTS: Ten inflammation-associated genes, including INS, were significantly under-expressed in both endotypes and 48 genes were more highly expressed. A different set of 13 genes associated with the development, activation and migration of lymphocytes was uniquely overexpressed in the pancreas of people developing diabetes at younger age. CONCLUSIONS: The results provide evidence that histologically defined type 1 diabetes endotypes differ in their immunopathology and identify inflammatory pathways specifically involved in disease developing at a young age, essential for a better understanding of disease heterogeneity.

Identification of Tetraspanin-7 as a Target of Autoantibodies in Type 1 Diabetes.

The presence of autoantibodies to multiple-islet autoantigens confers high risk for the development of type 1 diabetes. Four major autoantigens are established (insulin, glutamate decarboxylase, IA2, and zinc transporter-8), but the molecular identity of a fifth, a 38-kDa membrane glycoprotein (Glima), is unknown. Glima antibodies have been detectable only by immunoprecipitation from extracts of radiolabeled islet or neuronal cells. We sought to identify Glima to enable efficient assay of these autoantibodies. Mouse brain and lung were shown to express Glima. Membrane glycoproteins from extracts of these organs were enriched by detergent phase separation, lectin affinity chromatography, and SDS-PAGE. Proteins were also immunoaffinity purified from brain extracts using autoantibodies from the sera of patients with diabetes before SDS-PAGE. Eluates from gel regions equivalent to 38 kDa were analyzed by liquid chromatography-tandem mass spectrometry for protein identification. Three proteins were detected in samples from the brain and lung extracts, and in the immunoaffinity-purified sample, but not in the negative control. Only tetraspanin-7, a multipass transmembrane glycoprotein with neuroendocrine expression, had physical characteristics expected of Glima. Tetraspanin-7 was confirmed as an autoantigen by demonstrating binding to autoantibodies in type 1 diabetes. We identify tetraspanin-7 as a target of autoimmunity in diabetes, allowing its exploitation for diabetes prediction and immunotherapy.

Influence of HLA-DR and -DQ alleles on autoantibody recognition of distinct epitopes within the juxtamembrane domain of the IA-2 autoantigen in type 1 diabetes.

AIMS/HYPOTHESIS: Insulinoma-associated protein 2 (IA-2) is a major target of autoimmunity in type 1 diabetes. When first detected, IA-2-autoantibodies commonly bind epitopes in the juxtamembrane (JM) domain of IA-2 and antibody responses subsequently spread to the tyrosine phosphatase domain. Definition of structures of epitopes in the JM domain, and genetic requirements for autoimmunity to these epitopes, is important for our understanding of initiation and progression of autoimmunity. The aims of this study were to investigate the contribution of individual amino acids in the IA-2 JM domain to antibody binding to these epitopes and the role of HLA genotypes in determining epitope specificity. METHODS: Regions of the JM domain recognised by autoantibodies were identified by peptide competition and inhibitory effects of alanine substitutions of residues within the JM region. Antibody binding was determined by radioligand binding assays using sera from patients genotyped for HLA-DRB1 and -DQB1 alleles. RESULTS: Patients were categorised into two distinct groups of JM antibody reactivity according to peptide inhibition. Inhibition by substitutions of individual amino acids within the JM domain differed between patients, indicating heterogeneity in epitope recognition. Cluster analysis defined six groups of residues having similar inhibitory effects on antibody binding, with three clusters showing differences in patients affected or unaffected by peptide. One cluster demonstrated significant differences in antibody binding between HLA-DRB1*04 and HLA-DRB1*07 patients and within DRB1*04 individuals; antibody recognition of a second cluster depended on expression of HLA-DQB1*0302. CONCLUSIONS/INTERPRETATION: The results identify amino acids contributing to distinct epitopes on IA-2, with both HLA-DR and HLA-DQ alleles influencing epitope specificity.

Relationships between major epitopes of the IA-2 autoantigen in Type 1 diabetes: Implications for determinant spreading.

Diversification of autoimmunity to islet autoantigens is critical for progression to Type 1 diabetes. B-cells participate in diversification by modifying antigen processing, thereby influencing which peptides are presented to T-cells. In Type 1 diabetes, JM antibodies are associated with T-cell responses to PTP domain peptides. We investigated whether this is the consequence of close structural alignment of JM and PTP domain determinants on IA-2. Fab fragments of IA-2 antibodies with epitopes mapped to the JM domain blocked IA-2 binding of antibodies that recognise epitopes in the IA-2 PTP domain. Peptides from both the JM and PTP domains were protected from degradation during proteolysis of JM antibody:IA-2 complexes and included those representing major T-cell determinants in Type 1 diabetes. The results demonstrate close structural relationships between JM and PTP domain epitopes on IA-2. Stabilisation of PTP domain peptides during proteolysis in JM-specific B-cells may explain determinant spreading in IA-2 autoimmunity.

HLA-DR4-associated T and B cell responses to specific determinants on the IA-2 autoantigen in type 1 diabetes.

Autoantibodies to IA-2 in type 1 diabetes are associated with HLA-DR4, suggesting influences of HLA-DR4-restricted T cells on IA-2-specific B cell responses. The aim of this study was to investigate possible T-B cell collaboration by determining whether autoantibodies to IA-2 epitopes are associated with T cell responses to IA-2 peptides presented by DR4. T cells secreting the cytokines IFN-γ and IL-10 in response to seven peptides known to elicit T cell responses in type 1 diabetes were quantified by cytokine ELISPOT in HLA-typed patients characterized for Abs to IA-2 epitopes. T cell responses were detected to all peptides tested, but only IL-10 responses to 841-860 and 853-872 peptides were associated with DR4. Phenotyping by RT-PCR of FACS-sorted CD45RO(hi) T cells secreting IL-10 in response to these two peptides indicated that these expressed GATA-3 or T-bet, but not FOXP3, consistent with these being Th2 or Th1 memory T cells rather than of regulatory phenotype. T cell responses to the same two peptides were also associated with specific Abs: those to 841-860 peptide with Abs to juxtamembrane epitopes, which appear early in prediabetes, and those to peptide 853-872 with Abs to an epitope located in the 831-862 central region of the IA-2 tyrosine phosphatase domain. Abs to juxtamembrane and central region constructs were both DR4 associated. This study identifies a region of focus for B and T cell responses to IA-2 in HLA-DR4 diabetic patients that may explain HLA associations of IA-2 autoantibodies, and this region may provide a target for future immune intervention to prevent disease.

Failure to detect anti-idiotypic antibodies in the autoimmune response to IA-2 in Type 1 diabetes.

The concept that immune responses to self antigens are regulated by anti-idiotypic networks has attracted renewed interest following reports of circulating factors within IgG fractions of serum that impair detection of autoantibodies with autoantigen. Thus, preclearance of sera with bead-immobilised monoclonal autoantibodies to the Type 1 diabetes autoantigen GAD65, or prebinding of serum antibodies to protein A Sepharose prior to addition of antigen, increases immunoreactivity detected in serum samples consistent with the trapping on the beads of anti-idiotypic antibodies that block antibody binding to the autoantigen. The aim of this study was to investigate the presence of anti-idiotypic antibodies to another major target of autoantibodies in Type 1 diabetes, IA-2. As previously observed for GAD65, preadsorption of serum samples with immobilised monoclonal IA-2 autoantibody, or prebinding to protein A Sepharose, resulted in substantial increases in subsequent immunoprecipitation of radiolabeled IA-2 in a proportion of samples. However, control experiments indicated that the increases seen on pre-incubation with immobilized autoantibodies were caused by displacement of the antibody by serum IgG, whereas impaired detection of immunoreactivity in liquid-phase radiobinding assays was the result of formation of insoluble complexes that bind poorly to protein A. The results emphasise the importance of direct demonstration of specific binding of antibodies to the idiotype in the study of idiotypic networks in autoimmunity. Variability between patients in formation of insoluble immune complexes has implications for the design and standardization of autoantibody assays for diabetes prediction.

Somatostatin secreted by islet delta-cells fulfills multiple roles as a paracrine regulator of islet function.

OBJECTIVE: Somatostatin (SST) is secreted by islet delta-cells and by extraislet neuroendocrine cells. SST receptors have been identified on alpha- and beta-cells, and exogenous SST inhibits insulin and glucagon secretion, consistent with a role for SST in regulating alpha- and beta-cell function. However, the specific intraislet function of delta-cell SST remains uncertain. We have used Sst(-/-) mice to investigate the role of delta-cell SST in the regulation of insulin and glucagon secretion in vitro and in vivo. RESEARCH DESIGN AND METHODS: Islet morphology was assessed by histological analysis. Hormone levels were measured by radioimmunoassay in control and Sst(-/-) mice in vivo and from isolated islets in vitro. RESULTS: Islet size and organization did not differ between Sst(-/-) and control islets, nor did islet glucagon or insulin content. Sst(-/-) mice showed enhanced insulin and glucagon secretory responses in vivo. In vitro stimulus-induced insulin and glucagon secretion was enhanced from perifused Sst(-/-) islets compared with control islets and was inhibited by exogenous SST in Sst(-/-) but not control islets. No difference in the switch-off rate of glucose-stimulated insulin secretion was observed between genotypes, but the cholinergic agonist carbamylcholine enhanced glucose-induced insulin secretion to a lesser extent in Sst(-/-) islets compared with controls. Glucose suppressed glucagon secretion from control but not Sst(-/-) islets. CONCLUSIONS: We suggest that delta-cell SST exerts a tonic inhibitory influence on insulin and glucagon secretion, which may facilitate the islet response to cholinergic activation. In addition, delta-cell SST is implicated in the nutrient-induced suppression of glucagon secretion.