Identification of the beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes

Type 1 diabetes is an autoimmune disease in which autoreactive T cells attack and destroy the insulin-producing pancreatic beta cells. CD8+ T cells are essential for this beta cell destruction, yet their specific antigenic targets are largely unknown. Here, we reveal that the autoantigen targeted by a prevalent population of pathogenic CD8+ T cells in nonobese diabetic mice is islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Through tetramer technology, IGRP-reactive T cells are readily detected in islets and peripheral blood directly ex vivo. The human IGRP gene maps to a diabetes susceptibility locus, suggesting that IGRP also may be an antigen for pathogenic T cells in human type 1 diabetes and, thus, a new, potential target for diagnostic and therapeutic approaches.     

IGRP与1型糖尿病的关系

1型糖尿病是由T细胞介导的自身免疫性疾病.许多研究表明,抗原特异性的CD4~+和CD8~+T细胞在1型糖尿病的发病过程中起重要作用.胰岛细胞特异性葡萄糖-6-磷酸酶催化亚基相关蛋白(IGRP)作为1型糖尿病的主要自身抗原之一,是葡萄糖-6-磷酸酶蛋白家族中的一员,在人和非肥胖糖尿病鼠胰腺中均有表达.它在胰岛β细胞的代谢过程中起重要作用,但是确切机制仍不清楚.IGRP的研究将有助于探索1型糖尿病诊断和治疗的新方法.     

IA-2beta, but not IA-2, is induced by ghrelin and inhibits glucose-stimulated insulin secretion

Ghrelin is a newly discovered peptide and an endogenous ligand for growth hormone (GH) secretagogue (GHS) receptor. It has been shown to possess various central and peripheral effects, including GH secretion, food intake, and gastric and cardiac effects. Ghrelin and the GHS receptor are expressed also in pancreatic islets. We have identified several ghrelin-induced genes by PCR-select subtraction methods, among which is a beta-cell autoantigen for type 1 diabetes, IA-2beta. Administration of ghrelin increased IA-2beta mRNA in mouse brain, pancreas, and insulinoma cell lines (MIN6 and betaTC3). However, the expression of IA-2, another structurally related beta-cell autoantigen, was not induced by ghrelin. Administration of ghrelin or overexpression of IA-2beta, but not overexpression of IA-2, inhibited glucose-stimulated insulin secretion in MIN6 insulinoma cells and, moreover, inhibition of IA-2beta expression by the RNA interference technique ameliorated ghrelin's inhibitory effects on glucose-stimulated insulin secretion. These findings strongly suggest that inhibitory effects of ghrelin on glucose-stimulated insulin secretion are at least partly due to increased expression of IA-2beta induced by ghrelin. Our data demonstrate the link among ghrelin, IA-2beta, and glucose-stimulated insulin secretion.     

Deletion of the gene encoding the islet-specific glucose-6-phosphatase catalytic subunit-related protein autoantigen results in a mild metabolic phenotype

Aims/hypothesis Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP, now known as glucose-6-phosphatase, catalytic, 2 [G6PC2]) has recently been identified as a major autoantigen in mouse and human type 1 diabetes. Strategies designed to suppress expression of the gene encoding G6PC2 might therefore be useful in delaying or preventing the onset of this disease. However, since the function of G6PC2 is unclear, the concern with such an approach is that a change in G6PC2 expression might itself have deleterious consequences. Methods To address this concern and assess the physiological function of G6PC2, we generated G6pc2-null mice and performed a phenotypic analysis focusing principally on energy metabolism. Results No differences in body weight were observed and no gross anatomical or behavioural changes were evident. In 16-week-old animals, following a 6-h fast, a small but significant decrease in blood glucose was observed in both male (−14%) and female (−11%) G6pc2 ^−/− mice, while female G6pc2 ^−/− mice also exhibited a 12% decrease in plasma triacylglycerol. Plasma cholesterol, glycerol, insulin and glucagon concentrations were unchanged. Conclusions/interpretation These results argue against the possibility of G6PC2 playing a major role in pancreatic islet stimulus secretion coupling or energy homeostasis under physiological conditions imposed by conventional animal housing. This indicates that manipulating the expression of G6PC2 for therapeutic ends may be feasible. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorised users.     

ICA69 is expressed equally in the human endocrine and exocrine pancreas

Summary Islet cell autoantigen 69 kDa (ICA69) has been reported as a polypeptide antigen expressed in pancreatic beta cells, and autoimmunity against this antigen has been associated with insulin-dependent diabetes mellitus. We have studied the cell type specificity and ontogeny of ICA69 gene expression in man. The ICA69 gene was expressed in all adult human tissues. The level of expression was three-to five-times higher in the pancreas than in the brain, liver, intestine, kidney, spleen, lung or adrenal glands. Pancreatic ICA69 expression increased with age, adult levels being five times higher than the levels present at 13 weeks of gestation. Total RNA from four separate preparations of isolated human islets revealed levels of ICA69 mRNA similar to those found in the pancreas as a whole, although another islet antigen, glutamic acid decarboxylase 65, was highly enriched in the islets. In situ hybridization and immunohistochemical staining of sections of the fetal and adult pancreas revealed expression of the ICA69 gene and protein throughout the acinar, ductal, and islet tissue, but not in the mesenchyme. Analysis of ICA69 mRNA levels in human cell lines indicated expression in neural, endothelial and epithelial cells, but not in fibroblasts. In conclusion, ICA69, although highest in the pancreas, is widely distributed in other human tissues, excluding connective tissue. Within the human pancreas, ICA69 is not enriched in the islets or in the beta cells.Abbreviations GAD Glutamic acid decarboxylase - BSA bovine serum albumin - GLUT2 glucose transporter 2 - bp base pair - PBS phosphate buffered saline - IDDM insulin-dependent diabetes mellitus - HAEC human aortic endothelial cells - ICA islet cell autoantibodies     

胰岛人类白细胞抗原-A2限制性T淋巴细胞表位体外筛选方法的建立

目的 应用表位多肽与人类白细胞抗原(HLA)Ⅰ类分子结合力和解离率分析建立新型T淋巴细胞表位体外筛选方法.方法 采用基于矩阵算法的SYFPEITHI和BIMAS数据库预测6种胰岛细胞自身抗原[包括谷氨酸脱羧酶65(GAD65)、胰岛素瘤相关抗原2(IA-2)、前胰岛素原(PPI)、胰岛特异性葡萄糖6-磷酸酶催化亚基相关蛋白(IGRP)、胰岛淀粉样多肽(IAPP)、神经胶质纤维酸性蛋白(GFAP)]的表位序列,根据预测的结合力指数和已有数据分析筛选并合成15个HLA-A2限制性候选表位多肽.采用HLA-A2转基因的T2细胞检测候选肽与HLA-A2的分子结合力,通过多肽/HLA复合物解离率实验分析复合物的稳定性.采用单因素方差分析进行数据统计.结果 T2细胞肽结合力分析显示,15个候选表位多肽中,IGRP152～160、IGRP215～223、IGRP228-236、PPI2～10、胰岛素B10～18、IA-2172～180、GFAP143～151与HLA-A2的分子结合力＞80%.肽/HLA复合物解离率分析显示,上述结合力较强的7个表位多肽中,胰岛素B10-18、IGRP228～236、GFAP143～151、IA-2 172～180 4 h解离率低于20%.结论 本实验建立的候选多肽结合力与解离率相结合的T淋巴细胞表位体外筛选方法有助于减少研究中目标肽数量,推进1型糖尿病T淋巴细胞诊断方法的研究.     

Effect of proinflammatory cytokines on gene expression of the diabetes-associated autoantigen IA-2 in INS-1 cells

Cytokines released from activated antigen-presenting cells and T-lymphocytes are crucially involved in the pathogenesis of type 1 diabetes. Previous studies have shown that proinflammatory cytokines play an important role in the induction of autoimmunity and beta-cell damage. Inhibition of insulin expression has been described, but their effects on other major target autoantigens, such as the tyrosine phosphatase-like protein IA-2, is not known. In the present study, we established sensitive real-time RT-PCR to measure IA-2, insulin, and inducible nitric oxide (NO) synthase (iNOS) mRNA expression. Rat insulinoma INS-1 cells were stimulated with IL-1beta, TNF-alpha, interferon (IFN)-gamma, and IL-2 as well as with two combinations of these cytokines (C1: IL-1beta + TNF-alpha + IFN-gamma; C2: TNF-alpha + IFN-gamma). Treatment with IL-1beta, TNF-alpha, or IFN-gamma alone caused a significant down-regulation of IA-2 and insulin mRNA levels in a time and dose-dependent manner, whereas IL-2 had no effect. Exposure to cytokine combinations strongly potentiates the inhibitory effects. Incubation of cells with C1 and C2 for 24 h induces a significant inhibition of IA-2 mRNA levels by 78% and 58%, respectively. Under these conditions, an up to 5 x 10(4)-fold increase of iNOS gene expression was observed. The hypothesis that the formation of NO is involved in IA-2 regulation was confirmed by the finding that the coincubation of C1 with 4 mM L-N(G)-monomethyL-L-arginine, an inhibitor of the iNOS, partly reversed the down-regulation of IA-2. Further, incubation with the synthetic NO-donor S-nitroso-N-acetyl-D-L-penicillamine significantly decreased IA-2 mRNA level to 51% of basal levels. In conclusion, we have demonstrated for the first time that IL-1beta, TNF-alpha, and IFN-gamma exert a strong inhibitory effect on expression of the diabetes autoantigen IA-2. The action of IL-1beta may be partly mediated by the activation of the NO pathway.     

T cell reactivity to DR*0401- and DQ*0302-binding peptides of the putative autoantigen IA-2 in type 1 diabetes.

Type 1 diabetes is thought to be an autoimmune disease mediated by T lymphocytes recognizing critical islet cell antigens. Recently, the tyrosine phosphatase like protein IA-2 was suggested as a putative autoantigen in type 1 diabetes since autoantibodies are detected in sera of diabetic patients and prediabetic subjects. Similarly, T cell responses of peripheral blood lymphocytes of type 1 diabetic patients to this protein have been described. Only very few data is available about immunodominant epitopes of IA-2 recognized by T cells. We have studied T cell responses in type 1 diabetic patients and age and partly HLA matched controls to IA-2 peptides designed to bind HLA risk alleles of IDDM as DR*0401 and DQ*0302. Both diabetic patients and controls responded to IA-2ic and some of the peptides. Three peptides of the C-terminal region of IA-2 were recognised by T cells of a fraction of diabetic patients but at least two of these peptides triggered also T cell responses in DR*0401/DQ*0302-matched controls. Most peptides bound to different HLA alleles ("promiscous binders"). The identification of autoantigenic epitopes may offer clues to related sequences e.g. of viral origin what relates to models of diabetes pathogenesis ("molecular mimicry"). Secondly, the design of antigen- or even epitope-specific immune intervention strategies aiming at tolerization of disease specific T cells in type 1 diabetes may profit from the knowledge of immunodominant T cell epitopes of a putative autoantigen.     

Thymus function and autoimmunity

PURPOSE: Thymus is the site of T-cell development and is essential for the induction of self-tolerance, by deletion of autoreactive T lymphocytes (negative selection) and by generation of regulatory T cells. Defect of the selection mechanism of both types of lymphocytes lead to autoimmune diseases. CURRENT KNOWLEDGE AND KEY POINTS: Elimination of potentially self-reactive T cells in the thymus requires the intrathymic expression of ubiquitous and "tissue-specific" antigens. Some thymic antigen expressions are dependent on AIRE expression. Mutations in the AIRE gene that are associated with the absence of autoantigen expression in the thymus, defects in the peptide presentation or in apoptosis can allow autoreactive T cells to escape negative selection, and are associated with autoimmune diseases. Recent data are now available concerning the thymic selection of autoreactive regulatory T cells. The Foxp3 gene was recently shown to be predominantly expressed in regulatory T cells and could be a more specific marker of regulatory T cells than phenotypic markers. FUTURE PROSPECTS AND PROJECTS: Animal models show that regulatory T cells injection or intrathymic inoculation of antigen lead to immunological tolerance in autoimmunity and transplantation. These novel strategies could be used in human.