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Fate determination for autoreactive T cells relies on a series of avidity-dependent interactions during T cell selection, represented by two general types of signals, one based on antigen expression and density during T cell development, and one based on genes that interpret the avidity of TCR interaction to guide developmental outcome. We used proinsulin-specific HLA class II tetramers to purify and determine transcriptional signatures for autoreactive T cells under differential selection in type 1 diabetes (T1D), in which insulin (INS) genotypes consist of protective and susceptible alleles that regulate the level of proinsulin expression in the thymus. Upregulation of steroid nuclear receptor family 4A (NR4A) and early growth response family genes in proinsulin-specific T cells was observed in individuals with susceptible INS-VNTR genotypes, suggesting a mechanism for avidity-dependent fate determination of the T cell repertoire in T1D. The NR4A genes act as translators of TCR signal strength that guide central and peripheral T cell fate decisions through transcriptional modification. We propose that maintenance of an NR4A-guided program in low avidity autoreactive T cells in T1D reflects their prior developmental experience influenced by proinsulin expression, identifying a pathway permissive for autoimmunity.

Background: Islet-antigen-specific CD4+ T cells are known to promote auto-immune destruction in T1D. Measuring T-cell number and function provides an important biomarker. In response to this need, we evaluated responses to proinsulin and GAD epitopes in a multicentre study., Methods: A tetramer-based assay was used in five participating centres to measure T-cell reactivities to DR0401-restricted epitopes. Three participating centres concurrently performed ELISPOT or immunoblot assays. Each centre used blind-coded, centrally distributed peptide and tetramer reagents., Results: All participating centres detected responses to auto-antigens and the positive control antigen, and in some cases cloned the corresponding T cells. However, response rates varied among centres. In total, 74% of patients were positive for at least one islet epitope. The most commonly recognized epitope was GAD270-285. Only a minority of the patients tested by tetramer and ELISPOT were concordant for both assays., Conclusions: This study successfully detected GAD and proinsulin responses using centrally distributed blind-coded reagents. Centres with little previous experience using class II tetramer reagents implemented the assay. The variability in response rates observed for different centres suggests technical difficulties and/or heterogeneity within the local patient populations tested. Dual analysis by tetramer and ELISPOT or immunoblot assays was frequently discordant, suggesting that these assays detect distinct cell populations. Future efforts should investigate shared blood samples to evaluate assay reproducibility and longitudinal samples to identify changes in T-cell phenotype that correlate with changes in disease course., (Copyright © 2011 John Wiley & Sons, Ltd.)

Background & Aims: Autoimmune pancreatitis (AIP) underlies 5%-11% of cases of chronic pancreatitis. An association between AIP and the human leukocyte antigen (HLA)-DRB1*0405/DQB1*0401 haplotype has been reported, but linkage disequilibrium has precluded the identification of predisposing HLA gene(s). We studied the role of single HLA genes in the development of AIP in transgenic mice., Methods: CD4(+) T-cell-negative I-Abeta chain(-/-) (Ab0) mice develop AIP spontaneously, likely due to dysregulation of CD8(+) T- cell responses. We generated Ab0 nonobese diabetic (NOD) mice transgenic for HLA-DR*0405, leading to rescue of CD4(+) T cells; we compared their susceptibility to AIP with HLA-DQ8 or HLA-DR*0401 (single) transgenic, or HLA-DR*0405/DQ8 (double) transgenic mice., Results: CD4(+) T-cell-competent HLA-DR*0405 transgenic Ab0 NOD mice develop AIP with high prevalence after sublethal irradiation and adoptive transfer of CD90(+) T cells, leading to complete pancreatic atrophy. HLA-DR*0405 transgenic mice can also develop unprovoked AIP, whereas HLA-DR*0401, HLA-DQ8, and HLA-DR*0405/DQ8 transgenic Ab0 NOD controls all remained normal, even after irradiation and adoptive transfer of CD90(+) T cells. Pancreas histology in HLA-DR*0405 transgenic mice was characterized by destructive infiltration of the exocrine tissue with CD4(+) and CD8(+) T cells, B cells, and macrophages. Mice with complete pancreatic atrophy lost weight, developed fat stools, and had reduced levels of serum lipase activity., Conclusions: Because HLA-DR*0405 expression fails to protect mice from AIP, the HLA-DRB1*0405 allele appears to be an important risk factor for AIP on the HLA-DRB1*0405/DQB1*0401 haplotype. This humanized mouse model should be useful for studying immunopathogenesis, diagnostic markers, and therapy of human AIP., (Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Circulating CD4 T cells specific for peptide epitopes of proinsulin and other autoantigens are markers of autoimmune beta cell destruction in type 1 diabetes, while the role of CD8 T cells is still largely unknown. Here we show that CD8 T cells of a diabetic patient--after rechallange with proinsulin peptides--secrete IFNgamma and granzyme B, markers of their effector capacity. On the other hand, CD8 T cells of the same patient in a "cross-talk" with proinsulin-specific CD4 T cells suppress their proliferation. If confirmed in larger numbers of subjects with beta-islet cell autoimmunity, these results may help us to understand the role of CD8 cells in disease progression and extend our knowledge of disease pathogenesis.

Recently, we have identified proinsulin (P-Ins)(73-90) as an immunodominant T cell epitope of HLA-DRB1*0401 (DR4) subjects with beta-islet cell autoimmunity and of HLA-DR4/CD4 double-transgenic mice immunized with human P-Ins. We have compared the fine specificities of one human CD4 T cell clone and two mouse T cell hybridoma clones recognizing this epitope, and, although these three clones all recognized the same core region (LALEGSLQK), there were major differences in how they interacted with the peptide (p)/HLA complex, reflecting the fact that human P-Ins is a foreign antigen in the mouse and an autoantigen in the type 1 diabetes patient. The human T cell clone was forkhead transcription factor 3 (Foxp3)-positive, a marker for regulatory T cell lineages, and secreted predominantly IL-5, IL-10, and low levels of IFNgamma in response to P-Ins(73-90). This finding is compatible with the previously detected regulatory cytokine pattern in subjects with beta-cell autoimmunity. However, added N- or C-terminal amino acids drastically changed HLA and tetramer binding capacity as well as T cell reactivity and the cytokine phenotype of the P-Ins(73-90)-specific human CD4 T cell clone, suggesting a potential for this P-Ins epitope as a target for therapeutic intervention in HLA-DR4-positive humans with beta-islet cell autoimmunity or recent-onset type 1 diabetes.

Background: Preservation of beta cell function is a central goal in type 1 diabetes (type 1 DM) immune intervention. The characterization of individuals with recovery from established type 1 DM should provide insight into regulatory mechanisms of beta cell autoimmunity., Methods: We studied a patient with antibody-positive type 1 DM with complete recovery of beta cell function for an observation period of 60 months. Using a preproinsulin (PPI) peptide library approach and in vitro cytokine profiling, cellular autoimmunity was characterized in peripheral blood mononuclear cells (PBMC) and CD4(+) T-helper cell subsets., Results: A predominant secretion of interleukin-10 (IL-10) was detected in the patient's PBMC, mostly attributable to naïve and recently primed CD45(+)RA(+) T cells, with limited PPI epitope recognition. In contrast to a cohort of patients with permanent type 1 DM, interferon-gamma secretion was low in PBMC and CD45(+)RA(+), but not in CD45(+)RA(-) insulin-reactive T lymphocytes. Autoantibodies against islet cells, tyrosine phosphatase IA-2, GAD65 and insulin were positive at diabetes onset, but gradually declined during follow-up., Conclusions: Our observations support the concept that IL-10-dependent regulatory CD4(+) T-cell pathways are involved in beta cell recovery after the onset of hyperglycemia in autoimmune type 1 DM.

A variable number of tandem repeats (VNTR) polymorphism upstream of the insulin promoter is strongly associated with type 1 diabetes. The short class I alleles are predisposing and the long class III alleles are protective. As a possible mechanism for this effect, we previously reported a two- to threefold higher insulin transcription from class III than from class I chromosomes in thymus where insulin is expressed at low levels, presumably for the purpose of self-tolerance. In this article, we confirm this finding with independent methodology and report studies testing the hypothesis that class III alleles are associated with T-cell tolerance to (pro)insulin. Cytokine release in vitro after stimulation with 21 overlapping preproinsulin epitopes was assessed in blood mononuclear cells as well as naive and memory CD4+ T-cell subsets from 33 individuals with the high-risk DRB1*04, DQ8 haplotype (12 type 1 diabetic patients, 11 healthy control subjects, and 10 autoantibody-positive subjects). No significant differences between genotypes (24 I/I subjects versus 10 I/III or III/III subjects) were observed for gamma-interferon, tumor necrosis factor-alpha, or interleukin (IL)-4. By contrast, the I/III + III/III group showed a significant threefold higher IL-10 release in memory T-cells for whole proinsulin and the immunodominant region. Given that IL-10 is a marker of regulatory function, our data are consistent with the hypothesis that higher insulin levels in the thymus promote the formation of regulatory T-cells, a proposed explanation for the protective effect of the class III alleles.

(Prepro)insulin is considered a central antigenic determinant in diabetic autoimmunity. Insulin has been used to modify diabetes development in NOD mice and prediabetic individuals. We have recently shown that (prepro)insulin can adversely promote diabetes development in murine type 1 diabetes. Based on these findings we have developed experimental autoimmune diabetes (EAD), a new mouse model characterized by (1) CD4(+)/CD8(+) insulitis, induced by (2) (prepro)insulin DNA vaccination, leading to (3) beta cell damage and insulin deficiency in (4) RIP-B7.1 transgenic mice (H-2(b)). EAD develops rapidly in 60-95% of mice after intramuscular, but not intradermal ("gene gun"), vaccination; and DNA plasmids expressing insulin or the insulin analogues glargine, aspart, and lispro are equally potent to induce EAD. Similar to NOD mice, diabetes is adoptively transferred into syngeneic recipients by spleen cell transplantation in a dose-dependent fashion. We have devised a two-stage concept of EAD in which T cell activation and expansion is driven by in vivo autoantigen expression, followed by islet damage that requires beta cell expression of costimulatory B7.1 for disease manifestation. Taken together, EAD is a novel, genetically defined animal model of type 1 diabetes suitable to analyze mechanisms and consequences of insulin-specific T cell autoimmunity.

In animal models autoreactive CD8(+) T cells are crucial in the development of type 1 diabetes (T1D); however, their role in human T1D is still not known. To address the role of CD81 T cells we performed a pilot study by investigating CD8(+) T cell-mediated cytokine secretion after in vitro stimulation with 94 preproinsulin (PPI) peptides. We were able to show that CD8(+) T cells contribute to a strong IFNgamma reactivity against PPI in human T1D. Further investigations defining epitope specificity, cytokine secretion, and cytotoxic capacity are important to clarify their role in T1D development.

Active T cell recognition of islet antigens has been postulated as the pathogenic mechanism in human type 1 diabetes, but evidence is scarce. If T cells are engaged, they are expected to display increased clonal size and exhibit a T helper (Th)1/Th2 differentiation state. We used a peptide library that covers tyrosine phosphatase IA-2, a target antigen expressed in pancreatic beta cells, to probe 8 diabetic patients and 5 HLA-matched controls. When tested in a high resolution IFNgamma/IL-4 double color ELISPOT assay directly ex vivo, the number of IA-2-reactive IFNgamma producing cells was 17-fold higher in patients than in controls and IL-4 producing cells were not present. An average of 9 peptides was recognized in the patients vs. one in the controls. Determinant recognition primarily involved CD4+ cells and showed high variability among the patients. Furthermore, anti-CD28 antibody signal enhances quantitative assessment of effector T cells in T1D patients. In vitro expansion with peptides and IL-2 results in detection of responding cells in the controls and loss of disease specificity of the T cell response. Together these data provide strong evidence for the active targeting of IA-2 by Th1 memory effector cells in human type 1 diabetes., (Copyright 2004 Elsevier Ltd.)

In type 1 diabetes, humoral and cell-mediated responses to insulin and proinsulin are detectable. Autoantibodies to insulin are associated with impending disease in young individuals and are used as predictive markers to determine disease risk. The aim of this study was to investigate whether different cytokine patterns of cellular reactivity to insulin might serve as additional specific markers of disease maturation and might improve disease prediction in individuals at risk. We correlated T and B cell responses to insulin in subjects with increased genetic risk (HLA-DRB1*04, DQB1*0302) for diabetes with or without islet autoantibodies (Ab+ subjects and controls, respectively) and HLA-matched patients. Peripheral blood mononuclear cells were stimulated with 15 overlapping proinsulin peptides (16-mer), and proinflammatory Th1 (IFNgamma) and anti-inflammatory Th2 (IL-4) cytokines were analyzed. We observed a simultaneous increase in IL-4 and IFNgamma secretion in early islet autoimmunity of Ab+ subjects, but not in insulin-treated T1D patients. Furthermore, the increase in IL-4 secretion in Ab+ subjects was associated with insulin autoantibody responses. There was no correlation of either IFNgamma or IL-4 secretion with insulin antibody responses in patients already treated with exogenous insulin. In conclusion, our findings reveal that quantification of cytokine responses to proinsulin in peripheral blood may prove to be a promising specific marker of diabetes progression and could, in addition to insulin autoantibodies, be used in the prediction of type 1 diabetes.

In human type 1 diabetes (T1D) autoantibodies to insulin precede clinical disease, while little is known about the contribution of insulin-specific T lymphocytes-in particular, T helper (Th) subsets. Here we have studied the in vivo primed cytokine response to preproinsulin in peripheral blood mononuclear cells (PBMCs) and two major Th cell subsets-CD45RO+ memory cells and CD45RA+ naive/resting cells-in 35 individuals with HLA-DRB1*04, DQB1*0302 diabetes risk marker: 12 patients with T1D, 12 autoantibody-positive (Ab+) individuals, and 11 healthy controls. Cytokine secretion (TNF-alpha, IFN-gamma, IL-2, IL-4, IL-5, and IL-10) was measured in the supernatants of the cultures stimulated with 21 overlapping preproinsulin peptides as well as proinsulin and insulin. In Ab+ individuals our results reveal higher IL-4 levels in CD45RO+ memory cells and higher IL-5 levels in CD45RA+ naive/resting cells, while higher IL-2 production was found in PBMCs. In contrast, in PBMCs of T1D patients higher IFN-gamma and IL-10 secretion was found. Our data delineate characteristic cytokine patterns in peripheral T lymphocytes from patients at different stages of the T1D development.

Antibody response to insulin and to its precursor ProInsulin is associated with increased risk for type 1 diabetes (T1D), though little is known about T cell reactivity to this molecule. In the present study from peripheral blood mononuclear cells (PBMC), in vivo primed CD45RO+ memory T helper (Th) cells were enriched and their reactivity to eight overlapping ProInsulin peptides and to protein was analyzed. Individuals with high risk HLA-DRB1*04, DQB1*0302 alleles were investigated: relatives of patients with T1D having humoral markers of islet cell autoimmunity (autoantibody positive, Ab+; n=11), patients with T1D (n=8), and healthy control individuals (n=16). The ProInsulin epitope which was most frequently recognized in all the tested individuals was C-peptide (C) 18-A-chain (A)1. In Ab+ relatives the responses to this epitope and to two additional parts of the ProInsulin, B-chain (B) 11-C24 and C28-A21, was observed. In T1D patients who have already been treated with insulin, response to peptide B20-C4 and to the entire insulin molecule predominates. Our findings suggest that the spontaneous memory Th cell response to ProInsulin in individuals with high risk HLA alleles is predominantly directed to one epitope which maps to the central, C-peptide region. In individuals with humoral markers of islet cell autoimmunity and in patients with T1D, spread response to distinct ProInsulin regions was observed., (Copyright 2002 Elsevier Science Ltd.)

Type 1 diabetes (IDDM) is a T cell mediated autoimmune disease which in part is determined genetically by its association with major histocompatibility complex (MHC) class II alleles. The major role of MHC molecules is the regulation of immune responses through the presentation of peptide epitopes of processed protein antigens to the immune system. Recently it has been demonstrated that MHC molecules associated with autoimmune diseases preferentially present peptides of other endogenous MHC proteins, that often mimic autoantigen-derived peptides. Hence, these MHC-derived peptides might represent potential targets for autoreactive T cells. It has consistently been shown that humoral autoimmunity to insulin predominantly occurs in early childhood. The cellular immune response to insulin is relatively low in the peripheral blood of patients with IDDM. Studies in NOD mice however have shown, that lymphocytes isolated from pancreatic islet infiltrates display a high reactivity to insulin and in particular to an insulin peptide B 9-23. Furthermore we have evidence that cellular autoimmunity to insulin is higher in young pre-diabetic individuals, whereas cellular reactivity to other autoantigens is equally distributed in younger and older subjects. This implicates that insulin, in human childhood IDDM and animal autoimmune diabetes, acts as an important early antigen which may target the autoimmune response to pancreatic beta cells. Moreover, we observed that in the vast majority of newly diagnosed diabetic patients or individuals at risk for IDDM, T cell reactivity to various autoantigens occurs simultaneously. In contrast, cellular reactivity to a single autoantigen is found with equal frequency in (pre)-type 1 diabetic individuals as well as in control subjects. Therefore the autoimmune response in the inductive phase of IDDM may be targeted to pancreatic islets by the cellular and humoral reactivity to one beta-cell specific autoantigen, but spreading to a set of different antigens may be a prerequisite for progression to destructive insulitis and clinical disease. Due to mimic epitopes shared by autoantigen(s), autologous MHC molecules and environmental antigens autoimmunity may spread, intramolecularly and intermolecularly and amplify upon repeated reexposure to mimic epitopes of environmental triggers.