Human Type 1 Diabetes Is Associated with T Cell Autoimmunity to Zinc Transporter 8

Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, Aurora, CO 80045, USA.
The Journal of Immunology (Impact Factor: 4.92). 04/2011; 186(10):6056-63. DOI: 10.4049/jimmunol.1003815
Source: PubMed


Recently we demonstrated that zinc transporter 8 (ZnT8) is a major target of autoantibodies in human type 1 diabetes (T1D). Because the molecules recognized by T1D autoantibodies are typically also targets of autoreactive T cells, we reasoned that this would likely be the case for ZnT8. To test this hypothesis, IFN-γ-producing T cells specific for ZnT8 in the peripheral blood of 35 patients with T1D (<6 mo after onset at blood draw) and 41 age-matched controls were assayed by ELISPOT using a library of 23 overlapping dipeptide pools covering the entire 369 aa primary sequence. Consistent with our hypothesis, patients showed significantly higher T cell reactivity than the matched controls, manifest in terms of the breadth of the overall response and the magnitude of responses to individual pools. Therefore, the median number of pools giving positive responses (stimulation index ≥ 3) in the control group was 1.0 (range, 0-7) compared with 6.0 (range, 1-20; p < 0.0001) for the patients. Similarly, the median stimulation index of positive responses in controls was 3.1 versus 5.0 in the patients (p < 0.0001). Individually, 7 of 23 pools showed significant disease association (p < 0.001), with several of the component peptides binding the disease associated HLA-DR3 (0301) and -DR4 (0401) molecules in vitro. We conclude that ZnT8 is also a major target of disease-associated autoreactive T cells in human T1D, and we suggest that reagents that target ZnT8-specific T cells could have therapeutic potential in preventing or arresting the progression of this disease.

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    • "In addition to being a target of autoantibodies , ZnT8 was also identified as a major AAg of disease associated autoreactive CD4+ T cells in human T1DM [48]. "
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    ABSTRACT: Type 1 diabetes mellitus (T1DM) is characterized by recognition of beta cell proteins as self-antigens, called autoantigens (AAgs), by patients' own CD4+ and CD8+ T cells and/or the products of self-reactive B cells, called autoantibodies. These AAgs are divided into two categories on the basis of beta-cell-specificity. The list of the targets associated with beta cell-specific AAgs is continuously growing. Many T1DM-associated AAgs are well characterized and have important clinical applications for disease prediction, diagnosis, and antigen-specific tolerance immunotherapy. Identification of T1DM-associated AAgs provides insight into the pathogenesis of T1DM and to understanding the clinical aspects of the disease. Since many excellent reviews have covered the previously identified T1DM-associated AAgs exhaustedly, here we only focus on several recently discovered T1DM-AAgs (PDX1, ZnT8, CHGA, and IAAP).
    Preview · Article · May 2013 · American Journal of Translational Research
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    • "The C-allele (encoding ZnT8-325R) confers a minor risk of type 2 diabetes and has been associated with reduced insulin secretion (3), but no direct evidence that this polymorphism alters ZnT8 expression is available. Alternatively, the polymorphism at position 325 could alter the T-cell epitope repertoire (8), thereby modifying the influence of HLA-A*24 on humoral autoimmunity (24), but so far, no association has been found between the breadth of the ZnT8 CD4 T-cell responses in patients and ZnT8A (25). "
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    ABSTRACT: The HLA-A*24 allele has shown negative associations with autoantibodies to islet antigen-2 (IA 2) and zinc transporter 8 (ZnT8) in patients with established type 1 diabetes. Understanding how this HLA class I allele affects humoral islet autoimmunity gives new insights into disease pathogenesis. We therefore investigated the epitope specificity of associations between HLA-A*24 and islet autoantibodies at disease onset. HLA-A*24 genotype and autoantibody responses to insulin (IAA), glutamate decarboxylase (GADA), IA-2, IA-2β and ZnT8 were analysed in samples collected from patients with recent-onset type 1 diabetes. After correction for age, gender, and HLA class II genotype, HLA-A*24 was shown to be a negative determinant of both IA-2A and ZnT8A. These effects were epitopespecific. Antibodies targeting the protein tyrosine phosphatase domains of IA-2 and IA-2β, but not the IA-2 juxta-membrane region were less common in patients carrying HLA-A*24 alleles. The prevalence of ZnT8A specific or cross-reactive with the ZnT8 tryptophan-325 polymorphic residue, but not those specific to arginine-325 was reduced in HLA*A24 positive patients. No associations were found between HLA-A*24 and IAA or GADA. Association of an HLA class I susceptibility allele with altered islet autoantibody phenotype at diagnosis suggests CD8 T-cell and/or natural killer cell mediated killing modulate humoral autoimmune responses.
    Full-text · Article · Feb 2013 · Diabetes
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    • "Type 1 diabetes (T1D) is caused by autoimmune destruction of insulin-producing islet β cells [14], [15], [16], [17], [18]. Accordingly, T1D patients display IFN-γ-producing islet antigen-specific T cells in the blood [19], [20], [21]. T1D-associated islet antigens include GAD65 [8], [22], [23], (prepro)insulin (PPI) [24], [25], [26], insulinoma associated-2 (IA-2) [27], islet-specific glucose-6-phosphate catalytic subunit-related protein (IGRP) [28], [29], and zinc transporter-8 (ZnT8) [30]. "
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    ABSTRACT: Determination of antigen-specific T cell repertoires in human blood has been a challenge. Here, we show a novel integrated approach that permits determination of multiple parameters of antigen-specific T cell repertoires. The approach consists of two assays: the Direct assay and the Cytokine-driven assay. Briefly, human PBMCs are first stimulated with overlapping peptides encoding a given antigen for 48 hours to measure cytokine secretion (Direct assay). Peptide-reactive T cells are further expanded by IL-2 for 5 days; and after overnight starvation, expanded cells are stimulated with the same peptides from the initial culture to analyze cytokine secretion (Cytokine-driven assay). We first applied this integrated approach to determine the type of islet-antigen-specific T cells in healthy adults. Out of ten donors, the Direct assay identified GAD65-specific CD4(+) T cells in three adults and zinc transporter 8 (ZnT8)-specific CD4(+) T cells in five adults. The intracytoplasmic cytokine staining assay showed that these islet-antigen-specific CD4(+) T cells belonged to the CD45RO(+) memory compartment. The Cytokine-driven assay further revealed that islet-antigen-specific CD4(+) T cells in healthy adults were capable of secreting various types of cytokines including type 1 and type 2 cytokines as well as IL-10. We next applied our integrated assay to determine whether the type of ZnT8-specific CD4(+) T cells is different between Type 1 diabetes patients and age/gender/HLA-matched healthy adults. We found that ZnT8-specific CD4(+) T cells were skewed towards Th1 cells in T1D patients, while Th2 and IL-10-producing cells were prevalent in healthy adults. In conclusion, the Direct assay and the Cytokine-driven assay complement each other, and the combination of the two assays provides information of antigen-specific T cell repertoires on the breadth, type, and avidity. This strategy is applicable to determine the differences in the quality of antigen-specific T cells between health and disease.
    Full-text · Article · Feb 2013 · PLoS ONE
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