Autoantibodies and associated T-cell responses to determinants within the 831-860 region of the autoantigen IA-2 in Type 1 diabetes

Diabetes Research Group, Division of Reproduction & Endocrinology, 2nd Floor Hodgkin Building, King's College London, Guy's Campus, London, United Kingdom.
Journal of Autoimmunity (Impact Factor: 8.41). 06/2009; 33(2):147-54. DOI: 10.1016/j.jaut.2009.04.002
Source: PubMed


B-cells influence T-cell reactivity by facilitating antigen presentation, but the role of autoantibody-secreting B-cells in regulating T-cell responses in Type 1 diabetes is poorly defined. The aims of this study were to characterise epitopes on the IA-2 autoantigen for three monoclonal antibodies from diabetic patients by amino acid substitutions of selected residues of IA-2, establish contributions of these epitopes to binding of serum antibodies in Type 1 diabetes and relate B- and T-cell responses to overlapping determinants on IA-2. The monoclonal antibodies recognised overlapping epitopes, with residues within the 831-860 region of IA-2 contributing to binding; substitution of Glu836 inhibited binding of all three antibodies. Monoclonal antibody Fab fragments and substitution of residues within the 831-836 region blocked serum antibody binding to an IA-2 643-937 construct. IL-10-secreting T-cells responding to peptides within the 831-860 region were detected by cytokine-specific ELISPOT in diabetic patients and responses to 841-860 peptide were associated with antibodies to the region of IA-2 recognised by the monoclonal antibodies. The study identifies a region of IA-2 frequently recognised by antibodies in Type 1 diabetes and demonstrates that these responses are associated with T-cells secreting IL-10 in response to a neighbouring determinant.

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    • "To identify more clearly the epitope region affected by substitutions of the core cysteine, we investigated the effect of lysine substitution at E836. This substitution was previously shown to abolish binding of monoclonal antibody 96/3 and reduce binding of sera from patients with type 1 diabetes (23,24). The mutation E836K in IA-2ic and IA-2 PTP resulted in modest reductions in binding by patient sera (median 10%, range −0.8–93%, P < 0.001; and 18%, range −16–98%, P < 0.001, respectively) (Fig. 4). "
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    ABSTRACT: Cysteines are thought integral to conformational epitopes of islet antigen-2 (IA-2) autoantibodies (IA-2A), possibly through disulfide bond formation. We therefore investigated which cysteines are critical to IA-2A binding in patients with newly diagnosed type 1 diabetes. All 10 cysteines in the intracellular domain of IA-2 were modified to serine by site-directed mutagenesis, and the effects of these changes on autoantibody binding in comparison with wild-type control were investigated by radiobinding assay. Mutation of the protein tyrosine phosphatase (PTP) core cysteine (C909) in IA-2 caused large reductions in autoantibody binding. In contrast, little or no reduction in binding was seen following substitution of the other cysteines. Modification of the core cysteine (C945) in IA-2β also greatly reduced autoantibody binding. Lysine substitution of glutamate-836 in IA-2 or glutamate-872 in IA-2β resulted in modest reductions in binding and identified a second epitope region. Binding to IA-2 PTP and IA-2β PTP was almost abolished by mutation of both the core cysteine and these glutamates. The core cysteine is key to the major PTP conformational epitope, but disulfide bonding contributes little to IA-2A epitope integrity. In most patients, at disease onset, >90% of antibodies binding to the PTP domain of IA-2 recognize just two epitope regions.
    Full-text · Article · Sep 2012 · Diabetes
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    • "The evidence for molecular mimicry between an IA-2 (and GAD) epitope and the rotavirus VP7 protein has been further detailed by showing strong binding of both autoantigen and viral peptides to HLA-DRB1*04 and cross recognition by IA-2-specific T cells [92]. The 831–860 region of IA-2 frequently recognized by autoantibodies has been shown to be recognized by IL-10-secreting T cells from T1D patients [93]. Following identification of DRA1*0101/DRB1*0401-retricted IGRP23-35 and IGRP247-259 and DRA1*0101/DRB1*0301-retricted IGRP13-25 and IGRP226-238 epitopes, IGRP-specific CD4+ T cells have been detected in more than 80% of DRB1*0401 or DRB1*0301 healthy and T1D subjects [94]. "
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    ABSTRACT: Type 1 diabetes (T1D) is an autoimmune disease driven by the activation of lymphocytes against pancreatic β-cells. Among β-cell autoantigens, preproinsulin has been ascribed a key role in the T1D process. The successive steps that control the activation of autoreactive lymphocytes have been extensively studied in animal models of T1D, but remains ill defined in man. In man, T lymphocytes, especially CD8(+) T cells, are predominant within insulitis. Developing T-cell assays in diabetes autoimmunity is, thus, a major challenge. It is expected to help defining autoantigens and epitopes that drive the disease process, to pinpoint key functional features of epitope-specific T lymphocytes along the natural history of diabetes and to pave the way towards therapeutic strategies to induce immune tolerance to β-cells. New T-cell technologies will allow defining autoreactive T-cell differentiation programs and characterizing autoimmune responses in comparison with physiologically appropriate immune responses. This may prove instrumental in the discovery of immune correlates of efficacy in clinical trials.
    Full-text · Article · Jul 2011 · Clinical and Developmental Immunology
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    ABSTRACT: The aim of the current study was to investigate whether autoantigen directed T-cell reactivity relates to beta-cell function during the first 78 weeks after diagnosis of type 1 diabetes. 50 adults and 49 children (mean age 27.3 and 10.9 years respectively) with recent onset type 1 diabetes who participated in a placebo-controlled trial of immune intervention with DiaPep277 were analyzed. Secretion of interferon (IFN)-gamma, interleukin (IL)-5, IL-13 and IL-10 by single peripheral mononuclear cells (PBMC) upon stimulation with islet antigens GAD65, heat shock protein 60 (Hsp60) protein-tyrosine-phosphatase-like-antigen (pIA2) or tetanus toxoid (TT) was determined applying ELISPOT; beta-cell function was evaluated by glucagon stimulated C-peptide. Multivariate regression analysis was applied. In general, number of islet antigen-reactive cells decreased over 78 weeks in both adults and children, whereas reactivity to TT was not reduced. In addition, there was an association between the quality of immune cell responses and beta-cell function. Overall, increased responses by IFN-gamma secreting cells were associated with lower beta-cell function whereas IL-5, IL-13 and IL-10 cytokine responses were positively associated with beta-cell function in adults and children. Essentially, the same results were obtained with three different models of regression analysis. The number of detectable islet-reactive immune cells decreases within 1-2 years after diagnosis of type 1 diabetes. Cytokine production by antigen-specific PBMC reactivity is related to beta-cell function as measured by stimulated C-peptide. Cellular immunity appears to regress soon after disease diagnosis and begin of insulin therapy.
    No preview · Article · Oct 2009 · Journal of Autoimmunity
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