Detection of GAD65-Reactive T-Cells in Type 1 Diabetes by Immunoglobulin-Free ELISPOT Assays

Abstract

OBJECTIVE -To investigate the prevalence of ␤-cell autoantigen-reactive peripheral T-cells in type 1 diabetes, we developed an immunoglobulin-free enzyme-linked immunospot (ELIS-POT) assay and assessed its usefulness for diagnosing this disease. RESEARCH DESIGN AND METHODS-Cellular immune responses to ␤-cell autoantigens were studied both by immunoglobulin-free proliferation assays and ELISPOT assays in 33 patients with type 1 diabetes and 15 patients with type 2 diabetes, compared with 23 healthy control subjects. Autoantibodies against GAD65 and IA-2 were measured by radioimmunoassay.RESULTS -Significant proliferative responses to GAD65 were observed in 10 of 31 (32.3%) type 1 diabetic patients (P Ͻ 0.05), whereas GAD65-reactive ␥-interferon (IFN-␥)-secreting cells were detected in 22 of 33 patients (66.7%) by ELISPOT assay (P Ͻ 0.001). Of patients negative for both GAD65 and IA-2, five of six (83.3%) showed IFN-␥ positivity in ELISPOT and two of five (40.0%) showed significant proliferation against GAD65.CONCLUSIONS -Using a newly developed ELISPOT assay, GAD-reactive T-helper 1 cells in PBMC of type 1 diabetic patients could be identified at a higher frequency than by the proliferation assay. Therefore, the immunoglobulin-free ELISPOT assay is an excellent tool for detecting T-cell reactivity to autoantigens with greater specificity and, in combination with ␤-cell autoantibody determination, will improve the diagnosis of type 1 diabetes. Diabetes Care 25:1390 -1397, 2002S everal studies of type 1 diabetes have suggested that the destruction of pancreatic ␤-cells is caused by inflammatory cellular immune responses mediated by T-helper 1 (Th1) cells, which secrete ␥-interferon (IFN-␥) (1-3). In diabetic animal models, a correlation between disease onset and Th1 cytokine production has been observed, and IFN-␥ in particular plays an important role in ␤-cell destruction (4 -6). On the other hand, autoantibodies against GAD65, insulin, IA-2/ICA512, and other islet cell antigens have been used not only to diagnose but also to predict the onset of type 1 diabetes (7-9). However, differences in age at onset of diabetes as well as racial differences have been reported to influence patients' autoantibody status (10,11). In general, circulating levels of autoantibodies are considerably lower in patients with a long disease duration than in patients at the time of onset or in the prediabetic period (12). According to a Japanese multicenter study, positivity for GAD antibody was found in only 35.4% of 921 Japanese type 1 diabetic patients studied, but among those with newly diagnosed diabetes, 50.3% were GAD antibody positive (13). Japanese type 1 diabetic subjects, therefore, seemed to present with lower antibody levels than Caucasians. In addition, it has been suggested that the presence of GAD antibody can identify a subset of latent autoimmune diabetes in adults (LADA) and predict incipient insulin dependency (14,15). Indeed, the patient's autoantibody status is essential for the diagnosis and prediction of type 1 di...

T lymphocyte response against pancreatic beta cell antigens in fulminant Type 1 diabetes

“…The ELISPOT assay was performed as previously described [10]. In brief, nitro-cellulose-bottomed 96-well microtitre plates were coated with 15 µg/ml of mouse antihuman IFN-γ monoclonal antibody (mAb; 1-DIK; Mabtech, Stockholm, Sweden) or mouse anti-human IL-4 mAb (IL-4-I; Mabtech AB) in sterile coating buffer (0.1 mol/l carbonatebicarbonate buffer, pH 9.6) overnight at 4°C.…”

“…In the previous report, six of seven Type 1 diabetic patients with no diabetes-related antibodies also had GAD-reactive T-helper-1 cells [10]. To investigate whether T cell autoimmune responses are involved in fulminant Type 1 diabetes, we evaluated the presence of islet cell antigen-reactive T cells using an ELISPOT assay in fulminant Type 1 diabetic patients.…”

“…We have recently reported that an immunoglobulinfree ELISPOT assay can detect T cell reactivity to islet cell antigens in 66.7% of Type 1 diabetic patients, with good reproducibility [10]. In the previous report, six of seven Type 1 diabetic patients with no diabetes-related antibodies also had GAD-reactive T-helper-1 cells [10].…”

T Cell Epitope Mapping Study With Insulin Overlapping Peptides Using ELISPOT Assay in Japanese Children and Adolescents With Type 1 Diabetes

“…We previously developed an immunoglobulin-free ELISPOT assay, which had a low background, and reported that this method could detect GAD-specific T cells in human T1D (10,11). In this report, ELISPOT assay was performed using serial overlapping insulin peptides in Japanese patients with T1D.…”

Autoimmunity and Immunotherapy of Type 1 Diabetes

“…In fact, the binding of phogrin autoantibodies could be totally blocked if adding ICA512 to sera positive for both ICA512 and phogrin, while the binding of ICA512 antibodies cannot be fully blocked with phogrin (Savola, 2000). IA-2As can be evaluated by radioligand-binding assay and ELISA (Bonifacio et al, 2001;Chen et al, 2005a;Kotani et al, 2002); whereas, RIAs performed much better than ELISAs, as was found for GAD65A assays (Verge et al, 1998).…”