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

Detection of GAD65-Reactive T-Cells in Type 1 Diabetes by Immunoglobulin-Free ELISPOT Assays Reiko Kotani , MD , Masao Nagata , MD, PHD , Hiroaki Moriyama , MD, PHD , Maki Nakayama , MD , Katsumi Yamada , MD , Shahead Ali Chowdhury , MD , Sagarika Chakrabarty , MD , Zhenzi Jin , MD , Hisafumi Yasuda , MD, PHD and Koichi Yokono , MD, PHD Division of Internal and Geriatric Medicine, Department of Development and Aging, Kobe University Graduate School of Medicine, Kobe, Japan Abstract OBJECTIVE —To investigate the prevalence of β-cell autoantigen-reactive peripheral T-cells in type 1 diabetes, we developed an immunoglobulin-free enzyme-linked immunospot (ELISPOT) assay and assessed its usefulness for diagnosing this disease. RESEARCH DESIGN AND METHODS —Cellular immune responses to β -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 < 0.05), whereas GAD65-reactive γ-interferon (IFN-γ)-secreting cells were detected in 22 of 33 patients (66.7%) by ELISPOT assay ( P < 0.001). Of patients negative for both GAD65 and IA-2, five of six (83.3%) showed IFN-γ 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 β-cell autoantibody determination, will improve the diagnosis of type 1 diabetes. ELISPOT, enzyme-linked immunospot IFN-γ γ-interferon IL-4, interleukin-4 mAb, monoclonal antibody PBMC, peripheral blood mononuclear cell PHA, phytohemagglutinin SI, stimulation index Th1, T-helper 1 MW, molecular weight Footnotes Address correspondence and reprint requests to Masao Nagata, MD, PhD, Division of Internal and Geriatric Medicine, Department of Development and Aging, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: nagata{at}med.kobe-u.ac.jp . Received for publication 2