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

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.