EVALUATION OF GLUTAMATE DECARBOXYLASE (GAD65)-REACTIVE T-CELLS IN PATIENTS WITH NEW-ONSET TYPE 1 DIABETES

Abstract

<h3>Introduction</h3> Glutamate decarboxylase (GAD65 isoform) is a key pancreatic islet auto-antigen in type 1 diabetes (T1D). GAD65 antibody is routinely assayed as a diagnostic serological marker in T1D, but blood circulating antigen specific T cells (ASCs) are not. This study evaluates GAD65-reactive ASCs in patients with new-onset T1D. <h3>Methods</h3> GAD65 antibody-positive patients who provided informed consent were included in the study NCT05207995 (n=18). Whole blood was mixed with AIM-V media and monensine (1:1) and cultured for 6h with the addition: 1) PBS – negative control (NC), 2) pool of peptides, covering sequence of GAD65, 3) PMA – positive control (PC). Counts (%) of CD3⁺IFN-γ⁺ ASCs were assayed by FACS. <h3>Results</h3> The number of CD3⁺IFN-γ⁺ cells was > 20% in PC and <0.003% in NC in all samples (n = 18). Only 2 of 18 patients (11%) were positive for GAD65-reactive ASCs. There were 0.002% CD3⁺IFN-γ⁺ in NC, 0.021% after GAD65 peptide pool stimulation in the blood sample of one person and 0.001% CD3⁺IFN-γ⁺ in NC, with 0.025% in a GAD65 stimulated sample in the second one. <h3>Conclusion</h3> Patients up to 12 months after newly diagnosed T1D were positive for GAD65-reactive ASCs in 11% of cases whereas all of them have GAD65 antibody. The dynamic analysis of autoreactive ASCs together with antibody detection can be used as biomarker for tracing disease progression and immunotherapy effectiveness during cell therapy of T1D based on tolerogenic dendritic cells primed with GAD65 peptides.