1489-P: GAD65_247-266 Is a Major Th1 Epitope in Japanese Patients with Acute-Onset Type 1 Diabetes

Introduction & Objective: Autoreactive T cell responses in type 1 diabetes (T1D) may vary depending on race. In Japan, T1D is classified into three subtypes based on disease progression: acute onset (AT1D), slowly progressive (SP1D), and fulminant (FT1D). The aim of this study was to compare the features of islet antigen-specific CD4⁺ T cell (Th) responses among these subtypes and determine the Th epitope in Japanese AT1D patients.

Methods: Blood mononuclear cells from 20 AT1D, 17 SP1D, and 18 FT1D patients, and 17 nondiabetics (NDs) were stimulated with GAD65, preproinsulin (PPI), IGRP, and ZnT8 peptide clusters (4-6 peptides/cluster) in the presence of IL-2. Subsequently, intracytoplasmic cytokine staining (ICS) was performed to analyze cytokine expression on specific T cells (cluster analysis). After identifying the feature of Th cell responses in each T1D subtype, another stimulation and ICS were performed using single peptides to identify Th cell epitopes (epitope analysis).

Results: In the cluster analysis, the frequencies of GAD65- and PPI-specific Th1 cells were significantly higher in AT1D patients (P = 0.007 and P = 0.004, respectively) than in NDs, whereas GAD65- and IGRP-specific Th2 cells were more prevalent in SP1D patients (P = 0.010 and P = 0.030, respectively) than in NDs. Notably, FT1D patients displayed significantly less Tr1 cells specific for all four antigens. Epitope analysis, focusing on GAD65- and PPI-specific Th1 responses in AT1D patients, revealed that 3, 16, 3, and 3 out of 20 patients displayed positive Th1 responses (stimulation index > 3.0) under GAD65_115-127, GAD65_247-266, PPI_C19-A3, and PPI_C22-A5 stimulation, respectively. In addition, the frequencies of GAD65_247-266-specific Th1 cells were significantly higher than those specific for other epitopes (P < 0.001).

Conclusion: The phenotypes of islet antigen-specific Th cells were distinct among the three T1D subtypes. Furthermore, GAD65_247-266 was identified as a major Th1 epitope in Japanese AT1D patients.