T cell response pattern to glutamic acid decarboxylase 65 (GAD65) peptidesof newly diagnosed type 1 diabetic patients sharing susceptible HLA haplotypes

Autoantibodies and autoreactive T lymphocytes directed against several panc reatic beta cell proteins such as GAD65 have been identified in the circula tion before and at the onset of clinical type 1 (insulin-dependent) diabete s. Using GAD65 synthetic peptides, we studied the proliferative response of peripheral blood mononuclear cells (PBMC) either from recently diagnosed t ype 1 diabetic patients, of whom the majority share the disease-associated HLA class II haplotype (DR4-DQB1*0201 or DR3-DQB1*0302), or from HLA-matche d control subjects. We found that 67% (14/21) of the type 1 diabetic patien ts and 39% (9/23) of the control subjects exhibited a positive proliferativ e response. Compared with control subjects, however, PBMC from diabetic pat ients proliferated more frequently (P < 0.05) in the presence of peptide po ols from the C-terminal region of GAD65 (amino acids 379-585). Diabetic pat ients with the same HLA-DQ or HLA-DR alleles showed partially identical T c ell reactivity, but no clear correlation could be made between MHC class II specificity and T cell epitopes because of multiple combinations of class II alleles. In addition, by flow cytometry, we studied the direct binding o f GAD65 peptides to MHC class II molecules of Epstein-Barr virus (EBV)-tran sformed B (EBV-B) cells obtained from a diabetic patient. We found that 11 GAD peptides were able to bind to the highly susceptible haplotype DRB1*030 1/0401-DQA1*0301/0501-DQB1*0302/0201 on the surface of EBV-B cells in parti al correlation with the results obtained in the proliferation assays.