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.