Characterization of self-glutamic acid decarboxylase 65-reactive CD4 + T-cell clones established from Japanese patients with insulin-dependent diabetes mellitus

To investigate autoimmunity to glutamic acid decarboxylase (GAD) 65 in Japanese patients with insulin-dependent diabetes mellitus (IDDM, type I diabetes), we established seven CD4 + T-cell clones, by stimulating peripheral blood mononuclear cells (PBMC) of six IDDM patients, using a mixture of overlapping human GAD65 peptides. No GAD65 autoreactive T-cell clones were evidenced in four healthy controls. Specificities of T-cell clones were as follows: (a) two clones specific to GAD65 p111-131 (residue 111 to 131) + DR53 (DRB4∗0103); (b) one clone specific to GAD65 p413–433 + DR1 (DRB1∗0101); (c) two clones specific to GAD65 p200–217 + either DR9 (DRB1∗0901) or DR8 (DRB1∗0802); and (d) two clones specific to GAD65 p368–388 + DP2 (DPA1∗01 or 0201-DPB1∗0201). Two DR53-restricted and one DR1-restricted T-cell clones, responded to a recombinant human GAD65 protein, and showed cytotoxicity against B lymphoblastoid cell lines pre-pulsed with the peptides. Six T-cell clones exhibited the Th1-like phenotype. Interestingly, two DR53-restricted T-cell clones killed a Fas-deficient B lymphoblastoid cell line, thereby indicating that cytotoxicity was not completely dependent on a Fas-Fas ligand interaction. Thus, the T-cell epitopes were mapped in a limited portion of GAD65 protein, with a tendency to be restricted by disease-associated HLA-DR, but not DQ molecules.