Immunization With the Larger Isoform of Mouse Glutamic Acid Decarboxylase (GAD⁶⁷) Prevents Autoimmune Diabetes in NOD Mice

The 65-kDa isoform of glutamic acid decarboxylase (GAD₆₅) has been implicated in autoimmune diabetes in NOD mice, but the role of the 67-kDa GAD isoform (GAD₆₇) is less clear. We found that immunization of 4-week-old NOD mice with purified recombinant mouse GAD₆₇ prevented or significantly delayed the onset of diabetes. To further explore this phenomenon, we characterized anti-GAD₆₇ immune responses in naive and GAD-immunized NOD mice. Anti-GAD₆₇ antibodies titers were relatively low in naive mice at all ages, but a single immunization with GAD₆₇ at 4 weeks induced high titers of anti-GAD antibodies by 6 weeks of age. In both 4-week-old and diabetic NOD mice, there were significant endogenous T-cell proliferative responses against purified recombinant mouse GAD₆₇. These T-cell proliferative responses were blocked by anti-I-A_NOD and anti-CD4 antibodies. To characterize the anti-GAD T-cell responses in the NOD mice, we established T-cell lines and T-cell clones which recognized GAD₆₇, and we used recombinant subfragments of GAD to localize the predominant T-cell epitopes in GAD₆₇. T-cells from naive NOD mice proliferated in response to all GAD subfragments, whereas T-cells from diabetic mice responded primarily to the COOH-terminal 83 amino acids of GAD₆₇. These results suggest that GAD₆₇ is an autoantigen in IDDM and immunization of prediabetic NOD mice with GAD₆₇ can prevent the onset of diabetes.