GAD65-Specific Autoantibodies
GAD65 autoantibodies (GAD65Ab) are highly prevalent in type 1 diabetes, but their functional role in the pathogenesis of the disease and their relationship to T-cell reactivity to GAD65 is still unclear. We tested the hypothesis that GAD65Ab modulate presentation of GAD65 to T-cells. T-cell hybridoma T33.1, which recognizes the GAD65 274-286 epitope in the context of HLA-DRB1*0401, was incubated with antigen-presenting cells exposed to recombinant human GAD65 alone or complexed with GAD65Ab or GAD65Ab sera. Stimulation of the T33.1 hybridoma was greatly enhanced by multiple GAD65Ab sera. The enhancement effect was most prominent with sera from patients with high GAD65 autoantibody levels. Sera from GAD65Ab subjects had no effect. The correlation between T-cell stimulation and GAD65Ab levels was not absolute, suggesting that other variables such as autoantibody recognition of different regions of GAD65 and variable effects on processing of the 274-286 epitope may contribute. Uptake of antibody-complexed GAD65 was Fc receptor (FcR)-mediated because the enhancement of presentation was inhibited by monoclonal antibodies against FcR. Our results support the hypothesis that GAD65Ab modulate presentation of GAD65 to T-cells. Increased antigen uptake and heterogeneity in the autoantibody specificity may provide a mechanism for antibody-facilitated T-cell response influencing the progression of type 1 diabetes.

GAD65 autoantibodies (GAD65Ab) are common in newly diagnosed diabetic patients and often appear years before the clinical onset of the disease . GAD65Ab are also found in some individuals without type 1 diabetes and in patients with other diseases, such as stiff-man syndrome (SMS) or polyendocrine autoimmune disorders (PE) . There are differences in epitope specificity of GAD65Ab between type 1 diabetic patients and individuals with other autoimmune disorders . GAD65Ab in type 1 diabetes predominantly recognize conformation-dependent epitopes in the middle and COOH-terminal parts of GAD65 . In contrast, GAD65Ab in SMS and PE rarely display binding to COOH-terminal epitopes, but in SMS, the high-titer GAD65Ab instead recognize short linear epitopes in the NH₂-terminal part of the molecule . These findings suggest that the epitopes may be disease specific, although some recent studies have failed to demonstrate any disease-specific epitope profile that could differentiate between GAD65Ab subjects who progress to type 1 diabetes, PE, or SMS . However, a maturing autoantibody response over time has been shown to spread from the middle region of GAD65 (EP-1) to the COOH-terminal part of the molecule (EP-2) , and a high level of GAD65Ab seems to better predict the progression to type 1 diabetes .

In addition to their direct effector functions, antibodies may play a significant role in the processing and presentation of T-cell epitopes to T-cells. Antibodies increase the efficiency of antigen capture by antigen-presenting cells (APCs), including antigen-specific B-cells, Fc receptor (FcR)-positive monocytes/macrophages, and dendritic cells . It has been shown that processing antigen-antibody complexes can substantially lower the threshold for T-cell response: in a complexed form, a 10- to 10,000-fold lower amount of antigen can be stimulatory . In addition, several studies have explored the mechanism by which antibodies bound to antigens may modulate processing T-cell determinants . Most studies that have demonstrated that specific antibodies can modulate the presentation of antigen to T-cells have used a well-defined antigen-monoclonal antibody system, e.g., tetanus , hepatitis B surface antigen , or hen egg lysozyme . A direct link between the biochemical and functional antigen-antibody interaction within APCs was provided in a series of experiments using tetanus-specific T-cell clones . An important observation was that a high-affinity antibody does not dissociate from the antigen at mildly acidic pH levels in the processing compartment . Subsequent studies showed that antibody bound to a tetanus-toxoid antigen suppressed the presentation of some T-cell epitopes while enhancing the generation of others . Recently, a similar phenomenon was reported in experimental autoimmune thyroiditis , in which thyroglobulin-specific monoclonal antibodies altered the processing of a nondominant pathogenic T-cell epitope from thyroglobulin. Similar interactions between B-cell- and T-cell-mediated immune responses may be present in type 1 diabetes because the presence of high-titer GAD65Ab may affect CD4 T-cell responses to GAD65. We tested the hypothesis that polyclonal GAD65Ab present in type 1 diabetic patients modify the processing of an immunodominant T-cell epitope from GAD65.