Longitudinal changes in epitope recognition of autoantibodies against glutamate decarboxylase 65 (GAD65Ab) in prediabetic adults developing diabetes

Abstract

We analysed the beta cell-specific autoimmunity reflected in autoantibodies to the smaller isoform of glutamate decarboxylase (GAD65Ab) in the prediabetic period of GAD65Ab-positive healthy adults who developed Type 2 diabetes (T2D) during a follow-up period of 10 years. We found that of the adults that tested GAD65Ab-positive at baseline (n=25), six developed T2D and one developed Type 1 diabetes (T1D). Of the subjects that tested GAD65Ab-negative at baseline (n=2209), 81 developed T2D, one developed T1D and four developed unclassified diabetes, indicating that the risk for GAD65Ab-positive healthy adults to develop diabetes is increased sixfold. The GAD65Ab epitopes were characterized in a competition radioligand binding assay using recombinant Fab derived of GAD65-specific monoclonal antibodies. We observed that the GAD65Ab epitope specificities in the prediabetic period changed dynamically. Specifically, the binding to a middle and a C-terminal epitope increased during the follow-up period (P=0 x 03), causing a significant increase in the number of epitopes recognized (P=0 x 03). These findings are similar to previous observations of dynamic changes in the prediabetic period of schoolchildren at high risk for T1D development. However, the character of the epitopes differs between the two populations, suggesting differences in the beta cell-specific autoimmune response in the prediabetic period of patients with latent autoimmune diabetes in adults (LADA) and T1D.

Fig. 1

Flow-chart demonstrating the development of diabetes among glutamate decarboxylase antibody (GAD65Ab)-positive (upper panel) and -negative (lower panel) individuals.

Fig. 2

Glutamate decarboxylase (GAD65Ab) at baseline and follow-up. GAD65Ab titres are shown in (a). Serum samples were analysed for GAD65Ab epitope binding using rFab DPC (b), DPA (c), b96·11 (d), DPD (e) and N-GAD65 mAb (f). Binding in the presence of competing rFab is presented as percentage relative to binding to GAD65 in the absence of competitor (set as 100%). The cut-off for unspecific binding is indicated at 80% by the dashed line. Individual subjects are presented by the same symbol throughout this figure. To allow clear identification of all symbols, overlapping or partially overlapping symbols are shown in parallel positions.

Fig. 3

Epitope numbers recognized by glutamate decarboxylase (GAD65Ab)-positive adults that developed Type 2 diabetes during follow-up. Individuals are presented with the same symbols as in Fig. 2. To allow clear identification of all symbols, overlapping or partially overlapping symbols are shown in parallel positions.

Fig. 4

Glutamate decarboxylase (GAD65Ab) specificities and recognized epitope numbers by the GAD65Ab-positive adult who developed Type 1 diabetes during follow-up. GAD65Ab were analysed at baseline and at follow-up. GAD65Ab titres are shown in (a). Serum samples were analysed for GAD65Ab epitope binding (b) using rFab DPA (black square), b96·11 (black triangle), DPC (black diamond), DPD (black circle) and N-GAD65 mAb (white circle). Binding in the presence of competing rFab is presented as percentage relative to binding to GAD65 in the absence of competitor (set as 100%). Epitope numbers recognized by the individual at baseline and at follow-up (c).