To characterize glutamic acid decarboxylase 65 (GAD65)-specific T cell responses in patients with GAD65 antibody (GAD65Ab) -associated disorders (AD).

High-titer GAD65 antibodies are found in patients with stiff-person syndrome (SPS), cerebellar ataxia (CA), and epilepsy. Given the intracellular localization of the antigenic target, a direct pathogenic role for GAD65 autoantibodies is unlikely. Instead, the autoantibody may be a biomarker for the existence of pathogenic anti-GAD65 autoreactive CD8⁺ T cells.

PBMC-derived dendritic cells (DCs) from GAD65Ab+ patients with SPS (n=5), epilepsy (n=4), or CA (n=4) were pulsed with full-length GAD65 proteins or with 15-mer peptides from GAD65. T cell activation was measured via cytometric bead array, FluoroSpot, and flow cytometry. GAD65-peptide-MHC restricted patient T cells were identified by tetramer labeling. Direct cytotoxicity was measured by coculture with MHC class I haplotype matched GAD65⁺ target cells.

CD8⁺ T cells from GAD65AbAD patients were activated by exposure to DCs pulsed with full-length GAD65 (p <0.01, Mann-Whitney test; n=10). Activation was particularly apparent in patients with an epilepsy phenotype (p <0.01, n=4), compared to SPS or CA. We found 3 peptides of interest (GAD65_{211-225,241-255,451-465}) that consistently caused a 10-30% increase in CD69⁺CD8⁺ T cells and a 2-fold elevation of CD8⁺IL2⁺ T cells in all GAD65AbAD patients. Using NetMHCpan4.2, we further identified 8 different 9mer peptides with high predicted affinity for HLA-C*03:03/04 and HLA-C*07:01/02. These allotypes were present in 50% and 100% of GAD65⁺ patients in our cohort, respectively. Two of these peptides showed high binding to HLA-C and subsequently demonstrated tetramer labeling of CD8⁺ T cells in GAD65AbAD patients. Lastly, patient CD8⁺ T cells induced cytotoxicity and apoptosis of GAD65-expressing target cells.

We demonstrated that both full-length GAD65 protein and specific GAD peptides can activate CD8⁺ cytotoxic T cells isolated from patients with GAD65AbAD.