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=10), epilepsy (n=4), CA (n=10), or Type-I diabetes (n=14) 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. Using NetMHCpan4.2, we further identified 33 different 9mer peptides with high predicted affinity for HLA subtypes among patients and those affinities were further tested empirically using MHC I monomers. Thirty-one of 33 peptides showed high binding to 11 HLA alleles identified among patients and were subsequently utilized for tetramer labeling of CD8⁺ T cells in GAD65AbAD patients. Lastly, patient CD8⁺ T cells induced cytotoxicity and apoptosis of GAD65-expressing target cells in live-cell imaging assay.

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