Abstract Details

GAD65 antibodies of any titer are found in patients with DM1 while high-titer GAD65 antibodies can be detected in CSF and serum from patients with neurological autoimmunity like SPS. However, given its intracellular localization a direct pathogenic role for GAD65 autoantibodies is not expected and these antibodies likely serve as a surrogate marker for cell-mediated autoimmunity. 

Investigate glutamic acid decarboxylase 65 (GAD65)-specific T cell responses in patients with stiff-person syndrome (SPS) and type-I diabetes mellitus (DM1).

We utilized full-length GAD67, GAD65 proteins, and 226 overlapping 15-mer peptides covering full sequence of the two proteins to treat autologous PBMC-derived dendritic cells (DCs) from patients with SPS (n=5), DM1 (n=4), and healthy controls (n=12). We further co-cultured autologous T cells with DCs and measured the T cell activation via cytometric bead array, FluoroSpot, and Flow Cytometry. Direct cytotoxicity was measured by coculturing patient CD8+T cells with GAD65+ target cells via live cell imaging.

Using the peptidome methodology, we identified the same 5/46 built peptide pools that elicited T cell activation by multiple metrics and measured the capacity of individual peptides within these pools to activate patient T cells in both DM1 and SPS. We found 10-30% increase in the CD8+CD69+ T cell subpopulations and 5-time elevation of CD8+IL-2+ T cells after irritation from peptides of interest. Predicted affinity for patient HLA class I allotypes was analyzed by NetMHCpan4.1. Lastly, we report the capacity of patient CD8+ T cells to induce direct cytotoxicity and eliminate GAD65+  target cells within 48 hours (p <0.01, Student’s T test).

We identified and demonstrated specific GAD65 peptides can activate cytotoxic T cells in patients with SPS and DM1. Importantly, our research provides evidence for cytotoxic T cell pathogenic pathways and potential peptide sequence for counteracting combination between cytotoxic T cell receptors and MHC-I presented antigens in SPS.