Thirty million PBMCs in culture medium at a concentration of 150 million/ml were incubated with 50 nM dasatinib for 10 min at 37°C. The cells were next stained with 250 nM PE-labeled tetramers at room temperature for 120 min, followed by antibody staining for 20 min at 4°C with CD4 APC (clone RPA-T4, eBioscience), CD45RO FITC (clone UCHC1, eBioscience), and a combination of CD14 PerCP (clone MφP9, BD Pharmingen) and CD19 PerCP (clone SJ25C1, BD Pharmingen) to exclude B cells and monocytes from the analysis. Cells were washed twice and incubated with anti-PE magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°C for another 20 min. The cells were washed again and enriched with a Miltenyi magnetic column. Samples were labeled with ViaProbe (BD Biosciences) and analyzed on a BD FACSCalibur flow cytometer. Frequencies were calculated by dividing the number of tetramer-positive cells in the bound fraction by the number of total CD4+ T cells in the sample. For ex vivo costaining of GAD65250–266-specific cells with DQ8cis and DQ8trans tetramers, PBMCs from DQ8+DQ2 participants were first stained with PE-labeled DQ8trans/GAD65250–266 tetramers. After enrichment, tetramer-positive cells were stained again with APC-labeled DQ8cis/GAD65250–266 tetramers at 37°C for 1 hour.

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