Mice were intracardially perfused with 50 ml of phosphate-buffered saline (PBS). The forebrain and cerebellum were dissected, and spinal cords were collected from the spinal canal. CNS tissue was cut into pieces and digested with collagenase D (2 μg/ml; Roche Diagnostics) and deoxyribonuclease I (DNase I; 1 μg/ml; Sigma-Aldrich) at 37°C for 20 to 30 min while rotating. Mononuclear cells were isolated by passing the tissue through a 200-mesh cell filter membrane, followed by 80%/40% Percoll gradient centrifugation. Mononuclear cells were carefully removed from the interface, washed with PBS, and resuspended in culture medium for further analysis. For cytokine analysis, mononuclear cells were stimulated for 5 hours with phorbol 12-myristate 13-acetate and ionomycin (both from Sigma-Aldrich) in the presence of brefeldin A (eBioscience) and then subjected to flow cytometry analysis to detect intracellular IL-17A, IFN-γ, and Foxp3.

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