At day 9, 18 and 28 p.i., cell suspensions from spleen and lymph node were prepared respectively by mechanical disruption of tissues and isolated using Mouse Lymphocyte Separation Medium (DAKEWE, China). Mononuclear cells (MNCs) were cultured in RPMI-1640 medium and stimulated with 10μg/ml MOG35-55 for 48 hours. For intracellular IFN-γ, IL-17A and Eomes staining, cells were re-stimulated with eBioscience Cell Stimulation Cocktail (eBioscience, USA) for additional 5 hours. Cells were fixed and permeabilized using eBioscience Foxp3 staining kit (eBioscience, USA), and stained with anti-CD4 Ab (RM4-5; eBioscience), anti-IFN-γ Ab (XMG1.2; eBioscience), anti-IL-17A Ab (TC11-18H10.1, BioLegend) and anti-Eomes Ab (Dan11mag; eBioscience) according to the manufacturer's instructions. For flow cytometric analysis of JNK1/2 (pT183/pY185,), mice splenocytes were re-stimulated with PMA (Sigma, 400 nM), and Ionomycin (Sigma, 250 ng/ml) at 37˚C for 15 minutes according to the manufacturer's instructions and previous study 15. Then cells were fixed in 1× BD Phosflow™ Lyse/Fix Buffer (10 minutes at 37˚C) and permeabilized in BD Phosflow™ Perm Buffer III on ice for 30 minutes. Cells were then stained with anti-JNK (pT183/pY185) ab (562481, BD Bioscience). Appropriate fluorescein-conjugated, isotype-matched, irrelevant mAbs were used as negative controls. Cells were acquired on Attune NxT Flow Cytometer (Thermo Fisher Scientific, USA) and analyzed by FlowJo VX Software (FlowJo, LLC).

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