To induce EAE in Rag1−/− mice, naive CD4+ T cells (CD4+CD25-CD62LhiCD44lo) from WT and RORγtcreCxxc1fl/fl or dLckcreCxxc1fl/fl mice were sorted by the Mouse CD4 Naïve T cell Enrichment Kit (no. 8804-6824-74, Invitrogen) and intravenously transferred into Rag1−/− mice at 2 × 106 cells per mouse. Two days later, the recipient mice were subjected to EAE induction.

Female age-matched Rag1−/− mice (8 to 10 weeks old) were immunized with an emulsion containing the MOG peptide MOG35–55 (200 μg per mouse; MEVGWYRSPFSRVVHLYRNGK; Sangon) in an equal amount of complete Freund’s adjuvant (200 μl per mouse; no. 7027, Chondrex Inc.). Pertussis toxin (200 ng per mouse; no. 181, List Biological Laboratories) was administered intravenously 0 and 2 days after induction. Clinical evaluation was performed daily using a five-point scale: 0, no clinical signs; 1, limp tail; 2, paraparesis (weakness, incomplete paralysis of one or two hind limbs); 3, paraplegia (complete paralysis of two hind limbs); 4, hind limb and fore limb paralysis; and 5, moribund or death.

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