Mice were anesthetized with a mixture of tiletamine and xylazine (10 mL/kg, intraperitoneal (i.p.)). Subsequently, EAE was induced in mice using Myelin Oligodendrocyte Glycoprotein peptide (MOG) 35–55 (MEVGWYRSPFSRVVHLYRNGK; % peak area by high-performance liquid chromatography (HPLC) ⩾ 95, AnaSpec, EGT Corporate Headquarters, Fremont, CA, USA) as reported by Paschalidis et al.24 (link) In brief, mice were immunized subcutaneously in the flank with 300 µL of emulsion (300 µg of (MOG) 35–55 in Complete Freund’s Adjuvant (CFA) with 300 µg of heat-killed Mycobacterium tuberculosis H37Ra (Difco Laboratories Sparks, MD, USA)). An i.p. injection of Bordetella pertussis toxin (500 ng in 100 µL; Sigma-Aldrich, Milan, Italy) was administered immediately after (MOG) 35–55 injection and after 48 h. After 14 days of EAE induction, active encephalitogenic responses in EAE-induced mice were identified with the visible pathological signs such as tail flaccidity and loss of hind legs movement.