Animal maintenance and experiments were in accordance with National Institutes of Health (NIH) guidelines and were approved by the Institutional Animal Care and Use Committee of the Rush University of Medical Center, Chicago, IL. Adoptively transferred EAE was induced in 4- to 5-week-old female SJL/J mice (Harlan Sprague Dawley, Indianapolis, IN), as described (1618, 40, 41). Donor mice were immunized subcutaneously with 400 μg of bovine MBP and 60 μg of M. tuberculosis in IFA (1618, 41). Animals were euthanized 10 to 12 days after immunization, and the draining lymph nodes were harvested and single-cell suspensions were cultured in RPMI 1640 supplemented with 10% FBS, MBP (50 μg/ml), 50 μM 2-mercaptoethanol, 2 mM l-glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml). On day 4, cells were harvested and resuspended in Hanks’ balanced salt solution. A total of 2 × 107 viable cells in a volume of 200 μl were injected into the tail vein of naïve mice. Pertussis toxin (150 ng per mouse; Sigma-Aldrich) was injected intraperitoneally on 0 dpt of cells. Animals were observed daily for clinical symptoms. Experimental animals were scored by a masked investigator, as follows: 0, no clinical disease; 0.5, piloerection; 1, tail weakness; 1.5, tail paralysis; 2, hindlimb weakness; 3, hindlimb paralysis; 3.5, forelimb weakness; 4, forelimb paralysis; 5, moribund or death. To induce chronic EAE, C57BL/6 mice were immunized with 100 μg of MOG35-55, as described (40). Mice also received two doses of pertussis toxin (150 ng per mouse) on 0 and 2 days post-immunization.

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