Mog35 55 peptide

For adoptive transfer EAE, age- and sex-matched donor mice were subcutaneously immunized with 150 µg MOG_35–55 peptide (Genemed Synthesis, Inc.) emulsified in complete Freund’s adjuvant (5 mg/ml heat-killed M. tuberculosis), followed by an intraperitoneal (IP) injection of 250 ng of Bordetella pertussis toxin (List Biological Laboratories, Inc.) in 200 µl of PBS at 0 and 2 days postimmunization. Ten days postimmunization, spleens and lymph nodes were collected and mechanically disrupted to generate a single-cell suspension. For TH17-EAE, the cells were cultured at 2.5 × 10⁶ cells/ml for 72 h and stimulated with 10 µg/ml MOG_35–55, 10 ng/ml IL-23, and 10 µg/ml IFN-γ antibody in complete RPMI media (23 (link)). For TFH-EAE, cells were cultured with 10 µg/ml MOG_35–55, 20 ng/ml IL-6, 20 ng/ml IL-21, 10 µg/ml IFN-γ antibody, 10 µg/ml IL-4 antibody, and 20 µg/ml TGF-β antibody in complete RPMI media as previously described (24 (link)). On Day 3, cells were collected and 5 × 10⁶ cultured cells were transferred into healthy recipient mice by IP injection.
Mice were monitored daily for clinical signs. Paralysis was assessed using a standard clinical score ranging from 0 to 5 with scores corresponding to the following phenotypes: 0, no disease; 1, loss of tail tone; 2, partial hind-limb paralysis; 3, complete hind-limb paralysis; 4, forelimb paralysis; and 5, moribund/dead.

EAE was induced by subcutaneous immunization with 100 µg of myelin oligodendrocyte glycoprotein (MOG_35–55) peptide (Genemed Synthesis Inc., San Antonio, TX, USA) emulsified in 25 µL of Complete Freund’s Adjuvant (Sigma-Aldrich, St. Louis, MO, USA) containing 4 mg/mL of Mycobacterium tuberculosis (Difco, Detroit, MI, USA). Mice also received 2 intraperitoneal doses (150 µg each) of Bordetella pertussis toxin (Sigma-Aldrich); one following immunization and the other 48 h later. EAE clinical severity was evaluated daily by body weight loss and clinical score determinations. The following score system was adopted: 0—no symptoms; 1—limp tail; 2—loss of hip tone; 3—partial hind leg paralysis; and 4—complete hind leg paralysis. The percentage of weight loss and the maximum clinical score was calculated considering the highest values observed in all animals during the experiment.

MOG35–55 peptide (MEVGWYRSPFSRVVHLYRNGK) was synthesized by Genemed Synthesis Inc. (San Antonio, Texas, USA). Mice were immunized subcutaneously with 100 μg of MOG35–55 peptide emulsified in 25 μL of Complete Freund's Adjuvant (CFA) containing 4 mg/mL of Mycobacterium tuberculosis. Mice also received 2 intraperitoneal doses, 0 and 48 hours after immunization, of 200 ng of Bordetella pertussis toxin (Sigma-Aldrich Corporation, St. Louis, MO, USA). EAE clinical assessment was daily performed according to the following criteria: 0, no symptoms; 1, limp tail; 2, hind legs weakness; 3, partially paralyzed hind legs; 4, complete hind leg paralysis; and 5, complete paralysis/death. The % of weight loss and the maximum clinical score were calculated considering the highest body weight loss and the highest clinical score that each animal reached during the experiment, independently of the period, and the result was expressed as the mean per experimental group.