Study design

B lymphocyte dependent rhMOG EAE model: Female CD19TdTomato mice at 6-10 weeks of age were immunized subcutaneously with 200 µg rhMOG (1- 120), kindly provided by the lab of Dr. Jennifer Gommerman, in a 100 µl emulsion of incomplete Freund’s adjuvant (Thermofisher Scientific) supplemented with 4 mg/ml Mycobacterium tuberculosis (Thermofisher Scientific). Mice also received two intra-peritoneal doses of pertussis toxin (400 ng; List Biological Labs, Inc.) the day of immunization and 2 days later. The rhMOG consists of 120 amino acids of the extracellular portion of human MOG prepared as described by Galicia et al., 2013. The scoring system used was as follows: 0, unaffected; 1, limp tail; 2, slow righting-reflex; 3, partial hindlimb paralysis; 4, complete hindlimb paralysis; and 5, moribund/death.

Anti-ALCAM treatment of CD19TdTomato Red mice: After EAE induction, mice were injected intraperitoneally every day from day 8 to 18 with anti-ALCAM (250 µg; monoclonal MAB656; R&D Systems) or isotype control antibody (250 µg; mouse IgG1; MAB002, R&D Systems). Surgery for intravital 2 photon microscopy was performed on days 12/13 and 19/20 (d.p.i) on representative animals from each group. Mice were then sacrificed for immune cells analyses in lymph nodes, spleen and CNS by flow cytometry. Injections, scoring, surgeries and analysis were assessed by an investigator ‘blinded’ to the identity of the treatment group.

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