Mepazine acetate

Mice were randomly treated with either mepazine or control solution to eliminate possible cage effects. Mepazine acetate (Chembridge, San Diego, CA, USA) was solubilized in 0.7 × PBS at a concentration of 2 mg/ml. Mice were injected i.p. twice daily with 8 mg/kg starting at day 7 post-immunization, or between days 14 and 17 when they reached a clinical score of 2. For adoptive transfer EAE, donor mice were injected with mepazine or vehicle from the day of immunization until the isolation of splenocytes.

Eight-week-old C57BL/6 mice (Charles River France) were treated from day 0 to day 12, either via p.o. (MLT-943) or i.p. (mepazine) dosing. Mepazine acetate (Chembridge, San Diego, CA, United States) was solubilized and dosed in PEG200/water (30/70 v/v). Dosing volume was 10 ml/kg bodyweight for both MLT-943 and mepazine. Some animals were euthanized at the end of the treatment period for analysis of lymphoid organs, while other animals were kept until day 19 to analyze the recovery of Treg cells after cessation of the treatment. All mice were bled on day -1 for assessment of baseline Treg levels and activation markers on Treg cells in blood. Blood samples were collected along the treatment and recovery period to monitor changes in Tregs levels over time. Treg frequencies in blood and spleen as well as the expression of CTLA-4, TNFR2, KLRG1, and CD25 on Tregs was assessed by flow cytometry (see below). All animal studies were performed in accordance with the animal experimentation laws and guidelines laid down by the Swiss Federal and Cantonal Authorities.