FIGURE 5.

Immune cell function in vivo in female C57BL/6J C‐EAE is not directly affected by digoxin. 8‐week‐old female C57BL/6J mice remained naïve, or were primed with OVA_323‐339, or primed with MOG_35‐55, followed by treatment (Tx) i.p. with vehicle (VEH) or digoxin (DIG) 0.3 mg/kg qd for 5 days beginning at d15 post‐priming. Clinical scores were assessed until d20 (a). At d20, splenocytes from the various groups (n = 4 mice/group) were re‐activated in culture with MOG_35‐55 and cytokines in the culture supernatants were assessed for levels of the indicated cytokines using Luminex analysis (b). Female C57BL/6J recipient mice (8‐weeks‐old, n = 24 mice/group) received 3 × 10⁶ in vitro peptide re‐activated splenocytes labeled with CFSE from MOG_35‐55‐primed donor mice and were treated (Tx) i.p. with vehicle (VEH) or digoxin (DIG) 0.3 mg/kg qad from day 0–16; mean clinical scores (c) and day of onset of disease symptoms (d). At initial disease peak (day 17), mice were sacrificed and the numbers of donor MOG_35‐55‐specific CFSE⁺ T cells in the brains and spleens of the recipient mice (n = 6 mice/group) were assessed (e). In addition, splenocytes from the recipient mice (n = 6 mice/group) were stimulated in culture with MOG_35‐55 and cytokine production in culture supernatants were assessed by Luminex assay (f). Flow cytometric analysis of forebrain/spinal cord OL lineage cells (pre‐OL/OL, O4⁺, measured as fold change vs. vehicle), of n = 4 mice/group from panel a with actively‐induced C‐EAE, was assessed at day 20 (g). (ns, not significant).