Administration of ISLAD suppresses MOG35-55–induced EAE by downregulation of pathogenic T cells. (A) The graph shows ex vivo downregulation of encephalitogenic MOG35-55–reactive T cells following ISLAD administration. C57Bl mice were injected for EAE induction with MOG35-55 peptide. Ten days after immunization, draining LNCs from each treatment group (n = 4 mice per group) were pooled and analyzed for their ex vivo recall proliferative response to MOG35-55. The LNCs from each treatment group were cultured for 72 hours in microtiter wells in triplicate (0.5 × 10⁶ per well) in the absence or presence of MOG35-55 (2.5 or 5 μg/mL). [H3] thymidine was added for the last 18 hours. Results are the means ± SD. The graphs in (B-D) present the secretion of pro-inflammatory cytokines from cultured LNCs derived from PBS- or ISLAD-administered EAE-mice. LNCs from each treatment group were cultured (5 × 10⁶/mL) in the absence or presence of MOG35-55 (5 μg/mL) for 24 hours, and the supernatants were collected for detecting the secreted IFN-γ (B), TNF-α (C), and IL-12 (D) by ELISA. Results are the mean ± SD. (E) Clinical EAE was induced in mice that were administered PBS (♦) or 0.5 mg/kg of ISLAD (▪), n = 10 mice per group. Following the encephalitogenic challenge, mice were observed and scored for clinical severity of the disease. The daily rated clinical score is the mean ± SE. Results are from a representative experiment (out of 2 experiments). *P < .05; **P < .01.