Prevention and treatment of EAE with CD4⁺CD25⁺ RMTCs. (A) Prophylaxis of EAE. EAE was induced using a dual immunization regimen with MBP89-101 followed by the administration of pertussis toxin.¹⁵  To determine the clinical impact of the CD4⁺CD25⁺ RMTCs on encephalomyelitis, 1 × 10⁶ flow-cytometrically purified CD4⁺CD25⁺ RMTCs (▴), control cells, including CD4⁺CD25^- RMTCs (⬡), CD4⁺CD25⁺ non-Tg T cells (), or CD4⁺CD25^- non-Tg T cells (▪), or saline (PBS; ) was intravenously administered into SJL/J mice at the time of disease induction. Clinical course was monitored. (B) Dose-response relationship of CD4⁺CD25⁺ RMTCs; indicates 1 × 10⁶ cells; ▪, 0.5 × 10⁶; ▴, 0.25 × 10⁶; and ⬡, 0.125 × 10⁶. indicates PBS. (C) Delayed treatment of EAE. EAE was induced as above and 1 × 10⁶ Tg (▴) or non-Tg CD4⁺CD25⁺ T cells () were administered 11 days later to measure the activity of RMTCs at the time of early disease development. indicates PBS. (D) Treatment of adoptive transfer EAE. Draining LN cells from MBP89-101–immunized mice were briefly cultured in vitro and 25 × 10⁶ cells intravenously transferred into naive mice. On the same day, 1 × 10⁶ CD4⁺CD25⁺ RMTCs or control cells were administered intravenously through a separate site and disease was monitored clinically. Symbols indicate same information as in panel A. (E) Treatment of EAE after epitope spread. Treatment with 1 × 10⁶ Tg or non-Tg cells was delayed until 31 days after disease induction (vertical line). At this time, T-cell response was detected not only against the initiating MBP89-101 epitope, but also against pathologic PLP139-151 and PLP178-191 epitopes.¹²  Mean score for each arm was adjusted at the time of treatment to exclude animals not treated due to a moribund status or death. Symbols indicate same information as in panel A. Plots A-E show mean clinical score from 4 to 6 animals per treatment group. Each plot is representative of 2 to 3 independent experiments. A 2-sided t-test analysis of mean daily clinical score from the time of first disease symptoms (A-D) or treatment (E) to the end of observation showed a statistically significant beneficial effect of the CD4⁺CD25⁺ RMTCs compared with each control group in the experiments shown in panels A and C-E (P < .05). In the experiment shown in panel B, a statistically significant benefit (P < .05) was observed for doses equal to or greater than 5 × 10⁵. At a dose of 2.5 × 10⁵, a beneficial effect was observed from days 30 to 45 only (P < .05).