(A) Splenic CD1d^hiCD5⁺ or non-CD1d^hiCD5⁺ B cells were purified from naive mice or mice with EAE (day 10) by cell sorting. RNA was isolated from purified splenic B cells. Values represent relative mean IL-10 transcripts normalized to GAPDH transcript levels (mean ± SEM, n ≥ 6 mice per group) as quantified by real-time PCR analysis. Significant differences between sample means are indicated; *P < 0.05, **P < 0.01. Similar results were obtained in at least 2 independent experiments. (B) Representative results showing splenic CD19⁺ B cells from Cd20^–/– mice sorted into regulatory CD1d^hiCD5⁺ and nonregulatory CD1d^hiCD5⁺ B cell subsets. (C–E) Wild-type recipient mice that had been treated with CD20 or control mAb 7 days before MOG immunization (arrowheads) were given either purified CD1d^hiCD5⁺ or non-CD1d^hiCD5⁺ B cells from (C) naive Cd20^–/– mice 2 days before immunization, (D) naive Il10^–/–Cd20^–/– mice 2 days before immunization, or (E) naive Cd20^–/– mice 14 days after immunization. (F) Wild-type recipient mice treated with CD20 or control mAb 14 days after MOG immunization (arrowhead) were also given purified CD1d^hiCD5⁺ B cells from naive Cd20^–/– mice. Values represent (mean ± SEM) from more than 5 mice in each group, with similar results obtained in 2 independent experiments. Significant differences between the means of EAE clinical scores are indicated: *P < 0.05 (CD20 mAb treated plus CD1d^hiCD5 versus mAb treated); **P < 0.05 (CD20 mAb treated plus CD1d^hiCD5 versus CD20 mAb treated plus non–CD1d^hiCD5⁺ treated); ^†P < 0.05 (CD20 mAb treated plus CD1d^hiCD5 versus control mAb treated).