B6 mice were treated with CD20 or control mAb (250 μg) before or after MOG immunization (days –7, 7, 14, or 21) and scored daily for EAE disease severity. Arrowheads indicate the day of mAb injection. Values represent (mean ± SEM) EAE clinical scores from more than 5 mice in each group, with similar results obtained in 3 independent experiments. Significant differences between CD20 and control mAb–treated groups are indicated; *P < 0.05.

(A and B) Representative lumbar spinal cord sections harvested 18 days after MOG immunizations (n ≥ 4 mice per group) show (A) inflammation (H&E staining) and (B) demyelination (Luxol Fast Blue staining). Upper panels are low magnification (scale bar: 0.5 mm). Lower panels are high magnification (scale bar: 0.01 mm). (A) Arrowheads indicate inflammatory foci. (B) Yellow traced areas indicate demyelination. (C) Bar graphs indicate (mean ± SEM) numbers of inflammatory foci and (D) demyelinated area, with significant differences between CD20 (black bars) and control mAb–treatments (white bars) indicated; *P < 0.05, **P < 0.005. Similar results were obtained in at least 2 independent experiments.

B6 mice were treated with CD20 or control mAb 7 days before or 14 days after MOG immunization. Representative depletion of B cells from the (A) bone marrow, (B) blood, (C–F) spleen, (G) peripheral lymph nodes, and (H) peritoneal cavity 18 days after MOG immunization (n ≥ 4 mice per group) as determined by immunofluorescence staining with flow cytometry analysis. Within in the histograms, numbers indicate relative percentages of lymphocytes within the indicated gates. Bar graphs indicate (mean ± SEM) numbers of blood (per ml) and tissue B cells following mAb treatment. Within the bar graphs, numbers indicate the percentage of B cells of each phenotype found in CD20 mAb–treated mice (black bars) relative to the numbers of B cells found in control mAb–treated littermates (white bars). Significant differences between CD20 versus control mAb–treated mice are indicated; *P < 0.05, **P < 0.01. Data are representative of at least 2 independent experiments. MZ, marginal zone.

B6 mice were treated with CD20 (closed circles) or control (open circles) mAb 7 days before or 14 days after MOG immunization. Sera were collected 18 days (peak phase) and 28 days (recovery phase) after MOG immunization, with IgM and IgG MOG-specific antibody levels quantified by ELISA. Horizontal bars indicate mean values. Dashed lines indicate mean MOG-specific antibody values for unimmunized mice (n = 6). Significant differences between CD20 and control mAb–treated groups are indicated; *P < 0.01, **P < 0.001. Similar results were obtained in 2 independent experiments.

B6 mice were treated with mAb 7 days before or 14 days after MOG immunizations. CNS-infiltrating mononuclear cells were isolated from CNS tissue pooled from more than 3 mice per experiment, while splenocytes and lymph node lymphocytes were isolated from individual mice. (A) Representative histograms showing MOG-specific T cells following CD20 or control mAb treatments as determined by immunofluorescence staining with flow cytometry analysis. CD4⁺ T cells were staining with MOG/IAb or control tetramers 18 days after MOG immunizations and assessed by immunofluorescence staining with flow cytometry analysis. Representative MOG-specific Teff (MOG/IAb-tetramer⁺CD4⁺FoxP3^–) and Treg (MOG/IAb-tetramer⁺CD4⁺FoxP3⁺) frequencies are shown within the indicated CD4⁺ T cell quadrants. Numbers indicate percentages of CD4⁺ T cells within each quadrant. Bar graphs indicate numbers (mean ± SEM, n ≥ 4 experiments) of CNS-infiltrating CD4⁺ T cells and Teff and Tregs, and the ratio of Teff/Tregs following CD20 (black bars) or control mAb (white bars) treatments. (B) IL-17 and IFN-γ production by CNS-infiltrating CD4⁺ T cells 18 days after MOG immunization as determined by intracellular cytokine staining with flow cytometry analysis. Numbers indicate percentages of CD4⁺ T cells within the indicated quadrants. Bar graphs indicate numbers (mean ± SEM, n ≥ 3 experiments) of CNS-infiltrating IL-17⁺ and IFN-γ⁺ CD4⁺ T cells following CD20 (black bars) or control mAb (white bars) treatment. (A and B) Significant differences between sample means are indicated; *P < 0.05, **P < 0.01. Similar results were obtained in at least 2 independent experiments.

B6 mice were treated with CD20 or control mAb 7 days before or 14 days after MOG immunizations. (A) Eighteen days after MOG immunizations, CD4⁺ T cells were purified from superficial lymph nodes and incubated with MOG peptide plus mitomycin C–treated B cells from control mAb–treated EAE mice (n ≥ 3 mice per group). Values indicate (mean ± SEM) [³H]-thymidine (TdR) uptake from triplicate cultures. (B) Seventeen days after MOG immunizations, CFSE-labeled TCR^MOG CD4⁺Thy1.1⁺ T cells were transferred into Thy1.2 congenic recipients. Four days later, CNS-infiltrating cells were stained for CD4/Thy1.1 expression and analyzed for CFSE dilution by flow cytometry analysis. Representative frequencies of dividing CFSE-labeled cells are shown (gated on CD4⁺Thy1.1⁺ cells). (C) Seventeen days after MOG immunizations, CFSE-labeled TCR^MOG CD4⁺ T cells were transferred into mice. Four days later, superficial lymph node cells were stained for Vβ11/CD4 expression and analyzed for CFSE dilution by flow cytometry. Representative frequencies of dividing CFSE-labeled cells are shown (gated on CD4⁺Vβ11⁺CFSE⁺ cells). (B and C) Bar graphs indicate (mean ± SEM) numbers of dividing TCR^MOG T cells following CD20 (black bars) or control mAb (white bars) treatments. Numbers indicate percentages of CFSE-labeled CD4⁺ T cells. (A–C) Significant differences between CD20 versus control mAb treatment are indicated; *P < 0.05, **P < 0.001. Similar results were obtained in at least 2 independent experiments.

Representative flow cytometry analysis of CD4⁺ T cell subsets 18 days after MOG immunizations (n ≥ 4 mice per group): (A) naive, CD4⁺CD44^–CD62L⁺; activated, CD4⁺CD44⁺CD62L⁺; memory CD4⁺CD44⁺CD62L^–; (B) and Treg, CD4⁺CD25⁺FoxP3⁺. Numbers show relative percentages of lymphocytes within each quadrant. Bar graphs indicate T cell numbers (mean ± SEM) following CD20 (black bars) or control (white bars) mAb treatments. Similar results were obtained in at least 2 independent experiments.

(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).