Regulatory B cells (B10 cells) and regulatory T cells have independent roles in controlling experimental autoimmune encephalomyelitis initiation and late-phase immunopathogenesis

Abstract

Experimental autoimmune encephalomyelitis (EAE) is a T lymphocyte-mediated autoimmune disease of the CNS. Significant roles for B cells and a rare IL-10-producing CD1d(high)CD5(+) regulatory B cell subset (B10 cells) have been identified during the initiation and progression of EAE. Whether and how the regulatory functions of B10 cells and FoxP3(+) T regulatory cells (Tregs) overlap or influence EAE immunopathogenesis independently has remained unanswered. This study demonstrates that the number of endogenous or adoptively transferred B10 cells directly influenced EAE pathogenesis through their production of IL-10. B10 cell numbers expanded quickly within the spleen, but not CNS following myelin oligodendrocyte glycoprotein(35-55) immunization, which paralleled B10 cell regulation of disease initiation. The adoptive transfer of myelin oligodendrocyte glycoprotein(33-35)-sensitized B10 cells into wild-type mice reduced EAE initiation dramatically. However, B10 cells did not suppress ongoing EAE disease. Rather, Treg numbers expanded significantly within the CNS during disease progression, which paralleled their negative regulation of late-phase disease. Likewise, the preferential depletion of B10 cells in vivo during disease initiation enhanced EAE pathogenesis, whereas Treg depletion enhanced late-phase disease. B10 cells did not regulate T cell proliferation during in vitro assays, but significantly altered CD4(+) T cell IFN-gamma and TNF-alpha production. Furthermore, B10 cells downregulated the ability of dendritic cells to act as APCs and thereby indirectly modulated T cell proliferation. Thus, B10 cells predominantly control disease initiation, whereas Tregs reciprocally inhibit late-phase disease, with overlapping B10 cell and Treg functions shaping the normal course of EAE immunopathogenesis.

Figure 1

B10 cell and Treg cell numbers increase during EAE. (A) CD1d^hiCD5⁺ B cell frequencies and numbers increase during the course of EAE. Splenocytes were isolated from mice before, and 7, 14, 21 or 28 days after MOG_35-55 immunization and analyzed for CD1d, CD5, and CD19 expression by immunofluorescence staining with flow cytometry analysis. Representative results demonstrate the frequency of CD1d^hiCD5⁺ B cells within the indicated gates among total CD19⁺ B cells. Bar graphs indicate mean (±SEM) percentages and numbers of CD1d^hiCD5⁺ B cells. (B) IL-10⁺ B10 cell frequencies and numbers increase during the course of EAE. Splenocytes were cultured with L+PIM for 5 h, stained with CD19 mAb, permeabilized, and stained using IL-10 mAb with flow cytometry analysis. Representative results demonstrate the frequency of IL-10-producing cells within the indicated gates among total CD19⁺ B cells. Bar graphs indicate mean (±SEM) percentages and numbers of B cells that produced IL-10. (C) B cell IL-10 transcript expression during EAE. RNA was isolated from splenic CD19⁺ B cells purified from mice before, and 7, 14, 21 or 28 days after MOG_35-55 immunization by MACS beads (purities >99%). Values represent relative mean IL-10 transcript levels normalized to GAPDH transcript levels (±SEM) as quantified by real-time reverse transcription PCR analysis. (D) CFA immunization does not affect IL-10⁺ B10 cell frequencies or numbers. Spleen B10 cell frequencies were examined before and after immunization with CFA emulsified in an equal volume of PBS as outlined in (B). (E) CD4⁺CD25⁺FoxP3⁺ T cell frequencies and numbers increase during the course of EAE. Splenocytes were stained with CD4 and CD25 mAbs, permeabilized, and stained using FoxP3 mAb with flow cytometry analysis. Representative results demonstrate the frequency of CD4⁺CD25⁺FoxP3⁺ T cells within the indicated gates among total CD4⁺ T cells. Bar graphs indicate mean (±SEM) percentages and numbers of CD4⁺CD25⁺FoxP3⁺ T cells. (A–E) Bar graphs indicate results from one of two independent experiments with ≥5 mice in each group. Horizontal dashed lines are provided for reference to naïve mice. Significant differences between means of naïve and immunized mice are indicated; *p<0.05, **, p<0.01.

Figure 2

B10 cell frequencies significantly influence EAE severity. (A) CD1d^hiCD5⁺ B cell frequencies and numbers in wild type, hCD19Tg, CD19^−/−, and IL-10^−/− mice. Representative results demonstrate the frequency of spleen CD1d^hiCD5⁺ B cells within the indicated gates among total CD19⁺ or CD20⁺ B cells. Bar graphs indicate mean (±SEM) percentages and numbers of CD1d^hiCD5⁺ B cells in one of two independent experiments with 3 mice in each group. (B) IL-10⁺ B10 cell frequencies and numbers in wild type, hCD19Tg, CD19^−/−, and IL-10^−/− mice. Representative results demonstrate the frequency of spleen IL-10-producing cells within the indicated gates among total CD19⁺ or CD20⁺ B cells. Bar graphs indicate mean (±SEM) percentages and numbers of B cells that produced IL-10. (A–B) Values in each group represent results from 6 mice from two pooled independent experiments. Significant differences between means from wild type and other mouse groups are indicated; *p<0.05, **, p<0.01. (C) EAE progression and severity in wild type, CD19^−/−, and hCD19Tg mice immunized with MOG_35-55 on day 0 and scored daily thereafter for EAE disease severity. Values represent mean (±SEM) EAE clinical scores from ≥5 mice in each group, with similar results obtained in 2 independent experiments. Significant differences between wild type and mutant mice groups are indicated; *p<0.05 (wild type versus hCD19Tg mice), †p<0.05 (wild type versus CD19^−/− mice). (D) Adoptively transferred CD1d^hiCD5⁺ B cells reduce EAE severity in CD19^−/− mice. Splenic CD1d^hiCD5⁺ or CD1d^loCD5⁻ B cells were purified from naïve wild type or IL-10^−/− mice by cell sorting. Wild type and CD19^−/− recipient mice were either given PBS, purified CD1d^hiCD5⁺ or CD1d^loCD5⁻ B cells 1 day (arrowheads) before MOG_35-55 immunizations. Values represent mean (±SEM) results from ≥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 (black diamond versus white diamond).

Figure 3

B10 cells and Treg cells cooperatively regulate EAE severity. (A) B10 cell depletion in vivo. Spleen CD1d^hiCD5⁺ and IL-10⁺ B cell frequencies were determined in wild type mice 7 days after MB22-10 or control mAb treatments (250 μg/mouse). Upper panels, representative results demonstrate the frequency of CD1d^hiCD5⁺ B cells within the indicated gates among total CD19⁺ B cells. Lower panels, representative results demonstrate the frequencies of IL-10⁺ cells within the indicated gates among total CD19⁺ B cells. Bar graphs indicate mean (±SEM) percentages and numbers of CD1d^hiCD5⁺ or IL-10⁺ B cells from 6 mice in each group from two pooled independent experiments. Significant differences between MB22-10 or control mAb treatment groups are indicated; **, p<0.01. (B) Early B10 cell deletion exacerbates EAE disease severity. Mice were treated with MB22-10 (closed circles) or control (open circles) mAb (250 μg, arrowheads) from day −7 (days −7, 0, 7, 14, 21), from day 7 (days 7, 14, 21), from day 14 (days 14, 21), or from day 21, as indicated. Values represent mean (±SEM) EAE clinical scores from ≥5 mice in each group, with similar results obtained in 2 independent experiments. Significant differences between MB22-10 and control mAb-treated groups are indicated; *p<0.05. (C) Treg cell deletion. Representative results demonstrate spleen CD4⁺CD25⁺FoxP3⁺ Treg cell frequencies within the indicated gates among total CD4⁺ T cells and Treg cell numbers in wild type mice 7 days after initial denileukin diftitox or PBS treatments. Denileukin diftitox was administrated 1, 3 or 6 days before analysis or 1, 4 and 7 days before analysis as indicated. Bar graphs indicate mean (±SEM) percentages and numbers of CD4⁺CD25⁺FoxP3⁺ Treg cells from 6 mice in each group from two pooled independent experiments. Significant differences between denileukin diftitox or PBS treatment groups are indicated; *p<0.05, **, p<0.01. (D) Late Treg cell deletion exacerbates EAE disease severity. Mice were treated with denileukin diftitox (closed circles) or PBS (open circles) before (days 1, 4, 7, 10, 13, arrowheads) or after EAE onset (days 14, 17, 20, 23, 26, arrowheads). Values represent mean (±SEM) EAE clinical scores from ≥5 mice in each group, with similar results obtained in 2 independent experiments. Significant differences between denileukin diftitox and PBS treatment groups are indicated; *p<0.05.

Figure 4

Changes in B10 and Treg cell frequencies and numbers within tissues during the course of EAE. Mononuclear cells were isolated from (A) CNS tissue, (B) inguinal and axillary lymph nodes draining the site of MOG immunization, or (C) blood before, and 7, 14, 21 or 28 days after MOG_35-55 immunizations (left panels). Representative results demonstrate the frequencies of IL-10-producing cells after L+PIM stimulation for 5 h within the indicated gates among total CD19⁺ B cells, as well as CD4⁺CD25⁺FoxP3⁺ Treg cell frequencies among total CD4⁺ T cells. Bar graphs indicate mean (±SEM) frequencies and numbers of B cells that produced IL-10 or CD25⁺FoxP3⁺CD4⁺ Treg cells from 6 mice in each group from two pooled independent experiments. Horizontal dashed lines are provided for reference to naïve mice. (D) Treg cells dominate CNS tissues after EAE development. Bar graphs indicate mean (±SEM) percentages of IL-10⁺ B10 cells relative to total IL-10⁺ B10 cells plus Treg cells within various tissues. (A–D) Significant differences between means of naïve mice and mice with EAE are indicated; *p<0.05, **, p<0.01. Similar results were obtained in at least 2 independent experiments.

Figure 5

The adoptive transfer of MOG_35-55-sensitized B10 cells can reduce EAE disease severity in wild type mice. (A–B) Representative B10 cell purification results for adoptive transfer experiments. Purified spleen B cells from naïve mice or mice with EAE (day 28) were separated into CD1d^hiCD5⁺CD19⁺ and CD1d^loCD5⁻CD19⁺ B cell populations. The isolated cells were cultured with L+PIM for 5 h, or were cultured with agonistic CD40 mAb for 48 h with L+PIM added during the final 5 h of culture. B10 cell frequencies in the stimulated cell cultures were determined by immunofluorescence staining with flow cytometry analysis. (C) The adoptive transfer of purified spleen CD1d^hiCD5⁺ B cells reduces EAE disease severity. Wild type recipient mice were given either PBS, CD1d^hiCD5⁺ B cells, or CD1d^loCD5⁻ B cells from naïve wild type mice, mice with EAE (day 28), or IL-10^−/− mice with EAE (day 28) 1 day before MOG_35-55 immunizations or 7 or 14 days after MOG_35-55 immunization as indicated (arrowheads). In some cases, the purified B cell populations were stimulated as indicated with agonistic CD40 mAb for 48 h, with LPS added during the final 5 h of culture to induce B10pro cell maturation and thereby expand B10 cell frequencies. Values represent means (±SEM) from ≥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 (black diamond versus white circle).

Figure 6

Treg cell and leukocyte infiltration into the CNS following the adoptive transfer of B10 cells. Wild type recipient mice were given PBS, or either CD40/LPS-stimulated CD1d^hiCD5⁺ or CD1d^loCD5⁻ B cells from mice with EAE (day 28) 1 day before MOG_35-55 immunizations as in figure 5C. (A) Mononuclear cells within CNS tissues or spleen 18 days after MOG_35-55 immunizations. Bar graphs indicate mean (±SEM) numbers of CD4⁺ T cells or frequencies/numbers of CD4⁺CD25⁺FoxP3⁺ Treg cells among total CD4⁺ T cells (n≥5 mice per group). Significant differences between groups of mice receiving CD1d^hiCD5⁺ B cells or PBS are indicated; **p<0.01. Similar results were obtained in at least 2 independent experiments. (B–D) EAE histopathology following the adoptive transfer of B10 cells. Representative lumbar spinal cord sections were harvested 18 days after MOG_35-55 immunizations (n≥5 mice per group) with (B) inflammation (H&E staining) and (C) demyelination (Luxol Fast Blue staining) demonstrated. Scale bar is 0.5 mm. B) Arrowheads indicate inflammatory foci. C) Yellow traced areas indicate demyelination. (D) Bar graphs indicate mean (±SEM) numbers of inflammatory foci and percentages of spinal cord sections that were demyelinated, with significant differences between groups of mice receiving CD1d^hiCD5⁺ B cells or PBS indicated; **p<0.01. Similar results were obtained in at least 2 independent experiments.

Figure 7

B10 cells alter T cell cytokine profiles, but not T cell proliferation. Purified splenic CD1d^hiCD5⁺ or CD1d^loCD5⁻ B cells from mice with EAE (day 28) were stimulated with agonistic CD40 mAb for 48 h, with LPS added during the final 5 h of culture. (A) B10 cell effects on T cell proliferation. CFSE-labeled TCR^MOG CD4⁺ T cells were cultured alone or with CD40/LPS-stimulated CD1d^hiCD5⁺ or CD1d^loCD5⁻ B cells in the presence of MOG_35-55 (25 μg/ml) for 72 h. After 72 h, the cultured cells were stained for CD4 and Thy1.1 expression and analyzed for CFSE dilution by flow cytometry. Representative frequencies of dividing CFSE-labeled cells are shown (gated on CD4⁺Thy1.1⁺CFSE⁺ cells). Bar graphs (left) indicate CFSE geometric mean fluorescence of the entire histogram, which is inversely proportional to cell divisions. Bar graphs (right) indicate mean (±SEM) frequencies of dividing TCR^MOG CD4⁺ T cells (CD1d^hiCD5⁺ B cell group, closed bars; CD1d^loCD5⁻ B cell group, open bars) from 6 mice in each group from two pooled independent experiments. (B) B10 cells alter CD4⁺ T cell cytokine production. TCR^MOG CD4⁺ T cells were cultured with CD40/LPS-stimulated CD1d^hiCD5⁺ or CD1d^loCD5⁻ B cells from wild type or IL-10^−/− mice with EAE (day 28) in the presence of MOG_35-55 (25 μg/ml) for 72 h, with PMA, ionomycin, and BFA added during the final 5 h of culture. For negative controls, TCR^MOG CD4⁺ T cells were cultured with CD40/LPS-stimulated CD1d^loCD5⁻ B cells in the presence of MOG_35-55 (25 μg/ml) for 72 h, with BFA alone (without PMA/ionomycin) added during the final 5 h of culture. Cytokine production by TCR^MOG CD4⁺ T cells was determined by intracellular cytokine staining with flow cytometry analysis. Numbers indicate percentages of T cells within the indicated gates among total CD4⁺ T cells. Bar graphs indicate mean (±SEM) percentages of cytokine producing CD4⁺ T cells from 6 mice in each group from two pooled independent experiments. (C) Representative IL-10R expression (thick line) by splenic CD11c⁺ DCs, CD11b^hi macrophages, CD19⁺ B cells, CD4⁺ T cells, and CD8⁺ T cells from wild type mice before or 7 days after MOG_35-55 immunization. Gray histograms represent isotype-matched control mAb staining. Bar graphs indicate average mean linear fluorescence intensities (±SEM) of IL-10R expression by each cell type from 6 mice in each group from two pooled independent experiments. (D) B10 cells inhibit the ability of DCs to activate CD4⁺ T cells. Splenic CD1d^hiCD5⁺ or CD1d^loCD5⁻ B cells from mice with EAE (day 28) were purified by cell sorting, and cultured with agonistic CD40 mAb for 48 h, with LPS added during the final 5 h of culture. Purified splenic DCs from mice with EAE (day 10) were cultured alone or with CD40/LPS-stimulated CD1d^hiCD5⁺ or CD1d^loCD5⁻ B cells in the presence of MOG_35-55 (25 μg/ml) for 72 h. DCs purified from the B cell co-cultures were then cultured with TCR^MOG CD4⁺ T cells for 72 h, or the T cells were cultured alone, with CD4 and Thy1.1 expression and CFSE dilution analyzed by flow cytometry. Mean frequencies of dividing CFSE-labeled CD4⁺Thy1.1⁺CFSE⁺ cells are indicated (±SEM) in each histogram from 6 mice in each group from two pooled independent experiments. Bar graphs indicate CFSE geometric mean fluorescence of the entire histogram, which is inversely proportional to cell divisions. Significant differences between groups indicated; *p<0.05.