Regulatory B cells control T-cell autoimmunity through IL-21-dependent cognate interactions

Abstract

B cells regulate immune responses by producing antigen-specific antibodies. However, specific B-cell subsets can also negatively regulate T-cell immune responses, and have been termed regulatory B cells. Human and mouse regulatory B cells (B10 cells) with the ability to express the inhibitory cytokine interleukin-10 (IL-10) have been identified. Although rare, B10 cells are potent negative regulators of antigen-specific inflammation and T-cell-dependent autoimmune diseases in mice. How B10-cell IL-10 production and regulation of antigen-specific immune responses are controlled in vivo without inducing systemic immunosuppression is unknown. Using a mouse model for multiple sclerosis, here we show that B10-cell maturation into functional IL-10-secreting effector cells that inhibit in vivo autoimmune disease requires IL-21 and CD40-dependent cognate interactions with T cells. Moreover, the ex vivo provision of CD40 and IL-21 receptor signals can drive B10-cell development and expansion by four-million-fold, and generate B10 effector cells producing IL-10 that markedly inhibit disease symptoms when transferred into mice with established autoimmune disease. The ex vivo expansion and reinfusion of autologous B10 cells may provide a novel and effective in vivo treatment for severe autoimmune diseases that are resistant to current therapies.

Figure 1

IL-21 induces regulatory B10 cell function. a, IL-21 induces B10 cell IL-10 production and secretion. purified spleen CD19⁺ B cells from wild type mice were cultured in medium alone or containing the indicated cytokines or LPS. To visualize IL-10-competent B cells, LPS, PMA, ionomycin and monensin (L+PIM) were added to the cultures 5 h before the cells were stained for cell surface CD19 and cytoplasmic IL-10 expression and analyzed by flow cytometry. Representative histograms show IL-10⁺ cell frequencies within the indicated gates, with background staining shown for cells cultured with monensin (Mon.) alone. Bar graphs indicate mean (±s.e.m.) I L-10⁺ B cell frequencies or culture supernatant fluid IL-10 concentrations at 48 or 72 h from three independent experiments using individual mice. b, IL-21 induces CD1d^hiCD5⁺ B cell IL-10 production. Purified spleen CD1d^hiCD5⁺ or CD1d^loCD5⁻ B cells from wild type mice were cultured with media alone or containing IL-21 for 48 h before IL-10⁺ B cell frequencies were assessed as in (a). c, B10 cells express IL-21R. CD19⁺ splenocytes purified from wild type mice were cultured with L+PIM for 5 h before cell surface CD19 and IL-21R, and cytoplasmic IL-10 staining to identify IL-10-competent B10 cells (dot plot, left panel). Representative IL-21R expression by IL-10⁺ and IL-10⁻ B cells from wild type mice is shown in comparison with control B cells from IL-21R^−/− mice (gray histograms). Results represent three independent experiments using individual mice. d, IL-21R expression is required for B10 cell expansion in vivo following MOG immunization. B10 cell numbers were assessed in wild type, IL-21R^−/− or CD19^−/− mice 7 days after saline (PBS) or MOG_35-55 immunization. Representative flow cytometry histograms are shown. Bar graphs indicate mean (±s.e.m.) B10 cell frequencies (≥3 mice per group). a and d, Significant differences between sample means are indicated; *, p<0.05; **, p<0.01.

Figure 2

B10 cells require IL-10, IL-21R, CD40, and MHC-II expression to regulate EAE severity. a, One day before CD19^−/− or wild type (WT) mice were immunized with MOG_35-55 on day 0, the CD19^−/− mice received PBS or purified spleen CD1d^hiCD5⁺ or CD1d^loCD5⁻ B cells from either wild type, IL-10^−/−, IL-21R^−/−, CD40^−/−, or MHC-II^−/− mice. Mice were scored daily thereafter for disease severity. The top two graphs show data from the same experiment, but were separated to facilitate visualization of the overlapping curves. b, B10 cells require MHC-II expression to regulate EAE severity in wild type mice treated with CD20 or control mAb 7 days before MOG_35-55 immunization on day 0. The mice also received PBS or purified CD1d^hiCD5⁺ B cells from either CD20^−/− or MHC-II^−/−CD20^−/− mice 1 day before MOG_35-55 immunization. The two graphs are from the same experiment, but were separated to facilitate visualization of the overlapping curves. c, Activated MHC-II^−/− B10 cells do not reduce disease severity in wild type mice. Purified CD1d^hiCD5⁺ B cells from wild type or MHC-II^−/− mice were cultured with agonistic CD40 mAb for 48 h to induce B10pro cell maturation, with LPS added during the final 5 h of culture. Wild type mice were given either PBS or CD1d^hiCD5⁺ B cells 1 day before MOG_35-55 immunization on day 0. a–c, Values represent mean (±s.e.m.) symptom scores from ≥3 mice in each group, with similar results obtained in three independent experiments. Significant differences between sample means are indicated; *, p<0.05.

Figure 3

B10 cell expansion and regulation of T cell-mediated autoimmunity. a, B10 cells require IL-10, IL-21R, CD40 and MHC-II expression to regulate antigen-specific T cell proliferation in vivo. CD19^−/− recipient mice were given PBS as a control, or purified CD1d^hiCD5⁺ or CD1d^loCD5⁻ B cells from naïve wild type (WT), IL-10^−/−, IL-21R^−/−, CD40^−/− or MHC-II^−/− mice, or wild type mice with EAE (day 28) 1 day before MOG_35–55 immunization on day 0. Four days after immunization, dye (CFSE)-labeled TCR^MOG CD4⁺Thy1.1⁺ T cells were transferred into CD19^−/− recipient mice. Five days later, peripheral lymph node CD4⁺Thy1.1⁺ T cells were analyzed for proliferation, with representative flow cytometry analysis of CFSE dilution show. Bar graphs indicate mean (±s.e.m.) numbers of divided TCR^MOG T cells. b, B10 cells require IL-10, IL-21R, CD40 and MHC-II expression for their regulation of antigen-specific T cell cytokine production. Purified CD1d^hiCD5⁺ B cells from the indicated mice were transferred into CD19^−/− recipient mice 1 day before MOG35–55 immunization on day 0, with TCR^MOG Thy1.1⁺CD4⁺ T cells transferred on day 4. Fourteen days later, lymph node Thy1.1⁺CD4⁺ T cells were analyzed for IL-17 and IFN-γ production by intracellular cytokine staining, with representative flow cytometry results shown. (a–b) Bar graphs indicate mean (±s.e.m.) frequencies of divided or cytokine-expressing cells, with three mice in each group. Significant differences between sample means are indicated: *, p<0.05; **, p<0.01. c, Model for autoantigen (Ag)-specific B10 cell function. B cells capture autoantigens that trigger appropriate BCR signals (step 1) and promote IL-10-competent B10_pro cell development. During immune responses (step 2), B10_pro cells present peptides to antigen-specific T cells through cognate interactions that induce T cell activation and CD40/CD154 interactions. Activated T cells may produce IL-21 locally, which binds to proximal B10 cell IL-21R (step 3). IL-21R signals induce B10 cell IL-10 production and effector function (B10eff, step 4), which may negatively regulate antigen-specific T cell function (step 5).

Figure 4

IL-21 drives ex vivo regulatory B10 cell expansion. a, B10 cell development in vitro. Purified spleen B cells were cultured on NIH-3T3-CD154/BLyS cell monolayers with exogenous IL-4 for 4 days, then cultured on fresh NIH-3T3-CD154/BLyS cells with exogenous IL-21 for 3 or 5 days as indicated, isolated, cultured with monensin for 5 h and stained for cytoplasmic IL-10 expression. Representative IL-10⁺ B cell frequencies within the indicated gates are shown. Similar results were obtained in ≥10 experiments. b, IL-21 drives B10 cell expansion in vitro. B cells cultured as in (a) were harvested each day. Bar values represent mean (±s.e.m.) B cell and B10 cell numbers, or B10 cell frequencies (solid line) from three independent experiments. c, IL- 21-induced B10 cells express CD5. B cells cultured for 9 days as in (a) were stained for CD5 and CD19 expression. CD5⁺ or CD5⁻ B cells were then purified and cultured with monensin for 5 h before cytoplasmic IL-10 staining. Results represent three independent experiments. d, B10 effector cells inhibit EAE initiation and progression. CD5⁺CD19⁺ or CD5⁻CD19⁺ cells were isolated as in (c) and adoptively transferred into wild type mice on days −1, 7, 14 or 21 (arrows) before/after MOG immunization and EAE induction as in figure 2. e, B10 cell expansion in vitro requires IL-21R and CD40 expression, and in vivo BCR signaling. Purified spleen B cells isolated from wild type, IL-21R^−/−, CD40^−/−, MHC-II^−/−, CD19^−/−, or MD4 mice were cultured as in (a), with mean (±s.e.m.) cell numbers quantified after culture. Values represent means (±s.e.m.) of three independent experiments. IL-10⁺ B cell frequencies in the cultures are shown in parentheses. f, B10 effector cells require IL-10 and MHC-II expression to inhibit EAE. B cells from IL-10^−/− or MHC-II^−/− mice were cultured as in (a), separated into CD5⁺ or CD5⁻ cells as in (c) and adoptively transferred into wild type mice before MOG35–55 immunization as in (d). (d, f) Values represent mean (±s.e.m.) symptom scores from ≥3 mice in each group, with similar results obtained in three independent experiments. (b, d, e) Significant differences between sample means are indicated: *, p<0.05; **, p<0.01.