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IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases

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Abstract

B lymphocytes have critical roles as positive and negative regulators of immunity. Their inhibitory function has been associated primarily with interleukin 10 (IL-10) because B-cell-derived IL-10 can protect against autoimmune disease and increase susceptibility to pathogens1,2. Here we identify IL-35-producing B cells as key players in the negative regulation of immunity. Mice in which only B cells did not express IL-35 lost their ability to recover from the T-cell-mediated demyelinating autoimmune disease experimental autoimmune encephalomyelitis (EAE). In contrast, these mice displayed a markedly improved resistance to infection with the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium as shown by their superior containment of the bacterial growth and their prolonged survival after primary infection, and upon secondary challenge, compared to control mice. The increased immunity found in mice lacking IL-35 production by B cells was associated with a higher activation of macrophages and inflammatory T cells, as well as an increased function of B cells as antigen-presenting cells (APCs). During Salmonella infection, IL-35- and IL-10-producing B cells corresponded to two largely distinct sets of surface-IgM+CD138hiTACI+CXCR4+CD1dintTim1int plasma cells expressing the transcription factor Blimp1 (also known as Prdm1). During EAE, CD138+ plasma cells were also the main source of B-cell-derived IL-35 and IL-10. Collectively, our data show the importance of IL-35-producing B cells in regulation of immunity and highlight IL-35 production by B cells as a potential therapeutic target for autoimmune and infectious diseases. This study reveals the central role of activated B cells, particularly plasma cells, and their production of cytokines in the regulation of immune responses in health and disease.

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Figure 1: B cells secrete IL-35 upon activation by TLR4 and CD40.
Figure 2: IL-35 expression by B cells is required for recovery from EAE.
Figure 3: B-cell-derived IL-35 enhances susceptibility to Salmonella.
Figure 4: IL-10 and IL-35 are expressed by CD138hi plasma cells during Salmonella infection.

Accession codes

Accessions

Gene Expression Omnibus

Data deposits

The gene array data have been deposited in NCBI’s Gene Expression Omnibus database with the accession number GSE35998.

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Acknowledgements

We thank H. Schliemann, H. Ruebsamen, M. Spadaro and D. Jenne for assistance and support. We thank M. Loehning for providing IL-12 p40-deficient mice, and S. Akira for providing TLR2- and TLR4-deficient mice. We thank O. Neyrolles for help with some of the in vivo experiments. We thank E. Schott for help with the AST/ALT measurements. S.F. is supported by grants from the Deutsche Forschungsgemeinschaft (SFB-650, TRR-36, TRR-130, FI-1238/02), Hertie Stiftung, and an advanced grant from the Merieux Institute. C.D and T.D. are supported by the Deutsche Forschungsgemeinschaft (SFB-650, Do491/7-2, 8-2). P.B. and L.J. are supported by INRA. A.B.-O. is supported by a CIHR/MSSC New Emerging Team grant in Clinical Autoimmunity. Work in S.M.A.’s laboratory was supported by grants from the UK Medical Research Council and the Wellcome Trust. E.M. is supported by the Clinical Competence Network for Multiple Sclerosis and SFB-TR128.

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Contributions

P.S., T.R. and V.L. performed most of the experiments, the data analysis, and edited the manuscript. R.A.O., U.S., E.H., S.R., V.D.D., Y.J., C.D., R.L., L.J., P.B., S.W., I.S., Y.M., M.D.L., R.C.M., S.W., M.N., K.H., E.M., A.G., J.R.G., K.H., A.A.K., T.D., A.B.-O., S.H.E.K. and S.M.A. contributed to some experiments. L.J., P.B., A.G. and J.R.G. performed the microarray data analysis. T.D., S.H.E.K. and S.M.A. helped with the writing of the manuscript. S.M.A. helped with the design of some experiments. S.F. designed the study, performed some experiments and wrote the manuscript.

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Correspondence to Simon Fillatreau.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Gene array analysis and IL-35 secretion by B cells activated in vitro.

a, EAE was induced in B T l r 2 - / - (squares), B T l r 4 - / - (triangles) and BWT (circles) mice (see Fig. 1). Animals showing a body weight loss >20% and/or sustained front leg impairment were killed in accordance with ethical regulations. Survival curves were compared using Wilcoxon test. b, In the top panels, splenic B cells from C57BL/6 mice were stimulated with lipopolysaccharides (LPS), peptidoglycan (PGN) or CpG DNA oligonucleotides for 72 h, as indicated, and IL-10 concentrations in supernatants were determined by Bio-Plex. Results from stimulated cells were compared to unstimulated B cells (RPMI) using one-way ANOVA followed by Bonferroni post-test (***P < 0.001; **P < 0.01; *P < 0.05; mean ± s.e.m.); in the bottom panels, splenic B cells were simultaneously activated with anti-CD40 (clone FGK-45; in indicated amounts) and LPS (2 µg ml−1), PGN (2 µg ml−1) or CpG (2 µg ml−1). Supernatants were collected after 72 h and analysed for IL-10 content by Bio-Plex. Data shown are pooled from three independent experiments (mean ± s.e.m.). Results from B cells stimulated with LPS, CpG or PGN alone were compared to B cells co-stimulated with anti-CD40 using one-way ANOVA followed by Bonferroni post-test (***P < 0.001; **P < 0.01; *P < 0.05). c, Affymetrix MG 430 2.0 whole genome arrays were performed in quadruplicates for naive B cells, B cells activated by TLR4 for 24 h and 72 h, and B cells activated by TLR4 plus CD40 for 24 h and 72 h (20 arrays in total). To obtain genes significantly regulated upon stimulation with LPS, the expression profiles of naive B cells, B cells activated with LPS for 24 h, and B cells activated with LPS for 72 h were compared to each other, generating three lists of differentially expressed Affymetrix IDs (referred to as genes in this figure), whose union provided a set of n = 9,703 Affymetrix IDs differentially expressed during LPS activation. A similar analysis yielded a set of n = 10,456 Affymetrix IDs differentially expressed during LPS plus anti-CD40 activation. The union of these two sets was computed to obtain the list of Affymetrix IDs modulated during B-cell stimulation with LPS or LPS plus anti-CD40 (n = 12,933). To focus on genes differentially expressed between B cells activated with LPS or LPS plus anti-CD40, the expression profiles of B cells activated with LPS or LPS plus anti-CD40 for 24 h were compared to each other. A similar analysis was done for B cells activated for 72 h. The intersection of these two sets provided the list of Affymetrix IDs differentially expressed between LPS-activated and LPS-activated plus anti-CD40-activated B cells at both 24 h and 72 h (this was done to identify the Affymetrix IDs with long-term differential expression) (n = 436). The intersection of the set n = 12,933 with the set n = 436 gave a list of 417 Affymetrix IDs, which was further filtered on Gene Ontology to extract the Affymetrix IDs corresponding to secreted factors, yielding a final list of 7 genes. d, Signal intensities of p35, Ebi3 and p40 mRNA expression were calculated using the values of the Affymetrix arrays. Data show mean ± s.e.m. e, f, Splenic murine B cells were stimulated with LPS (1 µg ml−1), PGN (10 µg ml−1), CpG ODN 1826 (1 µg ml−1), and anti-CD40 (clone FGK-45; 10 µg ml−1), alone or in combination, as indicated, for 24 h or 72 h. p35 (e) and Ebi3 (f) mRNA expression was then quantified by real-time PCR. Data compile three independent experiments (mean ± s.e.m.). For statistical analysis, one-way ANOVA test followed by Bonferroni post-test was performed comparing each activated B-cell sample to naive B cells (***P < 0.001; **P < 0.01; mean ± s.e.m.). gi, Splenic murine B cells were stimulated for 72 h with anti-IgM (F(ab′)2 goat anti-mouse IgM; Jackson ImmunoResearch; 5 µg ml−1), LPS (1 µg ml−1) and L47 cells (5 × 104 cells, irradiated), alone or in combinations, as indicated. g, h, Levels of p35 and Ebi3 mRNA expression were quantified by real-time PCR. Data show compilation of three independent experiments (mean ± s.e.m.). i, Splenic murine B cells were activated as indicated for 72 h, and treated with GolgiStop for the last 4 h of culture. B-cell lysates were separated on SDS–PAGE gel and blotted with anti-EBI3 antibody. Data show representative results from three independent experiments. j, Splenic B cell from C57BL/6 and p28 mice were stimulated with LPS (1 µg ml−1), CpG (1 µg ml−1), PGN (10 µg ml−1), anti-CD40 (10 µg ml−1), alone or in combination, as indicated, for 72 h. p28 concentrations in culture supernatants were determined by ELISA. Data shown (mean ± s.em) are pooled from four independent experiments.

Extended Data Figure 2 Characterization of immune reponses during EAE in mice lacking IL-35 expression in B cells.

a, EAE was induced in B p 35 - / - (black diamonds, n = 5) and corresponding BWT mice (white triangles, n = 4). Data show clinical EAE scores (mean ± s.e.m.) from two independent experiments. Cumulative disease scores were compared using two-tailed unpaired t-test. b, EAE was induced in B p 2 8 - / - (grey squares; n = 10) and corresponding BWT mice (dark grey circles, n = 10). Splenocytes were collected from mice on day 28 after EAE induction, and re-stimulated individually for 48 h with MOG35–55. Supernatants were analysed by ELISA to determine concentrations of IFN-γ (middle) and IL-17 (right). Data (mean ± s.e.m.) are pooled from two independent experiments. c, EAE was induced in B p 35 - / - (triangles), B E b i 3 - / - (diamonds), B p 4 0 - / - (squares), and BWT (circles) mice (see Fig. 2). Animals showing a body weight loss > 20% and/or sustained front leg impairment were killed in accordance with ethical regulations. Survival curves were compared using Wilcoxon test. d, Splenocytes were collected from B p 35 - / - , B p 4 0 - / - , and BWT mice on day 28 after EAE induction (see Fig. 2a), and pooled before re-stimulation for 48 h with MOG35–55. Culture supernatants were analysed by ELISA to determine concentrations of IFN-γ (left) and IL-17 (right). Data show representative results from two independent experiments. Statistical analysis was performed using the two-way-ANOVA test followed by a Bonferroni post-test (*P < 0.05, **P < 0.01; ***P < 0.001; mean ± s.e.m.). Results of analysis are shown for BWT versus B p 35 - / - comparison. ej, EAE was induced in B p 35 - / - (grey squares; n = 8) and corresponding BWT mice (dark grey circles; n = 8) and mice were killed at day 12 post-EAE induction to analyse accumulation of immune cells in CNS. Data show clinical EAE scores (mean ± s.e.m.) (e). fh, Mononuclear cells were isolated from CNS (brain plus spinal cord) of individual mice shown in (e), and numbers of total cells (f), CD4+ T cells (g) and CD11b+Ly6G macrophages (h) were quantified by flow cytometry. Data shown (mean ± s.e.m.) are pooled from two independent experiments. Results were compared using two-tailed unpaired t-test with Welch’s correction in case of inequal variances. i, j, Representative FACS plots of CD4+ T cells and CD11b+Ly6G macrophages, respectively. k, Frequencies of Foxp3+ Treg cells among CD4+CD8 T cells in CNS at day 12 post-EAE induction (left panel), and in spleen at day 10 post-EAE induction (right panel) as determined by flow cytometry. Data shown in the left panel (mean ± s.e.m.) are pooled from two independent experiments with 10 mice per group in total. Data shown in the right panel (mean ± s.e.m.) are representative of three independent experiments with 9 mice per group in total. Results were compared using two-tailed unpaired t-test. P values >0.05 are indicated by ns. l, Treg suppression assay. CD4+CD25+ T cells (Treg) were FACS-sorted from pooled spleen and lymph nodes of B p 35 - / - and corresponding BWT mice on day 10 post-EAE induction, and then co-cultured at the indicated ratios with 2 × 104 CD4+CD25 T cells (Teff) isolated from naive C57BL/6 mice in presence of 1 × 105 irradiated splenocytes and 0.1 μg ml−1 anti-CD3 (clone 145-2C11). Proliferation was measured after 64 h of culture by incorporation of [3H]thymidine, and plotted as suppression percentage defining as 100% the proliferation values obtained for control cultures without Treg cells. Data (mean ± s.e.m.) are pooled from two independent experiments.

Extended Data Figure 3 Lack of IL-35 production by B cells results in increased activation of B cells during EAE.

a, EAE was induced in B p 35 - / - (n = 6) and corresponding BWT mice (n = 6). Mice were killed on day 10 after immunization and splenic B cells were analysed by flow cytometry for surface expression of antigen-presenting molecules (MHC II), co-stimulatory molecules (CD80, CD86), and activation markers (CD44, CD69). MFI, mean fluorescence intensity. Data (mean ± s.e.m.) are pooled from two independent experiments. Results were compared using two-tailed unpaired t-test with Welch’s correction in case of inequal variances. b, Representative histogram plots showing expression of these molecules on wild-type and p35-deficient B cells. Dead cells were excluded using propidium iodide. c, EAE was induced in B p 35 - / - and corresponding BWT mice. B cells and CD4+CD25 T cells (Teff) were isolated from pooled lymph nodes and spleens on day 10 after immunization. To test the APC function of B cells (see Fig. 2), 5 × 105 B cells from B p 35 - / - or BWT mice were then cultured with 1 × 104 Teff cells from B p 35 - / - or BWT mice in presence of MOG35–55, as indicated. Culture supernatants were collected after 48 h, and analysed by Bio-Plex to determine concentrations of IFN-γ (left) and IL-6 (right). Data (mean ± s.e.m.) are pooled from two independent experiments. Results were compared using two-way ANOVA followed by Bonferroni post-test (*P < 0.05). Results of analysis are shown for comparison, BWT B cells plus BWT T cells versus B p 35 - / - B cells plus B p 35 - / - T cells for IL-6.

Extended Data Figure 4 Bacterial burden and T-cell activation in mice lacking IL-35 production by B cells during Salmonella infection.

a, B p 35 - / - , B E b i 3 - / - , B p 4 0 - / - and corresponding BWT mice were infected intravenously with virulent Salmonella strain SL1344 (100 c.f.u.). Bacterial loads in spleens (top) and livers (bottom) were determined on day 6 post-infection (p.i). Data (mean ± s.e.m.) are pooled from two independent experiments. Numbers of mice analysed: B p 35 - / - (n = 6) and corresponding BWT mice (n = 8), B E b i 3 - / - (n = 5) and corresponding BWT mice (n = 5), B p 4 0 - / - (n = 6) and corresponding control BWT mice (n = 5). Data were analysed with two-tailed unpaired t-test. P values > 0.05 are indicated by ns. b, Top shows liver histochemistry of total infiltrating cells (haematoxylin and eosin; H&E), T cells (CD3), and macrophages (F4/80) at day 6 p.i.; bottom, numbers of inflammatory foci per liver section on day 6 p.i. Data (mean ± s.e.m.) are pooled from two independent experiments. Numbers of mice analysed: B p 35 - / - (n = 6) and corresponding BWT mice (n = 8), B E b i 3 - / - (n = 7) and corresponding BWT mice (n = 5), B p 4 0 - / - (n = 7) and corresponding BWT mice (n = 8). Data were compared using two-tailed unpaired t-test with Welch’s correction in case of inequal variances. P values > 0.05 are indicated by ns. c, B p 2 8 - / - (n = 6) and corresponding BWT (n = 6) mice were infected intravenously with attenuated Salmonella (strain SL7207). On day 21 p.i. bone marrow (BM) cells were isolated from individual mice, and re-stimulated for 6 h with heat-killed Salmonella before staining for surface CD4, and intracellular CD40L or IFN-γ. (top). Representative FACS plots showing CD40L and IFN-γ expression by CD4+ T cells, after gating on CD4+ cells. Bottom, frequencies of CD40L+ (left) and IFN-γ+ (right) cells among CD4+ T cells in BM (mean ± s.e.m.). Data are pooled from two independent experiments. Data were compared using two-tailed unpaired t-test. P values > 0.05 are indicated by ns. d, Representative FACS plot shows Foxp3 expression by splenic CD4+ T cells from a BWT mouse on day 21 p.i. with attenuated Salmonella (strain SL7207); (top right) frequency of Foxp3+ Treg cells among CD4+ T cells in spleens of naive B p 35 - / - (n = 5) and corresponding BWT mice (n = 5), B p 4 0 - / - (n = 7) and corresponding BWT mice (n = 7); (bottom) frequency of Foxp3+ Treg cells among CD4+ T cells on day 21 p.i. with attenuated Salmonella (strain SL7207) in spleens of B p 35 - / - (n = 8) and corresponding BWT mice (n = 8), B E b i 3 - / - (n = 10) and corresponding BWT mice (n = 10), B p 4 0 - / - (n = 15) and corresponding BWT mice (n = 9), B p 2 8 - / - (n = 6) and corresponding BWT mice (n = 6). Data (mean ± s.e.m.) are pooled from two independent experiments. Data were analysed with two-tailed unpaired t-test. P values >0.05 are indicated by ns. e, Mice were infected as in (d) and frequency of activated CD44hiCD62Llo CD4+ and CD8+ T cells were determined by flow cytometry. Representative FACS plot shows expression of CD44 and CD62L by live (PI negative) CD4+ T cells. Graphs show frequencies of CD44hiCD62Llo cells among CD4+ T cells (left) and CD8+ T cells (right). Data (mean ± s.e.m.) are pooled from two independent experiments. Numbers of mice analysed: B p 35 - / - (n = 14) and corresponding BWT mice (n = 12), B E b i 3 - / - (n = 10) and corresponding BWT mice (n = 10), B p 4 0 - / - (n = 15) and corresponding BWT mice (n = 9), B p 2 8 - / - (n = 6) and corresponding BWT mice (n = 6).

Extended Data Figure 5 Role of IL-35 and CD40 expression by B cells during Salmonella infection.

a, B p 35 - / - , B E b i 3 - / - , B p 4 0 - / - and their corresponding BWT mice were infected with attenuated Salmonella (strain SL7207). Relative titres of Salmonella-reactive IgM and IgG in serum were determined by ELISA. Data (mean ± s.e.m.) are pooled from three independent experiments. Numbers in brackets indicate the numbers of mice analysed for each measurement. b, B p 35 - / - (n = 17) and corresponding BWT mice (n = 16) were immunized with 200 µg NP-OVA in alum intraperitoneally. Relative titres of NP(15)-BSA-reactive antibodies in serum were determined by ELISA. Data (mean ± s.e.m.) are pooled from three independent experiments. Results were compared using two-tailed unpaired t-test. P values >0.05 are indicated by ns. c, Mice were infected with 100 c.f.u. of virulent Salmonella. Upper left shows survival curves of B C d 4 0 - / - (n = 9) and corresponding BWT mice (n = 16). Data are pooled from two independent experiments. Survival curves were compared using Wilcoxon test. Upper right, numbers of inflammatory foci per liver section were determined on day 5 p.i. Lower panel, bacterial loads in liver (left) and spleen (right) were determined on day 5 p.i. Data (mean ± s.e.m.) are pooled from two independent experiments. Numbers of mice analysed: B C d 4 0 - / - (n = 12), and corresponding BWT mice (n = 13). Results were analysed using two-tailed unpaired t-test with Welch’s correction.

Extended Data Figure 6 Characterization of IL-10- and IL-35-expressing plasma cells during Salmonella infection.

a, b, C57BL/6 mice were infected intravenously with attenuated Salmonella (strain SL7207; 107 c.f.u.). On indicated days p.i., CD138hi plasma cells and CD19+CD138 B cells were isolated from spleen through magnetic and FACS procedures. a, Strategy for isolation of CD138hi plasma cells and CD19+CD138 B cells: splenocytes were stained with anti-CD138-PE followed by labelling with magnetic anti-PE microbeads, and magnetic separation on autoMACS (Miltenyi Biotech). The obtained CD138-positive and CD138-negative fractions were stained for CD19, CD138 and CD11b/CD11c/TCR-β/DAPI. CD138hiCD11bCD11cTCR-βDAPI and CD19+CD138CD11bCD11cTCR-βDAPI cells were then isolated from CD138-positive and CD138-negative fraction, respectively, by FACS, as bulk or single cells. Numbers in FACS plots indicate the percentages of CD19+CD138 B cells and CD138hi plasma cells in total splenocytes on day 3 after challenge (left plots), after magnetic isolation (middle plots), and after FACS isolation (right plots). b, Single CD138hi cells were isolated on indicated days, and analysed by single cell-PCR using a mix of primers for β-actin, Il10, p35 and Ebi3. Only cells giving a positive signal for β-actin were included in the calculations shown. Data show frequencies of CD138hi cells that gave a positive signal for Il10, both p35 and Ebi3, or Il10 and p35 and Ebi3. Numbers of cells tested: day 1 (181 cells; 86% positive for β-actin), day 3 (156 cells; 79% positive for β-actin) and day 8 (163 cells; 97% positive for β-actin). Cells were obtained from two independent experiments. c, d, CD138hi plasma cells and CD19+CD138 B cells were enriched by magnetic isolation (as described in a) from spleen of C57BL/6 mice on day 3 after infection with attenuated Salmonella (SL7207; 107 c.f.u.). c, Data show expression levels of selected surface receptors on live (DAPI) CD138hi plasma cells and CD19+CD138 B cells. Cells were gated as in a (middle panels), and analysed by flow cytometry. d, Levels of CD1d and Tim-1 expressed by CD138hi cells were compared to those found respectively on CD1dhi and CD1dlo B cells, and on Tim1+ and Tim1 B cells from spleen of naive C57BL/6 mice. Data show representative results from 2–5 independent experiments.

Extended Data Figure 7 Characterization of plasma cells during Salmonella infection.

a, CD138hi plasma cells and CD19+CD138 B cells were magnetically enriched from spleens of C57BL/6 mice on day 3 after infection with attenuated Salmonella (SL7207; 107 c.f.u.). CD19+CD138 B cells (P1) were then isolated from the CD138-negative fraction by FACS. CD138intCD22+ (P2), CD138hiCD22+ (P3), and CD138hiCD22 (P4) were isolated from the CD138-positive fraction by FACS. b, Frequencies of IgM- and IgG-secreting cells among B cells (CD19+CD138) and each of these plasma cell subsets (CD138intCD22+, CD138hiCD22+, CD138hiCD22) were determined by ELISPOT assay. A compilation of two independent experiments is shown (mean ± s.e.m.). c, Blimp1, Irf4, Pax5, Il10, Ebi3 and p35 mRNA expression was quantified by real-time PCR in these populations. Naive splenic B cells (naive CD19+) were isolated from unchallenged C57BL/6 mice by magnetic selection. Data show the compilation of three independent experiments (mean ± s.e.m.). d, Plasma cells accumulate in splenic red pulp aggregates adjacent to white pulp areas on day 3 after Salmonella infection. C57BL/6 mice were infected intravenously with attenuated Salmonella (SL7207; 107 c.f.u.), and spleens were harvested 3 days later. Spleen sections (7 μm) were stained with: top panels: anti-Ig-κ (clone 187.1; green), anti-Igλ (clone SL136; green), anti-CD90 (clone T24; red) and anti-CD22 (clone OX-97; blue); middle panels: anti-Ig-κ (clone 187.1; red), anti-Igλ (clone SL136; red), anti-CD11b (clone M1/70; green), and anti-CD22 (clone OX-97; blue); bottom panels: anti-Ig-κ (clone 187.1; red), anti-Igλ (clone SL136; red), anti-CD169 (clone MOMA-1; green), and anti-CD22 (clone OX-97; blue).

Extended Data Figure 8 Production of IL-10 and IL-6 by plasma cells and B cells after Salmonella infection.

a, CD138hi plasma cells and CD19+CD138 B cells were isolated from spleen of C57BL/6 mice on day 3 after infection with attenuated Salmonella (SL7207; 107 c.f.u.), as described in Extended Data Fig. 6. Isolated cells were activated for 24 h as indicated with LPS (1 µg ml−1), anti-CD40 (10 µg ml−1), and anti-IgM (5 µg ml−1). IL-10 (left) and IL-6 (right) concentrations were determined by Bio-Plex. Data shown are pooled from 2–5 independent experiments (mean ± s.e.m.). Results were compared using two-tailed unpaired t-test. P values > 0.05 are indicated by ns. b, c, CD138hi plasma cells were isolated from spleen of C57BL/6 or IL-10–eGFP mice on day 3 after infection with attenuated Salmonella (SL7207; 107 c.f.u.). b, Cells were analysed by flow cytometry directly after isolation to quantify IL-10–eGFP expression (ex vivo), and 24 h after re-stimulation with PMA plus ionomycin. Data show representative FACS plots and histogram overlays. Numbers in the plots indicate the percentages of IL-10–eGFP-positive cells among total live (PI) cells. c, Cells isolated from C57BL/6 and IL-10–eGFP mice were re-stimulated for 24 h, as indicated with LPS (1 μg ml−1), anti-CD40 (10 μg ml−1), anti-IgM (5 µg ml−1), IL-4 (20 ng ml−1), IL-5 (20 ng ml−1), IL-6 (20 ng ml−1) and IL-21 (20 ng ml−1). Cells were analysed as in b. Data shown are pooled from 2–4 independent experiments (mean ± s.e.m.). Results were compared between condition RPMI and activated conditions (left) or between condition with added cytokine and condition LPS plus anti-CD40 (right) using one-way ANOVA followed by Bonferroni post-test (*P < 0.05, **P < 0.01; ***P < 0.001). dg, Expression of IL-6 by IL-10-expressing CD1dhi B cells. d, CD19+CD1dhi and CD19+CD1dlo cells were isolated from spleen of naive C57BL/6 mice according to CD19 and CD1d expression using magnetic and FACS procedures, and stimulated for 24 h, as indicated. IL-6 and IL-10 concentrations in culture supernatants were determined by Bio-Plex. e, f, CD1dhi B cells were isolated from naive IL-10–eGFP mice and stimulated with LPS (2 μg ml−1) for 48 h. IL-6 and IL-10 concentrations in culture supernatants were determined by Bio-Plex (e), and cells were analysed by flow cytometry to determine the frequency of IL-10–eGFP-expressing cells (f). g, CD1dhi B cells were isolated from naive IL-10–eGFP mice, and stimulated with LPS (2 μg ml−1) for 48 h. IL-10–eGFP and IL-10–eGFP+ cells were then separated by FACS and analysed by real time PCR for expression levels of Il10 and Il6 mRNA. Data shown are pooled from two independent experiments (mean ± s.e.m.).

Extended Data Figure 9 IL-35 and IL-10 production by plasma cells during EAE and Salmonella infection.

a, B cells and plasma cells were isolated from spleens of mice on days 14 and 28 after EAE induction through magnetic and FACS procedures. Splenocytes were stained with anti-CD138-PE followed by labelling with anti-PE microbeads, and magnetic separation. The obtained CD138-positive and CD138-negative fraction were stained for CD19, CD138, and CD11b/CD11c/CD90/DAPI. CD138+CD11bCD11cCD90DAPI and CD19+CD138CD11bCD11cCD90DAPI cells were then isolated from CD138-positive and CD138-negative fractions, respectively, by FACS, as bulk cells. Naive splenic B cells (naive CD19+) were isolated from unchallenged C57BL/6 mice by magnetic selection. Blimp1, Irf4 and Pax5 mRNA expression was quantified by real-time PCR. Data compile 2 independent experiments (mean ± s.e.m.). b, Production of IgM and IgG by plasma cells and B cells isolated from mice during EAE was determined by ELISPOT. A compilation of 3 independent experiments for day 14, and 2 independent experiments for day 28 is shown (mean ± s.e.m.). c, Left, CD19+CD138 B cells (B cell) and CD138+ plasma cells (plasma cell) were isolated from spleens of mice on days 14 and 28 after EAE induction through magnetic and FACS procedures, as described in a, and stimulated for 24 h with PMA plus ionomycin (PMA plus iono) or kept in medium (). IL-10 concentrations were determined by Bio-Plex. Data are pooled from 4 independent experiments for day 14 and 2 independent experiments for day 28 (mean ± s.e.m.). Right, B cells (CD19+CD138) and CD138+ plasma cells (CD138+) were isolated from spleens of mice on day 14 after EAE induction through magnetic isolation. For this, splenocytes were stained with anti-CD138-PE followed by labelling with anti-PE microbeads, and magnetic separation to yield CD138+ plasma cells. B cells were obtained from the CD138-negative fraction after further depletion of remaining CD138+ cells, and positive selection using anti-CD19 magnetic microbeads. Purities were above 95%. Cell lysates were separated on SDS–PAGE gel and sequentially blotted with anti-EBI3, anti-p35, and anti-actin antibodies. Data show representative results of two independent experiments. d, e, Mice in which B cells cannot co-express IL-10 and IL-35 do not develop exacerbated EAE. d Left, B p 35 - / - (50%) Il 10 - / - (50%) were obtained by reconstituting lethally irradiated recipient mice with BM cells from JHT mice (80%), IL-10-deficient mice (10%), and p35-deficient mice (10%). BWT mice were produced using C57BL/6 BM instead of JHT BM cells. B p 35 - / - and B I l 1 0 - / - mice were obtained as outlined in Extended Data Fig. 10. Right, EAE was induced in B p 35 - / - (50%) Il 10 - / - (50%) mice (grey diamonds, n = 10), B p 35 - / - (grey squares, n = 10), B I l 1 0 - / - (white diamonds, n = 5) and BWT (dark grey circles, n = 10) mice. Data show clinical EAE scores. e, Splenocytes were harvested from B p 35 - / - (50%) Il 10 - / - (50%) and BWT mice on day 28 after EAE induction, and re-stimulated individually for 48 h with MOG35–55. Culture supernatants were analysed by ELISA to determine concentrations of IFN-γ and IL-17. Data (mean ± s.e.m.) are pooled from two independent experiments. f, Production of IL-10 by IL-35-deficient B cells in vitro. Splenic B cells from naive C57BL/6, p35−/−, and p35−/−Ebi3−/− mice were stimulated with LPS (1 µg ml−1), anti-CD40 (10 µg ml−1), alone or in combination, as indicated, for 72 h, and IL-10 concentrations were determined by Bio-Plex. Data (mean ± s.e.m.) are pooled from three independent experiments. Results were compared between C57BL/6 cells and cells from mutant mice using one-way ANOVA followed by Bonferroni post-test (*P < 0.05). gi, IL-35 is not necessary for production of IL-10 by CD138hi plasma cells during Salmonella infection. CD138hi plasma cells were isolated from spleen of C57BL/6, IL-10–eGFP, p35−/−, and p35−/−IL-10–eGFP mice on day 3 after infection with attenuated Salmonella (107 c.f.u.) using a combination of magnetic and FACS methods. Cells were analysed by flow cytometry (g) to determine the frequency of IL-10–eGFP-expressing cells after re-stimulation as indicated for 24 h (h). Numbers in the plots indicate the percentages of IL-10–eGFP-positive cells among total live (PI) cells. Results were compared using two-tailed unpaired t-test. i, CD138hi plasma cells isolated from C57BL/6 and p35−/− mice were re-stimulated for 24 h as indicated. IL-10 concentrations were determined by Bio-Plex. Data are pooled from two independent experiments (mean ± s.e.m.). Results were compared using two-tailed unpaired t-test with Welch’s correction. P values >0.05 are indicated by ns.

Extended Data Figure 10 Generation and validation of bone marow chimaera.

a, Scheme for the generation of B X - / - and corresponding control BWT mice. Left, B X - / - mice with a deficiency in gene X restricted to B cells were obtained by reconstituting lethally irradiated recipient mice with a mixture of BM cells from B-cell-deficient JHT mice (80%) and from gene X-deficient (X−/−) mice (20%). The table indicates the contribution of JHT and X−/− BM cells to relevant haematopoietic cell types in the resulting chimaera, taking into account that JHT mice carry wild-type (WT) alleles of gene X. In B X - / - mice, B cells only developed from X−/− BM cells and therefore were all X−/−. In contrast, all other haematopoietic lineages developed from both X−/− and JHT BM cells. As the JHT and X−/− BM cells were injected in a ratio 80:20, most haematopoietic cells other than B cells carried a wild-type genotype for X in B X - / - mice. Right, the corresponding control BWT mice were obtained by reconstituting lethally irradiated recipient mice with a mixture of BM cells from WT C57BL/6 mice (80%) and from X−/− mice (20%). The table indicates the contribution of WT and X−/− BM cells to relevant haematopoietic cell types in the resulting chimaera. In BWT mice, all haematopoietic lineages, including B cells, developed from WT (80%) and X−/− (20%) BM cells. Therefore B cells mostly had a wild-type genotype in BWT mice, which also controlled for possible effects owing to the 20% X−/− haematopoietic cells other than B cells present in B X - / - mice (for example, 20% X−/− T cells, and so on). b, Top, representative FACS plots of isolated B cells, CD4+ T cells and CD11b+ cells purified from naive B E b i 3 - / - and BWT mice, using magnetic and FACS-based methodologies; (bottom) genomic DNA was extracted from the sorted cell populations, including the non-labelled B220CD4CD11b cells (indicated as Non-T,B,M). Genomic DNA samples from tail biopsies of C57BL/6 and Ebi3−/− mice were used as controls. The amounts of genomic DNA present in each sample were equilibrated using the Mog gene as standard. Using equilibrated samples, the wild-type Ebi3 allele was detected by PCR in all DNA preparations except for B cells from B E b i 3 - / - mice, and the tail biopsy from Ebi3−/− mice. c, Reconstitution of the B-cell compartment in BM chimaeric mice. B p 35 - / - , B Ebi3 −/− , B p 4 0 - / - and corresponding BWT controls were killed after reconstitution. Data show the percentage of CD19+ B cells in spleen, as determined by flow cytometry. Data are pooled from 2–3 independent experiments. Data (mean ± s.e.m.) include B p 35 - / - (n = 6) and corresponding BWT mice (n = 7), B E b i 3 - / - (n = 7) and corresponding BWT mice (n = 9), B p 4 0 - / - (n = 7) and corresponding BWT mice (n = 7). Comparisons of B-cell reconstitutions between B X - / - and corresponding BWT mice were done with two-tailed unpaired t-test with Welch’s correction (P values > 0.05 are indicated as ns). d, Representative FACS plots of B cells from BWT and B p 35 - / - mice. Cells are gated on live (PI) spleen cells.

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Shen, P., Roch, T., Lampropoulou, V. et al. IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases. Nature 507, 366–370 (2014). https://doi.org/10.1038/nature12979

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