Interleukin-35 induces regulatory B cells that suppress autoimmune disease - PubMed (original) (raw)

Interleukin-35 induces regulatory B cells that suppress autoimmune disease

Ren-Xi Wang et al. Nat Med. 2014 Jun.

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Abstract

Interleukin-10 (IL-10)-producing regulatory B (Breg) cells suppress autoimmune disease, and increased numbers of Breg cells prevent host defense to infection and promote tumor growth and metastasis by converting resting CD4(+) T cells to regulatory T (Treg) cells. The mechanisms mediating the induction and development of Breg cells remain unclear. Here we show that IL-35 induces Breg cells and promotes their conversion to a Breg subset that produces IL-35 as well as IL-10. Treatment of mice with IL-35 conferred protection from experimental autoimmune uveitis (EAU), and mice lacking IL-35 (p35 knockout (KO) mice) or defective in IL-35 signaling (IL-12Rβ2 KO mice) produced less Breg cells endogenously or after treatment with IL-35 and developed severe uveitis. Adoptive transfer of Breg cells induced by recombinant IL-35 suppressed EAU when transferred to mice with established disease, inhibiting pathogenic T helper type 17 (TH17) and TH1 cells while promoting Treg cell expansion. In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor comprising the IL-12Rβ2 and IL-27Rα subunits. As IL-35 also induced the conversion of human B cells into Breg cells, these findings suggest that IL-35 may be used to induce autologous Breg and IL-35(+) Breg cells and treat autoimmune and inflammatory disease.

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Figures

Figure 1

Figure 1. IL-35 induced regulatory B cells (Breg)

(a) Schematic of the cDNA constructs used to genetically engineer IL-12p35 (p35), Ebi3 and IL-35 (p35/Ebi3) recombinant proteins. (b) Coomassie blue gels of the recombinant proteins characterized on reducing SDS or non-reducing polyacrylamide gels. (c) Detection and characterization of p35, Ebi3 or IL-35 recombinant proteins by immunoprecipitation/Western blot analysis under reducing condition. (d) Characterization of 2 independently generated batches of the purified rIL-35 preparations by Western blotting under non-reducing conditions with anti-p35 or anti Ebi3 Abs. (e, f, g) Purified B220+CD19+ B cells from C57BL/6 mouse spleen were stimulated with LPS (5μg/ml) in medium containing 50 ng/ml pMIB, p35, Ebi3 or rIL-35 for 72 h. Lymphocyte proliferation was analyzed by [3H]-thymidine incorporation assay (e) and IL-10 production was analyzed by ELISA (f) or intracellular cytokine staining assay (g). (h, i) WEHI-279 B-cells were cultured in medium containing 50 ng/ml pMIB, p35, Ebi3 or rIL-35. Lymphocyte proliferation was analyzed by [3H]-thymidine incorporation assay (h) and IL-10 production was analyzed by ELISA (i). (j) CD19+ primary B cells were stimulated with rIL-35 (50ng/ml) and the purified Breg cells were co-cultured (1:5) for 3 days with freshly isolated CD19+ B cells in medium containing LPS. Proliferation was analyzed by the [3H]-thymidine incorporation assay. Results represent at least 3 independent experiments (*P<0.05; **P<0.01; ***P < 0.001; ****P<0.0001).

Figure 2

Figure 2. IL-35 preferentially induced CD5+CD19+B220lo Breg cells and a unique IL-35-producing Breg subpopulation (IL-35+Breg)

(a) Purified CD19+ B cells were stimulated for 3 days in medium containing 50ng/ml pMIB or rIL-35 and numbers in quadrants indicate the percent of B220+ B cells expressing Ebi3, p35 and/or IL-10. (b) IL-10− or IL-10+ B cells enriched with a Breg Isolation Kit (see Methods section) were analyzed by intracellular cytokine staining assay. (c,d) Activated B-cells were stimulated with rIL-35 and subjected to chromatin immunoprecipitation (ChiP) assay (c) or RT-PCR (d). (e) Purified B cells were stimulated for 3 days in medium containing pMIB or IL-35 and expression of B220, CD19, CD5, Foxp3 or Tim-1 and/or IL-10 was analyzed by intracellular cytokine staining assay or cell surface FACS analysis. (f, g) C57BL/6 mice were injected with LPS (15 μg/mouse) and/or rIL-35 (1μg/mouse) and after 4 days splenic cells were analyzed by FACS. (h) Absolute numbers of B220loCD19+CD5+ or B220loCD19+CD5+IL-10+ cells in the spleen. Results are representative of at least 3 independent experiments (****P<0.05; **P<0.01; ***P < 0.001).

Figure 3

Figure 3. rIL-35 induced in vivo expansion of Breg cells and suppressed EAU development

EAU was induced in C57BL/6 mice and eyes were analyzed at day-21 post-immunization by funduscopy or histology. (a) Fundus images of the retina (top): Black-arrow, inflammation with blurred optic-disc margins (papilledema); Blue-arrows, retinal vasculitis; White-arrows, retinal/choroidal infiltrates. H&E histological sections: Scale bar, 500 μM. OPN, optic nerve; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE/CH retinal pigmented epithelial and choroid. Open white-arrow, lymphocytes; black-arrowhead, enlarged retinal blood vessels; Blue-asterisk, retinal-folds. EAU scores were based on changes at the retina, optic nerve disc and choroid (ONLINE METHODS). (b-d) Intracellular-cytokine analysis of IL-17- or IFN-γ-expressing T cells in draining LN (b) or IL-10-producing B cells in spleen (c, d) on day-21 post-immunization. Spleen cells isolated from control, pMIB, rIL-35-treated EAU mice were also analyzed for IL-35 (p35 and Ebi3) expression by RT-PCR (e) or intracellular cytokine staining assay (f). (g) B cells from untreated, pMIB-treated or rIL-35-treated EAU mice were re-activated ex-vivo with IRBP/anti-CD40 and CD19+ B-cells were isolated on a cell sorter. The purified CD19+ B cells were co-cultured with uveitogenic LN and spleen cells for 3 days in medium containing IRBP and the cells (1×107) were then transferred into naïve syngeneic mice. Fundus images of the eyes and EAU scores recorded 10 days after adoptive transfer. Data represents at least 3 independent experiments (**P< 0.01; ***P <0.001; ****P< 0.0001).

Figure 4

Figure 4. Breg cells suppressed EAU by inducing Breg/IL-35+Breg and Treg cells while inhibiting Th17/Th1

(a) Purified rIL-35-induced Breg cells and IL-10negative B cells were derived as described (Supplementary methods). WT mice with EAU were treated with 1×106 Breg or IL-10− B cells. (b) Eyes were analyzed by fundoscopy or histology 21 days after disease induction and characteristic features of EAU are as described in Fig. 3a. Scale bar, 500 μM. (c) EAU scores were determined as described in Fig. 3a, 3b. (d, e) Cells from spleen or draining LN were gated on CD19 or CD4, respectively and the percentage of IL-10-, p35- or Ebi3-expressing B cells (d) or Foxp3-, IFN-γ or IL-17-expressing CD4+ T cells (e) was determined by intracellular-cytokine staining assay. (f, g) B cells from the pMIB- or rIL-35-treated mice (described in a) were co-cultured with IRBP-stimulated uveitogenic LN cells (5:1) for 3 days and the cells (1×107) were transferred into naïve CD45.1 congenic mice. Ten days after adoptive transfer, recipient (CD45.1) and donor (CD45.2) cells were analyzed for IL-10-producing B cells (gated on CD19) in spleen (f) or Foxp3-expressing T cells (gated on CD4) in LN (g). (h, i) Purified human CD19+ B cells were cultured for 3 days with PMA in medium with or without rIL-35 and analyzed by FACS or RT-PCR (h) or [3H]-thymidine-incorporation assay (i). Data is representative of analysis of PBMC from 10 donors. Other results represent at least 3 independent experiments (*P<0.05; **P<0.01; ***P < 0.001; ****P<0.0001).

Figure 5

Figure 5. IL-35-signaling is required for suppressive functions of Breg and i35-Breg cells

(a) EAU was induced in WT, p35KO, IL-12Rβ2KO or IL-10KO mice. Fundus images (top panels) and H&E-stained sections (bottom panels) of eyes enucleated 21 days after disease induction. Scale bar, 500 μM. (b) Analysis of IL-17- or IFN-γ-expressing T cells in the draining LN. (c) B cells from indicated EAU mice were re-activated ex-vivo with IRBP/anti-CD40 in medium containing pMIB or rIL-35 and percentage of Breg cells was determined by intracellular cytokine assay. (d) CD19+ B cells from (c) were co-cultured with draining LN cells from WT EAU mice (1:5) for 4 days in medium containing IRBP or pMIB and analyzed by [3H]-thymidine incorporation assay. (e, f) Ex-vivo activated B-cells from (c) were co-cultured with uveitogenic LN T cells (1:5) for 3 days and cells were then transferred to naïve congenic CD45.1+ mice. Fundus images and EAU scores were obtained 10 days after adoptive transfer (e) and the percentage of CD4+CD45.2+ T cells, IFN-γ- or IL-17-expressing CD4+CD45.2+ T cells in the draining LNs was determined by cell surface and intracellular cytokine staining (f). (g, h) Balb/c or Ebi3KO (Balb/c background) mice were immunized with IRBP/CFA and eyes enucleated on day-21 post-immunization were analyzed by fundoscopy or histology and red-arrowheads indicate blood vessels. Scale bar, 500 μM (g). (h) IL-17, IFN-γ or IL-10-expressing LN CD4+ T cells or IL-10-producing splenic B cells were analyzed by intracellular cytokine staining assay. (i) EAU was induced in WT or muMT mice by active immunization with IRBP/CFA and the mice were treated with rIL-35 as described (Supplementary Methods) and eyes examined by fundoscopy on day 21 post-immunization. Results represent at least 3 independent experiments (*P<0.05; **P<0.01; ***P < 0.001).

Figure 6

Figure 6. IL-35 mediates its biological effects on B-cells by activating STAT1 and STAT3 pathways through an IL-35 receptor comprising of IL-12Rβ2 and IL-27Rα

(a) WEHI-279 B-cell line were transduced with control siRNA or IL-12Rβ1-, IL-12Rβ2-, IL-27Rα- or gp130-specific siRNA and after 3 days total RNA was analyzed by RT-PCR. (b, c) siRNA-treated B-cells were cultured in medium containing pMIB or rIL-35 for 3 days and B-cell proliferation (b) or rIL-35-induced production of IL-10 (c) was assessed by [3H]-thymidine incorporation assays or ELISA, respectively. (d, e) Primary B cells from WT, IL12Rβ2KO or IL27RαKO mice were stimulated by LPS in the presence of pMIB or rIL-35 and analyzed by [3H]-thymidine incorporation assay (d) or intracellular cytokine IL-10 staining assay (e). (f) WEHI-279 cells were cultured overnight in medium containing rIL-35 and co-expression of IL-27Rα and IL-12Rβ2 was detected by Western blotting and immunoprecipitation using anti-IL-27Rα anti-Ebi3 or control isotype-specific IgG Abs. (g) Primary B cells were transduced with IL-12Rβ1-, IL-12Rβ2-, IL-27Rα-, gp130 or both IL-12Rβ2- and IL-27Rα-siRNA. The cells were then stimulated in presence of pMIB or rIL-35 and expression of p35 and Ebi3 was detected by RT-PCR analysis. (h, i) Primary T cells (h) or B cells (i) from WT C57BL/6 mice were stimulated with anti-CD3/CD28 or LPS, respectively, and cells were then washed and starved for 2 h in serum free medium (0.5% BSA). Cells were then stimulated for 30 minutes with pMIB, rIL-35 or IL-12 and analyzed for STAT activation by Western blotting. Data represent at least 3 independent experiments (*P<0.05; **P<0.01; ***P < 0.001; ****P<0.0001).

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