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Interleukin-35 induces regulatory B cells that suppress autoimmune disease.

Wang RX, Yu CR, Dambuza IM, Mahdi RM, Dolinska MB, Sergeev YV, Wingfield PT, Kim SH, Egwuagu CE - Nat. Med. (2014)

Bottom Line: 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.In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor comprising the IL-12Rβ2 and IL-27Rα subunits.

View Article: PubMed Central - PubMed

Affiliation: 1] Molecular Immunology Section, Laboratory of Immunology, National Eye Institute (NEI), National Institutes of Health (NIH), Bethesda, Maryland, USA. [2] Laboratory of Immunology, Institute of Basic Medical Sciences, Beijing, China.

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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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).
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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).

Mentions: To study the potential regulatory role of IL-35 in autoimmune diseases and examine whether it can be used to treat uveitis, we genetically engineered and produced mouse IL-35 in insect cells (Fig. 1a). Details of the production and purification of the mouse recombinant IL-35 (rIL-35) are presented (Supplementary methods/Supplementary Fig.1). Single chain Ebi3 or p35 migrated as 33 kDa monomeric protein on denaturing SDS gels while rIL-35 migrated as ~67 kDa heterodimeric protein on native, non-denaturing gel (Fig.1b). rIL-35 was further purified by two cycles of FPLC (Supplementary Fig.1a,1b) and characterized by SDS-PAGE (Supplementary Fig.1c). Accurate mass determination was obtained by sedimentation equilibrium analysis (Supplementary Fig.1d,1e). Western blotting and coimmunoprecipitation analyses using anti-Flag and anti-V5 Abs revealed specific association of Ebi3 with p35 as a stable p35:Ebi3 heterodimeric complex (Fig.1c), consistent with a previous study18. As control for functional studies we used pMIB, an unfractionated heterogeneous collection of irrelevant secretome of the insect cells. Western blot analysis of the pMIB control established that pMIB does not exhibit immunoreactivity to p35, Ebi3, Flag or V5 epitope (Fig.1c). Identity of the heterodimer was derived from dual reactivity with anti-p35 and Ebi3 monoclonal antibodies (Fig.1d). In line with a previous report15, we demonstrated that the heterodimeric protein is biologically active by showing that rIL-35 suppressed T-cell proliferation (Supplementary Fig.2a).


Interleukin-35 induces regulatory B cells that suppress autoimmune disease.

Wang RX, Yu CR, Dambuza IM, Mahdi RM, Dolinska MB, Sergeev YV, Wingfield PT, Kim SH, Egwuagu CE - Nat. Med. (2014)

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).
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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).
Mentions: To study the potential regulatory role of IL-35 in autoimmune diseases and examine whether it can be used to treat uveitis, we genetically engineered and produced mouse IL-35 in insect cells (Fig. 1a). Details of the production and purification of the mouse recombinant IL-35 (rIL-35) are presented (Supplementary methods/Supplementary Fig.1). Single chain Ebi3 or p35 migrated as 33 kDa monomeric protein on denaturing SDS gels while rIL-35 migrated as ~67 kDa heterodimeric protein on native, non-denaturing gel (Fig.1b). rIL-35 was further purified by two cycles of FPLC (Supplementary Fig.1a,1b) and characterized by SDS-PAGE (Supplementary Fig.1c). Accurate mass determination was obtained by sedimentation equilibrium analysis (Supplementary Fig.1d,1e). Western blotting and coimmunoprecipitation analyses using anti-Flag and anti-V5 Abs revealed specific association of Ebi3 with p35 as a stable p35:Ebi3 heterodimeric complex (Fig.1c), consistent with a previous study18. As control for functional studies we used pMIB, an unfractionated heterogeneous collection of irrelevant secretome of the insect cells. Western blot analysis of the pMIB control established that pMIB does not exhibit immunoreactivity to p35, Ebi3, Flag or V5 epitope (Fig.1c). Identity of the heterodimer was derived from dual reactivity with anti-p35 and Ebi3 monoclonal antibodies (Fig.1d). In line with a previous report15, we demonstrated that the heterodimeric protein is biologically active by showing that rIL-35 suppressed T-cell proliferation (Supplementary Fig.2a).

Bottom Line: 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.In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor comprising the IL-12Rβ2 and IL-27Rα subunits.

View Article: PubMed Central - PubMed

Affiliation: 1] Molecular Immunology Section, Laboratory of Immunology, National Eye Institute (NEI), National Institutes of Health (NIH), Bethesda, Maryland, USA. [2] Laboratory of Immunology, Institute of Basic Medical Sciences, Beijing, China.

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.

Show MeSH
Related in: MedlinePlus