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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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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).
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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).

Mentions: To examine whether ex-vivo generated Breg cells can be used to treat ongoing uveitis, we generated highly enriched Breg cells (>93% IL-10+ B-cells) and IL-10− B-cells (<1% IL-10+ B-cells) by sorting (Fig.4a). We induced EAU by active immunization and on day-4 post-immunization intravenously injected the purified Breg-cells or IL-10− B-cells (1×106/mouse). Fundoscopy and histological analysis of the eyes 21 days post-immunization show that the untreated mice or mice that received IL-10− B-cells developed full-blown EAU (Fig.4b,4c). In contrast, mice treated with Breg cells had relatively low EAU scores with very few cells in the vitreous and no evidence of retinal folds (hallmark of severe EAU) (Fig.4b,4c). More than 31.1% of the Breg-cells generated during EAU were IL-35+Bregs and as many as ~64.4% of the Breg-cells in spleen of rIL-35-treated mice were IL-35+Bregs (Fig.4d; lower panels). The increase in Breg and IL-35+Breg cells was accompanied by substantial increase in Foxp3+CD4+ T-cells (8.36% versus 2.81%) and a corresponding decrease in Th1 and Th17 levels in the LN (Fig.4e), suggesting that Breg and Treg cells may synergize to suppress uveitis. Similar experiments using congenic mouse strains suggest that Breg cells might inhibit EAU by inducing expansion of endogenous Breg and Treg cells while inhibiting pathogenic Th17/Th1 in recipient mice (Fig.4f,4g). Of particular significance to therapy, human recombinant IL-35 induced human Breg cells (Fig.4h) and inhibited human B-cell proliferation (Fig.4i).


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)

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).
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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).
Mentions: To examine whether ex-vivo generated Breg cells can be used to treat ongoing uveitis, we generated highly enriched Breg cells (>93% IL-10+ B-cells) and IL-10− B-cells (<1% IL-10+ B-cells) by sorting (Fig.4a). We induced EAU by active immunization and on day-4 post-immunization intravenously injected the purified Breg-cells or IL-10− B-cells (1×106/mouse). Fundoscopy and histological analysis of the eyes 21 days post-immunization show that the untreated mice or mice that received IL-10− B-cells developed full-blown EAU (Fig.4b,4c). In contrast, mice treated with Breg cells had relatively low EAU scores with very few cells in the vitreous and no evidence of retinal folds (hallmark of severe EAU) (Fig.4b,4c). More than 31.1% of the Breg-cells generated during EAU were IL-35+Bregs and as many as ~64.4% of the Breg-cells in spleen of rIL-35-treated mice were IL-35+Bregs (Fig.4d; lower panels). The increase in Breg and IL-35+Breg cells was accompanied by substantial increase in Foxp3+CD4+ T-cells (8.36% versus 2.81%) and a corresponding decrease in Th1 and Th17 levels in the LN (Fig.4e), suggesting that Breg and Treg cells may synergize to suppress uveitis. Similar experiments using congenic mouse strains suggest that Breg cells might inhibit EAU by inducing expansion of endogenous Breg and Treg cells while inhibiting pathogenic Th17/Th1 in recipient mice (Fig.4f,4g). Of particular significance to therapy, human recombinant IL-35 induced human Breg cells (Fig.4h) and inhibited human B-cell proliferation (Fig.4i).

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