Nature Medicine doi: /nm.3554

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6 SUPPLEMENTARY FIGURES LEGENDS Supplementary Figure 1: Generation, purification and characterization of recombinant mouse IL-35 (ril-35). High-Five insect cells expressing high levels of the bicistronic vector encoding the p35 and Ebi3 cdnas were identified by drug (Blasticidin S at 100 µg/ml) selection and presence of the vector (HBM-p35-Flag-IRES-HMB-Ebi3-V5-His) sequence was verified by DNA sequencing. The ril-35 was secreted and the His-tagged protein purified by affinity chromatography using Ni-NTA (Invitrogen) followed by two consecutive cycles of gel filtration using Sephacryl S-200 HR Hiprep 16/60 and Superose-6 HR 10/30 (GE HealthCare, NJ), respectively. (a) SDS-PAGE under non-reducing conditions of fractions collected from first cycle of gel filtration. (b) Chromatogram of Superose-6 gel filtration: peak fractions used for non-reducing SDS-PAGE (c) and sedimentation equilibrium analyses (d-e). (e) Equilibrium profiles of non-reduced and (d) reduced ril-35 (e). Panels are absorbance (bottom panel) and residuals (upper panel). Open circles show UV absorbance gradients in the centrifuge cell. The solid line indicates the calculated fit for an ideal single species. Residuals show the difference in the fitted and experimental values as a function of radial position. In (a) and (c) identities of protein bands were derived from Western blots using anti-il-12p35 or Ebi3 (Figure 1). The presence of carbohydrate accounts for the anomalous migration of non-reduced ril-35 (c) and also the overestimation of mass using sedimentation equilibrium (d). Supplementary Figure 2: IL-35 induced the conversion of lymphocytes into IL-10- producing functional Treg and Breg cells. (a-c) Naive CD4 + T cells were stimulated with anti- CD3/CD28 in medium containing p35, Ebi3 or ril-35. On the 3 rd day ril-35-mediated inhibition of proliferation was assessed by [ 3 H]-thymidine incorporation assay (a), and induction of IL-10

7 production was assessed by intracellular cytokine-staining assay (b) or ELISA (c). Numbers in quadrants indicate percentages of IL-10 or IFN-γ-expressing CD4 + T-cells detected by the intracellular cytokine-staining assay. (d) Primary mouse B cells were stimulated with LPS for 3 days in presence of ril-35, with or without, anti-il-10 neutralizing antibodies or isotypematched control antibodies. Proliferation was analyzed by [ 3 H]-thymidine incorporation assay. (e, f) Naive CD4 + T-cells were stimulated with anti-cd3/cd28 in medium containing TGF-β or IL- 35 (e) and ril-35-induced Tregs were washed and co-cultured (1:1) with naïve syngeneic T-cells for 3 days in medium containing anti-cd3/cd28 (f) and inhibition of T-cell proliferation was assessed by incorporation of [ 3 H]-thymidine after 72 hours of TCR stimulation. (g, h, i) Primary mouse B cells were stimulated with LPS for 3 days in presence of ril-35 and/or anti-p35 or Ebi3 antibodies and then analyzed by the [ 3 H]-thymidine incorporation assay (g), intracellular IL-10 staining assay (h) or qpcr (i). Data represents at least 3 independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < ). Supplement Figure 3: LPS activated B cells secrete functional IL-35 (a) Purified CD19 + B cells were activated for 2 days with LPS (5µg/ml) in medium containing ril-35 (100ng/ml) and immunophenotype of the B cells was determined by cell surface staining with anti-cd19, -CD1d and intracellular IL-10 staining. (b) B cells were activated for 2 days with LPS without ril-35. Culture supernatants were subjected to immunoprecipitation with anti- Ebi3 followed by western blotting with anti-p35 or anti-ebi3 antibody. (c) B cells were activated for 2 days with LPS plus ril-35 and whole cell lysates were subjected to immunoprecipitation with anti-ebi3 followed by western blotting with anti-p35 antibody. Data represent at least 3 independent experiments.

8 Supplementary Figure 4: Characterization of receptor and STAT pathways utilized by IL- 35 in B-cells. (a, b) Inhibition of lymphocyte proliferation or IL-10 production by IL-35 did not require gp130. B-cells from 3 human subjects were stimulated with PMA in presence of IL-6 or rhil-35 plus neutralizing anti-human gp130 antibodies or isotype antibody and after 3 days cell proliferation was analyzed by the [ 3 H]-thymidine incorporation assay (a) and production of IL-10 was analyzed by the intracellular cytokine-staining assay (b). (c, d) Primary B-cells from WT C57BL/6, STAT1KO or STAT4KO mice were stimulated by LPS in medium containing pmib or ril-35. STAT3 was depleted in primary B cells by use of STAT3-specific shrna and the cells were similarly stimulated. The requirement of STAT1, STAT3 and/or STAT4 was assessed by [ 3 H]-thymidine incorporation assay (c) or intracellular cytokine staining assay (d). Data are representative of at least 3 independent experiments (**P < 0.01, ***P < 0.001, ****P < ). Supplementary Figure 5: p35 blocks IL-6-induced STAT3 activation and enhanced STAT1 activation by IL-27. Mouse CD19 + B-cells were activated with LPS for two days, washed and starved for 2 hours in serum-free medium. Cells were then cultured for 15 min in medium containing p35 and or IL-6, IL-12, IL-27 and then subjected to Western blot analysis.