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Additional Supporting Information may be found in the online version of this article.

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HEP_25906_sm_SuppFig1.tif1317KSupplementary Figure 1. VSIG4 is expressed on KCs, but not on LSECs and regulatory DCs. (A) Liver MNCs that were enriched by centrifugation at 50 x g for 5 min were overlaid onto 25%/50% Percoll gradient and subjected to centrifugation at 1,800 x g for 30 min. Each cell fraction was harvested and stained with anti-VSIG4 and indicated surface markers, analyzed by flow cytometry, and represented as dot plots (A) and histograms (B).
HEP_25906_sm_SuppFig2.tif3966KSupplementary Figure 2. No difference in apoptotic cell percentage in livers between VSIG4 WT and KO mice. Formalin-fixed livers from VSIG4 WT and KO mice (B6 background) that were treated or untreated with ConA (15 mg/kg, for 24 h) were stained for apoptotic cells using the TUNEL staining kit, and observed by light microscopy (x200). For positive control, liver sections were treated with DNase I to fragment DNA. Data are representative of three independent experiments.
HEP_25906_sm_SuppFig3.tif768KSupplementary Figure 3. VSIG4 fusion protein binds to T- and NKT-cells. Purified liver MNCs were incubated with control Ig or VSIG4.Ig (30 μg/test) in the presence of mouse IgG or 14G8 (30 μg/test) for 30 min, and then stained with PE-cy5-conjugated anti-TCR-ß and PE-conjubated anti-NK1.1. Bound VSIG4.Ig was detected with FITC-conjugated anti-human IgG in cells gated on TCR-ß+NK1.1+ (NKT cells) and TCR-ß+NK1.1- (T-cells), and analyzed by flow cytometry.
HEP_25906_sm_SuppFig4.tif757KSupplementary Figure 4. Adoptively-transferred KCs were localized in liver. B6 WT KCs (3 x 106) were labeled with 1 μM CFSE and adoptively transferred into B6 WT mice. Seven days later, KCs were isolated and CFSE positive cells were analyzed by flow cytometry.
HEP_25906_sm_SuppFig5.tif1156KSupplementary Figure 5. No difference in surface phenotypes and cytokine production between VSIG4 WT and KO KCs. (A) Isolated KCs were stained for surface phenotype markers (MHC class II, CD80, CD86, and B7-H1) and analyzed by flow cytometry. Data are representative of three independent experiments. (B) KCs (106) were cultured in 24-well plates for 24 h. Levels of IL-10 and TGF-β in culture supernatants were measured by ELISA. Data represent means ± SD and are representative of three independent experiments.
HEP_25906_sm_SuppFig6.tif1233KSupplementary Figure 6. No difference in cellular composition in livers between VSIG4 WT and KO mice. (A) Liver MNCs were stained with FITC-conjugated NK1.1 and PE-conjugated TCR-? and analyzed by flow cytometry. Numbers indicate the percentage of gated populations among total MNCs. Data are representative of seven independent experiments. (B) F4/80-, CD11b- and CD11c-positive populations of liver MNCs were analyzed by flow cytometry. Numbers in the histograms indicate the percentage of positive populations compared with isotype controls. Data are representative of three independent experiments. (C) CD4+ FoxP3+ Treg cells in MNCs were analyzed by flow cytometry. Data are representative of six independent experiments. (D) Liver MNCs were isolated from VSIG4 WT and KO mice (B6 background), stained with FITC-, PE-, PE-cy5- or APC-conjugated anti-TCR-ß, anti-NK1.1, anti-CD44 and anti-CD62L and analyzed by flow cytometry. Data are representatives of four independent experiments.
HEP_25906_sm_SuppFig7.tif769KSupplementary Figure 7. T-cell production of immunosuppressive cytokines in the presence of VSIG4 WT or KO KCs. Balb/c WT splenic T-cells (105) were stimulated with anti-CD3 (1 μg/ml) in the presence of VSIG4 WT or KO KCs (104) for 2 days. Culture supernatants were assayed for cytokines by ELISA. Data represent means ± SD. Data are representative of two independent experiments.
HEP_25906_sm_SuppFig8.tif4366KSupplementary Figure 8. VSIG4 is downregulated in human autoimmune hepatitis liver. (A) Peritoneal macrophages (2 x 106) from Balb/c WT mice were treated with indicated concentration of ConA for 6 h. Total RNA was purified and subjected to RT-PCR for VSIG4, IFN-?, and ?-actin. Data are representative of two independent experiments. (B) Formalin-fixed sections of liver tissues from autoimmune hepatitis patients or from cholangiocarcinoma patients whose liver tissues did not show any hepatic pathology were used for immunohistochemistry. The sections were stained with rabbit anti-VSIG4 polyclonal antibody, developed as described in Supplementary materials, and subjected to microscopic examination (x 200).
HEP_25906_sm_SuppFig9.tif939KSupplementary Figure 9. Blocking antibody against C3b binding to VSIG4 does not interfere with VSIG4-mediated T-cell suppression. (A) Purified DO11.10 T-cells (105) were cocultured with peritoneal macrophages (104) from VSIG4 WT and KO mice in the presence of OVA323-339 (1 μg/ml) and indicated concentrations of mIgG1 or 14G8 mAb that blocks C3b binding to VSIG4 for 3 days. Culture supernatants were assay by ELISA for IL-2. Data are representatives of two independent experiments. (B) Balb/c WT mice were treated with control Ig or VSIG4.Ig (400 μg/mouse, n=5 per group) in the presence of mouse IgG or 14G8 (200 μg/mouse) 2 h before administration of a lethal dose of ConA (30 mg/kg). Survival was monitored (Gehan-Breslow-Wilcoxon test).
HEP_25906_sm_SuppInfo.doc48KSupporting Information

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