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HEP_25651_sm_SuppFig1.tif1191KSupporting Information Figure 1. Flow cytometry analysis of peripheral blood mononuclear cells isolated by Ficoll-Hypaque density gradient centrifugation. PBMCs were identified and gated through forward scatter and side scatter (A). Histograms show isotype control staining (B) and CD14 positive staining (C), followed by gating CD3- events. Monocytes and macrophages were defined as CD3-CD14+ cells.
HEP_25651_sm_SuppFig2.tif2312KSupporting Information Figure 2. Flow cytometry analysis of liver mononuclear cells. Each of 3 uninfected (upper panel) and HBV-infected (lower panel) human hepatocyte chimeric mice were transplanted with human PBMCs obtained from 3 healthy blood donors without HBs antigen vaccine. Mice liver mononuclear cells at 9 weeks were separated with antibodies for human CD45 and mouse H-2Db. Flow cytometry showed similar human PBMC populations between uninfected and HBV-infected mice.
HEP_25651_sm_SuppFig3A.tif1840KSupporting Information Figure 3A. Analysis of liver infiltrating cells by flow cytometry. After human PBMCs were defined as mouse H-2Db-human CD45+ cells, we further analyzed phenotypes of these cells obtained from the liver at day 2 (Suppl. Fig. 2) and day 7 (Suppl. Fig. 3). Liver mononuclear cells of uninfected (upper panel) and HBV-infected (lower panel) mice transplanted with human PBMC were separated with anti-human CD4 and CD8 antibody (left) or anti-human CD8 and HLA-A2 HBcAg tetramer (right) (A), anti-human CD3 and CD56 (left) or human CD3 and FasL (right) (B) and anti-human HLA-DR and CD123 (left) and HLA-DR and CD11c (right) (C). (D) The frequency of TRAIL and CD107a-positive cells in NK cells were analyzed in uninfected (upper panel) and HBV-infected mice (lower panel).
HEP_25651_sm_SuppFig3B.tif1016KSupporting Information Figure 3B. Analysis of liver infiltrating cells by flow cytometry. After human PBMCs were defined as mouse H-2Db-human CD45+ cells, we further analyzed phenotypes of these cells obtained from the liver at day 2 (Suppl. Fig. 2) and day 7 (Suppl. Fig. 3). Liver mononuclear cells of uninfected (upper panel) and HBV-infected (lower panel) mice transplanted with human PBMC were separated with anti-human CD4 and CD8 antibody (left) or anti-human CD8 and HLA-A2 HBcAg tetramer (right) (A), anti-human CD3 and CD56 (left) or human CD3 and FasL (right) (B) and anti-human HLA-DR and CD123 (left) and HLA-DR and CD11c (right) (C). (D) The frequency of TRAIL and CD107a-positive cells in NK cells were analyzed in uninfected (upper panel) and HBV-infected mice (lower panel).
HEP_25651_sm_SuppFig3C.tif487KSupporting Information Figure 3C. Analysis of liver infiltrating cells by flow cytometry. After human PBMCs were defined as mouse H-2Db-human CD45+ cells, we further analyzed phenotypes of these cells obtained from the liver at day 2 (Suppl. Fig. 2) and day 7 (Suppl. Fig. 3). Liver mononuclear cells of uninfected (upper panel) and HBV-infected (lower panel) mice transplanted with human PBMC were separated with anti-human CD4 and CD8 antibody (left) or anti-human CD8 and HLA-A2 HBcAg tetramer (right) (A), anti-human CD3 and CD56 (left) or human CD3 and FasL (right) (B) and anti-human HLA-DR and CD123 (left) and HLA-DR and CD11c (right) (C). (D) The frequency of TRAIL and CD107a-positive cells in NK cells were analyzed in uninfected (upper panel) and HBV-infected mice (lower panel).
HEP_25651_sm_SuppFig4A.tif1442KSupporting Information Figure 4A. Analysis of liver infiltrating cells by flow cytometry. After human PBMCs were defined as mouse H-2Db-human CD45+ cells, we further analyzed phenotypes of these cells obtained from the liver at day 2 (Suppl. Fig. 2) and day 7 (Suppl. Fig. 3). Liver mononuclear cells of uninfected (upper panel) and HBV-infected (lower panel) mice transplanted with human PBMC were separated with anti-human CD4 and CD8 antibody (left) or anti-human CD8 and HLA-A2 HBcAg tetramer (right) (A), anti-human CD3 and CD56 (left) or human CD3 and FasL (right) (B) and anti-human HLA-DR and CD123 (left) and HLA-DR and CD11c (right) (C). (D) The frequency of TRAIL and CD107a-positive cells in NK cells were analyzed in uninfected (upper panel) and HBV-infected mice (lower panel).
HEP_25651_sm_SuppFig4B.tif1907KSupporting Information Figure 4B. Analysis of liver infiltrating cells by flow cytometry. After human PBMCs were defined as mouse H-2Db-human CD45+ cells, we further analyzed phenotypes of these cells obtained from the liver at day 2 (Suppl. Fig. 2) and day 7 (Suppl. Fig. 3). Liver mononuclear cells of uninfected (upper panel) and HBV-infected (lower panel) mice transplanted with human PBMC were separated with anti-human CD4 and CD8 antibody (left) or anti-human CD8 and HLA-A2 HBcAg tetramer (right) (A), anti-human CD3 and CD56 (left) or human CD3 and FasL (right) (B) and anti-human HLA-DR and CD123 (left) and HLA-DR and CD11c (right) (C). (D) The frequency of TRAIL and CD107a-positive cells in NK cells were analyzed in uninfected (upper panel) and HBV-infected mice (lower panel).
HEP_25651_sm_SuppFig4C.tif621KSupporting Information Figure 4C. Analysis of liver infiltrating cells by flow cytometry. After human PBMCs were defined as mouse H-2Db-human CD45+ cells, we further analyzed phenotypes of these cells obtained from the liver at day 2 (Suppl. Fig. 2) and day 7 (Suppl. Fig. 3). Liver mononuclear cells of uninfected (upper panel) and HBV-infected (lower panel) mice transplanted with human PBMC were separated with anti-human CD4 and CD8 antibody (left) or anti-human CD8 and HLA-A2 HBcAg tetramer (right) (A), anti-human CD3 and CD56 (left) or human CD3 and FasL (right) (B) and anti-human HLA-DR and CD123 (left) and HLA-DR and CD11c (right) (C). (D) The frequency of TRAIL and CD107a-positive cells in NK cells were analyzed in uninfected (upper panel) and HBV-infected mice (lower panel).
HEP_25651_sm_SuppFig4D.tif637KSupporting Information Figure 4D. Analysis of liver infiltrating cells by flow cytometry. After human PBMCs were defined as mouse H-2Db-human CD45+ cells, we further analyzed phenotypes of these cells obtained from the liver at day 2 (Suppl. Fig. 2) and day 7 (Suppl. Fig. 3). Liver mononuclear cells of uninfected (upper panel) and HBV-infected (lower panel) mice transplanted with human PBMC were separated with anti-human CD4 and CD8 antibody (left) or anti-human CD8 and HLA-A2 HBcAg tetramer (right) (A), anti-human CD3 and CD56 (left) or human CD3 and FasL (right) (B) and anti-human HLA-DR and CD123 (left) and HLA-DR and CD11c (right) (C). (D) The frequency of TRAIL and CD107a-positive cells in NK cells were analyzed in uninfected (upper panel) and HBV-infected mice (lower panel).
HEP_25651_sm_SuppFig5A.tif753KSupporting Information Figure 5A. Time course of mice transplanted with human PBMC with DC depletion and analysis of liver infiltrating cells. (A) Time course of human serum albumin (upper panel) and HBV DNA (lower panel) before and one week after human PBMC transplantation are shown. Mice DC (closed circles) and NK cells (open triangles) were depleted with negative selection as described in the Methods section. Time courses of 3 mice infected with HBV and transplanted with human PBMC without depletion of DCs and NK cells (appeared in Figure 1C) are shown for comparison (shaded closed circle). (B) Liver-infiltrating cells at day 7 were analyzed by flow cytometry. After human PBMCs were defined as mouse H-2Db-human CD45+ cells (upper left panel), we further analyzed the phenotypes of these cells. DCs and activated NK cells are undetectable under these conditions.
HEP_25651_sm_SuppFig5B.tif1195KSupporting Information Figure 5B. Time course of mice transplanted with human PBMC with DC depletion and analysis of liver infiltrating cells. (A) Time course of human serum albumin (upper panel) and HBV DNA (lower panel) before and one week after human PBMC transplantation are shown. Mice DC (closed circles) and NK cells (open triangles) were depleted with negative selection as described in the Methods section. Time courses of 3 mice infected with HBV and transplanted with human PBMC without depletion of DCs and NK cells (appeared in Figure 1C) are shown for comparison (shaded closed circle). (B) Liver-infiltrating cells at day 7 were analyzed by flow cytometry. After human PBMCs were defined as mouse H-2Db-human CD45+ cells (upper left panel), we further analyzed the phenotypes of these cells. DCs and activated NK cells are undetectable under these conditions.
HEP_25651_sm_SuppFig6A.tif1229KSupporting Information Figure 6A. Flow cytometric analysis of liver infiltrating cells after depletion of DCs by clodronate. After human PBMCs were defined as mouse H-2Db-human CD45+ cells, we further analyzed the phenotypes of these cells. Mice livers were obtained from mice at day 4 (Suppl. Fig. 5) and day 7 (Suppl. Fig. 6). Liver mononuclear cells of uninfected (upper panel) and HBV-infected (lower panel) mice transplanted with human PBMCs were separated with anti-human CD4 and CD8 antibody (5A and 6A, left) or anti-human CD8 and HLA-A2 HBc Ag tetramer (5A and 6A, right), anti-human CD3 and CD56 (5B and 6B, left) or human CD3 and FasL (5B and 6B, right) and anti-human HLA-DR and CD123 (5C and 6C, left) and HLA-DR and CD11c (5C and 6C, right).
HEP_25651_sm_SuppFig6B.tif1842KSupporting Information Figure 6B. Flow cytometric analysis of liver infiltrating cells after depletion of DCs by clodronate. After human PBMCs were defined as mouse H-2Db-human CD45+ cells, we further analyzed the phenotypes of these cells. Mice livers were obtained from mice at day 4 (Suppl. Fig. 5) and day 7 (Suppl. Fig. 6). Liver mononuclear cells of uninfected (upper panel) and HBV-infected (lower panel) mice transplanted with human PBMCs were separated with anti-human CD4 and CD8 antibody (5A and 6A, left) or anti-human CD8 and HLA-A2 HBc Ag tetramer (5A and 6A, right), anti-human CD3 and CD56 (5B and 6B, left) or human CD3 and FasL (5B and 6B, right) and anti-human HLA-DR and CD123 (5C and 6C, left) and HLA-DR and CD11c (5C and 6C, right).
HEP_25651_sm_SuppFig6C.tif529KSupporting Information Figure 6C. Flow cytometric analysis of liver infiltrating cells after depletion of DCs by clodronate. After human PBMCs were defined as mouse H-2Db-human CD45+ cells, we further analyzed the phenotypes of these cells. Mice livers were obtained from mice at day 4 (Suppl. Fig. 5) and day 7 (Suppl. Fig. 6). Liver mononuclear cells of uninfected (upper panel) and HBV-infected (lower panel) mice transplanted with human PBMCs were separated with anti-human CD4 and CD8 antibody (5A and 6A, left) or anti-human CD8 and HLA-A2 HBc Ag tetramer (5A and 6A, right), anti-human CD3 and CD56 (5B and 6B, left) or human CD3 and FasL (5B and 6B, right) and anti-human HLA-DR and CD123 (5C and 6C, left) and HLA-DR and CD11c (5C and 6C, right).
HEP_25651_sm_SuppFig7A.tif986KSupporting Information Figure 7A. Flow cytometric analysis of liver infiltrating cells after depletion of DCs by clodronate. After human PBMCs were defined as mouse H-2Db-human CD45+ cells, we further analyzed the phenotypes of these cells. Mice livers were obtained from mice at day 4 (Suppl. Fig. 5) and day 7 (Suppl. Fig. 6). Liver mononuclear cells of uninfected (upper panel) and HBV-infected (lower panel) mice transplanted with human PBMCs were separated with anti-human CD4 and CD8 antibody (5A and 6A, left) or anti-human CD8 and HLA-A2 HBc Ag tetramer (5A and 6A, right), anti-human CD3 and CD56 (5B and 6B, left) or human CD3 and FasL (5B and 6B, right) and anti-human HLA-DR and CD123 (5C and 6C, left) and HLA-DR and CD11c (5C and 6C, right).
HEP_25651_sm_SuppFig7B.tif1839KSupporting Information Figure 7B. Flow cytometric analysis of liver infiltrating cells after depletion of DCs by clodronate. After human PBMCs were defined as mouse H-2Db-human CD45+ cells, we further analyzed the phenotypes of these cells. Mice livers were obtained from mice at day 4 (Suppl. Fig. 5) and day 7 (Suppl. Fig. 6). Liver mononuclear cells of uninfected (upper panel) and HBV-infected (lower panel) mice transplanted with human PBMCs were separated with anti-human CD4 and CD8 antibody (5A and 6A, left) or anti-human CD8 and HLA-A2 HBc Ag tetramer (5A and 6A, right), anti-human CD3 and CD56 (5B and 6B, left) or human CD3 and FasL (5B and 6B, right) and anti-human HLA-DR and CD123 (5C and 6C, left) and HLA-DR and CD11c (5C and 6C, right).
HEP_25651_sm_SuppFig7C.tif506KSupporting Information Figure 7C. Flow cytometric analysis of liver infiltrating cells after depletion of DCs by clodronate. After human PBMCs were defined as mouse H-2Db-human CD45+ cells, we further analyzed the phenotypes of these cells. Mice livers were obtained from mice at day 4 (Suppl. Fig. 5) and day 7 (Suppl. Fig. 6). Liver mononuclear cells of uninfected (upper panel) and HBV-infected (lower panel) mice transplanted with human PBMCs were separated with anti-human CD4 and CD8 antibody (5A and 6A, left) or anti-human CD8 and HLA-A2 HBc Ag tetramer (5A and 6A, right), anti-human CD3 and CD56 (5B and 6B, left) or human CD3 and FasL (5B and 6B, right) and anti-human HLA-DR and CD123 (5C and 6C, left) and HLA-DR and CD11c (5C and 6C, right).
HEP_25651_sm_SuppFig8.tif660KSupporting Information Figure 8. Effect of antibodies against IFN-gamma and IFN-alpha. Mice were given a single injection of 1x105 IU of IFN-gamma (A and B) or 1500 IU/g IFN-alpha (C and D). Mice were pre-treated with anti-human IFN-gamma mAb (B) or anti-human IFN-alpha mAb (D) as described in the Methods section. Six hours after injection, mice were sacrificed and liver samples were collected. RNA was extracted from liver samples by Sepa Gene RV-R (Sankojunyaku, Tokyo, Japan). Quantitation of ISGs (MxA, OAS1, PKR) was performed using real-time PCR Master Mix (TOYOBO, Kyoto, Japan) and TaqMan Gene Expression Assay primer and probe sets (PE Applied Biosystems, Foster City, CA). ISG expression levels are expressed relative to the endogenous RNA levels of the housekeeping reference gene glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Data are represented as mean ± SD (n = 3). *P < .001
HEP_25651_sm_SuppFig9.tif2785KSupporting Information Figure 9. Analysis of phenotype of the liver mononuclear cells in a patient with fulminant hepatitis B and of the peripheral mononuclear cells in a healthy blood donor by flow cytometry. Mononuclear cells in the livers of a patient with fulminant hepatitis B (upper panels) and a healthy blood donor (lower panels) were separated with anti-human CD8 antibody and HLA-A2 HBc-7Ag tetramer (left) or anti human CD3 and CD56 (middle) and anti-human CD3 and FasL (right). HBV-specific CTLs were detected in a fulminant hepatitis B patient but not in a healthy blood donor.
HEP_25651_sm_SuppFig10.tif328KSupporting Information Figure 10. The frequency of FasL-positive cells in NK cells. Chimeric mice with or without HBV-infection were transplanted with human PBMCs. The frequency of FasL-positive cells in NK cells in the mice livers at day 4 (left panel) and 7 (right panel) were analyzed by flow cytometry. The frequency of FasL-positive cells was significantly higher in HBV-infected mice compared to uninfected mice at day 4. Data are shown as mean ± SD (n = 3). *P < 0.01; NS, not significant.
HEP_25651_sm_SuppTables.doc29KSupporting Information Tables.

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