Additionally, RNA from these cells was harvested and qPCR was performed as previously CFTR activator described. Paired Student’s t tests were used to determine the statistical significance of the data in all figures except Fig. 6, where an unpaired Student’s t test used.

Statistical analysis was performed using Prism 4 v. 4.0c. P < 0.05 was considered significant. HCV clone, JFH-1, can infect Huh-derived hepatoma cells in cell culture and produce infectious virus.21-23 By using this system, we determined the effect of HCV-infected hepatocytes on the generation of suppressive CD33+ monocytes/macrophages. We first infected Huh7.5.1 cells with HCV (JFH-1) virus; infection was then confirmed by immunofluorescence

(IF) for the expression of HCV core protein (Fig. 1A). HCV-infected cells (HCV+ hepatocytes) were reseeded and cultured for 24 hours. After the coculture of HCV+ or HCV− hepatocytes with PBMCs for 7 days, CD33+ cells were subsequently selected and then cocultured with autologous see more CD4+ and CD8+ T cells. Interestingly, CD33+ cells cocultured with HCV+ hepatocytes significantly inhibited the production of IFN-γ by both CD4+ and CD8+ T cells using ELISA (Fig. 1B). In contrast, there was no significant difference in T-cell proliferation when T cells were cocultured with HCV+ hepatocytes as compared with those with HCV− hepaotcytes (data not shown). These results suggest that HCV impairs antiviral T-cell responses through the generation of suppressive CD33+ M/Mφ. To examine the effect of infectious virus on the generation of suppressive CD33+ cells, we treated PBMCs with varying doses of JFH-1 virus isolated using an Amicon filter system and CD33+ cells were then selected and cocultured with autologous CD4+ T cells. These results indicate that CD33+ cells are Docetaxel concentration not altered by

treatment with isolated virus (Supporting Fig. 1). Furthermore, CD33+ cells cocultured with HCV+ hepatocytes in the presence of a transwell modestly, but not significantly, suppress CD4+ T-cell activation as compared with control (Supporting Fig. 2), suggesting that close contact of CD33+ cells with HCV+ hepatocytes is required for optimal MDSC induction. Based on a report that CD33+ MDSCs, generated from human PBMCs following exposure to immunosuppressive factors for 7 days, suppress T-cell responsiveness, we assessed whether the immunomodulatory protein, extracellular HCV core, could induce MDSCs to impair T-cell responses.13 To this end, we treated human PBMCs with recombinant extracellular HCV core or control β-galactosidase (β-gal) for 7 days and then selected CD33+ cells using magnetic beads. The cells were then cocultured with CD4+ or CD8+ T cells for 3 days. HCV core-treated CD33+ cells inhibited the proliferation of both CD4 and CD8 T cells (Fig. 2A,B).