Treatment-induced and withIL-28 and IL-29 Modulate Dendritic CellsTable 1. Clinical characteristics of study individuals.Materials and Methods ReagentsRecombinant cytokines IL-2, IL-4, GM-CSF and IFNs (IL-29 and IL-28A) were from Peprotech (Rocky Hill, NJ), anti-IL-10 antibodies (clone JES3-9D7) from Biosource, anti-PD-1 antibody from eBioscience (San Diego, CA), carboxyfluorescein-succinimidylester (CFSE) was from 
Invitrogen (Carlsbad, CA), and 3Hthymidine was from PerkinElmer (Waltham, MA).ParameterValue 1676428 ?Treatment-naive SVR 48611 17 4 22611 0 16 4 4 0 16 10 6 NASH 4165 6 6 69631 0 12 1 1 0 12 9Age (years) Male Female AST (U/l) HCV viral load Liver biopsies performed Fibrosis Stage 1? Stage 3?42612 20 4 71623 1.92610660.366106 22 8 6Blood Donors and Cell CultureThe study was approved by the Committee for Protection of Human Subjects in Research at University of Massachusetts Medical School and all individuals provided written consent to participate. Patients’ characteristics are described in Table 1. Core liver biopsies from patients were collected in our clinic and snapfrozen until analysis. Liver RNA from control individuals (free of liver disease) was purchased from Origene (n = 3) and from Stratagene (n = 1). Blood plasma was separated by centrifugation; PBMC were separated by centrifugation in Ficoll gradient; monocytes were isolated by adherence to plastic, as previously described [1]. Serum and cells were Lixisenatide web paired in controls; liver tissue was not paired with serum or cells in controls due to the commercial origin of normal liver RNA. When possible, paired serum/liver samples were analyzed in HCV and SVR patients. To test the effect on DC generation, the IFN l (IL-29, IL-28A or their mixture) was added to adherent monocytes together with IL-4 and GM-CSF for 7 days. CD4+CD25+ (regulatory) T cells, CD4+CD252 (effector) T cells, CD16+CD56+ NK cells, BDCA-1+ myeloid dendritic cells, CD123+BDCA-2+ plasmacytoid dendritic cells, and total CD4+ T cells were purified using magnetic beads (Miltenyi Biotech and StemCell Technologies), following the manufacturer’s instructions.Liver inflammation 22 Score: 0? Score: 7?2 16A total of 24 HCV, 21 SVR, 20 controls (serum and/or cells), 4 control liver RNA and 12 NASH serum were analyzed in our manuscript as follows: N 18 HCV and 16 SVR pairs of blood and liver were analyzed for the data shown in Fig. 1. N 12 control serum and 4 control liver mRNA were analyzed for the data shown in Fig. 1; these samples were not paired. N 12 NASH blood (serum) were analyzed for the data shown in Fig. 1. N The cells from the same 18 HCV analyzed in Fig. 1 were analyzed in Fig. 3 for their DC allostimulatory capacity. N Additional 6 HCV, 5 SVR and 8 controls were recruited to perform the experiments shown in Fig. 3 in order to collect data for achieving sufficient statistical analysis power. N Control liver RNA was of commercial origin from individuals without known liver diseases. doi:10.1371/journal.pone.0044915.tDendritic Cells and T Cells Function Assaynatural HCV SVR [7,10], it is likely that in vivo IFN- l may be involved in anti-HCV innate immunity. Innate immunity is key to buy Fruquintinib antiviral defense. Innate immune defects have been identified in cHCV, including relative deficiency of circulating plasmacytoid dendritic cells (pDCs), altered expression of pathogen-recognition receptors, and a skewed monocytes/ DC cytokine profile towards enriched production of immunoregulatory cytokines and impaired production of IF.Treatment-induced and withIL-28 and IL-29 Modulate Dendritic CellsTable 1. Clinical characteristics of study individuals.Materials and Methods ReagentsRecombinant cytokines IL-2, IL-4, GM-CSF and IFNs (IL-29 and IL-28A) were from Peprotech (Rocky Hill, NJ), anti-IL-10 antibodies (clone JES3-9D7) from Biosource, anti-PD-1 antibody from eBioscience (San Diego, CA), carboxyfluorescein-succinimidylester (CFSE) was from Invitrogen (Carlsbad, CA), and 3Hthymidine was from PerkinElmer (Waltham, MA).ParameterValue 1676428 ?Treatment-naive SVR 48611 17 4 22611 0 16 4 4 0 16 10 6 NASH 4165 6 6 69631 0 12 1 1 0 12 9Age (years) Male Female AST (U/l) HCV viral load Liver biopsies performed Fibrosis Stage 1? Stage 3?42612 20 4 71623 1.92610660.366106 22 8 6Blood Donors and Cell CultureThe study was approved by the Committee for Protection of Human Subjects in Research at University of Massachusetts Medical School and all individuals provided written consent to participate. Patients’ characteristics are described in Table 1. Core liver biopsies from patients were collected in our clinic and snapfrozen until analysis. Liver RNA from control individuals (free of liver disease) was purchased from Origene (n = 3) and from Stratagene (n = 1). Blood plasma was separated by centrifugation; PBMC were separated by centrifugation in Ficoll gradient; monocytes were isolated by adherence to plastic, as previously described [1]. Serum and cells were paired in controls; liver tissue was not paired with serum or cells in controls due to the commercial origin of normal liver RNA. When possible, paired serum/liver samples were analyzed in HCV and SVR patients. To test the effect on DC generation, the IFN l (IL-29, IL-28A or their mixture) was added to adherent monocytes together with IL-4 and GM-CSF for 7 days. CD4+CD25+ (regulatory) T cells, CD4+CD252 (effector) T cells, CD16+CD56+ NK cells, BDCA-1+ myeloid dendritic cells, CD123+BDCA-2+ plasmacytoid dendritic cells, and total CD4+ T cells were purified using magnetic beads (Miltenyi Biotech and StemCell Technologies), following the manufacturer’s instructions.Liver inflammation 22 Score: 0? Score: 7?2 16A total of 24 HCV, 21 SVR, 20 controls (serum and/or cells), 4 control liver RNA and 12 NASH serum were analyzed in our manuscript as follows: N 18 HCV and 16 SVR pairs of blood and liver were analyzed for the data shown in Fig. 1. N 12 control serum and 4 
control liver mRNA were analyzed for the data shown in Fig. 1; these samples were not paired. N 12 NASH blood (serum) were analyzed for the data shown in Fig. 1. N The cells from the same 18 HCV analyzed in Fig. 1 were analyzed in Fig. 3 for their DC allostimulatory capacity. N Additional 6 HCV, 5 SVR and 8 controls were recruited to perform the experiments shown in Fig. 3 in order to collect data for achieving sufficient statistical analysis power. N Control liver RNA was of commercial origin from individuals without known liver diseases. doi:10.1371/journal.pone.0044915.tDendritic Cells and T Cells Function Assaynatural HCV SVR [7,10], it is likely that in vivo IFN- l may be involved in anti-HCV innate immunity. Innate immunity is key to antiviral defense. Innate immune defects have been identified in cHCV, including relative deficiency of circulating plasmacytoid dendritic cells (pDCs), altered expression of pathogen-recognition receptors, and a skewed monocytes/ DC cytokine profile towards enriched production of immunoregulatory cytokines and impaired production of IF.
