Agents to eradicate HCV infection, their mechanisms of action are not fully understood. Here we report that IFN- l expression is increased in cHCV patients, both in blood and liver. Our results provide novel evidence for the immunomodulatorydoi:10.1371/journal.pone.0044915.tIL-28 and IL-29 Modulate Dendritic CellsFigure 3. IFN-l treatment-induced inhibitory DCs phenotype is dependent on IL-10 and PD-1/PD-L1 ligand. Monocyte-derived DCs from healthy individuals were generated without (control) or with IFN-l 1676428 (combination of IL-29 and IL-28A, called IFN-l-DC) for 7 days, after which they were included in the mixed lymphocyte reaction (MLR) with KS-176 biological activity allogeneic CD4+ T cells for 4 days. Co-culture supernatants from indicated groups were analyzed for IL-2 (A), IL-12 (B) by ELISA; mean6SD pg/ml shown. Neutralizing a-IL-10 or a-PD-1 antibodies, or recombinant (r) IL-2 or IL-12 were added during MLR and T cell proliferation was analyzed as described in Methods; shown mean6SD cpm from n = 5 sets of experiments (C). Entire MLR co-culture was analyzed for PD-L1(D) or PD-1 (E) mRNA. DCs were analyzed for PD-L1 expression by flow cytometry using isotype or specific fluorescent antibodies (MFI Peptide M biological activity control 4146126 
vs 622698 IFN- l DCs) (F, left). T cells from MLR co-culture cells were analyzed for PD-1 expression by flow cytometry using isotype or specific fluorescent antibodies (MFI control 125627 vs 187639 IFN- l DCs) (F, right). * indicates p,0.05 compared to controls. doi:10.1371/journal.pone.0044915.gaction of IFN- l in anti-HCV immunity and add new complexity to the understanding of the anti-HCV potential of IFN- l.We found elevated levels of serum IFN- l upon cHCV; these data are in agreement with some [19] but not other [20] recentIL-28 and IL-29 Modulate Dendritic CellsFigure 4. IFN-l-DCs promote proliferation of existing but not de novo generation of regulatory T cells. (A,B) CD4+ T cells were labeled with CFSE and cultured alone (red) or co-cultured with control (blue) or IFN-l-DCs (green); at the end of culture CD4+CD25+ cells (A) or CD4+CD252 cells (B) were analyzed for CFSE content by flow cytometry; representative histogram of n = 8 is shown. (C,D) The DC/T cells co-culture was analyzed for FoxP3 mRNA (C) using specific primers in quantitative PCR or for the frequency of FoxP3+ cells by flow cytometry (D); data are shown as fold change compared to control DC/T cells co-culture (C) or as representative flow cytometry dot blots (D). (E) T cells were included into 2 
rounds of MLR. The first MLR was set up as described in Fig. 3 with CD4+ T cells as responders and control or IFN- l-exposed DCs as stimulators. At the end of this firstIL-28 and IL-29 Modulate Dendritic CellsMLR, the T cells were purified into CD4+CD252 and CD4+CD25+ populations using magnetic beads and equal numbers of cell types were included into a second round MLR with allogeneic normal DCs at indicated DC/T cell ratio. Proliferation during the second MLR was assessed by 3H-Td incorporation during the final 16 h of the 5-day co-culture. Mean6SD cpm values from n = 8 are shown. (F) IFN- l-exposed DCs were co-cultured with CD4+ (including CD25+ and CD25- populations) or CD4+CD252 or CD4+CD25+ T cells; T cell cultures without DCs were used as controls. The cocultures were analyzed by flow cytometry for the frequency of FoxP3+ cells (F). Representative set of flow cytometry dotblots of n = 4 with similar results is shown. * indicates p,0.05 compared to controls. doi:10.1371/journal.pone.Agents to eradicate HCV infection, their mechanisms of action are not fully understood. Here we report that IFN- l expression is increased in cHCV patients, both in blood and liver. Our results provide novel evidence for the immunomodulatorydoi:10.1371/journal.pone.0044915.tIL-28 and IL-29 Modulate Dendritic CellsFigure 3. IFN-l treatment-induced inhibitory DCs phenotype is dependent on IL-10 and PD-1/PD-L1 ligand. Monocyte-derived DCs from healthy individuals were generated without (control) or with IFN-l 1676428 (combination of IL-29 and IL-28A, called IFN-l-DC) for 7 days, after which they were included in the mixed lymphocyte reaction (MLR) with allogeneic CD4+ T cells for 4 days. Co-culture supernatants from indicated groups were analyzed for IL-2 (A), IL-12 (B) by ELISA; mean6SD pg/ml shown. Neutralizing a-IL-10 or a-PD-1 antibodies, or recombinant (r) IL-2 or IL-12 were added during MLR and T cell proliferation was analyzed as described in Methods; shown mean6SD cpm from n = 5 sets of experiments (C). Entire MLR co-culture was analyzed for PD-L1(D) or PD-1 (E) mRNA. DCs were analyzed for PD-L1 expression by flow cytometry using isotype or specific fluorescent antibodies (MFI control 4146126 vs 622698 IFN- l DCs) (F, left). T cells from MLR co-culture cells were analyzed for PD-1 expression by flow cytometry using isotype or specific fluorescent antibodies (MFI control 125627 vs 187639 IFN- l DCs) (F, right). * indicates p,0.05 compared to controls. doi:10.1371/journal.pone.0044915.gaction of IFN- l in anti-HCV immunity and add new complexity to the understanding of the anti-HCV potential of IFN- l.We found elevated levels of serum IFN- l upon cHCV; these data are in agreement with some [19] but not other [20] recentIL-28 and IL-29 Modulate Dendritic CellsFigure 4. IFN-l-DCs promote proliferation of existing but not de novo generation of regulatory T cells. (A,B) CD4+ T cells were labeled with CFSE and cultured alone (red) or co-cultured with control (blue) or IFN-l-DCs (green); at the end of culture CD4+CD25+ cells (A) or CD4+CD252 cells (B) were analyzed for CFSE content by flow cytometry; representative histogram of n = 8 is shown. (C,D) The DC/T cells co-culture was analyzed for FoxP3 mRNA (C) using specific primers in quantitative PCR or for the frequency of FoxP3+ cells by flow cytometry (D); data are shown as fold change compared to control DC/T cells co-culture (C) or as representative flow cytometry dot blots (D). (E) T cells were included into 2 rounds of MLR. The first MLR was set up as described in Fig. 3 with CD4+ T cells as responders and control or IFN- l-exposed DCs as stimulators. At the end of this firstIL-28 and IL-29 Modulate Dendritic CellsMLR, the T cells were purified into CD4+CD252 and CD4+CD25+ populations using magnetic beads and equal numbers of cell types were included into a second round MLR with allogeneic normal DCs at indicated DC/T cell ratio. Proliferation during the second MLR was assessed by 3H-Td incorporation during the final 16 h of the 5-day co-culture. Mean6SD cpm values from n = 8 are shown. (F) IFN- l-exposed DCs were co-cultured with CD4+ (including CD25+ and CD25- populations) or CD4+CD252 or CD4+CD25+ T cells; T cell cultures without DCs were used as controls. The cocultures were analyzed by flow cytometry for the frequency of FoxP3+ cells (F). Representative set of flow cytometry dotblots of n = 4 with similar results is shown. * indicates p,0.05 compared to controls. doi:10.1371/journal.pone.
