IFNAR-1 mRNA levels before and after treatment with IFN-alpha in PBMC from naive HCV-infected patients.

<p>Total cellular RNA was extracted and reverse-transcribed from PBMC of naive HCV-infected patients carrying different IL-28B rs12979869 genotypes CC, TT and CT before (<b>Panel A</b>) and after 3 h of exposure to 10³ IU/ml IFN-alpha (<b>Panel B</b>), then mRNA levels for IFNAR-1 were measured. Results are expressed as ratio to beta-actin (median, IQR).</p

Levels of IFNAR-1 mRNA in PBMC from the HCV-infected naïve subjects grouped according to their <i>IFNL3</i> and <i>IFNL4</i> genotype combinations.

<p>Panel A. Group 1: <i>IFNL3</i> CC and <i>IFNL4</i> TT/TT (<i>IFNL3</i> favourable, <i>IFNL4</i> favourable), n = 8; Group 2: <i>IFNL3</i> CT or TT and <i>IFNL4</i> TT/TT (<i>IFNL3</i> unfavourable and <i>IFNL4</i> favourable) n = 10; Group 3: <i>IFNL3</i> CT or TT and <i>IFNL4</i> TT/ΔG or ΔG/ΔG (<i>IFNL3</i> and <i>IFNL4</i> unfavourable) n = 14. The results are expressed as ratio to beta-actin (median, IQR). Levels of IP10 mRNA in PBMC from the various groups after 3h of exposure to 10³ IU/ml IFN-alpha2b <i>in vitro</i>, according to their <i>IFNL3</i> and <i>IFNL4</i> genotype combinations. Panel B. Group 1: <i>IFNL3</i> CC and <i>IFNL4</i> TT/TT (<i>IFNL3</i> favourable, <i>IFNL4</i> favourable) n = 6; Group 2: <i>IFNL3</i> CT or TT and <i>IFNL4</i> TT/TT (IFNL3 unfavourable and <i>IFNL4</i> favourable) n = 11; Group 3: <i>IFNL3</i> CT or TT and <i>IFNL4</i> TT/ΔG or ΔG/ΔG (<i>IFNL3</i> and <i>IFNL4</i> unfavourable) n = 11. The results are expressed as ratio to beta-actin, after subtraction of values from unexposed cultures (median, IQR). The range of IP10 mRNA levels in unexposed PBMC cultures was 0,375 to 0,967.</p

Levels of IFNAR-1 mRNA in PBMC from the HCV-infected naïve subjects after 3h of exposure to 10³ IU/ml IFN-alpha2b <i>in vitro</i>, according to their <i>IFNL3</i> and <i>IFNL4</i> genotype combinations.

<p>Group 1: <i>IFNL3</i> CC and <i>IFNL4</i> TT/TT (<i>IFNL3</i> favourable, <i>IFNL4</i> favourable) n = 6; Group 2: <i>IFNL3</i> CT or TT and <i>IFNL4</i> TT/TT (<i>IFNL3</i> unfavourable and <i>IFNL4</i> favourable) n = 11; Group 3: <i>IFNL3</i> CT or TT and <i>IFNL4</i> TT/ΔG or ΔG/ΔG (<i>IFNL3</i> and <i>IFNL4</i> unfavourable) n = 11. The results are expressed as ratio to beta-actin, after subtraction of values from unexposed cultures (median, IQR).</p

Characteristics of 32 treatment naive HCV-infected patients included in the study.

<p>Characteristics of 32 treatment naive HCV-infected patients included in the study.</p

Levels of IFNAR-1 mRNA in PBMC from HCV-infected naїve subjects carrying <i>IFNL4</i> ss469415590 TT/TT genotype vs patients carrying the ΔG allele.

<p>Results are expressed as ratio to beta-actin. The horizontal bar indicates median.</p

Time dependent induction of IFNAR-1 mRNA levels following treatment with IFN-lambda in PBMC from healthy donors.

<p>PBMC were exposed to control medium (▪) 10 ng/mL (▪) and 100 ng/mL (□) of IFN-lambda, then mRNA levels for IFNAR-1 were measured at different time points (0, 3, 6, 12 and 24 h). Results are expressed as ratio to beta-actin. Results from one representative experiment performed on PBMC from two healthy donors with IL-28B rs12979860 CC (<b>Panel A</b>) and TT (<b>Panel B</b>) genotype are shown.</p

IFN-α improves PhAg-induced IFN-γ production by Vγ9Vδ2 T-cells in HD and in non-human primates.

<p>(<b>A</b>) A quantitative analysis of IFN-γ production was performed <i>in vitro</i> by stimulating purified Vγ9Vδ2 T-cells from 6 HD by ELISA after medium (white boxes), IFN-α (grey boxes), PhAg (hatched boxes) and PhAg/IFN-α (dark grey boxes) stimulation. Statistical analysis was performed by Mann-Whitney test, **p<0.01 ***p<0.0001. (<b>B</b>) Plasma IFN-γ levels from <i>in vivo</i> PhAg (white dots, n = 4) and PhAg/IFN-α (black dots, n = 4) treated monkeys was quantified by ELISA test.</p

Chronic HCV infection induces an increase in activated/effectors Vγ9Vδ2 T-cells. (A)

<p>Representative flow cytometry panels on Vγ9Vδ2 T-cells frequency and differentiation profile are shown for one healthy donor and one HCV-infected patient. Differentiation was analyzed by monitoring CD27 and CD45RA expression. Naïve: CD45RA+CD27+; Central Memory: CD45RA-CD27+; Effector Memory: CD45RA-CD27-; Effectors: CD45RA+CD27-. (<b>B</b>) Statistical analysis of Vγ9Vδ2 T-cell differentiation profile from HD (white boxes, n = 35) and HCV (grey boxes n = 24) was performed by Mann-Whitney test. *p<0.05. (<b>C</b>) Representative flow cytometry panels on CD25 and CD69 expression on Vγ9Vδ2 T-cells are shown for one healthy donor and one HCV-infected patient. (<b>D</b>) Statistical analysis of CD25 and CD69 expression on Vγ9Vδ2 T-cells from HD (white boxes, n = 35) and HCV (grey boxes n = 24) was performed by Mann-Whitney test. *p<0.05.</p

IFN-α improves <i>in vitro</i> PhAg-induced IFN-γ production of Vγ9Vδ2 T-cells in HCV patients.

<p>(<b>A,B</b>) A quantitative analysis of IFN-γ production was performed in HD (n = 35, Panel A) and in HCV (n = 24, Panel B) by ELISA after medium (white boxes), IFN-α (grey boxes), PhAg (hatched boxes) and PhAg/IFN-α (dark grey boxes) stimulation. Statistical analysis was performed by Mann-Whitney test, **p<0.01 ***p<0.0001. (<b>C</b>) The percentage of increase in IFN-γ production after combined PhAg/IFN-α respect to single PhAg stimulation was compared between HD (white bar) and HCV patients (grey bar). Statistical analysis was performed by Mann-Whitney test, **p<0.01.</p

Chronic HCV infection induces a strong impairment in IFN-γ production. (A)

<p>A quantitative analysis of IFN-γ produced by unstimulated and PhAg-stimulated Vγ9Vδ2 T-cells from HD (n = 20, white boxes) and HCV (n = 24, grey boxes) was performed by ELISA assay. (<b>B</b>) The frequency of IFN-γ-producing Vγ9Vδ2 T-cells after PhAg stimulation was analyzed by intracellular staining and flow cytometry. Statistical analysis was performed by Mann-Whitney test, *p<0.05; ***p<0.0001. (<b>C</b>) Representative flow cytometry histograms of IFN-γ MFI (Median Fluorescence Intensity) produced by Vγ9Vδ2 T-cells after PhAg stimulation are shown for one healthy donor and one HCV-infected patient.</p