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 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

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

Main clinical features of Healthy Donors (HDs) and HCV patients.

<p>n.t.: not tested.</p

IFN-α and PhAg synergistically induce and stabilize IFN-γ-mRNA.

<p>(<b>A</b>) IFN-γ-mRNA levels in purified Vγ9Vδ2 T-cells were evaluated by TaqMan qRT-PCR after 18 hours of medium (white bar), IFN-α (grey bar), PhAg (hatched bar), and PhAg/IFN-α (black bar) stimulation (n = 4). Statistical analysis was performed by Mann-Whitney test. *p<0.05. (<b>B</b>) IFN-γ-mRNA stability over time was measured by adding actinomycin-D to cultures after 18 hours of stimulation with medium (black triangles), IFN-α (black squares), PhAg (white squares), PhAg/IFN-α (black circles). The number of copies of IFN-γ-mRNA was normalized in respect to β-actin. Results from one representative donor are shown. Times in brackets represent mRNA half life evaluated by linear regression.</p