Article first published online: 2 MAY 2011
Copyright © 2011 American Association for the Study of Liver Diseases
Volume 53, Issue 6, pages 1846–1853, June 2011
How to Cite
Petrovic, D., Stamataki, Z., Dempsey, E., Golden-Mason, L., Freeley, M., Doherty, D., Prichard, D., Keogh, C., Conroy, J., Mitchell, S., Volkov, Y., McKeating, J. A., O'Farrelly, C., Kelleher, D. and Long, A. (2011), Hepatitis C virus targets the T cell secretory machinery as a mechanism of immune evasion. Hepatology, 53: 1846–1853. doi: 10.1002/hep.24327
Potential conflict of interest: Nothing to report.
Supported by the Health Research Board of Ireland and Programme for Research in Third Level Institutions, Ireland; the MRC; and The Wellcome Trust. Current address for Lucy Golden-Mason: Division of Gastroenterology/Hepatology, Hepatitis C Center, University of Colorado Health Sciences Center and National Jewish Hospital, Denver, CO. Current address for Catherine Keogh and Cliona O'Farrelly: School of Biochemistry and Immunology, Trinity College Dublin, Ireland.
- Issue published online: 25 MAY 2011
- Article first published online: 2 MAY 2011
- Accepted manuscript online: 30 MAR 2011 11:31AM EST
- Manuscript Accepted: 18 MAR 2011
- Manuscript Received: 10 SEP 2010
Additional Supporting Information may be found in the online version of this article.
|HEP_24327_sm_suppinfoFig1.tif||4231K||Supporting Information Figure S1. Hepatic T cell numbers in endstage liver disease. Formalin fixed paraffin embedded liver tissue sections were stained for the pan-T cell antigen CD3 using a standard avidin:biotin peroxidase technique and counted as described in the methods section. Representative staining is shown for HCV-infected (A), Primary Biliary Cirrhosis (PBC) (B) and Alcoholic Liver Disease (ALD) (C); the negative control staining is also shown (D). The vast majority of T cells are located in the portal tract area and counts are similar in portal tract (E) and in the liver parenchyma (F) irrespective of disease state|
|HEP_24327_sm_suppinfoFig2.tif||57K||Supporting Information Figure S2. A. Inhibitory effect of HCV+ serum on IL-2 secretion is dose-dependent. PBMCs from normal donors were stimulated with plate-bound α-CD3/αCD28 for 24 hours following pre-incubation in the presence of serum (1/5 or 1/10 dilution) from normal donors (NS), chronic HCV-infected patients (PCR+) or from subjects who had spontaneously resolved HCV infection (PCR-) and the production of IL-2 by T cells quantified using ELISA. B. Illustrates the mean percentage recovery of IL-2 secretion in activated PBMC in the presence of BP (black) or SCR (white) for five independent experiments. IL-2 produced by cells incubated in the absence of peptide is shown in grey|
|HEP_24327_sm_suppinfoFig3.tif||1279K||Supporting Information Figure S3. Jurkat T cells were pre-incubated with 8 μg/ml blocking peptide (BP) or scrambled control peptide (SCR) for 1 hour at 4°C, in a final volume of 25 μl, followed by E2 protein (40 μg/ml) under the same conditions. Bound E2 was detected using 1 μg/ml of rat anti-E2 (clone 9/75) followed by an anti-rat pacific blue-labelled secondary antibody (Invitrogen) and quantified by flow cytometry. Panel A and B are representative of an individual experiment, Panel C shows the normalised mean of E2 binding for 2 independent experiments. BP significantly reduced the binding of E2 to Jurkat T cells (p=0.01), this was not observed with SCR control. Median fluorescence intensity (MFI)|
|HEP_24327_sm_suppinfoFig4.tif||245K||Supporting Information Figure S4. Inhibition of IL-2 secretion by E2 is concentration dependent. PBMc (1×106/ml) were incubated with the indicated concentrations of E2 overnight and then stimulated with anti-CD3 and anti_CD28 for 18 hours. IL-2 produced was quantified using ELISA. Results are representative of 4 independent experiments|
|HEP_24327_sm_suppinfoFig5.tif||137K||Supporting Information Figure S5. CD81 is co-stimulatory for IL-2 secretion when it is cross-linked by immobilized ligand but is inhibitory when bound by a soluble ligand. (A) Jurkat T cells produce IL-2 when activated using a combination of anti-CD3/anti-CD81 antibodies. This is inhibited when cells are pre-incubated in the presence of E2 (CD81 ligand). (B) PMA and Ionomycin-stimulated IL-2 secretion in Jurkat T cells is inhibited by pre-incubation of cells with E2 or soluble anti-CD81|
|HEP_24327_sm_suppinfoFig6.tif||4614K||Supporting Information Figure S6. E2 inhibits IL-2 secretion but not production of IL-2 protein in HuT 78 cells. Cells were stimulated in the presence or absence of E2 and production of IL-2 detected in non-permeabilized or permeabilized cells. When cells were pre-treated with E2 prior to PMA stimulation, IL-2 could only be detected in the cytosol of permeabilized cells. This immunofluorescence assay was developed by incubating the cells in the presence of an anti-IL-2 capture antibody (Figure S5)|
|HEP_24327_sm_suppinfoFig7.tif||3210K||Supporting Information Figure S7. Confirmation that detection of IL-2 in non-permeabilized cells is only that which is secreted on the cell surface (secreted) by three-dimensional confocal analysis. (A-C) non-permeabilized cells; (D-F) permeabilized cells. (A) PMA stimulated HuT 78 cells stained for IL-2 (B) 3D projection of the cells indicated by arrows in respective left image (C) 3D projection of IL-2 distribution in vertical and horizontal plane as indicated by the arrows. (D) intracellular staining for IL-2 in permeabilized HuT 78 (E) 3D projection of the cells indicated by arrows in respective left image (F) 3D projection of IL-2 distribution in vertical and horizontal plane as indicated by the arrows|
|HEP_24327_sm_suppinfoFig8.tif||103K||Supporting Information Figure S8. HCV E2 inhibits T cell secretion of Interferon-γ, TNFα and IL-10 from activated PBMCs. PBMCs were activated with α-CD3/α-CD28 following pre-incubation overnight with HCV E2. Cytokines were measured using Evidence Investigator multiplex analysis (A-C). mRNA levels were quantified in PBMCs in response to activation (with or without prior exposure to E2) (D-F)|
|HEP_24327_sm_suppinfoFig9.tif||2177K||Supporting Information Figure S9. A Anti-CD81 induces the translocation of PKC? to associate with lipid raft microdomains in HuT 78 T cells. Subcellular localization of PKC? (red) and lipid raft/Ganglioside GM-1 (green) in cells treated with a control IgG antibody (left panel) and anti-CD81 (right hand panel). B Ligation of CD81 with soluble monoclonal antibody inhibits PMA-stimulated IL-2 production in the HuT 78 T cell line. Cells (1×106/ml) were incubated with anti-CD81 overnight followed by PMA for 18 hours. Il-2 was quantified using ELISA|
|HEP_24327_sm_suppinfoFig10.tif||234K||Supporting Information Figure S10. Hepatic interferon-gamma protein was quantified by ELISA. Levels of interferon-gamma in endstage HCV-infected liver are increased when compared to those detected in normal donor liver. Circles represent individual patients and horizontal lines show median values. P<0.0001|
|HEP_24327_sm_suppinfoTable1.doc||44K||Supporting Information Table 1. Laboratory details of patients whose serum was used for experiments described in Figure 1|
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