Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats or blood samples of healthy blood donors as described (22
) and used fresh unless indicated otherwise. Sera of individuals who had cleared persistent HCV genotype 2 infection were preserved at −80°C. All subjects gave written informed consent for research testing under protocols approved by Institutional Review Boards of the National Institutes of Health.
Anti-CD3-FITC(UCHT1), anti-CD3-PE-Cy5(UCHT1), anti-CD3-Alexa700(UCHT1), anti-CD14-APC(M5E2), anti-CD16-Pacific Blue(3G8), anti-CD19-PE-Cy5 (HIB19), anti-CD25-FITC(M-A251), anti-CD69-PE(FN50), anti-IFN-γ-PE-Cy7(B27) (all from BD Pharmingen, San Jose, CA), anti-CD56-PE(AF12–7H3), anti-CD56-APC(AF12–7H3) (all from Miltenyi Biotec, Berglisch Gladbach, Germany), and anti-CD14-PE-Cy5(TÜK4) (Serotec, Raleigh, NC) were used for flow cytometry. Anti-CD16(3G8) and anti-CD81(JS-81) (all from BD Pharmingen) were used for NK cell stimulation and inhibition, respectively.
HCV JFH1 strain (genotype 2a) (18
) and the chimeric H-NS2/NS3-J virus, which expressed HCV core to NS2 proteins of genotype 1a (H77) sequence and the remaining nonstructural proteins of JFH1 sequence (23
), were produced as described (24
) by transfecting Huh7.5 cells (Apath, St. Louis, MO) with linearized RNA from plasmids (provided by Dr. T. Wakita, National Institute of Infectious Diseases, Tokyo, Japan and Dr. S. Lemon, University of Texas Medical Branch, Galveston, TX, respectively). Transfected Huh7.5 cells were cultured in DMEM with 10% fetal bovine serum (FBS; US Bio-Technologies, Pottstown, PA), 10 mM HEPES, 100 IU/ml penicillin, 100 µg/ml streptomycin and 2 mM L
-glutamine (Mediatech, Herndon, VA). Cell culture supernatants at the peak of HCV production (typically days 12 through 16 after transfection) were used to infect naïve Huh7.5.1 cells (provided by Dr. F. V. Chisari, Scripps Research Institute, La Jolla, CA) in 10 cm2
culture dishes at a multiplicity of infection of 0.006. The infected Huh7.5.1 cells were transferred into T175 flasks after 1–2 days and passaged every 3.5 days. The viability of HCV-infected Huh7.5.1 cells was assessed by trypan blue staining and lactate dehydrogenase release using the CytoTox96 assay (Promega Corp., Madison, WI).
Filtered (0.45 µm) supernatant of HCV-infected Huh7.5.1 cells containing HCV was used for NK cell experiments. HCV RNA, infectious titer and HCV core protein concentration were determined exactly as previously described (24
). To determine the HCV E2 concentration cell culture supernatants were incubated with 0.1% NP-40 for 15 minutes at 4°C followed by a Galanthus nivalis
antigen (GNA) lectin capture EIA (25
). Soluble JFH1 E2 of known concentration (provided by Dr. J. McKeating, University of Birmingham, UK) was included as calibrant. In experiments reported in – and supplementary figure 1
the JFH1 infectious titer was 4 × 103
ffu/ml (5 × 107
RNA copies/ml; 0.03 nmol HCVcore/l), comparable to those in the blood of infected patients (26
). Supernatant from uninfected Huh7.5.1 cells was used as control. To achieve an even higher viral titer (), 500 ml supernatant from a separate batch of infected Huh7.5.1 cells was concentrated using a Pellicon XL filter system (Millipore, Billerica, MA) with a pressure pump. This resulted in an increase of the JFH1 infectious titer to 2.39 × 105
ffu/ml (2 × 108
HCV RNA copies/ml, 2.99 nmol HCVcore/l, 12 ng HCV E2/ml). The infectious titer of the chimeric H-NS2/NS3-J virus was 2.93 × 105
ffu/ml (2 × 108
HCV RNA copies/ml, 3.4 nmol HCVcore/l, 37 ng HCV E2/ml). Concentrated supernatant from uninfected Huh7.5.1 cells served as control.
Anti-CD16 stimulates NK cell activation and IFN-γ production, but HCV does not
HCV does not inhibit NK cell cytotoxicity but crosslinking of CD81 by antibodies does
Fig. 5: High concentrations of HCV encoding either genotype 2a or genotype 1a structural proteins does neither activate nor inhibit NK cells
Detection of Antibodies against HCV JFH1 Structural Proteins
96-well EIA plates (Dynex Technologies, Chantilly, VA) were coated with 1 µg/ml GNA lectin (Sigma, St. Louis, MO) overnight at 4°C and incubated with 1.8 × 107
RNA copies HCV/well for 2h at room temperature. Fifty microliter HCV-RNA-negative serum of individuals who had cleared HCV genotype 2 or normal healthy donors were added at a 1:20 dilution. After 1h at room temperature, plates were washed three times with PBS and the captured antibodies were detected with mouse anti-human IgG-horseradish peroxidase (Serotec) and tetramethylbenzidine super sensitive 1-C (BioFX Laboratories, Owings Mills, MD) (25
). Optical density was determined at 450 nm with a microplate reader (Bio-Rad, Hercules, CA).
Huh7.5.1 cells were incubated with 5 × 107 RNA copies/ml HCV in the presence of 20% human serum. The percentage of HCV-infected Huh7.5.1 cells was assessed by flow cytometry using an anti-HCVcore antibody and a secondary, Alexa488-conjugated anti-mouse IgG (Invitrogen). Percent neutralization was calculated as (control infectivity – experimental infectivity) / control infectivity × 100.
NK Cell Isolation
NK cells were negatively isolated from PBMCs with the NK Cell Isolation Kit and autoMACS separator (Miltenyi Biotec) after lysis of red blood cells with ACK lysing buffer (Biosource, Rockville, MD). The purity of isolated CD3−CD56+ NK cells as assessed with anti-CD3-FITC, anti-CD56-PE, and anti-CD14-APC antibodies by flow cytometry was greater than 91% in all experiments.
Stimulation and Inhibition of NK Cells
cells/ml NK cells were cultured for 18h in RPMI medium containing 100 IU/ml penicillin, 100 µg/ml streptomycin and 2 mM L
-glutamine (Mediatech) and either 10% FBS or 20% human serum (complete medium) in (i) 96-well flatbottom culture plates (Corning Inc., Corning, NY) with the indicated concentration of HCV in Huh7.5.1 supernatant or with supernatant from uninfected Huh7.5.1 cells or in (24
) 96-well flat-bottom EIA plates (Immulux HB; Dynex Technologies) that had been coated with 1 µg/ml anti-CD16 and/or 10 µg/ml anti-CD81 in carbonate buffer (pH 9.5) overnight at 4°C. Human IL-12 (1.2 ng/ml, PeproTech, Rocky Hill, NJ), which is typically secreted by monocytes and DCs, was added in all stimulation conditions. As previously described (27
) and shown in , this suboptimal concentration IL-12 alone did not induce significant activation of resting NK cells but primed them to respond to activating stimuli.
To examine possible inhibitory effects of HCV and anti-CD81 on NK cell stimulation, NK cells were incubated with the indicated concentration of HCV or plate-bound anti-CD81 2h before, at the same time or 2h after stimulation with plate-bound anti-CD16 or 50 µg/ml poly(I:C) (InvivoGen, San Diego, CA) and 1.2 ng/ml recombinant human IL-12.
Analysis of NK cell Activation and IFN-γ Production
After 18h culture supernatants were frozen at −20°C and later tested with the Human IFN-γ Quantikine EIA Kit (R&D systems, Minneapolis, MN). Cells were washed, stained with anti-CD25-FITC, anti-CD69-PE, anti-CD3-PE-Cy5, and anti-CD56-APC antibodies and analyzed on a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA) using CellQuest v3.3 (BD Biosciences) and FlowJo v6.4.7 software (Tree Star, Ashland, OR).
Analysis of NK Cell Cytotoxicity
Thawed PBMCs were incubated in complete medium at 37°C in a CO2 incubator for 8h prior to NK cell isolation. Two thousand 51Cr-labeled (GE Healthcare, Piscataway, NJ) MHC class I negative K562 cells/well (ATCC, Manassas, VA) were plated in 96-well round-bottom plates (Nunc, Roskilde, Denmark) and in parallel in 96-well round-bottom EIA plates (Immulux HB; Dynex Technologies) coated or not coated with 10 µg/ml anti-CD81. NK cells were added at effector to target (E:T) ratios of 30:1, 15:1, 7.5:1, and 3.75:1 with or without 5 × 107 RNA copies/ml HCV in complete RPMI medium supplemented with 20% human serum with or without HCV JFH1-neutralizing antibodies. Virus was incubated with serum for 30 minutes prior to the use in NK cell assays. The amount of 51Cr released in the culture supernatant was determined after 3h, 5h and 7h using a scintillation counter (PerkinElmer, Wellesley, MA). Percent specific cytotoxicity was calculated as (experimental release – spontaneous release) / (maximum release – spontaneous release), and the mean cytotoxicity of at least three cultures per E:T ratio was calculated. Spontaneous release was less than 30% of maximum release in all assays.
Analysis of NK Cells in Stimulated PBMCs
PBMCs were stimulated at 2.5 × 106 cells/ml as described for NK cells. Brefeldin A (1 µl/ml, Golgi Plug; BD Biosciences) was added after 1h. PBMCs were stained with 50 µg/ml ethidium monoazide (EMA; Invitrogen Molecular Probes), anti-CD3-Alexa700, anti-CD14-PE-Cy5, anti-CD16-Pacific Blue, anti-CD19-PE-Cy5, and anti-CD56-APC antibodies after 18h. After fixation and permeabilization with BD Cytofix/Cytoperm solution (BD Biosciences), cells were stained with anti-IFN-γ-PE-Cy7, anti-CD25-FITC, and anti-CD69-PE antibodies and analyzed on a flow cytometer (LSR II; BD Biosciences) using FACSDiva v4.1.2 (BD Biosciences) and FlowJo v6.4.7 software. NK cells were identified by gating on singlets (FSC-A vs. FSC-H plot), lymphocytes (FSC-A vs. SSC-A plot) and finally on CD3−CD56+ NK cells after exclusion of CD14+, CD19+, and EMA+ (dead) cells.
Paired Student t test analysis was performed with GraphPad Prism (San Diego, CA). A two-sided P value of less than 0.05 was considered statistically significant.