Antibodies and Reagents.
Antibodies used were: anti-CD3 (clones OKT-3 and TR-66; American Type Culture Collection), anti-CD16 (3G8, American Type Culture Collection), anti-CD56 (B159.5, provided by G. Trinchieri, Schering Plough Research Institute, Dardilly, France), anti-CD81 (JS-81; BD PharMingen), anti-HLA class I (W6/32; Serotec), anti–HCV-E2 (provided by D. Rosa, Chiron S.p.A., Siena, Italy), anti–IFN-γ (B133.1 and B133.5), anti–TNF-α (B145.9 and B154.7) were both provided by G. Trinchieri. The antiphosphotyrosine (4G10; Upstate Biotechnology, Inc.), anti-p42/p44 erk-2, anti-phospho p42/p44 erk-2 (Cell Signaling Technology), and anti-CD3ζ chain (6B10.2; Santa Cruz Biotechnology, Inc.) were used for immunoprecipitation and Western blot analysis.
Cell Preparation and Cultures.
PBMCs were prepared from peripheral blood by Ficoll-Paque density gradient centrifugation, followed by 1-h incubation in plastic flask to remove adherent monocytes. Cultured NK cells were prepared as described previously (12
). All cultures were collected at days 8–10. NK cells (>98% CD56+
) were purified by depletion of the magnetically labeled CD3+
cells using MACS®
Separation Columns (Miltenyi Biotec). NK and T cell clones were generated and maintained as described previously (13
). In brief, PBMCs were stained with antibodies for CD3, CD56, killer Ig-like receptor (KIR)2DL/S1 (EB6, CD158a; Immunotech), KIR2DL/S2 (GL183, CD158b; Immunotech), KIR3DL1 (NKB1, DX9; Becton Dickinson), KIR3DL2 (NKAT4, DX31, provided by L. Lanier, University of California at San Francisco, San Francisco, CA) TCR Vα24 (C15; Immunotech), and/or TCR Vβ11 (C21; Immunotech) and the desired subpopulations were single-cell sorted using a FACSVantage™ SE®
cell sorter (Becton Dickinson) in 60-well Terazaki plates (Robbins Scientific). To expand NK T cells, cells were activated with α-GalCer (100 μg/ml in 100% DMSO) at the final concentration of 30–100 ng/ml (14
Antibody Coating and Cell Stimulation.
The following purified mAb were used in stimulation experiments: anti-CD16 (3G8), anti-CD3 (TR-66), anti CD81 (JS-81), anti E2 (291A2), anti-CD56 (B159.5), anti-HLA class I (W6/32). 96-well plates (Greiner) were coated as described previously (11
). The recombinant purified HCV E2 protein (10 μg/ml in PBS) was incubated in the coated anti-E2 mAb (10 μg/ml) plates for 1 h at 37°C. Prior to the addition of cells the excess HCV-E2 was removed by washing in PBS.
Antibodies used for flow cytometry were the following: anti-CD3, anti-CD56, anti CD16, NKB1 (KIR3DL1), anti–IFN-γ, and anti–IL-4 (all Becton Dickinson). Anti-CD158a (KIR2DL/S1), anti-CD158b (KIR2DL/S2), anti-TCR Vα24 (C15), and anti-TCR Vβ11 (C21) were from Immunotech. Cells were analyzed on a FACSCalibur™ flow cytometer (Becton Dickinson) using CELLQuest™ software.
Cytokine Production Assays.
Purified NK cells were cultured in 96-well coated plates for 24 and 48 h and the supernatants were assessed by ELISA for IFN-γ and TNF-α. The capture antibodies were anti–IFN-γ mAb B133.1 and the anti–TNF-α mAb B154.9, respectively, which were immobilized on ELISA plates. Detection of each cytokine was achieved using biotinylated anti–IFN-γ mAb B133.5 and anti–TNF-α mAb B154.7, streptravidin-conjugated peroxidase (Sigma-Aldrich), and the chromogenic substrate o-phenylenediamine dihydrochloride (Sigma-Aldrich). Spectrophotometric analysis was performed at 450 nm on a Spectramax 340 spectrophotometer (Molecular Devices) using Softmax Pro software (Molecular Devices).
N-carbobenzoxy-L-thiobenzyl Ester Esterase Assay.
The amount of secreted N-carbobenzoxy-L-thiobenzyl ester (BLT)-esterase was measured using 105
purified cultured NK cells in 100 μl of complete medium in a 96-well ELISA plate coated with the indicated mAb, as described previously (15
). After 4-h incubation at 37°C, 5% CO2
, 50 μl of cell-free supernatant fluids were added to 100 μl of BLT solution (0.2 mM BLT, 0.22 mM 5,5′-dithio-bis-[2-nitrobenzoic acid]; DTNB) in PBS. BLT-esterase activity was measured by absorbance at 405 nm. Total BLT-esterase activity was determined from lysed (freeze/thaw three times) untreated NK cells.
NK cell proliferation was assessed by 3[H] thymidine incorporation using 2 × 104 cells per well in mAb-coated 96-well plates. Cultures were pulsed after 48 h with Ci/well 3[H] thymidine, incubated for an additional 12 h, harvested onto filter plates (Packard Instrument Co.), and counts were analyzed using a Top Count NXT β counter (Packard Instrument Co.).
Immunoprecipitation and Immunoblot Analysis.
Purified NK cells (107 per sample for immunoprecipitation or 106 per sample for whole-cell extracts) were incubated on ice for 5 min with anti-CD16 mAb (3G8), anti-CD81mAb (JS-81, 10 μg/ml), or anti-CD56 mAb (B159.5, 10 μg/ml). Cells were then washed and incubated with goat anti–mouse IgG F(ab)′2 fragments (Sigma-Aldrich) at 30 μg/ml at 37°C for the indicated time. Cells were lysed in buffer containing 20 mM Tris-HCl, pH 7.4, 40 mM NaCl, 5 mM EDTA, 1 mM NaF, 20 mM Na4P207, 1 mM Na3VO4, 0.1% BSA, 1 mM PMSF, 5 μg/ml aprotinin, 10 μg/ml leupeptin, and 1% Triton X-100. In some experiments, whole cell extracts were resolved by SDS-PAGE. For immunoprecipitations, cell lysates were incubated for 18 h with the appropriate Ab (4G10 for phosphotyrosines, 6B10.2 antibody for the ζ-chain) and then for 1–2 h with protein A or G –Sepharose (Upstate Biotechnology, Inc.). The immunoprecipitates were resolved by SDS-PAGE and transfered to nitrocellulose membranes (Sigma-Aldrich). Tyrosine-phosphorylated proteins were detected with the 4G10 mAb, followed by sheep anti–mouse IgG coupled to horseradish peroxidase (Amersham Pharmacia Biotech) and the Supersignal detection system (Pierce Chemical Co.). The p42/p44 and phospho p42/p44 mitogen-activated protein kinases (MAPKs) were detected using Cell Signaling Technology antibodies, followed by donkey anti–rabbit IgG coupled to horseradish peroxidase (Amersham Pharmacia Biotech).