Supernatants of NK cells (1×106/mL) incubated in the presence of 1 μg of HPV16-VLPs or with positive and negative controls were stored at −80°C and were analyzed for TNF-α and IFN-γ production in a specific capture ELISA according to the manufacturer’s instructions (BioSource, Merelbeke, Belgium). NK cells (2×105/200 μL) were incubated with 10 μg of CFSE-VLPs or LYNX-VLPs in complete RPMI for 1 h at 4°C (binding step). After washing, cells were placed at 37°C for different incubation times. For the experiments investigating the entry pathway,

different reagents (Sigma) were added 1 h before the see more incubation with labeled VLPs: 2 μM of cytochalasin D, 25 μg/mL of chlorpromazine for the clathrin-dependent pathway, 25 μg/mL of nystatin for the caveolin-dependent pathway, or 1 U/mL heparinase Kinase Inhibitor Library cell assay II. For blocking experiments with anti-CD16 mAb (BD Biosciences, 1 μg/mL), cells were incubated with this antibody for 40 min before the addition of labeled VLPs. After incubation with fluorescent VLPs, 0.5×106 cells were incubated for 1 h with Hoechst 33342 DNA stain (10 μM, Acros Organics, Geel, Belgium). After washing, cells were deposited on a polylysine-coated coverslip, fixed with PAF (4%) and cell membranes were stained with phalloidin 633 (Invitrogen) for 45 min at room temperature in the dark. Then, coverslips were fixed with 20 μL of Mowiol (Hoechst GmbH, Frankfurt, Germany). Images were acquired using an

Olympus Fluoview FV1000 confocal system (Olympus, Aartselaar, Belgium) equipped with an Olympus IX81 inverted microscope (objective UPLSAPO 60X/NA 1.35). To check VLP conformation, 10 μL of each VLP pool were deposited on copper grids coated with a carbon film (EMS, 3-oxoacyl-(acyl-carrier-protein) reductase Brussels, Belgium). Grids were stained twice with 2% uranyl acetate (Fluka, Bornem, Belgium) for 1 min and washed with filtered demineralized water. To analyze VLP entry, NK cells were incubated with VLPs as described above for 10 min to 18 h. Cells were then centrifuged, fixed at room temperature in 4% glutaraldehyde (Laborimpex, Brussels,

Belgium) and post-fixed in 1% osmium tetroxide (Laborimpex) for 1 h at 4°C. Pellets were dehydrated with ethanol solutions (VWR International, Leuven, Belgium) and embedded in Epon (Serva, Breda, The Netherlands) and propylene oxide (Laborimpex) at 60°C. Ultrathin sections were stained with uranyl acetate (Fluka) and lead citrate (Leica, Groot Bijgaarden, Belgium). Grids were examined using a transmission electron microscope EM Jeol 100 CX II (Jeol, Zaventem, Belgium). A fluid-uptake assay with FITC-dextran was performed on cells (2×105/200 μL) incubated in the presence of HPV16–VLPs (10μg/mL) or positive and negative controls in complete RPMI containing FITC-dextran (1 mg/mL, average mol wt. 20 000, Sigma) at 37°C. After incubation, cells were washed three times and fixed (PAF 1%). Cells treated with 2 μM of cytochalasin D (Sigma) for 30 min before the incubation of VLPs were also used as controls.