For analysis of the expression of intracellular proteins, cells were permeabilized with the Cytofix/Cytoperm kit (BD Pharmingen), according to the manufacturers’ instructions, and incubated with Ab specific for GrzB (FITC-conjugated, BD Pharmingen), IFN-γ and Ki67 (PE conjugated, BD Pharmingen). Finally, paraformaldehyde-fixed
cells were studied by flow cytometry (FacsCanto; BD Biosciences or EPICS-XL, Beckman Coulter). Data were analyzed with FlowJo software. K562 target cells were added to the NK/APC cocultures 48 h after seeding, at an E:T ratio of 10:1. FITC-CD107a Ab (BD Pharmingen) was then added and cells were incubated for 5 h at 37°C. Monensin (Golgi-Stop, BD Pharmingen) Pim inhibitor was added for the last 4 h to prevent CD107a degradation. NK cells were then labeled with PE-Cy5-CD56 Ab (BD Pharmingen) and MΦs were excluded on the basis of CD14 staining (Beckman Coulter). Finally, the expression of CD107a by K562-stimulated NK cells was analyzed by flow cytometry. Supernatants of NK/MΦ cocultures were harvested 72-h postinfection and stored at −80°C.
Commercial ELISA kits were used for IFN-α (Bender MedSystems, Vienna, Austria) and CXCL11 (R&D Systems) detection, following the manufacturers’ instructions. NK, DCs, and MΦs were infected with LASV or MOPV at a MOI of 0.1. In coculture experiments, noninfected NK were added to LASV- or MOPV-infected APCs, at an NK-cell:APC ratio of 5:1. The culture supernatants were harvested and viral titers were
determined and expressed in focus-forming units per mL (FFU/mL) as described Selleck Ipatasertib previously [6, 8]. Twenty-four hours after infection, total RNAs was obtained from a coculture of 6 × 105 cells, using RNeasy kit® and DNA I digestion (both from Qiagen, Hilden, Phosphoglycerate kinase Germany). Reverse transcription was then carried out using SuperScript III® reverse transcriptase, RNaseOUT, first-strand buffer, DTT, oligodT, and dNTP mix (all from Invitrogen). The resulting cDNA was analyzed by real-time PCR (Taqman, Applied Biosystems, Foster Coty, USA) with Taqman Universal master mix and Taqman commercial primers and probes for IFN-γ, GrzB, FasL, and TRAIL (Applied Biosystems). The GAPDH gene was amplified in duplex, with commercial primers and probes (Applied Biosystems) for normalization of the results. Relative mRNA levels were then calculated as 2−ΔCt, Δ cycle threshold (Ct) = gene Ct − GAPDH Ct. Statistical analyses were performed with SigmaStat® software. Student’s t-tests and Mann-Whitney U-tests were carried out to analyze data from flow cytometry experiments, ELISA assays, and qRT-PCR. M. Russier held a fellowship from the Délégation Générale pour l’Armement (G. Vergnaud, the French Army). We thank C. Clegg and G. Lloyd for providing MOPV, and S. Becker for the AV strain of LASV. We also thank T. G. Ksiazek, P. E. Rollin, and P.