Anti cd123 apc

CD1c⁺ DCs and pDCs were incubated overnight at 37°C with different stimuli in a 96-well round-bottom plate, either separate or together at a 1:1 ratio, with each well containing equal cell numbers (50 × 10³ cells in 100 μL). After overnight culture, supernatants were taken and cells were stained with the following primary monoclonal antibodies: anti-human leukocyte antigen (HLA)-ABC-APC (BD Biosciences, 555555), anti-HLA-DR/DP/DQ-FITC (BD Biosciences, 555558), anti-CD80-PE (BD Biosciences, 557227), anti-CD83-FITC (BD Biosciences, 556910), anti-CD83-APC (BD Biosciences, 551073), anti-CD86-PE (BD Biosciences, 555658), anti-PDL-1-PE (BD Biosciences, 557924), anti-CD40-PE (Beckman Coulter, PN IM1936U), anti-CCR7-PE (Miltenyi Biotec, 130-093-621). Anti-CD11c-PE (BD Biosciences, 333149) or anti-CD123-APC (Miltenyi Biotec, 130-090-901) primary monoclonal antibodies were used to distinguish between DC subsets in co-cultures. Samples were measured on a FACSCalibur or FACSVerse (BD Biosciences) and analyzed by FlowJo software (TreeStar, Inc.). The results are depicted as geometric mean fluorescence intensity (MFI) normalized to the negative control. Supernatants were analyzed for IL-12p70 (Thermo Fisher Scientific, M122), TNF-α (eBioscience, 88-7346-88) and IFN-α (Bender Medsystems, BMS216MST) by sandwich ELISA.

Purity and phenotype of nDCs after immunomagnetic isolation were determined by flow cytometry with a FACSVerse® (BD biosciences, San Jose, CA) or MACS Quant® (Miltenyi Biotec). For this purpose, the following primary monoclonal antibodies and the appropriate isotype or fluorescence minus one controls were used: anti-CD1c-Viobright-FITC, anti-BDCA2-PE, anti-CD123-APC, anti-CD20-PE-Vio770, anti-CD45-APC-Vio770, anti-CD14-Viogreen, anti-FcεRI-BioBlue, anti-CD14-FITC, anti-CD15-PE, anti-CD56-APC, anti-CD3-VioBlue, anti-HLA-ABC-APC, anti-HLA-DR/DP/DQ-APC, anti-CCR7-APC, anti-CD80-APC, anti-CD83-APC, and anti-CD86-APC (all Miltenyi Biotec). Details are depicted in Supplementary Table 2. The purity of the nDC product was defined as the percentage of nDCs (sum of CD123⁺BDCA2⁺ pDC plus CD1c⁺CD20^- cDC2) of all viable cells in the nDC product. After 6 h of protamine/mRNA stimulation, cytokine production of nDCs was measured in the supernatant by cytometric bead array according to the manufacturer’s instruction (Miltenyi Biotec).

Peripheral Blood Mononuclear Cells (PBMCs) were separated from the blood of healthy adult donors on a Ficoll-Hypaque density gradient. pDCs were isolated from fresh PBMCs using the Human Plasmacytoid DC Negative Isolation Kit (StemCell Technologies) according to the manufacturer’s protocol. The enriched cells were assessed for more than 90% purity using the following antibodies: anti-CD123–APC, anti–BDCA-2–PE (Miltenyi Biotec) and anti-CD3-FITC (Becton Dickinson–PHArmingen). pDCs were cultured in RPMI 1640 (Invitrogen, Gaithersburg, MD, USA) containing 10% FCS and 1% penicillin-streptomycin at 37°C in a humidified 5% CO² chamber according to protocol.
CD56⁺ NK cells were isolated by negative selection from fresh PBMCs using the «EasySep NK depletion Kit» (StemCell Technologies). NK cell fraction (CD3⁻CD56⁺) was more than 95% pure, as assessed by flow cytometry (FACScalibur, BD) using FITC-conjugated anti-CD3 and APC-conjugated anti-CD56 antibodies. Contamination with myeloid cells, assessed with FITC-conjugated anti-CD14 antibodies, was consistently less than 1%. Purified NK cells were activated by a combination of PHA (10 μg/ml) (Sigma) and rhuIL-2 (10 μg/ml) (referred as aNK cells), before launching NK-DC coculture experiments.