Rhil 3

Human peripheral blood mononuclear cells (PBMCs) from a healthy donor were isolated from whole blood by Ficoll- Hypaque density centrifugation (Amersham-Pharmacia- Biotech). The cells were counted and plated at 2 × 10⁶ cells/well in 96-well V-bottom plates (Thermo-Fisher-Scientific), in 100 μL of RPMI (GibcoBRL, Invitrogen) supplemented with 10% fetal bovine serum (GibcoBRL, Invitrogen), or 100 μL of RPMI supplemented with 1:10 serafrom patients or controls. PBMCs were left unstimulated or were stimulated with 10 ng/μL of rhIL-3 or GM-CSF or 50 ng/μL of rhIL-3 (Miltenyi-Biotec) for 15 min at 37°C. Thereafter, cells were fixed and permeabilized with a fixation/permeabilization kit (eBioscience). Extracellular labeling was performed with antibodies anti CD14-Pacific Blue and anti CD4-FITC (Sony-Biotechnology, clones M5E2 and RPA-T4, respectively). Cell viability was determined with the Aqua Dead Cell Stain Kit (Thermo-Fisher-Scientific). STAT5 phosphorylation (p-STAT5) levels were assessed by intracellular staining with Phospho-Flow PE Mouse Anti-p-STAT5 (pY694) antibody (BD Biosciences). Data were collected with a Gallios flow cytometer (Beckman-Coulter) and analyzed with FlowJo software v.10.6.2 (Becton–Dickinson).

On day 4 of differentiation, EBs were harvested and transferred to factory media, consisting of X-VIVO 15 media (Lonza) supplemented with 2 mM GlutaMAX (Thermo Fisher Scientific), 10 U/ml Penicillin/Streptomycin (Thermo Fisher Scientific), 50 µg/ml Mercaptoethanol (Thermo Fisher Scientific), 100 ng/ml rhM-CSF (Miltenyi Biotech), and 25 ng/ml rhIL3 (Miltenyi Biotech). EBs were plated at a density of 1 EB/cm² in growth factor reduced matrigel (Corning) precoated cell culture vessels and myeloid factories were matured as described previously (25 (link)).

Monocyte subsets were induced to differentiate to one of four phenotypes following isolation: (1) mo-DCs by culturing in AIM-V medium supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gibco), 1000 U/mL recombinant human (rh) granulocyte–macrophage colony-stimulating factor (GM-CSF) (Berlex) and 1000 U/mL rhIL-4 (R&D Systems) for seven days; (2) pDCs by culturing in complete medium supplemented with 20 ng/mL rhIL-3 and 100 ng/mL rhFlt3 ligand (both from Miltenyi Biotec) for seven days; (3) classical (M1) macrophages by culturing in complete medium supplemented with 50 ng/mL rhGM-CSF for five days; or (4) alternative (M2) macrophages by culturing in complete medium supplemented with 100 ng/mL rh macrophage colony-stimulating factor (M-CSF) (Miltenyi Biotec) for five days. Differentiation studies were performed on tissue culture-treated plastic (Sigma).