Anti cd141

When the blood volume was sufficient (for 25 children—Supplementary Table), 100 μl of additional undiluted blood was stained ex-vivo with different monoclonal antibodies in order to identify monocyte and DC subsets as described (23 (link)). Briefly, fresh whole blood was stained for 30 min at room temperature in the dark with monoclonal antibodies to the following surface markers: CD3-FITC, CD19-FITC, CD56-FITC, CD141-PE, CD123-PerCpCy5.5, HLA-DR-PE-Cy7, CD16-APC, CD45-APC-H7, CD11c-V450, and CD14-V500 (all from BD Biosciences, except anti-CD141 from Miltenyi and anti-CD45 from Biolegend). After lysis of the red blood cells with 2 ml of BD-lysing solution (BD Biosciences), data were acquired on a FACSCanto II and analyzed with the Flowjo software. The median number of acquired CD45⁺ cells was 172,000 (25th−75th percentiles = 144,000–208,000). A sequential gating strategy was applied as described (23 (link)), allowing us to identify the three subsets of monocytes (CD14⁺CD16⁻, CD14⁺CD16⁺, CD14⁻CD16⁺) and of DCs (CD123⁺CD11c⁻ plasmacytoid DC (pDC), CD123⁻CD11c⁺CD141⁻ type 1 myeloid DC (mDC) or CD123⁻CD11C⁺CD141⁺ type 2 mDC). The results of the different monocyte subsets and of the pDC and mDC subsets are expressed as percentages among CD45⁺ cells. The results of type 1 and type 2 mDC are expressed as percentages among total mDC (CD123⁻CD11c⁺).

Human monocytes or DCs (1 × 10⁶ cells/condition) were cultured for 48 h with medium alone or with the conditioned media (CM) obtained from BECs or primary cells, such as PNECs and PAECs. In some experiments, after 24 h of culture, cells were stimulated with 100 ng/mL of lipopolysaccharide (LPS, DIFCO E. coli 055:B5) and/or with 0.5 μg/mL anti-CD141 (Miltenyi Biotec, Germany). Mouse IgG1 isotype control (0.5 μg/mL) (eBioscience, Switzerland) was used as a control. To induce an inflammatory environment, monocytes were cultured for 48 h with medium alone or with BEC-CM with 1 ng/mL of the following cytokines, designated as the inflammatory cocktail: human recombinant IL-6 (Invitrogen, Reinach, Switzerland), IL-8, IL-15, IL-1β, GM-CSF (R&D System, Abingdon, UK), and TNF-α (Roche, Basel Switzerland). For the control, cells were supplemented with 100 pg/mL of IL-6 (Invitrogen, Switzerland) and 500 pg/mL IL-8 (R&D System, UK).