Cd14 v500

Leucocytes subsets were identified using an antibody panel containing CD45‐PO (Life Technologies) for lymphocytes; CD123‐PerCPCy5.5 (BD Pharmingen), CD203c APC (Sony), HLA‐DR‐PB (Sony), and CD41‐PE‐Cy7 for basophils; and CD45‐PO (Life Technologies) and CD14‐APC‐H7 (BD Pharmingen) for monocytes. To distinguish between the three different subsets of dendritic cells (DCs), an antibody panel containing HLA‐DR‐PE‐CY7 (BioLegend), CD11c‐PB (BioLegend), and CD123 PerCP Cy5.5 (BD Pharmingen) was used for plasmacytoid dendritic cells (pDCs). For two subsets of myeloid dendritic cells (mDCs), CD14‐V500 (BD), HLA‐DR‐PE‐CY7 (BioLegend), CD1c‐APC‐Cy7 (BioLegend), and CD141‐APC (Miltenyi) were used. Leucocyte subset quantities were depicted as the percentage of cells within the total leucocyte measures/numbers.

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⁺).

Freshly isolated hHSC were stained with mAbs against CD19-APC-Cy7, CD146-FITC (BioLegend, London, UK), CD14-v500, CD45-FITC (BD Biosystems, Oxford, UK), CD68-FITC, Cytokeratin-FITC, CD3-PE-Cy7 (eBioscience, Hatfield, UK), CD56-ECD (Beckman Coulter, High Wycombe, UK), TRAIL-R1-PE, TRAIL-R2-PE, TRAIL-R3-PE and TRAIL-R4-PE (R&D Systems, Abingdon, UK) after FcR blocking reagent (Miltenyi Biotec, Surrey, UK) blocking. Appropriate isotype controls were used where necessary. NK cells were stained for CD56-ECD (Beckman Coulter, High Wycombe, UK), CD3-PE-Cy7 (eBioscience, Hatfield, UK), CD16-APC, TRAIL-PE. All cells were stained with Live/Dead® Cell viability stain (Invitrogen, California, USA). Data were acquired on BD^TM LSR II flow cytometer (Beckton Dickinson, New Jersey, USA) and analysed using FlowJo (Treestar Oregon, USA).