Cd16 apc

One-step staining of cell-surface antigens was performed using fluorochrome-conjugated primary antibodies as previously described (10 (link), 18 (link), 33 (link)). For the analysis of blood NK cells two antibody panels were constructed around CD56-FITC, CD16-APC, and CD3-PE-Cy7 (BD Bioscience, San Jose, CA) antibodies. NK cell activation receptors were evaluated with CD69-BV421, NKp30-BV711 (BD Bioscience), and NKp44-PerCP eFluor 710 (eBioscience, San Diego, CA) antibodies. Chemokine expression levels were tested using CXCR1-PE (R&D Systems, Minneapolis, MN), CXCR3-BV421, and CCR7-BV510 (BD Bioscience) antibodies. For the TINK analysis, Zombie NIR (BioLegend), CD45 BUV395, CD56 BV510, CD16 BUV737 (BD Bioscience), and CD3 PE-Vio770 (Miltenyi Biotec) antibodies were used. Suitable IgG controls were acquired from the same vendors. FACS analyses were performed using the BD LSRFortessa™ cell analyzer, and analyzed using FlowJo v10 (FlowJo, LLC; Ashland, OR) software.

Leukocyte Characterization: Peripheral blood mononuclear cells (PBMC) were prepared on a Ficoll gradient and the pellet was re-suspended with a dilution of 1 × 106 cells/ml in Fluorescence-activated cell sorting (FACS) buffer. Then, 100 μl of suspension were distributed in four tubes:

Tube 1: not stained, 100 μl PBMC suspension;

Tube 2: for CD3 assessment: 100 μl PBMC suspension, monoclonal antibodies (3 μl CD3 FITC—BD Bioscience 561806 + 40 μl FACS buffer);

Tube 3: for T and B cell characterization, 100 μl PBMC suspension, monoclonal antibodies (3 μl CD3 FITC + 3 μl CD4 PE—BD Bioscience 555347 + 2 μl CD8 PE-Cy7—BD Bioscience 557746 + 3 μl CD19 PE-Cy5—BD Bioscience 555414);

Tube 4: for monocyte (M1 and M2) and natural killer (NK) characterization, 3 μl CD3 FITC + 2 μl CD56 PE-Cy7—BD Bioscience 557747 + 3 μl CD16APC—BD Bioscience 561248 + 2 μl CD14APC-Cy7—BD Bioscience 561384 + 3 μl HLA-Dr PerCP—BD Bioscience 347402).

Incubation for staining was performed at 4°C for 30 min and the tubes were washed with 2 ml FACS buffer, spinned at 1,500 rpm × 5 min, dechanted, and re-suspended in 500 μl of FACS buffer.
The tubes were analyzed with FACSCanto II (BD, USA) and with BD FACSDiva v8.1. The gating strategy for the different subpopulations of leukocytes analyzed by flow cytometry is shown in Supplementary Figure 1.

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⁺).

To evaluate the leukocyte populations, 5 × 10⁵ cells of the thawed PBMCs were seeded in 96-well plates (Additional file 1: Figure S1) and stained with a panel of humans antibodies CD4-PE (Clone, RPA-T4 BD bioscience), CD8-BB515 (Clone, RPA-T8 BD bioscience), CD3-PeCy7 (Clone, SK7 BD bioscience), CD14-BV510 (Clone, M0P9 BD bioscience), CD16-APC (Clone, 3G8 BD bioscience) and CD56-BUV-395 (Clone, NCAM16.2 BD bioscience), according to manufacturer instructions. Cells were resuspended in 100 ul of PBS 1%FBS containing the mix of antibodies and incubated at 4°C for 30 min. After incubation, cells were washed twice with PBS and centrifuged at 400 g for 5 min at 4°C. Then, the cell pellet was resuspended in 200 ul of PBS 1%FBS. Samples were acquired at the LSRFortessa X20 flow cytometer (BD Biosciences) and analyzed using FlowJo v10.0.7 software (BD Biosciences). Gating strategy used to identify lymphoid and myeloid subsets is showed in additional file 1: Figure S1.

The following antibodies were used for flow cytometry (all anti-human): CD16-PE (BD Biosciences, 560995, clone 3G8), CD16-APC (BD Biosciences, 561248, clone 3G8) NKG2D-PE (BD Biosciences, 557940, clone 1D11), NKp44-PE (BD Biosciences, 558563, clone p44-8), NKp46-PE (BD Biosciences, 557991, clone 9E2), TRAIL-PE (BD Biosciences, 565499, clone YM366), FAS ligand-PE (BD Biosciences, 56426, clone NOK-1), NKG2A-PE (Beckman Coulter, IM3291U, clone Z199), CD158a,h (KIR2DL1, KIR2DS1)-PE (Beckman Coulter, A09778, clone EB6B), CD158b1/b2,j (KIR2DL2, KIR2DL3, KIR2DS2)-PE (Beckman Coulter, IM2278U, clone GL183), CD158e1(KIR3DL1)-BV421 (BioLegend, 312713, clone DX9), CD158a(KIR2DL1)-APC (Miltenyi, 130-120-584, clone REA284), CD158b1/b2,j (KIR2DL2, KIR2DL3, KIR2DS2)-PE-Cy5.5 (Beckman Coulter, A66900, clone GL183), CD158a,h(KIR2DL, KIR2DS1)-PE-Cy7 (Beckman Coulter, A66899, clone EB6B), CD158b2(KIR2DL3)-PE, (R&D systems, FAB2014P, clone 180701), CD155-PE (BioLegend, 337609, Clone SKII.4), HLA E-PE (BioLegend, 342603, Clone 3D12) MICA/MICB-PE (BD Biosciences, 558352, Clone 6D4), CD112-PE (BD Biosciences, 551057, Clone R2.525), ULBP2/5/6-PE (R&D Systems, FAB1298P, Clone 16590).