Cd16 clone 3g8

For baseline quantification of surface receptors, blood leukocytes were stained and analyzed using flow cytometry as previously described [16 (link)]. Whole blood samples were collected into K₂EDTA vacutainers (BD Biosciences) during the same blood draw described above. Samples were centrifuged at 400x g for 10 min at 4°C, plasma was removed, and cells were resuspended to the original volume in ice cold PBS containing 2.5 mM EDTA. Cells were kept on ice and in the dark for the remaining steps. Cells were incubated with a live/dead discriminator (Life Technologies) and an Fc-blocking reagent (TruStain FcX, BioLegend) for 15 min before an antibody cocktail was added for an additional 15 min. The antibody cocktail consisted of antibodies against CD16 (clone 3G8, BioLegend), CD14 (clone 61D3, eBioscience), CXCR2 (clone 5E8-C7-F10, eBioscience), activated CD11b (clone CBRM1/5, eBioscience), CD63 (clone H5C6, eBioscience), and CD66b (clone G10F5, eBioscience). Live, singlet cells were analyzed using a LSR Fortessa flow cytometer (BD Biosciences) and gated based on forward scatter, side scatter, and surface marker expression (neutrophils are defined as CD16^High FSC^High SSC^Mid, monocytes as CD14^High CD63^High FSC^Mid SSC^Mid, and NK cells as CD16^Mid FSC^Low SSC^Low).

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from healthy donors (Oklahoma Blood Institute). Monocytes were enriched from PBMCs using magnetic bead separation (Miltenyi Biotech). Purity was >90% by flow cytometry and viability was >99% by trypan blue exclusion post enrichment. Monocytes were stimulated with 10 ng/ml recombinant hIL-10 (Peprotech) or recombinant vIL-10 (R&D systems) for indicated times. STAT3 phosphorylation was detected by flow cytometry using antibodies directed against phospho-STAT3 Y705 (BD Biosciences). To inhibit signaling through IL-10R, monocytes were stimulated with hIL-10 or vIL-10 in the presence or absence of a neutralizing antibody to IL-10R1 (Clone: 37607, R&D systems), and STAT phosphorylation was measured as above. To differentiate monocytes into macrophages, cells were cultured with 50 ng/ml M-CSF (R&D systems). On day 6 cells were additionally stimulated with IFNγ (20 ng/ml, Peprotech), IL4 (20 ng/ml, Peprotech), hIL-10 (10 ng/ml), or vIL-10 (10 ng/ml) for 24 h. Surface markers were stained using following antibodies: CD14 Clone M5E2, CD163 Clone GHI/61, CD32 Clone FLI8.26 (BD Biosciences), CD16 Clone 3G8, CD64 Clone 10.1, HLA-DR Clone LN3, CD86 Clone IT2.2 (BioLegend). Cells were acquired on BD LSR II or BD Celesta (BD Biosciences) and data were analyzed using FlowJo (TreeStar, v10).

Flow cytometric data where acquired using a CytoFLEX (Beckman Coulter). Antibodies from Thermo Fisher Scientific were anti-HLA-E (clone 3D12; Thermo Fisher). Antibodies from BioLegend were: anti-pan-HLA class I (clone W6/32), CD3 (clone HIT3a), CD4 (clone RPA-T4), CD8 (clone SK1), CD56 (clone 5.1H11), CD16 (clone 3G8), CD57 (clone HNK-1), KIR2DL2/3 (CD158b, clone DX27), KIR2DL1/S1/3/5 (CD158a,h, clone HP-MA4), CD107a (clone H4A3), CD137 (clone 4B4-1), anti-NKG2D (CD314, clone 1D11), anti-NKp30 (clone P30-15), anti-NKp44 (clone P44-8), anti-NKp46 (clone 9E2). Antibodies from Miltenyi Biotec were anti-NKG2A (CD159a, clone REA110), anti-NKG2C (CD159c, clone REA205). Antibody stainings were performed in PBS (Thermo Fisher Scientific) supplemented with 0.5% BSA (Sigma-Aldrich) and 2 mM EDTA (Sigma-Aldrich), termed MACS buffer. Of note, the clone W6/32 does not recognize the sc-HLA-E due to the covalently linked N-terminus of the incorporated B2M (79 (link)), enabling discrimination between endogenous HLA class I and the sc-HLA-E. Analysis was performed using the CytExpert software (Beckman Coulter) and FlowJo V10.6.2 (Becton Dickinson).

To observe erythroid differentiation in human tissues, staining was performed with a combination of CD117/c-Kit (clone YB5.B8; eBioscience), CD34 (clone 581; BioLegend), CD38 (clone HIT2; BioLegend), CD36 (clone 5-271; BioLegend), CD71 (clone OKT9; eBioscience), and CD235a (clone HIR2; eBioscience). To observe monocytic and granulocytic differentiation, antibodies against CD117/c-Kit, CD34, CD38, CD15 (clone HI98; eBioscience), CD14 (clone HCD14; BioLegend), and CD16 (clone 3G8; BioLegend) were used.

Whole blood samples from COVID-19 patients were diluted with Tyrode’s/HEPES buffer at 1:5. Cells were stained with anti–human CD66b (clone G10F5, BioLegend), CD16 (clone 3G8, BioLegend), and CD40 (clone 5C3, BioLegend); platelet marker anti–human CD41 (clone HIP8, BioLegend); and platelet activation marker anti–human CD62P (clone AK4, BioLegend) for 10 minutes at RT in the dark. Cells were fixed with 1% paraformaldehyde for 10 minutes and then acquired by FACSCanto.

Primary human NK cells were treated and stimulated as described and incubated for 5 days. Stimulated cells were washed and resuspended in flow buffer (0.5% fetal bovine serum/PBS) before cell surface staining was performed at 4°C, with fluorophore-conjugated antibodies against the following proteins: CD56 (clone HCD56, Biolegend), CD16 (clone 3G8, Biolegend), NKG2D (clone 1D11, BD Biosciences), CD57 (clone HNK-1, Biolegend), NKG2A (clone 131411, R&D Systems), CD94 (clone DX22, Biolegend), and DNAM1 (clone TX25, Biolegend). Isotype-matched control antibodies conjugated with the appropriate fluorophores were used in parallel (mouse IgG1, κ isotype control, clone MOPC-21, Biolegend; mouse IgM, κ isotype control, clone MM-30, Biolegend; mouse IgG1, κ isotype control, clone MOPC-21, BD Biosciences; mouse IgG2A isotype control, clone 20102, R&D Systems). Staining was performed with appropriate combination of fluorophores. Cell viability was assessed using a Live/Dead stain (Zombie NIR™ Fixable Viability kit, Biolegend). Cells were then washed and resuspended in flow buffer before fixation with 2% paraformaldehyde/0.25% fetal bovine serum/PBS at 4°C. Cells were analyzed by flow cytometry, and data analysis was performed using FlowJo_v10 software (Tree Star). Cell debris was excluded, and cells were gated on live single CD56⁺ pNK cells.