Cd57 clone hnk 1

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).

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.