Cd56 clone hcd56

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.

Ex vivo expanded primary AML cells of 9 different patients were co-cultured with freshly isolated healthy donor NK cells, at an E:T ratio of 5:1 in an ex vivo long term culture system as described by Krupka and coworkers [29 (link), 54 (link)]. Antibodies were added at a final concentration of 10 nM. After 24 hours, cells were harvested, stained for CD16 (clone B73.1), CD56 (clone HCD 56), CD33 (clone WM53) and in concrete cases CD123 (clone 6H6; all antibodies from Biolegend) and analyzed by flow cytometry with a BD LSR II (Becton Dickinson, Heidelberg, Germany). The percentage of residual CD33 or CD123 positive cells in treated cultures relative to control cultures was used to determine licMAB-mediated cellular cytotoxicity. Patient characteristics are summarized in Supplementary Table S1.