Nkp30 af647

To assess degranulation, NK cells were co-incubated with opsonized target cell lines in the presence of GolgiPlug (1/1000 dilution, BD Biosciences), monensin (1/1000 dilution, Biolegend) and anti-CD107a PE mAb (clone H4A3, BD biosciences) or an isotype-matched control (PE-IgG1κ; clone MOPC-21, BD) for 4 h at 37°C. After incubation, cells were washed and stained with Zombie NIR viability dye (Biolegend), anti-CD56-AF488 mAb (clone HCD56, Biolegend), anti-CD107a-PE (clone H4A3) and anti-CD16-AF647 (clone; B.731, Biolegend) (42 (link)). To assess expression of other proteins, NK cells were co-incubated with opsonized target cell lines for 4 h, then washed and stained with Zombie NIR viability dye (Biolegend) and LFA-1-PE (Clone M24), NKp46-PercP-Cy5.5 (Clone 9E2) and NKp44-APC (Clone P44-8) or NKp30-AF647 (Clone P30-15) and NKG2D-PE (Clone 1D11, all from Biolegend). Isotype-matched control mAbs were also used accordingly (mouse IgG1 isotype control; clone MOPC-21, Biolegend). Finally, cells were fixed in 2% PFA/PBS and staining was assessed using a BD FACS LSRFortessa™ X-20 flow cytometer and analyzed by FlowJo V10 software (BD Biosciences). The gating strategy used for all flow cytometry assays is shown in Supplementary Figure 5.

All samples were stained with anti-CD56-APC, anti-CD3-FITC (Biolegend) and 7AAD (BD Biosciences). Transgene expression was detected by flow cytometry on 7AAD⁻ CD56(-APC)⁺ CD3(-PE)⁻ cells (Biolegend). For NK-cell receptor detection, samples were stained with DAPI, CD56-BV711, CD16-BV786, NKp30-AF647, NKp44-PE, NKp46-BV421 (Biolegend), NKG2D-APC (BD Biosciences), and NKG2A-PE (Miltenyi Biotec). CD3-BV650 and CD19-APC-Cy7 (Biolegend) markers were used as a gating exclusion strategy for the NK cell staining. Receptor expression was assessed on DAPI⁻ CD56(-BV711)⁺ CD3(-BV650)⁻ cells. To detect CAR-expression, cells were incubated with 2 μl Siglec2(CD22)-Fc chimera (50 mg/ml, R&D) for 30 min at 4°C, washed and stained with anti-Fc-PE (Jackson Immune).