Nkp46 percp cy5

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.

CD3-BV785 (clone 17A2, BioLegend #100232), NKp46-PerCP-Cy5.5 (clone 29A1.4, BioLegend #560800), Live/dead staining performed using Fixable Viability Dye 780 (eBioscience #65-0865-14).