Anti nkp30

All antibodies were purchased from BD Biosciences (San Jose, CA, USA) with the exception of phycoerythrin (PE)-conjugated anti-CD158a, anti-CD158b, anti-NKG2A, anti-NKG2D, anti-NKp30, anti-NKp44, anti-NKp46 and anti-TRAIL antibodies, which were purchased from R&D Systems (Minneapolis, MN, USA). NK subset frequencies and NK receptor expressions were analysed according to previously described protocols26 (link)50 (link). To detect NK cell activation, PBMCs were incubated with PE-conjugated anti-CD3, APC-conjugated anti-CD56 and PerCP-conjugated anti-HLA-DR, or FITC-conjugated anti-CD69 and anti-CD38. For intracellular staining, cells were permeabilised and stained with the corresponding intracellular antibodies, including those recognising perforin, granzyme A and B. Cells were then analysed using FACSCaliber or FACSCanto II and Flowjo software (TreeStar, Ashand, OR, USA).

Natural killer cell-mediated lysis of DLD-1 cells was assessed by xCELLigence System (Roche), a label-free real-time monitoring assay of adherent cell lysis (26 (link)). It measures electrical impedance across interdigitated micro-electrodes integrated on the bottom of tissue culture E-Plates. The electrode impedance, which is recorded as a dimensionless parameter denominated cell index (CI) value, provides quantitative information about the status of the adherent cells, including cell number, viability, and morphology. DLD-1 targets (15,000 cells) were seeded into the wells of 96 E-Plates in 100 μl of RPMI 1640 medium plus 10% human serum and their adhesion was monitored for 5 h. IL-2-activated purified NK cells were added in a volume of 50 μl/well at 1:1 E:T ratio. Cocultures were assessed by the system with a measurement of CI every 15 min for up to 300 min. Results expressed as CI, were normalized with RTCA Software (Roche, Applied Science, Mannheim, Germany) (nCI) and are expressed as % specific lysis:% lysis = (nCI (no effector) − nCI (effector))/nCi (no effector) × 100. In some cocultures, NK cells were pretreated with anti-NKp46, anti-NKp30, anti-NKG2D, or anti-DNAM-1 mAbs (R&D System).