Anti dnam 1

Freshly isolated PBMCs were stained with antibodies specific to the cell surface markers of T cells, B cells, and the NK lymphocyte lineage, including anti-CD3, CD16, anti-CD19, CD56, CD4, CD8, γδ TCR (BD Biosciences, San Jose, CA, USA), anti-NKG2D, and anti-DNAM-1 (R&D Systems, Minneapolis, MN, USA). The cells were also stained with NIR zombie dye (BioLegend, San Diego, CA, USA) to exclude dead cells from the analysis. The proportion of circulating regulatory T cells (cTreg) was assessed in cryopreserved PBMCs. The stained cells were analyzed using a BD Canto instrument (BD Biosciences), and the data were analyzed using the FlowJo software package (version 10.1, Tree Star, Ashland, OR, USA).

Target cells were loaded for 1 h with 50 μCi of ⁵¹Cr (PerkinElmer, Massachusetts), washed twice and incubated with fresh or activated NK cells at different E:T ratios (1:1, 3:1 and 10:1) in 200 μL of complete RPMI in 96-well U-bottom tissue culture plates at 37 °C in a 5 % CO₂.
After a 4-h incubation period, the supernatants were harvested and counted for released radioactivity in a gamma counter (CRC-55tW Capintec), within a ⁵¹Cr sensitivity energy window (300–400 keV). The specific lysis of target cells was calculated as follows: Percentage of specific lysis = (experimental release – spontaneous release)/(maximum release – spontaneous release) × 100. Spontaneous release was calculated from target cells without effector cells. Maximum release was determined by incubating target cells with 4 % SDS detergent. In all experiments, the spontaneous release was < 20 % of maximum release.
For NK cells blocking receptor experiments, activated NK cells were pre-incubated with 10 μg/mL of anti-NKG2D (clone 149810, R&D Systems), 10 μg/mL of anti-DNAM-1 (clone 102511, R&D Systems), and 0.5 μg/mL of anti-FasL (clone ZB4, Merck Millipore, Germany), individually or in combination, before co-culture with tumor target cells.