CD137 ligation on NK cells results in enhanced NKG2D expression that is involved in tumor cell killing. (A) NK cells purified from PBMCs of 11 individual donors were cocultured in the presence of expanded γδ T lymphocytes (4:1 ratio) on plates precoated with hIgG1. After 48 hours of culture, the expression of NKG2D was analyzed on NK cells. Dots represent individual values of NKG2D expression on gated NK cells. Horizontal lines represent average values of NKG2D expression in indicated groups. (B) Cytotoxic activity of NK cells purified after 48 hours of culture with in vitro expanded γδ T lymphocytes was measured in a standard 4-hour ⁵¹Cr-release assay against TU167 squamous cell carcinoma of the head and neck (SCCHN). Blocking anti-NKG2D antibodies or isotype control IgG were added into the wells containing purified NK cells and TU167 targets for the duration of the cytotoxicity test. Data are presented as mean ± SD of triplicate samples and are representative of 2 independent experiments. *P < .05 compared with isotype control. (C) CD137Ig (10 μg/mL) was added to wells containing NK cells and IPP+IL-2 expanded γδ T lymphocytes. After 48 hours of culture, cells were stained with anti-NKG2D mAb. The histograms depict NKG2D expression on gated CD56⁺CD3⁻ NK cells. Numbers in brackets indicate mean fluorescent intensity of NKG2D expression. (D) Purified NK cells were cultured with irradiated mock or CD137L-transfected P815 cells (4:1) on plates precoated with hIgG1. After 48 hours the expression of NKG2D was analyzed by fluorescence-activated cell sorting (FACS). Expanded γδ T lymphocytes were used as a positive control for NK-cell activation. Numbers in brackets indicate mean fluorescent intensity of NKG2D expression.