Blocking of CD137L partially inhibits γδ T lymphocyte–induced cytolytic activity of NK cells. (A) Purified NK cells were cocultured with IPP+IL-2 expanded γδT lymphocytes at a 4:1 ratio in the presence of immobilized hIgG1 (2.5 μg/mL) for 48 hours. In some groups, soluble CD137Ig fusion protein at 10 μg/mL was added to block CD137 receptor and ligand interactions. The bar diagram represents the percentage of cells expressing CD54 in gated CD3⁻CD56⁺ NK-cell population. Representative data from 1 of 4 independent experiments is shown. (B) Purified NK cells were cocultured with either mock (left histograms) or CD137L-transfected P815 (middle histograms) at a 4:1 ratio in the presence of immobilized hIgG1 (2.5 μg/mL). In some wells containing NK cells and CD137L-transfected P815 tumors, soluble CD137Ig fusion protein (10 μg/mL) was added (right histograms). After 48 hours of culture, cells were stained for CD54 and CD25. Histograms represent cells gated on CD56⁺CD3⁻ NK population. (C) Soluble CD137Ig fusion protein (10 μg/mL) was included during the culture of purified NK cells and γδ T lymphocytes (4:1 ratio) in hIgG1-precoated plates for 48 hours. Cytotoxicity of NK cells repurified after culture was analyzed in a standard 4-hour ⁵¹Cr-release assay against the TU167 SCCHN cell line. Data are presented as mean ± SD of triplicate samples and are representative of 4 independent experiments. *P < .05 compared with NK cells cultured in the presence of CD137Ig blocking. (D) NK cells were purified from PBMCs of healthy donors and cocultured with irradiated mock or CD137L-transfected P815 cells at a 4:1 ratio. Expanded γδ T lymphocytes were used as a positive control for NK-cell activation. After 48 hours of culture, NK cells were repurified and used as effectors against TU167 SSCHN target cells. Data are presented as mean ± SD of triplicate samples and representative of 3 independent experiments. *P < .05 compared with NK cells cultured with mock transfected P815.