ADAM17 inhibition increases cytokine production upon NK cell stimulation by antibody-coated target cells. Purified NK cells (n = 11) were incubated in media alone, with the HLA class I expressing B cell line Raji, or with Raji cells pre-incubated with 10 µg/mL rituximab at a 2:1 E:T ratio. (A) CD16 and CD62L were measured after the 5-hour assay. (B) Corresponding function for intracellular IFN-γ, TNF-α, CD107a expression, and direct ADCC is shown. Bars represent mean ± SEM. NK cells incubated without ADAM17 inhibitor were compared with NK cells incubated with ADAM17 inhibitor using the paired t test. Statistical significance is indicated as: *P ≤ .05; **P < .01; ***P < .001. (C) To test function after partial CD16 loss, purified NK cells (n = 4) were incubated with and without ADAM17 inhibitor, along with IL-12 and IL-18 activation overnight. NK cells were then tested against Raji cells in the presence of 10 µg/mL rituximab. Intracellular IFN-γ, TNF-α, CD107a expression, and ADCC were then measured. Bars represent mean ± SEM. (D) Purified NK cells (n = 5) pre-incubated with and without ADAM17 were cross-linked with plates adsorbed with antibodies directed to 2B4, 2B4 and NKp46, NKp46, and NKG2D overnight. CD16 expression was measured (left panel) prior to incubation with Raji cells coated with 10 μg/mL rituximab. Intracellular cytokine production (middle panels) and CD107a expression (right panel) were measured after 5 hours incubation with rituximab-coated Raji cells. Bars represent mean ± SEM. Statistical significance is indicated as: *P ≤ .05; **P < .01; ***P < .001.