CSA-exposed NK cells do not show Ca²⁺ oscillations, NFAT dephosphorylation, and/or nuclear translocation following engagement with K562 cells. (A) NK cells were cultured with IL-2 (100 U/mL) and IL-15 (10 ng/mL) with and without CSA for 7 days and used to assess dynamic intracellular calcium changes in individual cells (described in “Ca²⁺ imaging, Materials and methods”). Following the addition of K562 target cells, vehicle-treated (EtOH) NK cultures showed the expected calcium oscillations. In contrast, CSA-treated NK cells showed no oscillations. Results are representative of cells from 5 individual donors. Error bars are plus or minus standard error. (B) The addition of K562 cells leads to rapid NFAT dephosphorylation in control (EtOH)-treated cells, but not in CSA-exposed NK cells. Freshly isolated NK cells were cultured for 7 days with IL-2 (100 U/mL) and IL-15 (10 ng/mL), with and without CSA. Following this, NK cells were cocultured with K562 target cells (E/T = 1:1) for 5 and 10 minutes. Controls included K562 cells alone (left) and NK cells without the addition of K562 (time 0). Immediately at the time points stated (5 and 10 minutes), cells were lysed and subjected to Western blotting. Actin served as the loading control. Results are representative of cells from 3 individual donors. (C,D) Multispectral imaging demonstrating that NFAT nuclear translocation is inhibited following K562 engagement in CSA-treated NK cells, but not vehicle-treated control cultures. (C) Individual images of vehicle (EtOH) control-treated (top) and CSA-treated (bottom) cells showing nuclear NFAT translocation and nontranslocation after coculture with K562 cells (10 minutes), respectively. Shown are bright field, FL-1, FL-4, FL-2, and composite images from left to right. (D) NFAT versus 7-AAD similarity score for vehicle control–treated (top row) and CSA-treated (bottom row) NK cultures both at baseline (left columns) and following coculture with K562 cells (right columns) after 10 minutes. Gates identify cells that have not (left) and have (right) undergone NFAT translocation. Results are representative of 2 experiments on approximately 2500 events for each condition.