Cell lines

NK-92 cells (ATCC, Manassas, VA) were cultured in X-VIVO 10 (Lonza, Basel, Switzerland) supplemented with 5% FBS (WiCell, Madison, WI), 5% horse serum (Thermo Fisher, Waltham, MA). 

Nucleofection

NK cells were nucleofected on day 4, unless otherwise described for optimization experiments. Post-nucleofection recovery medium consisting of complete NK MACS medium (Miltenyi Biotec, Gaithersburg, MD) with 100 IU/mL IL-2 and 2 ng/mL IL-15 ± 0.6 µM M3814 (Selleck Chemicals, Houston, TX) was prepared, with M3814 added at 1.2X the working concentration to account for future dilution. For M3814-related optimization experiments, multiple aliquots of post-recovery medium were prepared with a corresponding concentration of M3814. Next, 150 µL of M3814 recovery medium was plated into individual wells of a 96-well round bottom plate for each replicate and set aside in the incubator. PB-NK cells were harvested and counted using the Countess II FL Automated Cell Counter (ThermoFisher, Waltham, MA) with 0.4% Trypan Blue viability stain (Thermo Fisher, Waltham, MA). 5e5 cells were aliquoted into individual 1.5-mL Eppendorf tubes for each nucleofection replicate. While the RNP complexes incubated, the cells were pelleted at 100 x g for 10 min, and 3 µg (1.5 µL) of dsDNA templates were aliquoted into PCR tubes for each replicate. Following incubation, the
RNP complexes were added to the dsDNA templates and were allowed to incubate for 3–5 min at room temperature. Cell pellets were resuspended in P3 buffer with 22% supplement (Lonza, Basel, Switzerland), and combined with the RNP+dsDNA mixture, for a total volume of 24 µL. Samples were added to the 24 µL reaction cuvettes (Lonza, Basel, Switzerland) and were nucleofected using the EH-100 program (Lonza Amaxa 4D Nucleofector, Basel, Switzerland), unless specified otherwise for optimization experiments. Immediately after nucleofection, 100 µL of pre-warmed recovery medium (no M3814) was added to each of the cuvette wells, and samples were rested at 37°C for 15 min. Finally, samples were transferred from the cuvette to the 96-well plate containing pre-warmed recovery medium with M3814. After 24 h, nucleofected NK cells were combined with irradiated K562-mb15-41BBL (100 Gy) at a 1:2 ratio in complete NK MACS medium with 100 IU/mL IL-2 and 2 ng/mL IL-15.