Transduction of expanded NK cells and NK-92 cells

NK cells from healthy donors or breast cancer patients were expanded for a minimum of 2 weeks prior to use in experiments. 1 × 10⁵ expanded NK cells were replenished at a 1:1 ratio with irradiated K562 mb-IL21 feeder cells, supplemented with 100U/mL rhIL-2 and seeded in a U-bottom 96-well plate. Alternatively, 1 × 10⁵ human NK-92 cells were plated in a U-bottom 96-well plate with NK-92 media containing 100 U/mL rh-IL2. Twenty-four hours after plating, expanded NK cells and NK-92 cells were resuspended in transduction medium containing polybrene (8 μg/mL; Sigma-Aldrich Canada Co.) and 500U/mL rhIL-2 or 200 U/mL rh-IL2, respectively. Cells were transduced with thawed lentivirus at MOIs between 2 and 8. The plates were immediately centrifuged at 1000 × g for 45 min at 32 C° as a spinfection step and further incubated at 37 C°, 5% CO₂ overnight. The transduced cells were maintained in NK or NK-92 cell media containing 100 U/mL rhIL-2 at 37°C and 5% CO₂ prior to use in functional assays. For NK cell gating strategy see Figure S6.