NK cells expanded with IL-15 are resistant to cytokine withdrawal. For expansion, 0.5 × 10⁶ to 2 × 10⁶ purified NK cells were cultured at a 1:10 ratio with irradiated EBV-transformed B cells in X-VIVO 20 medium supplemented with 10% heat-inactivated human AB serum, in the presence of recombinant human IL-2 (1000 IU/mL; Proleukin) or IL-15 (61 ng/mL). Fresh media supplemented with AB serum and cytokines (500 IU/mL for IL-2 or 30.5 ng/mL for IL-15) was added on day 5 and thereafter every 3 days and cells were harvested between 11 and 14 days. Expression levels of various activation markers on NK cells (A) freshly after expansion or (B) following 48 hours of cytokine withdrawal were measured by FACS. (C) Comparison of cytolytic capacity of IL2-NK (circles, dashed line) and IL15-NK (squares, solid line) cells against K562 target cells after expansion or cytokine withdrawal (48 hours). (D) NSG mice were injected (intraperitoneally) with 1 × 10⁶ to 5 × 10⁶ DiR-labeled IL-2– or IL-15–expanded NK cells. The liver was resected 4 to 5 days after injection of NK cells. Single-cell suspension of the liver was stained with a live-dead fixable aqua dead cell stain and anti-CD56 and thereafter acquired by flow cytometry. The frequency of NK cells calculated is based on viable CD56⁺/DiR⁺ cells. Each symbol represents 1 mouse injected with IL2-NK (n = 9) or IL15-NK (n = 8). Error bars show mean and SD and P value is calculated by a t test. (E) NK cells expanded with IL-2 or IL-15 were cultured in the same or alternative cytokines for 24 hours and expression intensities of various activating markers including CD25, CD69, NKp30, and DNAM-1 were measured by FACS. Results from 4 independent expansions were summarized and presented as mean ± SD. Representative histograms were chosen based on proximity to average values. *P < .05; **P < .01; ***P < .001; Mann-Whitney nonparametric U test.