Figure 1.

The combination of IL-21 supplementation at start of cultivation, IL-2 and irradiated EBV-LCL feeder cells enabled a long-lasting, pronounced expansion of NK cells. (A) NK cells were cultivated with IL-2 (black bars) or with IL-2 and 100 ng/mL IL-21 (gray bars) in the presence or absence of irradiated EBV-LCL feeder cells. The expansion of NK cells was measured after 7 d. Displayed are mean values and standard deviation of eight NK cell donors and statistical significance was tested by paired Student's t-test. (B) NK cells were labeled with CellTrace Violet Proliferation Dye and cultivated with IL-2 (dark gray histograms) or IL-2 and 100 ng/mL IL-21 (light gray histograms) in the presence or absence of irradiated EBV-LCL feeder cells. The dilution of CellTrace Violet corresponding to cell proliferation was analyzed by flow cytometry at day 3 and day 5. A representative donor out of three donors is shown. (C) NK cells were co-cultured with irradiated EBV-LCL feeder cells and IL-2 at different concentrations of IL-21. Mean NK cell fold expansion and standard deviation are displayed for three donors. (D) NK cells were co-cultured with irradiated EBV-LCL feeder cells and IL-2. IL-21 (100 ng/mL) was supplemented only at day 0 (gray dots) or permanently (gray squares), meaning at day 0, 7, 9, 11, 13, 16, 18, 20, and 26 when fresh medium was added. Mean NK cell expansion fold and standard deviation of six donors are shown at different time points. (E) NK cells were cultured with IL-2 and 100 ng/mL IL-21 was added at day 0. NK cells were co-cultured with irradiated EBV-LCL and re-stimulated with EBV-LCL at day 13, 26, and 39 (gray line and dots). In addition, the expansion without EBV-LCL restimulation at day 13, 26, or 29 was determined and monitored for 7 d (gray dashed line and triangles). Shown are mean and range of the NK cell expansion folds of six donors at different time points.