Purified NK cells were used in subsequent experiments. NK cell cytotoxicity was determined using the calcein release assay, a fluorometric assay comparable to the chromium release assay [8, 9]. Target K562 cells were labelled with 2 μg/ml calcein-AM for 1 h at 37°C with occasional shaking. Effector cells and target cells were co-cultured at the indicated effector-to-target (E : T) ratios and incubated at 37°C for 4 h. After incubation, 100 μl of the supernatant was transferred to a new plate. The fluorescence of the samples was measured with a Spectramax Gemini EM Proteasome inhibitor Fluorescence Microplate Reader (Molecular Devices, Sunnyvale, CA, USA)

(excitation filter 485 nm, emission filter 538 nm). The percentage lysis was calculated according to the formula [(experimental release − spontaneous release)/(maximum release − spontaneous release)] × 100. To investigate the effect of STAT-3 inhibitor JSI-124 on the viability of human NK cells, 1 × 106 primary purified or expanded NK cells were seeded per well in 24-well plates. JSI-124 was added at the indicated final concentrations (0, 0·05, 0·1, 0·2 and 0·5 μM). At the 24, 48 and 72 h time-points, cells were stained with 7-AAD, then analysed by flow cytometry. Primary NK cells were EPZ 6438 purified and incubated with 20 ng/ml of IL-21 with or without 0·1 μM of JSI-124 for 24 h, and were then lysed with 50 mM Tris-Cl (pH 6·8), 100 mM dithiothreitol, 2% sodium dodecyl sulphate (SDS) and 10% glycerol. Samples were analysed

by SDS-polyacrylamide gel electrophoresis (PAGE), followed by immunoblotting using the Chemo Glow chemiluminescent substrate (Alpha Innotech, San Leandro, CA, USA) according to the manufacturer’s instructions. Results are expressed as the mean ± standard deviation.

Statistical comparison was performed by Student’s t-test. P-values of less than or equal to 0·05 were considered significant. We engineered K562 cells to express mbIL-21 and CD137L, and used these cells to expand NK cells efficiently from the peripheral blood mononuclear cells (Fig. 1). For cell engineering, CD137L and mbIL-21 sleeping beauty expression vectors were harvested as described in Materials and methods, and then transfected into K562 cells, together with the sleeping beauty transferase SB11. CD137L was first transfected, and CD137L-positive K562 cells (CD137L-K562) were sorted by the flow cytometer; mbIL-21 was transfected Y-27632 2HCl subsequently into CD137L-K562 cells, and mbIL-21-positive CD137L-K562 (mbIL-21-CD137L-K562) cells were sorted. Isolated cells were stained with CD137L and IL-21 flow cytometer antibodies. Results showed that both CD137L and IL-21 were expressed clearly on the surface of mbIL-21-CD137L-K562 cells (Supporting Fig. S1). After constructing the mbIL-21-CD137L-K562, NK cell expansion was performed as described in Materials and methods. To evaluate NK cell purity, expanded cells were stained with CD3, CD56 and CD16 antibodies. Figure 2 was a representative of six different expansions.