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. 2017 Dec 8;7:17230. doi: 10.1038/s41598-017-17442-7

Figure 6.

Figure 6

Cytotoxicity of human PB-NK in NOG-IL-15 Tg mice. (a) In vitro CTL assay. Hu-PB-NK cells from NOG-IL-15 Tg or NOG-IL-2/IL-15 double Tg mice were purified at 4 or 6 weeks post-transfer and cultured for 48 h in the presence of the indicated cytokines. Freshly isolated hu-PB-NK cells were used as a control. The activated NK cells were co-cultured with K562 as a target at a 10:1 ratio for 4 h. The amount of lactate dehydrogenase (LDH) released in the supernatants was measured and CTL activity was calculated using the following formula: Cytotoxicity(%) = [(NK + K562 co-culture)OD490 − (K562 culture) OD490]/[(K562 Triton-X100-lysed) OD490 − (K562 culture) OD490]. The fresh PB-NK cells and in vivo-expanded NK cells in NOG-IL-15 Tg mice were derived from different donors. Averages from triplicate wells were used for the calculation. A representative result from two independent experiments is shown. The p-value was obtained using Student’s t-test (*p < 0.05). (b) Production of IFN-γ by NK cells. The supernatants from the 48 h in vitro culture after cytokine stimulation, which were described in (a), were used for quantitation of IFN-γ by ELISA. Averages from triplicate wells were indicated. A representative result from two independent experiments is shown. The p-value was obtained using Student’s t-test (*p < 0.05). N.D. means not detectable. (c) Suppression of K562 by hu-PB-NK cells in NOG-IL-15 Tg mice. K562 was subcutaneously inoculated into NOG-IL-15 Tg mice (n = 4) at 3 weeks after hu-PB-NK-cell transfer. Tumor growth was monitored periodically. NOG-IL-15 Tg mice without hu-PB-NK inoculation (n = 4) were used as controls. The results from two independent experiments are shown. *Indicates statisitical significance and the p-value was obtained using Student’s t-test. (d) Enhanced suppression of tumor growth in NOG-IL-2/IL-15 double Tg mice. Tumor growth was compared between NOG-IL-15 Tg (n = 5) and NOG-IL-2/IL-15 double Tg mice (n = 5). The experiments were conducted as described in (c). The results from two independent experiments are shown. The p-value was obtained by Student’s t-test. (e) Engraftment of hu-PB-NK cells in IL-2/IL-15 double Tg mice. PB was collected from mice used in (d) at 2 and 6 weeks post-NK cell transfer and analyzed by FACS. The p-value was obtained using Student’s t-test.