Figure 4.

Already three days after transfer into NSG mice, ex vivo-expanded NK cells changed their phenotype and lost their cytotoxicity and potential for degranulation, while they retained an enhanced ability to produce IFNγ and TNF-α. (A) Scheme for characterization of three types of NK cells from the same donor. First, freshly isolated, naive NK cells were analyzed (black). Second, NK cells were expanded ex vivo for 17 d by use of short-term IL-21 stimulation, IL-2 and irradiated EBV-LCL feeder cells (red). Third, NK cells were expanded in the same way for 14 d before they were transferred to tumor-bearing mice (30 × 10⁶ NK cells per mouse) (blue). Three days after the transfer, the transferred human NK cells were recovered from the mouse lungs and NK cells from 3–4 mice were pooled per donor (blue). (B) The differentially prepared NK cells, as described in A, were analyzed for the surface markers NKG2D, TRAIL, and DNAM-1 by flow cytometry. Histograms for one representative NK cell donor are shown (back) together with isotype controls (gray). (C) The flow cytometric analysis, as described in B, is applied for three different donors. For each marker the mean and standard deviation of all donors are shown for the mean fluorescence intensity (MFI) corrected by isotype subtraction (top) and the frequency of NK cells expressing the marker (bottom). (D) The differentially prepared NK cells, as described in A, were analyzed for cytotoxicity against K562 and SK-MEL-28 target cell lines at a 3:1 effector-to-target ratio in triplicates per donor. One out of two representative experiments is shown using two different NK cell donors. (E) The differentially prepared NK cells, as described in A, were analyzed for degranulation and production of IFNγ and TNF-α upon stimulation with PMA/Iono. Mean and standard deviation of three different NK cell donors are displayed. Statistical significance was tested by paired Student's t-test in all experiments.