Figure 3. Blocking IL-10 in NK92 cells did not increase cytotoxicity or IFN-γ secretion.
One million purified primary NK cells or NK92 parental cells were left untreated or treated with IL-2 (1000 units/ml) or the combination of IL-2 (1000 units/ml) and anti-CD16mAb (3 ug/ml) in the presence and absence of anti-IL-10mAb (10 μg/ml) and anti-IL-10RmAb (5 μg/ml). After an overnight incubation, treated primary human NK cells and NK92 cells were added to 51Cr labeled OSCSCs at different E:T ratios. NK cell cytotoxicity was determined using a standard 4 hour 51Cr release assay, and the lytic units 30/106 cells were determined using inverse number of NK cells required to lyse 30% of the target cells × 100. Minimum one of five representative experiments is shown in this figure. *The differences between IL-2 or IL-2+anti-CD16mAb treated primary NK cells and those mediated by IL-2 or IL-2+anti-CD16mAb treated NK92 cells is significant at p < 0.05. No significant differences could be obtained between untreated, IL-2 and/or anti-CD16mAb treated NK92 cells in the presence and absence of anti-IL-10mAb and anti-IL-10RmAbs A. Primary NK cells (1×105/ml) and NK92 (1×105/ml) were treated as described in Figure 3A. Afterwards, the supernatants from each of the NK cell samples or NK92 cells were removed and the levels of IFN-γ B. and IL-10 C. were determined using specific ELISAs. *The differences between IL-2+anti-CD16mAb treated primary NK cells and those mediated by IL-2+anti-CD16mAb treated NK92 cells is significant at p < 0.05 for IFN-γ, and the differences between untreated, IL-2 treated and IL-2+anti-CD16mAb treated primary NK cells and untreated, IL-2 treated and IL-2+anti-CD16mAb NK92 cells is significant at p < 0.05 for IL-10 secretion. No significant differences could be obtained for NK92 cells treated with isotype control antibody or anti-IL-10mAb and anti-IL-10RmAbs for IFN-γ secretion, whereas **p < 0.05 was obtained for differences of untreated, IL-2 treated and IL-2+anti-CD16mAb treated NK92 cells between isotype control treated and those of anti-IL-10mAb and anti-IL-10RmAbs for IL-10 secretion. Minimum one of five representative experiments is shown. Untreated, and IL-2 (1000 units/mL) +anti-CD16mAb (3 μg/ml) treated primary NK cells (1×105/ml) were co-cultured with and without monocytes and sAJ2 at (1:1:2, NK:monocyte:bacteria) in the presence or absence of anti-IL-10mAb (10 μg/ml) for 12–18 hours before the supernatants were collected and the level of IFN-γ secretion were determined using IFN-γ ELISA. Minimum one of three representative experiments is shown. *The difference between NK cells cultured with sAJ2+monocytes or IL-2+anti-CD16mAb+sAJ2+monocytes and those treated with either sAJ2+monocytes+anti-IL10 mAb or IL-2+anti-CD16mAb+sAJ2+monocytes+anti-IL-10 mAb are significant at p < 0.05 D. NK cells were treated and co-cultured with and without monocytes and sAJ2 as described in Figure 3D in the presence or absence of anti-IL-10mAb (10 μg/ml) for 12–18 hours before the supernatants were collected and the level of IL-10 secretion was determined using specific ELISA. Minimum one of three representative experiments is shown in this figure. *The difference between NK cells cultured with sAJ2+monocytes or IL-2+anti-CD16mAb+sAJ2+monocytes and those treated with either sAJ2+monocytes+anti-IL10 mAb or IL-2+anti-CD16mAb+sAJ2+monocytes+anti-IL-10 mAb are significant at p < 0.05 E. Primary NK cells (1×105/ml) and NK92 cells (1×105/ml) were left untreated or treated with IL-2 (1000 units/ml), or IL-2 (1000 units/ml) +anti-CD16mAb (3 μg/ml) with or without sAJ2 (1:2; NK:bacteria) and the secretion of IFN-γ was determined using specific ELISA. Minimum one of three representative experiments is shown. *The differences between IL-2 or IL-2+anti-CD16mAb with or without sAJ2 is significant at p < 0.05. **the difference between primary NK cells treated with IL-2 and/or anti-CD16mAb+sAJ2 and NK92 cells treated with IL-2 and/or anti-CD16mAb+sAJ2 are significant at p < 0.05 F.