Figure 3.

Supernatants and paraformaldehyde-fixed NK cells treated with IL-2 in combination with anti-CD16 mAb with and without monocytes increased resistance of OSCSCs and DPSCs to NK cell-mediated cytotoxicity. NK cells were purified from healthy donors and left untreated or treated with IL-2 (1000 units/ml), anti-CD16 mAb (3 μg/ml), or the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) in the presence or absence of autologous monocytes (1:1 ratio of NK:monocytes) for 24 h, after which the same amounts of supernatants from different treatments of NK cells were removed and added to OSCSCs for a period of 5 days (A) or DPSCs for a period of 7 days (B). NK supernatant-treated OSCSCs or DPSCs were then washed with 1× PBS, detached and labeled with ⁵¹Cr, and used in the cytotoxicity assay against freshly isolated NK cells. Untreated or IL-2-treated (1000 units/ml) NK cells were used to assess cytotoxicity against NK supernatant-treated target cells using a standard 4 h ⁵¹Cr release assay. Percent cytotoxicity was determined at different effector to target ratio, and the lytic units 30/10⁶ cells were determined using inverse number of NK cells required to lyse 30% of the tumor cells × 100. NK cells were purified from healthy donors and left untreated or treated with IL-2 (1000 units/ml), anti-CD16 mAb (3 μg/ml), or the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) in the presence or absence of monocytes (1:1 ratio of NK:monocytes) for 24 h. Treated NK samples were then washed and fixed with 2% paraformaldehyde for 15 min before they were either added to OSCSCs at 0.75:1 for 5 days (C) or DPSCs at 2:1 for 7 days (D). The fixed NK cells were then completely removed from OSCSCs and DPSCs and the target cell sensitivity to NK cell-mediated lysis were determined using a standard 4 h ⁵¹Cr release assay using freshly isolated and untreated or IL-2-treated (1000 units/ml) NK cells. Removal of fixed NK cells from stem cell cultures were assessed using microscopic observation. Percent cytotoxicity was determined at different effector to target ratio, and the lytic units 30/10⁶ cells were determined using inverse number of NK cells required to lyse 30% of the tumor cells × 100. One of minimum three representative experiments is shown in each of (A–D).