Figure 7.

Induction of split anergy mediated by LPS was observed in Human NK cells which resulted in a loss of their cytotoxic function but gained the ability to secrete high levels of IFN-γ, especially in the presence of autologous monocytes. Human NK cells were purified from healthy donors and were left untreated or treated with IL-2 (1000 U/mL), anti-CD16mAb (3 μg/mL), or the combination of IL-2 (1000 U/mL) and anti-CD16mAb (3 μg/mL) in the presence or absence of LPS (20 ng/mL) and autologous monocytes (NK cell:monocytes, 1:1) for 24–48 h. Afterward, the cytotoxicity against OSCSCs was assessed using a standard 4 h ⁵¹Chromium release assay. Percent cytotoxicity was obtained 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 (A). NK cells were prepared as described in Figure 5A. Monocytes were treated with IL-2 (1000 U/mL) and/or anti-CD16mAb (3 μg/mL) and LPS (20 ng/mL) for 24–48 h and used as controls. After the treatment period, the supernatants were removed from the co-cultures and the levels of IFN-γ cytokine were measured with specific ELISA (B). *P < 0.05 was obtained for the differences in cytotoxicity and IFN-γ secretion between NK cells cultured in media and those treated with LPS, monocytes, or the combination of LPS and monocytes. One of several representative experiments is shown in this figure.