Figure 1. Monocytes protected primary differentiated Oral Squamous Carcinoma Cells (OSCCs) and Oral Squamous Carcinoma Stem Cells (OSCSCs) against NK cell mediated cytotoxicity, but significantly augmented the secretion of IFN-γ in co-cultures of NK cells, monocytes and tumor cells.
OSCCs A. or OSCSCs C. at 1 × 106 cells/plate were co-cultured with and without irradiated monocytes (10 Gy) (monocytes: tumor cells ratio of 1:1) for 24–48 hours before they were removed from the plates, washed and labeled with 51Cr and used as targets in the cytotoxicity assays against NK cells. The NK cells from different donors were either left untreated or treated with anti-CD16mAb (3 μg/ml), IL-2 (1000 units/ml), or a combination of IL-2 (1000 units/ml) and anti-CD16mAb (3 μg/ml) for 24–48 hours before they were added to 51Cr labeled OSCCs or OSCSCs at different effector to target (E:T) ratios. Supernatants were removed after 4 hours of incubation and the released radioactivity counted by a γ counter. % cytotoxicity was determined at different E:T ratio, and LU30/106 cells were calculated using the inverse of the number of effectors needed to lyse 30% of the tumor cells × 100. Minimum one of twenty representative experiments is shown for each cell in this figure. *The difference between IL-2 activated NK cells with OSCCs or OSCSCs and IL-2+anti-CD16mAb treated NK cells with OSCCs or OSCSCs is significant at p < 0.05. **The difference between untreated or IL-2 treated NK cells cultured with OSCCs or OSCSCs with and without monocytes is significant at p < 0.05. 1 × 105 OSCCs B. or OSCSCs D. were co-cultured with and without irradiated monocytes at 1:1 ratio (OSCCs or OSCSCs:monocytes) for 24–48 hours before untreated or IL-2 (1000 units/ml) pre-treated or anti-CD16mAb (3 μg/ml) pre-treated, or a combination of IL-2 (1000 units/ml) and anti-CD16mAb (3 μg/ml) pre-treated NK cells at 1:1:1 ratios (NK:monocyte:tumor) were added. NK cells were pre-treated as indicated for 24–48 hours before they were added to the cultures of monocytes with tumors. After 24–48 hours of the addition of NK cells the supernatants were removed from the cultures and the levels of IFN-γ secretion were determined using a specific ELISA. Minimum one of twenty representative experiments is shown for each tumor type in this figure. *The difference between IL-2 activated NK cells incubated with OSCCs or OSCSCs and those of IL-2 treated NK cells cultured with OSCCs or OSCSCs with monocytes or IL-2+anti-CD16mAb treated NK cells cultured with and without OSCCs or OSCSCs with monocytes is significant at p < 0.05.