Figure 7.

Higher levels of NK cell-mediated ADCC was seen against OCs treated with a combination of denosumab and RANKL. Human OCs were generated as described in the Materials and Methods. OCs were left untreated or treated with denosumab (20 μg/ml) or RANKL (25 ng/ml) or a combination of denosumab (20 μg/ml) and RANKL (25 ng/ml) for 30 minutes. The unbounded antibodies were washed away, and the surface expression levels were analyzed after cells were stained with goat anti-human FITC using flow cytometry. IgG2 isotype antibodies were used as controls. One of three representative experiments is shown in the figure (A). OCs were generated as described in the Materials and Methods. Purified NK cells (1×10⁶ cells/ml) from healthy individuals were treated with IL-2 (1000 U/ml) for 18 hours and were as effectors in chromium release assay. OCs were labeled with ⁵¹Cr for an hour after which cells were washed to remove unbound ⁵¹Cr. ⁵¹Cr-labeled OCs were then left untreated or treated with denosumab (20 μg/ml) or RANKL (25 ng/ml) or a combination of denosumab (20 μg/ml) and RANKL (25 ng/ml) for 30 minutes. The unbounded antibodies were washed away, and the cytotoxicity against the OCs was determined using a standard 4-6 hour ⁵¹Cr release assay. The lytic units (LU) 30/10⁶ cells were determined using the inverse number of NK cells required to lyse 30% of OCs × 100 (n=3) (B). *(p value 0.01-0.05).