NK cells secrete large quantities of IFN-γ in response to Herceptin-coated human breast cancer cells and IL-12. (a) The MDA-468 and SKBR3 cell lines were cultured with purified NK cells in our in vitro coculture assay. Control wells contained tumor cells and NK cells supplemented with medium alone, Herceptin alone (Her), or rhuIL-12 alone. As a further control, some wells were treated with Herceptin F(ab′)₂ fragment prior to the addition of NK cells. Culture supernatants were harvested after 72 hours and analyzed for IFN-γ content by ELISA. *P < 0.01 versus medium, F(ab′)₂, Herceptin, IL-12, Her + IL-12 (MDA-468), and F(ab′)₂ + IL-12 (MDA-468 and SKBR3). (b) The SKBR3 (HER2 high-level expression) and HT-1080 (HER2 low-level expression) human breast cancer cell lines were used in our in vitro coculture assay. Supernatants were harvested at the indicated times (12–72 hours). **P < 0.05 versus medium, IgG, Herceptin, IL-12 (HT-1080 and SKBR3), and Her + IL-12 (HT-1080). (c) NK cells cultured in our in vitro coculture assay were harvested after 72 hours and processed for real-time PCR analysis of the indicated transcript (see Methods). Results are given as fold increase in cytokine transcript over baseline versus the SKBR3 cell line. ***P < 0.05 versus medium, Her, and IL-12.