Induction of IFN-γ production is NO independent and is partially mediated by NKG2D. (A) NK cells purified from naive mice were cultured in IL-12 in the absence or presence of Gr-1⁺CD11b⁺F4/80⁺ cells isolated from RMA-S tumor bearing mice at the indicated ratios. As indicated, L-NMMA, D-NMMA, or medium only were added. Supernatants were harvested after 24 hours and IFN-γ production was determined by ELISA. (B) Gr-1⁺CD11b⁺F4/80⁺ cells were purified from RMA-S tumor-bearing mice. Gr-1⁺CD11b⁺F4/80⁺ cells were cultured alone or cocultured with splenocytes (spl.) from naive C57BL/6 mice with ConA in the absence or presence of the NO inhibitor L-NMMA or its inactive isoform D-NMMA at the indicated ratios for 72 hours. Proliferation was determined by ³H-thymidine incorporation assay. (C) NK cells were cultured in IL-12 in the absence or presence of Gr-1⁺CD11b⁺F4/80⁺ cells from tumor-bearing mice at a 1:1 ratio using a standard plate or a transwell system. IFN-γ production was determined by ELISA. (D) Gr-1⁺CD11b⁺F4/80⁺ cells were sorted from PBL of tumor-bearing mice. Fab fragments of the anti-NKG2D mAb or of an isotype-matched control mAb were added to cocultures of NK cells and Gr-1⁺CD11b⁺F4/80⁺ cells. Data are shown as mean plus or minus SD of triplicates. One representative experiment of 3 experiments is shown. *P < .05. ND indicates not detectable.