Figure 3.

INAM in BMDC participates in DC-mediated NK activation. (A) Quantitative RT-PCR for INAM expression in WT, TICAM-1^−/−, IRF-3^−/−, and IRF-7^−/− BMDC stimulated with 10 µg/ml polyI:C. (B) Quantitative RT-PCR for INAM expression in WT BMDC stimulated by 100 ng/ml LPS, 10 µg/ml polyI:C, 1 µg/ml Pam3, 100 nM Malp-2, 10 µg/ml CpG, and 2,000 IU/ml IFN-α for 4 h. (C) BMDCs were transduced with Flag-tagged INAM-expressing lentivirus or control lentivirus. GFP expression in the BMDC was determined by flow cytometry, and subcellular localization of INAM was examined by immunofluorescence assay using anti-Flag mAb. Shaded peak, noninfected control; Blank peak, infected BMDC. Bar, 10 µm. (D) ELISA of IFN-γ induced by WT NK cells co-cultured with WT BMDC or IRF-3^−/− BMDC transfected with control lentivirus (CV) or INAM-expressing lentivirus (INAM) with/without 10 µg/ml polyI:C. (E) Cytotoxicity against B16D8 by NK cells co-cultured with BMDC transfected with control or INAM-expressing lentivirus with/without 10 µg/ml polyI:C for 24 h. (F) ELISA of IFN-γ induced by WT NK cells co-cultured with IRF-3^−/− BMDC transfected with control lentivirus (CV) or INAM-expressing lentivirus (INAM) with 10 µg/ml polyI:C. In some experiments, a transwell was inserted between the INAM-transduced BMDC and NK cells to separate the cells. (G) Quantitative RT-PCR for expression of INAM in BMDC transduced with INAM-shRNA (INAM) or scrambled shRNA (control) and cultured for 48 h. (H) IFN-γ production by WT NK cells determined using ELISA after coculturing with control or the shRNA transfected-BMDC (INAM) and 10 µg/ml polyI:C for 24 h. All data shown are means ± SD of triplicate samples from one experiment that is representative of three.