(A) Selective contribution of IRF5 in the suppression of lung metastasis of B16F1 cells. Number of metastasized colonies in lungs from wild-type (WT), Irf3−/−, Irf5−/−, or Irf7−/− mice 14 days after intravenous injection of 1 × 106 of B16F1 cells. Means are indicated as black bars. *p < 0.05 by Student's t test. (B) Representative images of lungs from WT or Irf5−/− mice 14 days after intravenous injection of 2 × 106 of B16F1 cells. (C) In vitro killing assay of immune cells from WT or Irf5−/− mice against B16F1 cells. Whole splenocytes (left panel), purified NK cells (middle panel), or NK-depleted splenocytes (right panel) from WT or Irf5−/− mice are mixed with 51Cr-labeled target B16F1 cells at the indicated ratios. 4 hr later, 51Cr radioactivity released from target cells was monitored. E/T: effector/target cell ratio. (D) Purified NK cells (WT; 1 × 105 cells) without or with 1 × 105, 2 × 105, or 4 × 105 of WT splenic CD11c+, CD11b+, T, or B cells were subjected to in vitro killing assay for B16F1 cells. Target cell lysis was measured by co-culturing target cells and myeloid cells, with (total values) or without NK cells (background values), and background values were subtracted from the total values. Each background lysis was less than 6% of maximum release. The calculated percentage of cytotoxicity was represented as Net lysis (%). In all in vitro killing assays, 1 × 104 of 51Cr-labeled B16F1 cells were used (C and D). All in vitro killing assays were performed at least three times with high reproducibility. Represented as means ± SD.