(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.
DOI: http://dx.doi.org/10.7554/eLife.04177.003

Tumor growth in WT and Irf5^−/− mice. Tumor volume of WT or Irf5^−/− mice was monitored at the indicated days after subcutaneous injection of 1 × 10⁵ of B16F1 cells. Represented as means ± SD.
DOI: http://dx.doi.org/10.7554/eLife.04177.004

(A) Tumor metastasis in bone marrow chimera mice. Chimera mice were generated using WT, Irf5^−/−, and C57BL/6-Ly5.1 (Ly5.1) mice. Numbers of metastasized colonies in lungs from the bone marrow chimera mice were counted 14 days after intravenous injection with 1 × 10⁶ of B16F1 cells. Means are indicated as black bars. *p < 0.05 by Student's t test. (B) Tumor metastasis in WT or Rag1^−/− mice. Numbers of metastasized colonies in lungs from WT or Rag1^−/− mice were counted 14 days after intravenous injection with 1 × 10⁶ of B16F1 cells. Means are indicated as black bars.
DOI: http://dx.doi.org/10.7554/eLife.04177.005

Requirement of IRF5 in myeloid cells for the enhancement of NK cell's in vitro killing activity. In vitro killing activities of purified NK cells (WT; 1 × 10⁵ cells) against B16F1 cells were monitored in the absence or presence of 1 × 10⁵, 2 × 10⁵, or 4 × 10⁵ of WT or Irf5^−/− splenic CD11b⁺ (left panel) or CD11c⁺ (right panel) cells. The percentage of cytotoxicity was calculated as described in the legend of and represented as Net lysis (%). Each background lysis was less than 6% of maximum release. Represented as means ± SD. *p < 0.05 by Student’s t test. In in vitro killing assays, 1 × 10⁴ of ⁵¹Cr-labeled B16F1 cells were used. All in vitro killing assays were performed at least three times and the results were highly reproducible.
DOI: http://dx.doi.org/10.7554/eLife.04177.006

The effect of various myeloid cells on the enhancement of NK cell killing activities. In vitro killing activities of purified NK cells (WT; 1 × 10⁵ cells) were monitored in the absence or presence of 1 × 10⁵, 2 × 10⁵, or 4 × 10⁵ of splenic CD8⁺CD11c⁺ (left panel), CD8^–CD11c⁺ (middle panel), or CD11c⁻CD11b⁺ (right panel) cells. CD11c⁻CD11b⁺ cells were purified from diphtheria toxin-treated CD11c-DTR mice. The percentage of cytotoxicity was calculated as described in the legend of and represented as Net lysis (%). Each background lysis was less than 5% of maximum release. Represented as means ± SD. 1 × 10⁴ of ⁵¹Cr-labeled B16F1 cells were used as target cells. All in vitro killing assays were performed at least three times and the results were highly reproducible.
DOI: http://dx.doi.org/10.7554/eLife.04177.007

(A) Nuclear translocation of IRF5 in WT or Dectin-1^−/− splenocytes (5 × 10⁷ cells) co-incubated with B16F1 cells (5 × 10⁵ or 1 × 10⁶ cells) or stimulated with curdlan (30 µg/ml) for 6 hr. Nuclear protein fraction from the culture was analyzed by immunoblotting for IRF5 and USF-2. USF-2 was used as a nuclear marker protein. (B) In vitro killing activity of whole splenocytes from WT or Dectin-1^−/− mice against B16F1 cells. (C) In vitro killing activity of purified NK cells (WT; 1 × 10⁵ cells) against B16F1 cells was assessed in the absence or presence of 1 × 10⁵, 2 × 10⁵, or 4 × 10⁵ of WT or Dectin-1^−/− splenic CD11b⁺ (left panel) or CD11c⁺ (right panel) cells. The percentage of cytotoxicity was calculated as noted in the legend of and represented as Net lysis (%). Each background lysis was less than 6% of maximum release. In all in vitro killing assays, 1 × 10⁴ of ⁵¹Cr-labeled B16F1 cells were used (B and C). All in vitro killing assays were performed at least three times with high reproducibility. Represented as means ± SD. *p < 0.05 by Student's t test. Of note, we could not found mRNA induction for typical inflammatory and cytotoxic mediators in the co-culture system, wherein the ratio of B16F1 cells, DCs, and NK cells is 1:30:10 (). As such, from these analyses, it seems unlikely that Dectin-1 signaling in DCs affects the expression of these molecules in NK cells. (D) Number of metastasized colonies in lungs from WT or Dectin-1^−/− mice intravenously injected with 1 × 10⁶ of B16F1 cells. Means are indicated as black bars. *p < 0.05 by Student's t test. (E) Representative images of lungs from WT or Dectin-1^−/− mice 14 days after intravenous injection of 2 × 10⁶ of B16F1 cells.
DOI: http://dx.doi.org/10.7554/eLife.04177.008

In vitro killing activity of WT or Myd88^−/− splenocytes against B16F1 cells. Represented as means ± SD. E/T: effector/target cell ratio. In in vitro killing assays, 1 × 10⁴ of ⁵¹Cr-labeled B16F1 cells were used. In vitro killing assays were performed at least three times, and the results were highly reproducible.
DOI: http://dx.doi.org/10.7554/eLife.04177.009

Nuclear translocation of IRF5 of WT splenocytes (5 × 10⁷ cells) co-incubated with B16F1 cells (1 × 10⁶ cells) for 6 hr in the absence or presence of 50 or 500 nM of FK506. Nuclear protein extracts from the culture were analyzed by immunoblotting for IRF5 and USF-2.
DOI: http://dx.doi.org/10.7554/eLife.04177.010

(A) In vitro killing activity of purified NK cells from WT or Dectin-1^−/− mice against B16F1 cells. Represented as means ± SD. E/T: effector/target cell ratio. In in vitro killing assays, 1 × 10⁴ of ⁵¹Cr-labeled B16F1 cells were used. In vitro killing assays were performed at least three times, and the results were highly reproducible. (B) qRT-PCR analysis of Dectin-1 mRNA in WT splenic CD11b⁺ cells, CD11c⁺ cells and NK cells, and MEFs. Results are presented as relative to the expression of Gapdh mRNA. Represented as means ± SD. ND, none detected. (C) Expression levels of Dectin-1 mRNA in myeloid cells from liver and lungs. CD11b⁺F4/80⁺ cells and CD11c⁺ cells from liver and lung were isolated by cell sorting. Results are presented as relative to the expression of Gapdh mRNA. Represented as means ± SD.
DOI: http://dx.doi.org/10.7554/eLife.04177.011

Tumor growth in WT and Dectin-1^−/− mice. Tumor volume of WT or Dectin-1^−/− mice was monitored at the indicated days after subcutaneous injection of 1 × 10⁵ of B16F1 cells. Represented as means ± SD.
DOI: http://dx.doi.org/10.7554/eLife.04177.012

Number of metastasized colonies in the lungs from WT, Irf5^−/−, or Dectin-1^−/− mice. Mice were intravenously injected with 1 × 10⁶ of B16F1 cells and, 14 days later, the number of metastasized colonies in lungs was measured. Means are indicated as black bars. *p < 0.05 (WT vs Irf5^−/−, WT vs Dectin-1^−/−, Irf5^−/− vs Dectin-1^−/−) by Student's t test.
DOI: http://dx.doi.org/10.7554/eLife.04177.013

NK-depleted WT, or NK-depleted Dectin-1^−/− mice were intravenously injected with 1 × 10⁶ of B16F1 cells. After 14 days, the numbers of metastasized colonies were counted in the lungs of each mouse. This result suggests that NK cells are indeed the effector cells and that Dectin-1 signaling in DCs and macrophages contributes to NK cell-mediated tumor killing. Means are indicated as black bars. NS, not significant.
DOI: http://dx.doi.org/10.7554/eLife.04177.014

(A) The population of splenic NK cells in Dectin-1^−/− mice. Splenocytes were isolated from WT and Dectin-1^−/− mice. The NK cell population (percentage of total cells) was then analyzed by flow cytometry using anti-DX5 and anti-CD3ε antibodies. Represented as means ± SD. NS, not significant. (B) IL12-induced IFN-γ production in NK cells. Purified NK cells (WT or Dectin-1^−/−; 5 × 10⁴ cells) were stimulated with 10 or 100 ng/ml of IL12. IFN-γ levels in the cultured supernatants were measured by ELISA 24 hr after IL12 stimulation. Represented as means ± SD. NS, not significant. ND, none detected. (C) Viral loads in the spleens of WT and Dectin-1^−/− mice after MCMV infection. WT, Dectin-1^−/−, and NK-depleted WT mice were intraperitoneally infected with MCMV (5 × 10³ pfu). Mice were sacrificed 3 days after infection and MCMV titers were then examined by plaque forming assay. Means are indicated as black bars; NS, not significant. (D) Survival of the WT (n = 4), Dectin-1^−/− (n = 4), and NK-depleted WT mice (n = 4) after MCMV infection. Mice were intraperitoneally infected with MCMV (3 × 10⁵ pfu) and their survival was monitored at the indicated periods.
DOI: http://dx.doi.org/10.7554/eLife.04177.015

NK cells (WT; 1 × 10⁵ cells) and WT or Dectin-1^−/− splenic CD11c⁺ cells (3 × 10⁵ cells) were co-cultured with B16F1 cells (1 × 10⁴ cells). Total RNA was isolated at time zero and 4 hr after the co-culture and then mRNA expression levels for Granzyme B (Gzmb), Perforin-1 (Prf1), IFN-γ (Ifng), IL-6 (Il6), and TNF-α (Tnf) were analyzed by qRT-PCR analysis. Results are presented relative to the expression of Gapdh mRNA. Represented as means ± SD.
DOI: http://dx.doi.org/10.7554/eLife.04177.016

(A) Binding of sDectin-1 to B16F1 cells (left panel) and primary mouse embryonic fibroblasts (MEFs; right panel). The cells (4 × 10⁵ cells) were incubated with human IgG1 Fc (control Fc) or sDectin-1 (fused with the Fc) for flow cytometric analysis. (B) Effect of N-glycosidase treatment (left panel) or O-glycosidase in combination with neuraminidase (right panel) on the sDectin-1 binding to B16F1 cells. The cells (4 × 10⁵ cells) were treated with either N-glycosidase (25 U/ml) or O-glycosidase (25 mU/ml) with neuraminidase (250 mU/ml) for 1 hr and then subjected to the sDectin-1 binding assay. These enzymatic reactions were performed under the conditions wherein these cells remain alive. (C) The effect of N-glycosidase or O-glycosidase treatment of B16F1 cells on in vitro killing activity of WT splenocytes. B16F1 cells were treated with or without N-glycosidase (25 U/ml) for 1 hr in RPMI medium (left panel) or treated with or without the combination of O-glycosidase (25 mU/ml) and neuraminidase (125 mU/ml) for 1 hr in RPMI medium (right panel) and then subjected to in vitro killing assay. ⁵¹Cr radioactivity released from target cells was measured. Represented as means ± SD. E/T: effector/target cell ratio. In in vitro killing assays, 1 × 10⁴ of ⁵¹Cr-labeled B16F1 cells were used.
DOI: http://dx.doi.org/10.7554/eLife.04177.017

The sDectin-1 binding to splenic CD11b⁺ cells, CD11c⁺ cells, NK cells, liver cells, or lung cells were examined as described in .
DOI: http://dx.doi.org/10.7554/eLife.04177.018

B16F1 cells (4 × 10⁵ cells) were treated with or without neuraminidase (250 mU/ml) for 1 hr and then subjected to the sDectin-1 binding assay.
DOI: http://dx.doi.org/10.7554/eLife.04177.019

B16F1 cells were treated with or without N-glycosidase (25 U/ml) for 1 hr in RPMI medium and then used as target cells for in vitro killing assay with purified NK cells from WT mice. ⁵¹Cr radioactivity released from the target cells was measured. Represented as means ± SD. E/T: effector/target cell ratio. In in vitro killing assays, 1 × 10⁴ of ⁵¹Cr-labeled B16F1 cells were used.
DOI: http://dx.doi.org/10.7554/eLife.04177.020

The supernatant of B16F1 cells treated with N-glycosidase for 1 hr was collected and incubated with protein G-conjugated sepharose bound without (blue peaks) or with (green peaks) sDectin-1. N-glycans remained in the supernatant was then analyzed by mass spectrometry. The intensities of the spectra were normalized to that of glycan corresponding to the peak (m/z; 1159.40).
DOI: http://dx.doi.org/10.7554/eLife.04177.021

Proposed structures of N-glycans detected by the mass spectrometric analysis () are depicted.
DOI: http://dx.doi.org/10.7554/eLife.04177.022