Absence of LTB4/BLT1 signaling promotes activation of various innate immune subpopulations in tumor tissues, accompanied with attenuated immune tolerance in KO/WGM mice. (A-E) One day after the FTC with WGM cells, tumors were excised from WT/WGM or KO/WGM mice, minced finely, and treated with collagenase for tissue dissociation. For assessment of innate immunity subpopulations, tumor infiltrating cells were subsequently analyzed by flow cytometry as described in “Analyses for tumor-infiltrating leukocytes.” The numbers of viable cells are shown. (F-H) For detection of mature DCs, cells were stained with anti-CD11c, anti-CD40, anti-CD86, and anti-CCR7 mAb and subjected to flow cytometric analysis. Results shown are representative 2-dimensional dot plots (top panel, F-G) and bar graphs depicting the rates of indicated tumor infiltrating mature DCs (bottom panel, F-G). Numbers in 2-dimensional dot plots reflect the frequencies of (F) CD40⁺ or CCR7⁺CD11c⁺cells, (G) CD40⁺CD86⁺, CD40⁺CCR7⁺, or CCR7⁺CD86⁺CD11c⁺cells, and (H) CD40⁺CD86⁺CCR7⁺CD11c⁺ cells relative to the total CD11c⁺ cells. (I) The concentrations of VEGF (n = 5 or 6), TGF-β (n = 3), and IL-10 (n = 9 or 10) in supernatants derived from smashed tumor tissue of WT/WGM or KO/WGM mice were measured by ELISA assay. (J) MLR assay evaluated by [³H]thymidine incorporation. Various numbers of MACS-sorted splenic (left panel)- or TDLNs (right panel)-derived CD11c⁺ DCs from WT/WGM or KO/WGM mice in early phase were 30 Gy irradiated as stimulator cells and incubated with 2 × 10⁵ MACS-sorted splenic CD3⁺ T cells (responder) harvested from splenic C57BL/6 mice at responder to stimulator (R:S) ratios as indicated for 4 days in triplicate. [³H]Thymidine was added 16 hours before the cells were harvested, and thymidine incorporation was assessed using TopCount NXT microplate scintillation counter. Cultures in the absence of DCs (T cells only) were used as a negative control. (K) CFSE-labeled MLR assay. A total of 100 000 MACS-sorted splenic CD11c⁺ DCs were harvested from WT/WGM or KO/WGM mice, irradiated with 30 Gy, and cocultured with CD3⁺ T cells at an R:S ratio of 2:1. After 6 days of coculture, the proliferation rate of CD3⁺CD4⁺ T cells (left panel) or CD3⁺CD8⁺ T cells (right panel) labeled with 2.0μM CFSE in the coculture was assessed by flow cytometric analysis. The histograms are gated on CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells. Cultures in the absence of DCs (T cells only) were used as a negative control. Bar graphs represent mean ± SEM. *Significant difference (P < .05). **Significant difference (P < .01). Representative data from 3 independent experiments or combined data from 2 independent experiments (D-E) with similar results are shown.