Increased neutrophils in B7-H4KO mice are required for resistance to infection but the bactericidal functions of neutrophils are normal. (A) Wild-type (WT) B6 mice or B7-H4KO (KO) mice of 6 to 9 weeks old at groups of 3 were used for all experiments. The mice were injected intraperitoneally with anti–Gr-1, anti-NK1.1, and anti-pDC mAb to deplete neutrophils, NK cells, and plasmatoid cells, respectively. Injection of carrageenan was used to deplete macrophages. Depletion of subset of cells was confirmed by flow cytometry analysis. After depletion, mice were challenged with 10⁶ CFUs Listeria by intraperitoneal injection. Two days after Listeria infection, mice were terminated and the liver Listeria load was evaluated by colony plating assay. Listeria colonies in each mouse were shown. The data are expressed as CFUs/gram of liver. Data are representative of at least 3 independent experiments for each treatment. Open symbols indicate control reagents; closed symbols, treatment by antibody or carrageenan. (B) Neutrophils from B7-H4 KO mice have normal capacity to intake and digest Listeria. Three mice of B7-H4 KO or littermate control mice were intraperitoneally injected with l mL 3% thioglycollate. Mice were terminated 4 to 5 hours after injection and peritoneal cells were harvested and incubated with Listeria for 10 minutes. Listeria infection was stopped by washing cells with medium containing antibiotics in large volumes. Neutrophils were then cultured. At indicated time points, cultured cells were lysed and the cell lysates were plated for CFU counting as described in “Listeria infection of neutrophils in vitro.” Eror bars indicate SD (n = 3 samples). (C) Normal respiratory burst and phagocytosis of neutrophils from B7-H4KO mice. Neutrophils were harvested as described in panel B. Neutrophils (1 × 10⁶) were incubated with 5 × 10⁷ of red-fluorescent microbeads and 25 μM H2DCFDA for 30 to 60 minutes at 37°C. Red and green fluorescence were analyzed by flow cytometry and the results were expressed as percentage of cells with oxidative and/or phagocytic capacity. The data are representative of 2 separate experiments. There is no statistically significant difference between neutrophils with both oxidative and phagocytic capacity from WT and B7-H4KO mice.