FIGURE 1.

MAP CobT induces phenotypical and functional maturation of DCs. A, recombinant MAP CobT was produced in BL21 cells and purified with NTA resin. The purified protein was then subjected to (a) SDS-PAGE and (b) Western blot analyses using 1:1000 mouse anti-His antibodies. B, DCs were treated for 24 h with 10 μg/ml of MAP CobT and 100 ng/ml of LPS. DCs were stained with annexin V-FITC/PI-PE and analyzed by flow cytometry. C, DCs were stimulated for 24 h with 100 ng/ml of LPS, or 5 or 10 μg/ml of MAP CobT, and analyzed for surface marker expression by two-color flow cytometry. Cells were gated to include CD11c⁺ cells. DCs were stained with anti-CD80, anti-CD86, or anti-MHC class I or anti-MHC class II. The percentage of positive cells is shown for each panel. Bar graphs show the mean ± S.E. of the percentages of each surface molecule on CD11c⁺ cells for three independent experiments. Statistical significance (*, p < 0.05; **, p < 0.01; or ***, p < 0.001) of treatments compared with controls is indicated. D, DCs were stimulated for 24 h with 100 ng/ml of LPS, or 5 or 10 μg/ml of MAP CobT, and the amounts of TNF-α, IL-6, IL-1β, IL-10, and IL-12p70 in the culture supernatant were measured by ELISA. All data are expressed as the mean ± S.E. (n = 3). Statistical significance (*, p < 0.05; **, p < 0.01; or ***, p < 0.001) is shown for treatments compared with the controls. E, dot plots of intracellular IL-12p70 and IL-10 in CD11c⁺ DCs. The percentage of positive cells is shown in each panel. F, endocytic activity of MAP CobT-treated versus untreated DCs. Endocytic activity at 37 or 4 °C was assessed by flow cytometry analyses as dextran-FITC uptake. The percentages of dextran-FITC⁺CD11c⁺ cells are indicated. Bar graphs show the mean ± S.E. of the percentage of dextran-FITC⁺CD11c⁺ cells for three independent experiments. Statistical significance (**, p < 0.01 or ***, p < 0.001) is indicated for treatments compared with controls.