Figure 5.

Arrest of DC maturation at the semimature stage. Immature monocyte-derived DCs were generated from human CD14⁺ monocytes by incubation with GM-CSF and IL-4 in the presence of MD-3 or isotype-matched control antibody (control Ab) from the beginning of culture. After 6 d, DCs were stimulated or not with LPS. (A) Expression levels of MHC class I and II, CD80, CD86, CD40, PD-L1, PD-L2, and CTLA-4 on their surface were compared by flow cytometry. Cumulative data showing mean fluorescent intensity (MFI) ± SE of MHC class I and II, CD80, CD86, and CD40 were obtained from four independent experiments. Data showing mean fluorescent intensity ± SE of PD-L1, PD-L2, and CTLA-4 are representative of two independent experiments in triplicate. (B) Representative cytokine levels in the culture supernatants of immature and LPS-treated monocyte-derived DCs in the presence of MD-3 or control antibody. Results are the mean ± SE of triplicate cultures, and data are representative of three independent experiments. (C) Humanized mice received MD-3 or control antibody three times before LPS (100 µg/mouse) administration. Splenocytes were isolated 1 d after LPS injection and stained with HLA-ABC, CD11c, CD80, and CD86 antibodies. Representative dot plots of CD80 and CD86 expression on gated CD11c⁺ DCs are shown at the left. Numbers indicate the percentage of cells in each quadrant. Cumulative data (n = 3) showing mean fluorescent intensity were obtained from three independent experiments (middle). The percentages of CD11c⁺ cells among HLA-ABC⁺ cells in spleens were also calculated (right). Error bars indicate SE.