DcR3-induced cell death is via the HBD. (A) Morphology of DcR3.Fc- or HBD.Fc-treated DCs. A total of 5 × 10⁵ CD14⁺ monocytes were incubated with GM-CSF (200 ng/mL) and IL-4 (10 ng/mL) in the presence of 10 μg/mL Fc-fusion proteins or IgG₁ for 6 days to differentiate them into DCs. Apoptotic cells were identified by adding trypan blue/PBS (0.1%) and Hoechst 33258 (1μg/mL) for 3 minutes, and observing them under phase-contrast and fluorescence microscopy, respectively. (B,C) Analysis of apoptotic cells by sub-G₁ peak (B) and annexin V–PE (FL-2) and 7-AAD (FL-3) staining (C). 5 × 10⁵ monocyte-derived DCs (moDCs) were cultured in the presence of 10 μg/mL Fc-fusion proteins or IgG₁ during the differentiation to DCs, followed by incubation with PI (0.01 mg/mL) or annexin V–PE/7-AAD (after 36 hours of Fc-fusion protein treatment), and analyzed by flow cytometry. The numbers in the quadrants represent the percentage from total cell population. (D) Kinetics of dead and apoptotic cells analyzed by annexin V–PE/7-AAD staining. *P < .05 compared with control groups at each time point. Data are representative of 3 independent experiments. Error bars represent SD of the mean..