Expression of death domain-associated receptors on DCs and ligand neutralization. (A) LPS-matured DCs were activated for 15 minutes with anti-CD40 (B-B20) or isotype control and stained for cell surface CD40 and for intracellular FADD (green), as described in “Materials and methods.” Cells were then analyzed by confocal microscopy. Colocalization is shown in the bottom panels. (B) Immature and LPS-matured DCs activated with anti-CD40 (B-B20) or isotype control for 2 hours were stained with anti-TNF-R1, anti-TNF-R2, anti-TRAIL, anti-DR4, and anti-DR5 (solid line) or with isotype control (dashed line). (C) TNF-α production by immature and LPS-matured DCs was measured by ELISA. Results are means ± SEM of 3 independent experiments. (D) To verify that anti-TNF-α was functional, immature DCs were treated for 30 minutes with 10 μg/mL neutralizing anti-TNF-α or control, then incubated for 24 hours with 50 ng/mL TNF-α. DCs were then stained for CD83 expression. Results are means ± SEM of 3 independent experiments. (E) Neutralization assays. DCs were preincubated for 2 hours with the indicated neutralizing antibodies or isotype control, then activated with anti-CD40 (BB-20) or an isotype control for 6 hours before analyzing apoptosis. Results are means ± SEM of 3 independent experiments. (F) Immature and LPS-matured DCs activated with anti-CD40 (B-B20) or isotype control for 2 hours were stained with anti-Fas, anti-Fas ligand, and anti-CD86 (control) (solid line) or with isotype control (dashed line). Results are representative of 3 independent experiments. (G) Immature or LPS-matured DCs were activated with increasing concentrations of anti-Fas antibody (CH11) and stained with annexin V-FITC and 7-AAD. Results are means ± SEM of 3 independent experiments. Jurkat cells were used as a positive control.