CD40 Ligation Conditions Dendritic Cell Antigen-Presenting Function Through Sustained Activation of NF-κB

Inhibition of NF-κB blocks functional MDDC maturation. A, p50, RelB, and RelA DNA binding activity in nuclear extracts from MDDC treated for 24 h with TNF-α or CD40L in the presence or absence of 1, 5, or 10 μM BAY 11-7082. B, MDDC treated for 24 h with TNF-α (left) or CD40L (right) in the presence or absence of 1, 5, or 10 μM BAY 11-7082 were stained with Abs to CD83, HLA-DR, and CD86 and analyzed by flow cytometry. Data are presented as change in mean fluorescence intensity from isotype control and are the mean of two separate experiments ± SEM. *, p < 0.05 vs MDDC stimulated with TNF-α or CD40L in absence of BAY. Open symbols indicate unstimulated MDDC controls. C, Varying numbers of MDDC treated for 24 h with TNF-α or CD40L in the presence or absence of 10 μM BAY 11-7082 were incubated with 10⁵ purified allogeneic T cells for 5 days, and T cell proliferation was measured by incorporation of [³H]thymidine. Data are the mean of triplicate wells ± SEM and are representative of two separate experiments.

Sustained induction of nuclear RelB and p50 translocation in MDDC treated with CD40L. A, Nuclear extracts prepared from MDDC stimulated with TNF-α or CD40L for various times were immunoblotted with anti-RelB and anti-p50. B, Quantitation of nuclear RelB and p50 by densitometry. Data are representative of two separate experiments.

Transcriptional activation of RelB by CD40L is prolonged. NF-κB mRNA levels from MDDC treated with TNF-α (open bars) and CD40L (filled bars) by real-time PCR. Results are normalized to GAPDH and expressed as fold increase relative to untreated control MDDC. Data are expressed as the mean ± SEM of two independent experiments using different donor MDDC.

Reduced degradation of IκBα in MDDC in response to TNF-α. A, Cytoplasmic extracts prepared from MDDC stimulated with either TNF-α or CD40L were immunoblotted with anti-IκBα or anti-phospho-IκBα (Ser³²). B, Quantitation of cytoplasmic IκBα by densitometry. Data are representative of two separate experiments.

Blocking IL-10 leads to sustained NF-κB translocation in response to TNF-α. A, Nuclear extracts from MDDC treated for 6 or 24 h with TNF-α in the presence or absence of anti-IL-10, anti-IL-12, or control Ig were immunoblotted with anti-RelB. B, Quantitation of nuclear RelB by densitometry. Data are representative of two separate experiments. C, RelB DNA binding activity in MDDC treated for 24 h with TNF-α in the presence or absence of anti-IL-10 and 10 μM BAY 11-7082. D, Varying numbers of MDDC treated for 24 h with TNF-α in the presence or absence of anti-IL-10 and 10 μM BAY 11-7082 were incubated with 10⁵ purified allogeneic T cells for 5 days, and T cell proliferation was measured by incorporation of [³H]thymidine. Data are the mean of triplicate wells ± SEM and are representative of two separate experiments.