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

Brendan John O’Sullivan and Ranjeny Thomas
J Immunol June 1, 2002, 168 (11) 5491-5498; DOI: https://doi.org/10.4049/jimmunol.168.11.5491
Brendan John O’Sullivan
Center for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland, Australia
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Ranjeny Thomas
Center for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland, Australia
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  • FIGURE 1.
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    FIGURE 1.

    Enhanced NF-κB activation is associated with APC function in MDDC treated with CD40L. A, Monocytes cultured for 48 h with GM-CSF and IL-4 were harvested and analyzed by FACS. Data are representative of at least four experiments. B, RelB DNA binding activity in nuclear extracts from MDDC treated for 24 h with increasing doses of TNF-α or CD40L was analyzed by ELISA. Data represent the mean of duplicate wells ± SEM and are representative of two separate experiments. C, Varying numbers of MDDC from the same donor were treated for 24 h with increasing doses of TNF-α, or CD40L were incubated with 105 purified allogeneic T cells for 5 days, and T cell proliferation was measured by incorporation of [3H]thymidine. Data represent the mean of triplicate wells ± SEM and are representative of two separate experiments.

  • FIGURE 2.
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    FIGURE 2.

    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.

  • FIGURE 3.
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    FIGURE 3.

    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.

  • FIGURE 4.
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    FIGURE 4.

    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.

  • FIGURE 5.
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    FIGURE 5.

    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.

  • FIGURE 6.
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    FIGURE 6.

    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.

  • FIGURE 7.
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    FIGURE 7.

    Modest effects of autocrine IL-12 on NF-κB in MDDC following CD40L. A, Varying numbers of MDDC treated for 24 h with CD40L in the presence or absence of anti-IL-10, anti-IL-12, or control Ig 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. B, NF-κB mRNA levels from MDDC treated with IL-12 for 24 h. Results are normalized to GAPDH and expressed as fold increase relative to untreated control MDDC. C, p50, RelB, and RelA DNA binding activity in nuclear extracts from MDDC treated for 24 h with IL-12. Data represent the mean of duplicate wells ± SEM and are representative of two separate experiments.

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    Table I.

    Cytokine production by MDDCa

    TreatmentIL-10 (pg/ml)IL-12 p70 (pg/ml)
    Control53 ± 125 ± 2
    CD40L54 ± 1445 ± 8
    TNF-α264 ± 1916 ± 4
    LPS462 ± 27208 ± 17
    • a Supernatants from 4 × 106 MDDC stimulated for 24 h with 100 U/ml TNF-α or 500 ng/ml CD40L were assayed for IL-10 and IL-12 p70 by ELISA.

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The Journal of Immunology: 168 (11)
The Journal of Immunology
Vol. 168, Issue 11
1 Jun 2002
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CD40 Ligation Conditions Dendritic Cell Antigen-Presenting Function Through Sustained Activation of NF-κB
Brendan John O’Sullivan, Ranjeny Thomas
The Journal of Immunology June 1, 2002, 168 (11) 5491-5498; DOI: 10.4049/jimmunol.168.11.5491

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CD40 Ligation Conditions Dendritic Cell Antigen-Presenting Function Through Sustained Activation of NF-κB
Brendan John O’Sullivan, Ranjeny Thomas
The Journal of Immunology June 1, 2002, 168 (11) 5491-5498; DOI: 10.4049/jimmunol.168.11.5491
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