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Blood, Vol. 110, Issue 5, 1540-1549, September 1, 2007
Noncanonical NF- B signaling in dendritic cells is required for indoleamine 2,3-dioxygenase (IDO) induction and immune regulation
Blood Tas et al.
110: 1540
Supplementary materials for Tas et al, Vol. 110, Issue 5, 1540-1549
Files in this Data Supplement:
- Table S1. Expression profile of CD40L-NBD T cells (PDF, 75.7 KB) -
Cells were analyzed for surface or intracellular expression of various known Treg markers and cytokines by flow cytometry. FoxP3 expression was quantified by quantitative PCR. Data indicate nonsignificant changes (except *: P<.05; see also Figure S4) and are expressed as change compared to control/MUT treated cells.
- Figure S1. Selective blockade of the canonical NF-κB pathway inhibits DC maturation (JPG, 86.7 KB)
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(A) NBD peptide blocks both lipopolysaccharide (LPS)- and CD40L-induced up-regulation of major histocompatibility complex (MHC) class II and co-stimulatory molecules. Immature DCs were cultured with LPS or CD40L in the presence or absence of NBD/MUT peptides. After 2 days, DCs were analyzed for the expression of cell surface molecules HLA-DR, CD83, and CD86 by flow cytometry. The filled histograms represent the specific expression of surface markers on DCs incubated with NBD and the open histograms represent control DCs incubated with MUT peptides. NBD peptide treatment abrogated the upregulation of HLA-DR (P<.05), CD83 (P<.05), and CD86 (P<.05) after LPS (upper panels) and CD40L stimulation (lower panels). In contrast, the MUT peptide did not influence CD40L-induced upregulation of HLA-DR, CD83, and CD86. These data show that inhibition of the canonical NF- B pathway by the NBD peptide alters the maturation state and costimulatory molecular expression of LPS- and CD40L- stimulated DCs to the same extent. Data are representative of at least five independent experiments. (B) Pro-inflammatory cytokine production by DC is dose-dependently inhibited by the NBD peptide. Immature DC were pre-incubated with the indicated concentrations of NBD/MUT peptides and matured for 2 days with LPS or CD40L. After 48 hours, the cells were thoroughly washed and stimulated with CD40L-expressing mouse plasmacytoma cells. Supernatants were harvested after 24 hours and secreted IL-12p70 was measured by ELISA. Results are expressed as mean plus or minus a SD from one experiment representative of three performed in triplicate.
- Figure S2. Transfection efficiency of siRNA in DC (JPG, 49.7 KB)
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(A) To evaluate transfection efficiency, immature DCs were incubated with FAM-labeled control RNA siRNA in Lipofectamine 2000 for 4 hours at 37°C. After that, serum concentration was adjusted to 10% by addition of Iscove modified Dulbecco medium (IMDM) containing 50% fetal calf serum (FCS). After 24 hours, transfected DCs were analyzed by flow cytometry for FAM content. The histogram shows fluorescence in the FL1-channel of untransfected DCs (black line), control siRNA transfected DC (grey line), and FAM-labeled RNA transfected DC (filled histogram). We routinely obtained transfection efficiencies of 90% to 95%. A representative example of more than five independent experiments is shown. (B) No effect of siRNA mediated knock-down of the noncanonical NF-B pathway in J558-mock stimulated DCs. Immature DCs were treated with control nonblocking siRNA (siC) or siRNA for the noncanonical NF-B pathway associated kinases NIK (siNIK) and IKK (siIKK). Subsequently, cells were matured for 2 days with J558-CD40L or J558-mock cells, extensively washed and lysed in sample buffer. Cell lysates were analyzed by western blotting for indoleamine 2,3 dioxygenase (IDO) content. One representative experiment of three is shown.
- Figure S3. siRNA treatment does not change the capacity of DCs to induce T-cell proliferation (JPG, 38.8 KB)
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Immature DCs were either untreated or treated with siRNA, incubated with NBD/MUT peptides, and matured for 2 days with CD40L. Subsequently, the cells were thoroughly washed, loaded with SEB, and used to stimulate naive CD4+ Th cells. The proliferative response was determined at day 5 of coculture by 3HTdR incorporation. Data are presented as mean counts per minute (cpm) plus a SD of triplicate cultures. Results are representative of three independent experiments.
- Figure S4. Blockade of the canonical NF-κB pathway in DC alters T-cell polarization (JPG, 88 KB)
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Naïve T cells that encounter CD40L-NBD DCs develop into Th2 cells. Naive CD4+ T cells were stimulated with SEB-loaded DCs that were earlier stimulated with LPS or CD40L in the absence or presence of NBD peptides. T cells from the MLR were expanded at day 5, using IL-2 and IL-15, followed by restimulation of the proliferated T cells after 12 days with phorbol-12-myristate-13-acetate (PMA)/ionomycin. Single-cell IL-4 and IFN- production was measured by intracellular flow cytometric analysis to determine the percentage of IL-4¬and IFN--producing T cells after DC instruction. Clearly, NBD treatment of DCs affected the cytokine balance of the effector T cells. Like for LPS-matured DCs, NBD treatment of CD40L-matured DCs resulted in a strongly reduced percentage of IFN--producing T cells. However, in contrast to LPS-NBD DC–derived T cells (LPS-NBD T cells), CD40L¬NBD DC–derived T cells (CD40L-NBD T cells) revealed a significant shift in the Th1/Th2 profile towards a more Th2-like phenotype demonstrated by an increase in IL-4 expression. Calculated over all experiments, the percentage of IFN-+ T cells dropped 62.5% (± 18.8 %), whereas the percentage of IL-4+ T cells increased dramatically (P<.001). When MUT-treated DCs were used to stimulate naive T cells, no difference was observed in the development of IL-4- or IFN--producing T cells compared to stimulation with untreated DC. These data demonstrate that CD40L-NBD DC block Th1 polarization and favor Th2 differentiation. This suggests that canonical NF-B inhibition by the NBD peptide, in combination with CD40L-induced noncanonical pathway-associated mechanisms, such as the induction of IDO, results in increased Th2 instruction by DC. Shown are flow cytometry plots from one representative experiment out of at least five, which were performed in duplicate.
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