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Blood, Vol. 106, Issue 10, 3457-3464, November 15, 2005
Distinct roles for the NF- B1 and c-Rel transcription factors in the differentiation and survival of plasmacytoid and conventional dendritic cells activated by TLR-9 signals
Blood O'Keeffe et al.
106: 3457
Supplemental materials for: O’Keeffe et al, Vol 106, Issue 10, 3457-3464
Files in this Data Supplement:
- Figure S1. Nuclear complexes in cDCs and pDCs following 12 hours of CpG-ODN stimulation (JPG, 47.5 KB)
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Two complexes, C1 and C2, are upregulated after 12 hours of CpG-ODN stimulation in both cDCs and pDCs. Antibody shifts of Rel/NF- B complexes in (A) conventional DCs and (B) plasmacytoid DCs. Preimmune sera (lane 1) or antibodies specific for NF-B1 (lane 2), c-Rel (lane 3), RelA (lane 4) and p52NF-B2 (lane 5) were first incubated with nuclear extracts from wt cDCs or pDCs stimulated for 12 hours with CpG-ODN prior to performing EMSA.
- Figure S2. The conventional DCs of nfkb1–/–c-rel–/– mice are activated in response to TLR9 ligands (JPG, 42.9 KB)
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Sorted cDCs from wt (light line) or nfkb1–/–c-rel–/– (dark line) mice were cultured for 16 hours in the presence of CpG1668-ODN. Cells were stained with antibodies against MHCII and CD80. Dashed line represents unstained control. Data are representative of three independent experiments.
- Figure S3. The increased cell death and lack of nfkb1–/–c-rel–/– pDC activation is independent of the nonhematopoietic environment of nfkb1–/–c-rel–/– mice (JPG, 69.0 KB)
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(A) Spleen pDCs from wt or nfkb1–/–c-rel–/– mice, or donor spleen pDCs from wt mice reconstituted with nfkb1–/–c-rel–/– BM, were stimulated for 20 hours with CpG1668-ODN. Cell death within the cultures (the percentage of cells in the lower left quadrants) was examined by propidium iodide (PI) incorporation, shown here plotted against the forward scatter profile (FSC) of the pDCs. (B) pDCs sorted from wt or nfkb1–/–c-rel–/– FLBM cultures were stimulated overnight with CpG2216 and stained with antibodies to MHCII. The level of MHCII staining is shown on stimulated wt FLBM pDCs (light black line), unstimulated FLBM nfkb1–/–c-rel–/– pDCs (dashed line) and stimulated FLBM nfkb1–/–c-rel–/– pDCs (heavy dark line). Data are representative of more than five independent experiments.
- Figure S4. The enforced expression of Bcl-2 does not rescue the defect in CD40 expression observed during activation of nfkb1–/–c-rel–/– pDCs (JPG, 46.0 KB)
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Sorted pDCs from wt bcl-2T or nfkb1–/–c-rel–/–bcl-2T FLBM cultures were stimulated for 40 hours with CpG1668, CpG2216, or HSV-disc. The level of expression of CD40 on wt bcl-2T pDCs (heavy black line), nfkb1–/–c-rel–/–bcl-2T pDCs (gray shaded) is shown. The light gray line depicts the level of CD40 on nfkb1–/–c-rel–/–bcl-2T pDCs incubated in media only. The dashed line is the nfkb1–/–c-rel–/–bcl-2T pDC-unstained control. Data are representative of three independent experiments.
- Figure S5. The enforced expression of Bcl-2 does not affect the production of IL-6 or IL-12p70 by pDCs lacking NF-κB1 and c-Rel (JPG, 30.3 KB)
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wt bcl-2T and nfkb1–/–c-rel–/–bcl-2T pDCs were stimulated in culture in the presence (+, open bars) or absence (–, closed bars) of CpG1668. The supernatants were harvested and examined for IL-6 or IL-12 expression by ELISA. Error bars represent the variation among replicate samples within an experiment. Data are representative of two independent experiments.
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