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Blood, Vol. 113, Issue 18, 4232-4239, April 30, 2009
Human plasmacytoid dendritic cells are unresponsive to bacterial stimulation and require a novel type of cooperation with myeloid dendritic cells for maturation
Blood Piccioli et al.
113: 4232
Supplemental materials for: Piccioli et al
Files in this Data Supplement:
- Figure S1 (JPG, 77.5 KB)
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(A) pDCs express CD40 upon exposure to S. aureus bacterial particles, only in presence of mDCs (striped bars), but not when cultured in isolation (black bars). Data collected from three independent experiments are plotted. (B and C) pDCs (white bars) do not take up bacterial particles upon either over night incubation with mDCs (B, black bars) using purified cells or 30 min incubation of PBMCs (C). Within PBMCs, pDCs were identified as BDCA-2 positive cells, while CD11c positive cells (black bars, mDCs and monocytes), were considered the phagocytic cells. B and C are two independent experiments. (D) pDCs (white bars) do not produce significant amounts of TNF-α in contrast of mDCs (black bars), upon exposure to the indicated live growing bacteria. Data collected from three independent experiments are plotted.
- Figure S2 (JPG, 63 KB)
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(A) IL-6 production, reported as an example representing all other cytokines, measured in the supernatants of mDC (black bars), pDC (white bars) and mDC-pDC (gray bars) cultures stimulated over night as indicated. Data collected from five independent experiments are plotted. The result is reported in log scale. (B and C) T-cell proliferation induced by cross-activated mDCs in presence of CpG (B) or pDCs in presence of LPS (C). Data obtained from three different donors tested are plotted. The points of 1/20 (B) or 1/40 (C) stimulator cell/T-cell ratio are shown. The donor 3 (red rectangles) is the one reported in the Fig. 2F, G.
- Figure S3 (JPG, 66.7 KB)
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(A) pDCs (left panel) and mDCs (right panel) increase significantly the CD40 expression in presence of sub-optimal concentration of LPS and CpG, when cultured together (striped bars) compared to the culture in isolation (black bars). Data from three donors tested are shown. The donor 3 (red rectangles) is the one reported in Fig. 3B. (B) The synergistic production of IP-10 in the supernatants of the mDC-pDC co-cultures (striped bars) compared with the ones of pDCs (white bars) and mDCs (black bars) cultured in isolation, stimulated with sub-optimal concentration of LPS and CpG. Data from three donors tested are shown. The donor 3 (red rectangles) is the one reported in Fig. 3C. The result is reported in log scale. BDL: below detection limit.
- Figure S4 (JPG, 64.6 KB)
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(A) Type-I interferons and TNF-α play a central role in mDC-pDC cross-talk. The CD40 expression of cross-activated mDCs (left panel) is partially decreased by blocking type-I interferons, while the one of cross-activated pDCs (right panel) is strongly decreased blocking TNF-α. Data collected from three independent experiments are plotted. (B) Cell contact is essential for the mDC-pDC cross-talk. The CD40 expression of cross-activated (black bars) mDCs (left panel) and pDCs (right panel) is dramatically reduced or completely shut down when the two cell types are cultured separated by a semi-permeable membrane (striped bars). Data collected from three independent experiments are plotted.
- Figure S5. CD40 expression (JPG, 55.9 KB)
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The CD40 expression of cross-activated pDCs (A) and mDCs (B) is revealed also stimulating PBMCs either with LPS (black bars) or with CpG type A (strong type-I interferons inducers, gray bars) or B (poor type-I interferons inducers, striped gray bars). Data from three donors tested are shown. The donor 3 (red rectangles) is the one reported in Fig. 5A.
- Figure S6. TLR2, but not TLR7 and TLR9 HEK293 transfectants respond to bacterial particles (JPG, 28.4 KB)
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Fold induction of NF-kB–driven luciferase expression controlled by NF-kB was determined in untransfected (red circles) or TLR2 (black diamonds), TLR7 (white diamonds) or TLR9 (gray diamonds) transfected HEK293 cells after exposure to the indicated amounts of fixed S. aureus cells. A representative experiment out of three performed is shown. Similar results were obtained using E. coli and Y. pestis (data not shown).
- Figure S7. Comparison of TLR2 expression in pDCs and mDCs (JPG, 68.9 KB)
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(A) Flow cytometry histograms of surface and intracellular TLR2 expression (thick line) compared with isotype control (thin line), in isolated mDCs (left panel) or pDCs (right panel) are shown. (B) Surface TLR2 expression was determined by flow cytometry for mDCs (blue line) and pDCs (red line) in total PBMCs. One representative experiment out of three is shown in A and B. (C) The TLR expression profile of isolated mDCs and pDCs obtained from five different donors was measured by quantitative PCR. The amount of TLR mRNA relative to the housekeeping gene GAPDH (left panel) and the relative TLR expression between mDCs and pDCs (right panel) are shown. Bars show geometric means from five independent experiments reported in a logarithmic scale.
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