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Blood, Vol. 113, Issue 1, 75-84, January 1, 2009
Imaging of plasmacytoid dendritic cell interactions with T cells
Blood Mittelbrunn et al.
113: 75
Supplemental materials for: Mittelbrunn et al
Files in this Data Supplement:
- Table S1. Analysis of MHC-II expression by in vitro and ex vivo pDC (PDF, 18.3 KB) -
The data represent the mean fluorescence intensities (MFI) and the geometric MFI of MHC-II surface expression levels by Flt3-L–derived immature pDC and ex vivo isolated pDC from peripheral lymph node from several independent experiments.
- Figure S1. In vitro differentiation and isolation of Flt3-L–derived plasmacytoid and conventional DCs (JPG, 36.8 KB)
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Representative flow cytometry density plots of pDC and cDC populations generated from Flt3-L–driven mouse bone marrow cultures. Left panel: DC-subpopulations, defined according to their differential expression of B220 and CD11b, after 7 days of differentiation. The percentage of each DC-population is indicated. Middle and right panels: cDC and pDC populations after negative selection by magnetic cell sorting.
- Figure S2. Phenotype of freshly isolated pDCs (JPG, 61.9 KB)
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pDCs were isolated from mesenteric and peripheral lymph nodes and expression of MHC-II, costimulatory and adhesion molecules was analysed by flow cytometry. Light grey histogram corresponds to negative control.
- Figure S3 (JPG, 113 KB)
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(A) (B) pSMAC at the IS between pDCs and T cells. Confocal analysis showing actin cytoskeleton forming a pSMAC-structure during IS formation by mature pDCs and cDCs (A) or immature pDCs (B) with antigen specific CD4+ T cells. OVA peptide-loaded DCs were conjugated with blue-labeled OT-II T cells and double stained for phalloidin (red) and Talin or Arp2/3 (green). Plates show DIC images and maximal projections of confocal sections. (C) pY174-Vav relocation to the IS between cDCs and T cells. Confocal analysis shows that pY174-Vav is localized throughout the contact zone during IS formation by CpG-matured cDCs and antigen specific CD4+ T cells. OVA peptide-loaded mature cDCs were conjugated with blue-labeled OT-II T cells and double stained for phalloidin (red) and pY174-Vav (green). Plates show the maximal projection of confocal sections and the DIC image. (D) Antigen specific signaling induced by DCs. Western blot analysis of LAT phosphorylation (pY191) in T cells interacting with immature and mature pDCs and cDCs in the presence or absence of OVA peptide. ipDC: immature pDC; mpDC: mature pDC; icDC, immature cDC; mcDC: mature cDC.
- Video 1. In vitro dynamics of immature pDC–T-cell interactions (MOV, 0.98 MB)
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Time-lapse video-microscopy showing interactions between naïve OT-II CD4+ T cells and immature pDCs. OVA peptide-loaded immature pDCs, isolated from Flt3-L–driven cultures, were seeded onto fibronectin-coated coverslips. OT-II T cells labeled with calcein (green) were added to the chambers. pDC–T-cell interactions are predominantly short-lived.
- Video 2. In vitro dynamics of immature cDC–T-cell interactions (MOV, 300 KB)
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Time-lapse video-microscopy showing interactions between naïve OT-II CD4+ T cells and immature cDCs. OVA peptide-loaded immature cDCs, isolated from Flt3-L–driven cultures, were seeded onto fibronectin-coated coverslips. OT-II T cells labeled with calcein (green) were added to the chambers. Short- and long-lived cDC–T-cell interactions can be observed.
- Video 3. In vitro dynamics of mature pDC–T-cell interactions (MOV, 347 KB)
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Time-lapse video-microscopy showing interactions between naïve OT-II CD4+ T cells and mature pDCs. OVA peptide-loaded mature pDCs, isolated from Flt3-L–driven cultures, were seeded onto fibronectin-coated coverslips. OT-II T cells labeled calcein (green) were added to the chambers. The proportion of long-lived pDC–T-cell interactions is increased after CpG maturation.
- Video 4. In vitro dynamics of mature cDC–T-cell interactions (MOV, 722 KB)
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Time-lapse video-microscopy showing interactions between naïve OT-II CD4+ T cells and mature cDCs. OVA peptide-loaded mature cDCs, isolated from Flt3-L–driven cultures, were seeded onto fibronectin-coated coverslips. OT-II T cells labeled with calcein (green) were added to the chambers. Long-lived cDC–T-cell interactions are frequently observed.
- Video 5. Intravital immature pDC–T-cell interactions (MOV, 821 KB)
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Two photon imaging of interactions between CMTMR-labeled OT-II T cells (red) and OVA peptide-loaded immature pDCs (green) in the spleen. Flt3-L− in vitro differentiated pDCs from GFP mice were i.v. injected into C57BL/6 recipients and 16h later naïve OT-II CD4+ T cells were i.v. transferred. After 4h, mice were anesthetised and the spleens were microsurgically exposed for intra-vital two-photon imaging. The interactions observed are predominantly short-lived.
- Video 6. Intravital mature pDC–T-cell interactions (MOV, 460 KB)
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Two photon imaging of interactions between CMTMR-labeled OT-II T cells (red) and OVA peptide-loaded immature pDCs (green) in the spleen. Mature Flt3-L− in vitro differentiated pDCs from GFP mice were i.v. injected into C57BL/6 recipients and 16h later naïve OT-II CD4+ T cells were i.v. transferred. After 4h, mice were anesthetised and the spleens microsurgically exposed for intra-vital two-photon imaging. Long-lived interactions are frequently observed.
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