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Results: 5

1.
Figure 4.

Figure 4. From: Plasmacytoid dendritic cell-specific receptor ILT7-Fc?RI? inhibits Toll-like receptor-induced interferon production.

Cross-linking of ILT7/FcɛRIγ activates ITAM-mediated signaling. (A) 2B4 T cell hybridoma expressing FcɛRIγ and ILT7 was cross-linked with mouse IgG1 or anti-ILT7 mAb and analyzed for NFAT-activated GFP expression. Cells cultured in medium alone were also analyzed. (B) Freshly isolated human pDCs were cross-linked with mouse IgG1, anti-ILT7, or anti-BDCA4 mAb and the cell lysates were analyzed by Western blotting. (C) The kinetics of intracellular calcium flux in human pDCs when cross-linked (arrow) by control (IgG1) or anti-ILT7 Ab. As indicated, cells were preincubated for 30 min with 50 μM of PP2, 50 μM of PP3, or 10 μM of Syk inhibitor (EMD Biosciences) before cross-linking.

Wei Cao, et al. J Exp Med. 2006 June 12;203(6):1399-1405.
2.
Figure 3.

Figure 3. From: Plasmacytoid dendritic cell-specific receptor ILT7-Fc?RI? inhibits Toll-like receptor-induced interferon production.

Anti-ILT7 mAb specifically stains peripheral blood pDCs. (A) Mouse 2B4 T cell hybridoma expressing human FcɛRIγ (top) was transduced with HA-ILT7 (bottom). The cells were stained with an isotype-matched control Ab (IgG1), anti-HA, or anti-ILT7 mAb. (B) Total PBMCs were stained with IgG1 (left) or anti-ILT7 (right) and anti-BDCA2 mAb. Representative results from the analysis of multiple healthy donors are shown. (C) Surface ILT7 was measured on freshly isolated, IL-3–treated, or CpG-treated pDCs at various times after culture. Mean fluorescence intensity of each sample is indicated (top right).

Wei Cao, et al. J Exp Med. 2006 June 12;203(6):1399-1405.
3.
Figure 2.

Figure 2. From: Plasmacytoid dendritic cell-specific receptor ILT7-Fc?RI? inhibits Toll-like receptor-induced interferon production.

ILT7 associates with FcɛRIγ. (A) BaF/3 cells stably transfected with FLAG-FcɛRIγ (top), -DAP12 (middle), or -DAP10 (bottom) were transduced with HA-ILT7 (center column) or with the vector alone (left column). Cells were stained for FLAG and HA and analyzed by flow cytometry. HA-tagged human KIR2DL4 or a mouse NKG2D variant was transduced into the BaF/3 cells as positive controls (right column). (B) Lysate from parental BaF/3 cells or cells expressing ILT7-HA and FcɛRIγ-FLAG was precipitated with IgG1, anti-HA, or anti-FLAG mAbs. Western blot (WB) was performed with anti-HA or anti-FcɛRIγ Ab. A fraction of the original lysate was run as control. Notably, ILT7-HA ran as two bands, likely reflecting differentially processed isoforms.

Wei Cao, et al. J Exp Med. 2006 June 12;203(6):1399-1405.
4.
Figure 1.

Figure 1. From: Plasmacytoid dendritic cell-specific receptor ILT7-Fc?RI? inhibits Toll-like receptor-induced interferon production.

Human pDCs preferentially express ILT7 and three transmembrane signaling adapters. (A) The relative expression of ILT family members on peripheral blood leukocytes was compared by plotting the values extracted from the gene expression database. A value <1 indicated the absence of gene expression. (B) The relative gene expression of ILTs on different cell types from three healthy donors was determined by quantitative RT-PCR analysis. The expression was normalized with the level of total PBMCs. The median expression is marked by a horizontal bar. (C) The expression levels of known transmembrane signaling adapters in pDCs were plotted using the values extracted from the gene expression database. (D) The relative expression of FcɛRIγ, DAP12, and DAP10 was determined from three healthy donors by quantitative RT-PCR analysis. The expression was normalized with S18 and multiplied by 1,000. The median expression is marked by a horizontal bar.

Wei Cao, et al. J Exp Med. 2006 June 12;203(6):1399-1405.
5.
Figure 5.

Figure 5. From: Plasmacytoid dendritic cell-specific receptor ILT7-Fc?RI? inhibits Toll-like receptor-induced interferon production.

ILT7 cross-linking inhibits TLR responses by pDCs. (A) Human pDCs were cross-linked with various Abs before stimulation by CpG (left) or Flu virus (right). The levels of the secreted IFNα and TNFα in a triplicate assay from a representative donor are shown. (B) Purified pDCs were cultured with CpG for 1 h (left) or 30 min (second panel from left) at 37°C before being cross-linked. Alternatively, CpG was added at the same time as (second panel from right) or 30 min after (right) cells were cross-linked by various mAbs. The amounts of IFNα and TNFα in the supernatant after 18 h of culture are shown. (C) The amounts of IFNα and TNFα were determined by intracellular staining of CpG-activated pDCs. The percentage of the double-positive cell population is indicated. (D) The amounts of type I IFN transcripts were determined by quantitative RT-PCR analysis. The expression is shown as the relative level of transcription compared with unstimulated pDCs.

Wei Cao, et al. J Exp Med. 2006 June 12;203(6):1399-1405.

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