American Society of Hematology

Figure 2

$Figure 2. Differential pDC maturation is triggered by different TLR7 ligands. GEN2.2 cells were untreated or stimulated with Flu or CL097 for 24 hours. (A) Production of proinflammatory cytokines was measured in culture supernatants by enzyme-linked immunoabsorbent assay and CBA. The mean and SD from duplicate values of 3 independent experiments are shown. (B) After 24 hours of culture, RNA was extracted, and RNA expression levels of type I IFNs: IFN-α1, -α2, -β1, and -ω1 were measured by quantitative PCR. Data shown are normalized to G6PDH and are representative of 2 independent experiments. (C) PBMCs from healthy donors were cultured in the absence or presence of Flu or CL097 for 3 hours. Secretion was blocked by adding brefeldin A for a further 4 hours. Intracellular IFN-α production was measured in HLA-DR+ BDCA4+ pDCs by flow cytometry. Dot plots show the percentage of IFN-α–producing cells among pDCs. Representative results from 3 different donors are shown. (D) GEN2.2 cells were cultured in the absence or presence of Flu or CL097 for 3 hours. Cells were lysed and proteins were extracted. The different NF-κB subunits were quantified in nuclear fractions with the use of the TransAM kit. The mean OD and SD from duplicate values of 3 independent experiments are shown. (E) GEN2.2 cells were cultured in the absence or presence of Flu or CL097 for 3 hours. Cells were immunostained for IRF7 and Evans blue colored. Immunofluorescence was assessed by microscopy. Representative images from 3 independent experiments are shown.$

Differential pDC maturation is triggered by different TLR7 ligands. GEN2.2 cells were untreated or stimulated with Flu or CL097 for 24 hours. (A) Production of proinflammatory cytokines was measured in culture supernatants by enzyme-linked immunoabsorbent assay and CBA. The mean and SD from duplicate values of 3 independent experiments are shown. (B) After 24 hours of culture, RNA was extracted, and RNA expression levels of type I IFNs: IFN-α1, -α2, -β1, and -ω1 were measured by quantitative PCR. Data shown are normalized to G6PDH and are representative of 2 independent experiments. (C) PBMCs from healthy donors were cultured in the absence or presence of Flu or CL097 for 3 hours. Secretion was blocked by adding brefeldin A for a further 4 hours. Intracellular IFN-α production was measured in HLA-DR⁺ BDCA4⁺ pDCs by flow cytometry. Dot plots show the percentage of IFN-α–producing cells among pDCs. Representative results from 3 different donors are shown. (D) GEN2.2 cells were cultured in the absence or presence of Flu or CL097 for 3 hours. Cells were lysed and proteins were extracted. The different NF-κB subunits were quantified in nuclear fractions with the use of the TransAM kit. The mean OD and SD from duplicate values of 3 independent experiments are shown. (E) GEN2.2 cells were cultured in the absence or presence of Flu or CL097 for 3 hours. Cells were immunostained for IRF7 and Evans blue colored. Immunofluorescence was assessed by microscopy. Representative images from 3 independent experiments are shown.

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