Figure 1.
Figure 1. Freshly isolated NK cells promote IFN-α and IL-6 release by pDCs. (A) Peripheral-blood–derived pDCs were cultured in the presence or in the absence of resting autologous NK cells at the NK/pDC ratios of 10:1 or 5:1, stimulated by CpG-A that was added at the concentrations of 5 μg/mL or 0.5 μg/mL. After 24 hours of culture, supernatants were harvested and analyzed for IFN-α content by specific ELISA. ▪, IFN-α concentrations in supernatants collected from cell cultures stimulated by CpG-A at the higher dose of 5 μg/mL; □, supernatants collected from cell cultures stimulated with 0.5 μg/mL; ▦, supernatants collected from unstimulated cell cultures (ie, in the absence of CpG-A). Histograms on the right show IFN-α release at the NK/pDC ratio of 5:1, and histograms on the left indicate IFN-α release at the NK/pDC ratio of 10:1. NK cells cultured alone did not produce IFN-α either in the presence or in the absence of CpG-A. A representative experiment of 10 performed is shown. (B) pDCs were precultured in the presence (right panels) or in the absence (left panels) of autologous resting NK cells for 18 hours and then cultured for 6 hours in the presence of brefeldin A with the indicated stimuli (medium, IL-3 + CpG-A, IL-3 + CpG-B). Then, TNF and IL-6 production by pDCs was evaluated by intracellular double immunofluorescence and cytofluorimetric analysis. The percentage of cytokine-producing pDCs is indicated in the corresponding square. (C) Supernatants collected from parallel NK/pDC cocultures run for 72 hours of culture (without the addition of brefeldin A) were analyzed for IL-6 content with Bioplex. As control, supernatants from cultures containing pDCs alone or NK cells plus IL-3 and CpG-A have also been analyzed. (D) The purity of pDCs after FACS is shown as a double fluorescence staining where pDCs are labeled with anti–BDCA4-PE and anti–CD19-FITC mAbs. (E) The purity of NK cells after FACS is shown as a double fluorescence staining where NK cells are labeled with anti–CD56-PC5 and anti–CD3-FITC. Data are representative of 6 independent experiments that gave comparable results.

Freshly isolated NK cells promote IFN-α and IL-6 release by pDCs. (A) Peripheral-blood–derived pDCs were cultured in the presence or in the absence of resting autologous NK cells at the NK/pDC ratios of 10:1 or 5:1, stimulated by CpG-A that was added at the concentrations of 5 μg/mL or 0.5 μg/mL. After 24 hours of culture, supernatants were harvested and analyzed for IFN-α content by specific ELISA. ▪, IFN-α concentrations in supernatants collected from cell cultures stimulated by CpG-A at the higher dose of 5 μg/mL; □, supernatants collected from cell cultures stimulated with 0.5 μg/mL; ▦, supernatants collected from unstimulated cell cultures (ie, in the absence of CpG-A). Histograms on the right show IFN-α release at the NK/pDC ratio of 5:1, and histograms on the left indicate IFN-α release at the NK/pDC ratio of 10:1. NK cells cultured alone did not produce IFN-α either in the presence or in the absence of CpG-A. A representative experiment of 10 performed is shown. (B) pDCs were precultured in the presence (right panels) or in the absence (left panels) of autologous resting NK cells for 18 hours and then cultured for 6 hours in the presence of brefeldin A with the indicated stimuli (medium, IL-3 + CpG-A, IL-3 + CpG-B). Then, TNF and IL-6 production by pDCs was evaluated by intracellular double immunofluorescence and cytofluorimetric analysis. The percentage of cytokine-producing pDCs is indicated in the corresponding square. (C) Supernatants collected from parallel NK/pDC cocultures run for 72 hours of culture (without the addition of brefeldin A) were analyzed for IL-6 content with Bioplex. As control, supernatants from cultures containing pDCs alone or NK cells plus IL-3 and CpG-A have also been analyzed. (D) The purity of pDCs after FACS is shown as a double fluorescence staining where pDCs are labeled with anti–BDCA4-PE and anti–CD19-FITC mAbs. (E) The purity of NK cells after FACS is shown as a double fluorescence staining where NK cells are labeled with anti–CD56-PC5 and anti–CD3-FITC. Data are representative of 6 independent experiments that gave comparable results.

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