Article Figures & Data
Figures
- FIGURE 1.
Generation of cytotoxic activity and IFN-γ production are differentially induced in NK cells by pDC and myeloid DC. A, Purified NK cells were cultured for 18 h alone (left) or with increasing concentrations (indicated as a percentage of DC added to NK cells) of purified pDC (middle), or myeloid DC (right and C) or with 25% pDC (B, ▦) or myeloid DC (B, □) in the presence or in the absence of inactivated influenza virus, poly(I:C), CpG2216, or CpG2006 at the concentrations indicated in Materials and Methods. Cytolytic activity against Daudi at the NK cells to target ratios of 7.5:1, 5:1, and 2.5:1 (triplets of each symbol in A; data shown are results obtained from one of three similar experiments) and IFN-γ production (B, mean ± SE, n = 3) and (C, one of two experiments with similar results) were evaluated after 18 h of coculture.
- FIGURE 2.
Cytolytic activity and CD69 expression induced in NK cells by activated pDC are dependent on type I IFN. A and E, pDC were kept on ice or cultured for 18 h with virus and washed before coculture with purified NK cells together or separately by a transwell. B, F, and G, pDC were pulsed for 1 h with virus, washed and further incubated in medium. After 18 h, pDC (B and F) or their supernatant fluids (B and G) were added to purified NK cells in the presence or in the absence of a mixture of anti-IFN-αβ Abs ± anti-TNF mAb. C, NK cells were cultured with or without pDC in the presence of virus ± anti-IFN-αβ abs or anti-IL-12 mAb. D, NK cells were incubated with medium or IL-2 (500 U/ml), or cultured in transwell with pDC that had been pulsed for 1 h with virus. After 18 h NK cells were washed and further cultured for 18 h with monocyte-derived DC at a 2:1 ratio. H and I, NK cells were incubated with or without IFN-α (500 U/ml) in the presence or in the absence of the mixture of anti-IFN-αβ abs (H), or of TNF or anti-TNF mAb (I). Cytolytic activity against Daudi (A–C), CD86 expression on mono-DC, as defined by cytofluorimetric side scatter and forward scatter parameters (D) and CD69 expression on CD16+ NK cells (E–I) were evaluated after 18 h. Mean fluorescence intensity for each histogram (D–I) is reported by number in brackets. Similar results were obtained in at least three different experiments.
- FIGURE 3.
Cytolytic activity and CD69 expression induced in NK cells by myeloid DC in the presence of poly(I:C) is dependent on type I IFN, whereas IFN-γ production is dependent on IL-12 secretion and cell contact. NK cells were cultured in the presence or in the absence of myeloid DC (10% in A and B, left, and C or with 1.25% in B, right) with or without poly(I:C). Cells were cocultured together or separately across a transwell (A–C, left), in the presence or in the absence of a mixture of anti-IFN-αβ Abs (A–C, right), anti-IFN-αβ abs plus anti-TNF mAb (A, right), or anti-IL-12 mAb (B and C, right). CD69 expression on CD16+ NK cells, mean fluorescence intensity in brackets (A), cytolytic activity (B) are shown, and are results from one of two similar experiments, and IFN-γ production (mean ± SE, n = 3) (C) were evaluated after 18 h.
- FIGURE 4.
IL-2-activated NK cells activate pDC. A, pDC from different donors (a, b, and c) or immature or LPS-treated monocyte-derived DC (donor d) were cultured with autologous or allogeneic IL-2-activated NK cells from different donors (a, b, and c) at the indicated NK cell to DC ratios. B and C, pDC were cultured in medium alone or with untreated or IL-2-activated NK cells (1:2 ratio) together (B, left) or separated by a transwell (B, right), in the presence of different concentrations of CpG2216 (C, top) (mean ± SE, n = 4) or CpG2006 (C, bottom) Results are from two different experiments. D, pDC were cultured with IL-2-activated NK cells or IL-2-treated CD4+ T lymphocytes (triplets of histograms represent 1:4, 1:2, 1:1 ratios) in the presence of different concentrations of CpG2216. At the CpG2216 concentration of 4 μg/ml only 1:4 and 1:2 ratios were performed. E, pDC were cultured with IL-2-activated NK cells (1:2 ratio) together or separated by a transwell, in the presence of 0.2 μg/ml CpG2216. Results are from two different experiments. After 18 h of culture pDC and monocyte-derived DC recovery (A), calculated as a percentage of DC cultured, respectively, with virus or medium alone, in the absence of NK cells, was evaluated by BDCA-4-FITC mAb for pDC or cytometric side scatter and forward scatter parameters for monocyte-derived DC, in the region of viable cells; CD83 expression (B) and mean fluorescence intensity shown in brackets on BDCA-4+ cells was evaluated by double immunofluorescence; IFN-α (C and D, top and E) and TNF (D, bottom) production were tested in the supernatants.
- FIGURE 5.
IL-2-activated NK cells interact with myeloid DC. A, Freshly separated myeloid DC were cultured in medium alone or with allogeneic untreated or IL-2-activated NK cells at the indicated NK cell to DC ratio. Mean ± SE, n = 3 (except for the 2:1 ratio, where n = 4) is shown. B, Myeloid DC were cultured with medium, GM-CSF, and LPS (left), autologous untreated or IL-2-activated NK cells (1:2 ratio) together (left) or separated by a transwell (right). C, Myeloid DC were cultured with autologous untreated or IL-2-activated NK cells (left) or with untreated or IL-2-treated CD4+CD56− T lymphocytes (right). After 18 h, DC recovery (A, top) was evaluated by cytofluorimetry as a percentage of viable HLA-DRhigh cells cultured in medium, without NK cells; CD80 expression on myeloid DC (A, bottom, B, and C) was evaluated by double immunofluorescence with CD80-PE and HLA-DR-FITC. Mean fluorescence intensity (B and C) is reported in brackets.
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