Figure 4.
DCs derived from monocytes primed by cell-to-cell contact with NK cells display a unique transcriptional program and enhanced antigen-presenting capacity. (A) Immature DCs derived from NK-cell–primed CD14+CD16− monocytes were obtained after NK-cell depletion and 5-day treatment with IL-4 and GM-CSF (NK-iDCs). Immature DCs derived from monocytes alone (iDCs) or primed by NK cells through a Transwell membrane (NK//iDCs) were used as controls. For some experiments, immature DCs were activated for 24 hours using TNF-α. (B) DCs at fifth day of differentiation, photographed at an original magnification of ×20 using an inverted microscope. After differentiation, iDCs were sorted by flow cytometry and total RNA was extracted for the determination of gene expression by microarray. (C) Transcriptional heterogeneity between DCs determined by principal component analysis (PCA) of differentially expressed genes (P < .005). (D) Heatmaps for differentially expressed genes (P < .005) of DCs derived from monocytes primed by NK cells through cell-cell contact (NK-iDCs) vs Transwell contact (NK//iDCs); control iDCs vs Transwell contact (NK//iDCs). (E) Transcriptional network upregulated in DCs derived from NK cell-primed monocytes (NK-iDCs). After differentiation, the surface phenotype of iDCs was analyzed by flow cytometry. (F) HLA-DR histograms and expression (MFI) of iDCs at different conditions. (G) Dot plot and frequency of CD86+ CD80+ iDCs at different conditions. Untreated and TNF-α–treated iDCs from different conditions were cocultured with purified, CFSE-stained naive allogeneic T cells for 5 days. Unstimulated T cells and T cells stimulated with α-CD3/α-CD28 beads were used as negative and positive proliferation controls, respectively. (H) Dot plots showing total CD3 T proliferation (CFSE dilution) induced by DCs from different conditions by the fifth day, assessed by flow cytometry; proliferation index obtained in each condition determined in FlowJo. (I) Frequency of IL-15RA+ and IL-6+ DCs derived from NK-primed monocytes, assessed by flow cytometry after 48 hours of coculture with purified naive allogeneic T cells. Error bars represent standard deviation. Data are representative of at least 2 or more independent experiments combined; n = 2-3 HDs per experiment. Statistical comparisons were performed with the unpaired Student t test with Welch correction. *P < .05, **P < .01, ***P < .001.

DCs derived from monocytes primed by cell-to-cell contact with NK cells display a unique transcriptional program and enhanced antigen-presenting capacity. (A) Immature DCs derived from NK-cell–primed CD14+CD16− monocytes were obtained after NK-cell depletion and 5-day treatment with IL-4 and GM-CSF (NK-iDCs). Immature DCs derived from monocytes alone (iDCs) or primed by NK cells through a Transwell membrane (NK//iDCs) were used as controls. For some experiments, immature DCs were activated for 24 hours using TNF-α. (B) DCs at fifth day of differentiation, photographed at an original magnification of ×20 using an inverted microscope. After differentiation, iDCs were sorted by flow cytometry and total RNA was extracted for the determination of gene expression by microarray. (C) Transcriptional heterogeneity between DCs determined by principal component analysis (PCA) of differentially expressed genes (P < .005). (D) Heatmaps for differentially expressed genes (P < .005) of DCs derived from monocytes primed by NK cells through cell-cell contact (NK-iDCs) vs Transwell contact (NK//iDCs); control iDCs vs Transwell contact (NK//iDCs). (E) Transcriptional network upregulated in DCs derived from NK cell-primed monocytes (NK-iDCs). After differentiation, the surface phenotype of iDCs was analyzed by flow cytometry. (F) HLA-DR histograms and expression (MFI) of iDCs at different conditions. (G) Dot plot and frequency of CD86+ CD80+ iDCs at different conditions. Untreated and TNF-α–treated iDCs from different conditions were cocultured with purified, CFSE-stained naive allogeneic T cells for 5 days. Unstimulated T cells and T cells stimulated with α-CD3/α-CD28 beads were used as negative and positive proliferation controls, respectively. (H) Dot plots showing total CD3 T proliferation (CFSE dilution) induced by DCs from different conditions by the fifth day, assessed by flow cytometry; proliferation index obtained in each condition determined in FlowJo. (I) Frequency of IL-15RA+ and IL-6+ DCs derived from NK-primed monocytes, assessed by flow cytometry after 48 hours of coculture with purified naive allogeneic T cells. Error bars represent standard deviation. Data are representative of at least 2 or more independent experiments combined; n = 2-3 HDs per experiment. Statistical comparisons were performed with the unpaired Student t test with Welch correction. *P < .05, **P < .01, ***P < .001.

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