American Society of Hematology

Figure 2

$Figure 2. VDR promotes TGF-β1–dependent LC differentiation. (A) Human infant foreskin assessed by immunofluorescence staining of cryosections for the expression of CD45 (blue), CD207 (red), and VDR (green). Immunostained sections were analyzed on a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss GmbH) using immersion oil as imaging media and a 40×1.3 NA oil immersion objective (Plan-Neofluar; Zeiss). FITC, PE, APC channels were acquired by sequential scanning using 488-, 543-, or 633-nm laser lines for excitation; HFT 488, HFT 488/543, or HFT 514/633 beam-splitters for separation; and BP 505-530 nm, BP 560-615 nm, or LP 650 nm and Photomultiplier (PMT) for detection of the signals. Scale bar, 50 μm. The inset shows an enlarged view of part of the figure. (B) CD34+CD45RA+CD19− umbilical cord blood cells were sorted by FACS and cultivated with the indicated cytokines. Cell numbers of sorted fractions ranged from 7.25 × 104 to 2.3 × 105 (mean, 1.3 × 105; n = 6). Cultures were initiated with 1.2 × 104 to 4.1 × 104 cells (mean, 2.3 × 104; n = 6). FACS diagrams represent day 7–generated cells analyzed for informative marker molecules (± TGF-β1). In the bar diagram, VDR mRNA levels were determined by quantitative real-time RT-PCR at 0, 6 and 24 hours. Values are normalized to HPRT (n = 4). (C) CD34+ cells were transduced with a retroviral vector encoding VDR-IRES-GFP or empty control vector (CTRL) and were then cultured in LC conditions (GM-CSF, SCF, FL, TNFα) in the presence (top panel) or absence (bottom panel) of TGF-β1. Day 7–generated GFP+ cells were analyzed for CD1a versus CD207. Bars represent the mean of percentages (± SEM) CD1a+CD207+ cells observed in 4 independent experiments. (D) CD34+ cells were transduced with a retroviral vector encoding VDR-IRES-GFP or empty control vector (CTRL) and were then cultured in moDC conditions (see Figure 1A). Day 7–generated GFP+ cells were analyzed for CD11b versus CD207. Data are representative of 3 independent experiments. (B-D) Each number in a quadrant represents the percentage of cells in the quadrant. (E) LCs were generated from CD34+ cells in the absence or presence of the VDR antagonist ZK159222 (10−6 M). At day 7 cells were harvested and analyzed for CD207 versus CD1a. Data are representative of 3 independent experiments.$

VDR promotes TGF-β1–dependent LC differentiation. (A) Human infant foreskin assessed by immunofluorescence staining of cryosections for the expression of CD45 (blue), CD207 (red), and VDR (green). Immunostained sections were analyzed on a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss GmbH) using immersion oil as imaging media and a 40×1.3 NA oil immersion objective (Plan-Neofluar; Zeiss). FITC, PE, APC channels were acquired by sequential scanning using 488-, 543-, or 633-nm laser lines for excitation; HFT 488, HFT 488/543, or HFT 514/633 beam-splitters for separation; and BP 505-530 nm, BP 560-615 nm, or LP 650 nm and Photomultiplier (PMT) for detection of the signals. Scale bar, 50 μm. The inset shows an enlarged view of part of the figure. (B) CD34⁺CD45RA⁺CD19⁻ umbilical cord blood cells were sorted by FACS and cultivated with the indicated cytokines. Cell numbers of sorted fractions ranged from 7.25 × 10⁴ to 2.3 × 10⁵ (mean, 1.3 × 10⁵; n = 6). Cultures were initiated with 1.2 × 10⁴ to 4.1 × 10⁴ cells (mean, 2.3 × 10⁴; n = 6). FACS diagrams represent day 7–generated cells analyzed for informative marker molecules (± TGF-β1). In the bar diagram, VDR mRNA levels were determined by quantitative real-time RT-PCR at 0, 6 and 24 hours. Values are normalized to HPRT (n = 4). (C) CD34⁺ cells were transduced with a retroviral vector encoding VDR-IRES-GFP or empty control vector (CTRL) and were then cultured in LC conditions (GM-CSF, SCF, FL, TNFα) in the presence (top panel) or absence (bottom panel) of TGF-β1. Day 7–generated GFP⁺ cells were analyzed for CD1a versus CD207. Bars represent the mean of percentages (± SEM) CD1a⁺CD207⁺ cells observed in 4 independent experiments. (D) CD34⁺ cells were transduced with a retroviral vector encoding VDR-IRES-GFP or empty control vector (CTRL) and were then cultured in moDC conditions (see Figure 1A). Day 7–generated GFP⁺ cells were analyzed for CD11b versus CD207. Data are representative of 3 independent experiments. (B-D) Each number in a quadrant represents the percentage of cells in the quadrant. (E) LCs were generated from CD34⁺ cells in the absence or presence of the VDR antagonist ZK159222 (10⁻⁶ M). At day 7 cells were harvested and analyzed for CD207 versus CD1a. Data are representative of 3 independent experiments.

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