Figure 1
Figure 1. LSCs program Lin−CD117+ progenitor differentiation into DC-like cells. (A) Differentiation of bone marrow–derived Lin−CD117+ progenitor on the monolayer of LSCs at different times. CoolSNAP cf camera (Roper Scientific Photometrics, Tucson, AZ) mounted on a microscope (Leica-DMIRB) and Meta Imaging Series 5.0 (Molecular Devices, Downingtown, PA) were used to acquire images. Original magnification, 40× objective. (B) Morphology of DC-like cells differentiated from progenitors under liver stroma (we refer to them as LRDCs) and cDCs under phase-contrast microscope, transmission electron microscope (Philips CM120; Philips, Eindhoven, The Netherlands), and Wright staining after stimulation with or without LPS. Original magnification, 40× objective (phase-contrast microscope). Scale bars equal 2 μm (transmission electron microscope); 100× oil objective (Wright staining). (C) Phenotype of LRDCs and cDCs. Open histograms represent isotype control; numbers in histograms indicate the geometric mean fluorescence. (D) Phenotype of LRDCs matured with 0 ng/mL LPS (black line), 1 ng/mL LPS (dashed line) or 100 ng/mL LPS (long dotted line). Short dotted line represents isotype control. (E) The phagocytosis ability of LRDCs was assessed by incubating with 100 mg/mL FITC-conjugated BSA at 37°C or 4°C for 4 hours. Numbers in histogram indicate geometric mean fluorescence. (F,G) Expression of IL-10 and IL-12p70 by LRDCs stimulated with LPS was measured by ELISA (F) and intracellular cytokine staining (G). (H) Antigen-presenting ability of LRDCs. CD4 T cells from DO11.10 × C57BL/6 F1 hybrid mice cocultured with LRDCs, LPS-stimulated LRDCs (LRDC-LPS), and cDCs in the presence of OVA323-339 peptide or cocultured with OVA protein–loaded LRDCs (OVAp-LRDC), OVA323-339 peptide–loaded LRDCs (OVA-LRDC)/cDC(OVA-cDC). cDCs or LRDCs were pulsed with 2 μM OVA323-339 peptide or 5 μM OVA protein at 37°C for 6 hours, then washed with cold PBS 3 times. After 5 days, the total number of live CD4 T cells was counted by FACS, and IL-2 was analyzed by ELISA. Data represent mean plus or minus SD of triplicate wells and are representative of at least 3 independent experiments with similar results. *P < .001.

LSCs program Lin−CD117+ progenitor differentiation into DC-like cells. (A) Differentiation of bone marrow–derived Lin−CD117+ progenitor on the monolayer of LSCs at different times. CoolSNAP cf camera (Roper Scientific Photometrics, Tucson, AZ) mounted on a microscope (Leica-DMIRB) and Meta Imaging Series 5.0 (Molecular Devices, Downingtown, PA) were used to acquire images. Original magnification, 40× objective. (B) Morphology of DC-like cells differentiated from progenitors under liver stroma (we refer to them as LRDCs) and cDCs under phase-contrast microscope, transmission electron microscope (Philips CM120; Philips, Eindhoven, The Netherlands), and Wright staining after stimulation with or without LPS. Original magnification, 40× objective (phase-contrast microscope). Scale bars equal 2 μm (transmission electron microscope); 100× oil objective (Wright staining). (C) Phenotype of LRDCs and cDCs. Open histograms represent isotype control; numbers in histograms indicate the geometric mean fluorescence. (D) Phenotype of LRDCs matured with 0 ng/mL LPS (black line), 1 ng/mL LPS (dashed line) or 100 ng/mL LPS (long dotted line). Short dotted line represents isotype control. (E) The phagocytosis ability of LRDCs was assessed by incubating with 100 mg/mL FITC-conjugated BSA at 37°C or 4°C for 4 hours. Numbers in histogram indicate geometric mean fluorescence. (F,G) Expression of IL-10 and IL-12p70 by LRDCs stimulated with LPS was measured by ELISA (F) and intracellular cytokine staining (G). (H) Antigen-presenting ability of LRDCs. CD4 T cells from DO11.10 × C57BL/6 F1 hybrid mice cocultured with LRDCs, LPS-stimulated LRDCs (LRDC-LPS), and cDCs in the presence of OVA323-339 peptide or cocultured with OVA protein–loaded LRDCs (OVAp-LRDC), OVA323-339 peptide–loaded LRDCs (OVA-LRDC)/cDC(OVA-cDC). cDCs or LRDCs were pulsed with 2 μM OVA323-339 peptide or 5 μM OVA protein at 37°C for 6 hours, then washed with cold PBS 3 times. After 5 days, the total number of live CD4 T cells was counted by FACS, and IL-2 was analyzed by ELISA. Data represent mean plus or minus SD of triplicate wells and are representative of at least 3 independent experiments with similar results. *P < .001.

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