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CD34+ Hematopoietic Progenitors From Human Cord Blood Differentiate Along Two Independent Dendritic Cell Pathways in Response to Granulocyte-Macrophage Colony-Stimulating Factor Plus Tumor Necrosis Factor α: II. Functional Analysis

Christophe Caux, Catherine Massacrier, Béatrice Vanbervliet, Bertrand Dubois, Isabelle Durand, Marina Cella, Antonio Lanzavecchia and Jacques Banchereau

Article Figures & Data

Figures

  • Fig. 1.

    Efficient naive T-cell proliferation is induced by CD1a-or CD14-derived DCs. Cord blood CD34+ HPCs were cultured in the presence of GM-CSF plus TNFα. After 5 to 6 days cells were collected, processed for double staining using anti-CD14–PE and anti-CD1a–FITC, and FACS-sorted into CD14CD1a+ and CD14+CD1a. Sorted cells were seeded in the presence of GM-CSF plus TNFα (1 to 2 × 105 cells/mL) for 6 to 7 additional days, with last medium changes being performed at day 10. (A and B) At the indicated time points, independent aliquots of cells were harvested and used, after irradiation (30 Gy), as stimulator cells for CD45RA+ CD4+ naive cord blood T cells (2 × 104 cells/well). For day 0, uncultured CD34+ HPCs were used as stimulator cells. T cells from the same batches were used each time after thawing. Proliferation of allogeneic T cells induced by 103 stimulator cells is shown (A). Proliferation of syngeneic T cells in absence or presence of 1 ng/mL SEA induced by 102 stimulator cells is shown in (B). (C and D) Cells were recovered at day 12 and used after irradiation (30 Gy) as stimulator cells for cord blood CD45RA+ CD4+ T cells (2 × 104 cells/well; C) or cord blood CD8+ T cells (2 × 104 cells/well; D). Proliferation was shown by 3H-TdR uptake after 5 days of culture. Results are expressed as mean cpm ± SD of triplicate cultures. Results of each panel are representative of three experiments or more.

  • Fig. 2.

    Both CD1a- or CD14-derived DC-induced T-cell priming are inhibited by anti-CD86 or IL-10. CD1a- and CD14-derived DC subsets were obtained as described in Materials and Methods and in the legend to Fig 1. At day 14, cells were used after irradiation (30 Gy) as stimulator cells (103 cells/well) for cord blood CD4+ T cells (2 × 104 cells/well). (A) Experiments were performed in the presence of 10 μg/mL of anti-CD80 (Mab 104), anti-CD86 (IT2-2), anti-CD80 plus anti-CD86, or isotype match control. (B) Experiments were performed in the presence or absence of IL-10 (50 ng/mL). Proliferation was shown by 3H-TdR uptake after 5 days of culture. Results are expressed as mean cpm ± SD of triplicate cultures. Results of each panel are representative of three experiments.

  • Fig. 3.

    Differential uptake of FITC dextran by CD1a- and CD14-derived DCs during maturation. Cord blood CD34+ HPCs were cultured in the presence of GM-CSF plus TNFα. After 5 to 6 days, cells were collected and half were processed for double staining, using anti-CD14–PE and anti-CD1a–FITC, and FACS-sorted into CD14+CD1a. Half of the cells were processed for double staining, using anti-CD14–FITC and anti-CD1a–PE, and FACS-sorted into CD14CD1a+. Using this procedure, the sorted populations were labeled with PE, thus, avoiding interferences with FITC dextran. Sorted cells were seeded in the presence of GM-CSF plus TNFα (1 to 2 × 105 cells/mL) for 6 to 7 additional days with last medium changes being performed at day 10. At the indicated time points, cells were processed for FITC dextran uptake. Cells were incubated in medium containing 0.1 mg/mL FITC dextran at 37°C for 15 minutes, washed with cold medium, and analyzed on a FACScan. For days 4 and 6, FITC dextran uptake was performed on total populations by double color fluorescence using anti-CD1a–PE or anti-CD14–PE. The same parameters of the FACScan were used during the kinetic. Results are representative of three independent experiments.

  • Fig. 4.

    Parameters of FITC dextran accumulation in day-6 CD1a-DC precursors and day-11 CD14-derived DCs. (A) MFI of histograms shown in Fig 3. The background fluorescence (cells pulsed at 4°C) was subtracted. (B and C) Cells were pulsed with 0.1 mg/mL FITC dextran at 37°C for various times, washed with cold medium, and analyzed on a FACScan. Uptake of FITC dextran was performed at day 6 on total population by double color fluorescence using, after pulse with FITC dextran, anti-CD1a–PE (CD1a+ cells), anti-CD14–PE (CD14+ cells), and anti-CD1a–PE plus anti-CD14–PE (CD14CD1a cells; B). Uptake of FITC dextran was performed at day 11 by single color fluorescence on the sorted populations (C). (D and E) Cells were pulsed with 0.1 mg/mL FITC dextran for 15 minutes at 37°C, washed with cold medium, and analyzed immediately or after culture at 37°C for various times in marker-free medium. Chase was performed at day 6 on total populations by double color fluorescence (D). Chase was performed at day 11 by single color fluorescence on the sorted populations (E). Results of each panel are representative of three experiments or more.

  • Fig. 5.

    FITC dextran and HRP uptake by the two DC subpopulations is mediated through receptors for mannose polymers. The experiments of FITC dextran uptake were performed on day-6 CD1a precursors by double color fluorescence (A and C) and on day-11 CD14-derived DCs (B and D; see legend to Figs 3 and 4). The uptake of HRP was performed on day-6 and day-11 CD1a+- and CD14-derived cells (E and F ). (A and B) Cells were pulsed for 15 minutes at 37°C with various concentrations of FITC dextran in the absence or in the presence of 1 mg/mL mannan. (C and D) Cells were pulsed for various times at 37°C with 0.1 mg/mL FITC dextran in the absence or in the presence of 1 mg/mL mannan. (E and F ) Cells were pulsed for 15 minutes at 37°C with various concentrations of HRP in the absence or in the presence of 1 mg/mL mannan. Results of each panel are representative of three experiments or more.

  • Fig. 6.

    FITC dextran and HRP uptake by the two DC subpopulations are mediated through mannose receptors. (A and B) The experiments were performed on day-6 CD1a precursors by double color fluorescence (A) and on day-11 CD14-derived DCs (B). Cells were pulsed for 15 minutes at 37°C with 0.1 mg/mL FITC dextran in the presence of medium, 10% supernatant MoAb anti-mannose receptor (anti-MR), 50% supernatant MoAb anti-MR, or 1 mg/mL mannan. (C) Cells were processed at day 6 for double staining using anti-MR shown by PE-conjugated antimouse Ig. Then, after saturation in 5% mouse serum, cells were stained with anti-CD14–FITC or anti-CD1a–FITC. A total of 20,000 events were acquired. Histograms show PE staining gated on CD1a+ cells (left) or on CD14+ cells (right). (D) CD1a- and CD14-derived DC subsets, obtained as described in Materials and Methods and in the legend to Fig 1, were processed at day 11 for single staining using anti-MR shown by PE-conjugated antimouse Ig. A total of 5,000 events were acquired. White histograms represent isotype matched control. Results of each panel are representative of three experiments or more.

  • Fig. 7.

    Only CD14-derived DCs display nonspecific esterase activity. The CD1a- (A, B, C, D, and E) and CD14- (F, G, H, I, and J) derived DC subsets were obtained as described in Materials and Methods and in the legend to Fig 1. Cells were processed at day 6 (A and F ), day 8 (B and G), day 10 (C and H), day 13 (D and I), and day 16 (E and J) for nonspecific esterase staining. Original magnification × 400. Results are representative of three experiments.

  • Fig. 8.

    Only CD14-derived DCs can induce CD40-activated naive B cells to differentiate into IgM secreting cells. The CD1a- and CD14-derived DC subsets were obtained as described in Materials and Methods and in the legend to Fig 1. A total of 104 highly purified IgD+ B cells were cultured over 2,500 irradiated CD40-L–transfected L cells in the presence or absence of DC subsets. Proliferation was measured by 3H-TdR incorporation at day 6. Supernatants were harvested after 15 days of culture and assayed for the presence of IgM. (A) DC subsets were recovered at day 12 and used as stimulator cells (3,000 cells/well) for IgD+ B-cell proliferation in the absence or presence of 20 U/mL IL-2. (B) DC subsets were recovered at day 12 and used as stimulator cells (3,000 cells per well) for IgD+ B cell differentiation in the absence or presence of 20 U/mL IL-2. (C) DC subsets were recovered at day 12, and 300, 1,000, and 3,000 cells/well were used as stimulator cells for IgD+ B-cell differentiation in the presence of 20 U/mL IL-2. (D) DC subsets were recovered at the time point indicated and used as stimulator cells (3,000 cells/well) for IgD+ B-cell differentiation in the presence of 20 U/mL IL-2. IgM levels are expressed as mean ± SD of triplicate cultures. Results of each panel are representative of three experiments or more.

  • Fig. 9.

    CD40 triggering upregulates CD25 specifically on CD14-derived DCs. The CD1a- (upper panels) and CD14- (lower panels) derived DC subsets were obtained as described in Materials and Methods and in the legend to Fig 1. At day 12, DC subsets were recovered and cultured over CD40-L–transfected L cells on control L cells (2 × 104 LCs for 1 × 105 DCs) in the presence of GM-CSF for 4 days. Then cells were processed for staining using anti-CD25–PE, anti-CD86–PE, and isotype match control. A total of 5,000 events were acquired. White histograms show phenotype over control L cells and black histograms show phenotype over CD40-L transfected L cells. Results are representative of three experiments.

  • Fig. 10.

    Only the CD14-derived DCs bind immune complexes. The CD1a- and CD14-derived DC subsets were obtained as described in Materials and Methods and in the legend to Fig 1. At days 6 and 12, DC subsets were recovered and were incubated at 4°C for 15 minutes with 5 μg/mL FITC-coupled streptavidin and 100 μg/mL mouse IgG1 MoAb anti-FITC. (A) Day 6, (B) day 12, and (C) day-12 CD14-derived cells were first incubated for 15 minutes with 50 μg/mL of a blocking MoAb against CD32 followed by 15 minutes with 5 μg/mL FITC-coupled streptavidin and 100 μg/mL mouse IgG1 MoAb anti-FITC. White histograms show FITC streptavidin alone. FITC-coupled isotype match controls were superposable to the white histograms.

  • Fig. 11.

    Evolution of functions during DC subpopulation maturation. The figure represents parameters of DC activities as a function of time. MFI of CD86 illustrates the progression of DC maturation. FITC dextran illustrates the capacity of antigen uptake. NSE illustrates the lysosomal activity. T-cell alloreaction illustrates the capacity to activate naive T cells. B-cell differentiation illustrates the capacity to induce naive B-cell differentiation in response to IL-2. To plot the different parameters on a same scale, values were calculated as the following ratio: (experimental value/maximal value) × 100; the maximal value being the maximal value obtained with either CD1a or CD14 populations. (A) The evolution of the CD1a-derived cells activities. (B) The evolution of the CD14-derived cells activities.