CD34⁺-derived int-DCs sustain viability of L-DCs.

(A) Schematic description of the experiment. CD34⁺progenitor cells were cultured in SCF, GM-CSF, and TNFα, and at day 6 the CD1a⁺CD14⁻ and the CD1a⁻CD14⁺ precursors were cell sorted. L-DCs were generated from the CD1a⁺CD14⁻ precursors in the presence of GM-CSF,25 and CD34⁺-derived int-DCs were generated from the CD1a⁻CD14⁺precursors by culture in M-CSF. L-DCs and CD34⁺-derived int-DCs were washed and cocultured for 2 days in medium lacking cytokines. (B) L-DCs expressed CD1a and RANK but lacked CD14 and TRANCE. White histograms represent isotype controls, and specific labeling is shown in gray. (C) Percentage cell viability of int-DCs and L-DCs after 2 days in cytokine-free medium: int-DCs and L-DCs alone, L-DCs with soluble TRANCE, coculture of in-DCs and L-DCs in the presence of IgG1 or RANK-Fc. Cell viability was determined by trypan blue exclusion of dead cells. The results are expressed as the means of 5 independent experiments with SD of the data. (D) Double-labeling of cells in the cocultures using anti–Bcl-2-FITC and anti–CD14-PE or anti–CD1a-PE. Percentages of labeled cells are indicated in the quadrants. The data are representative of 3 independent experiments.