Generation, isolation, and characterization of different DC subsets.

(A) A brief scheme for the preparation of each DC subset. Each subset of DCs was prepared as described in “Materials and methods.” CD1a⁺ DCs and CD14⁺ DCs were generated from CD34⁺ hematopoietic progenitor cells in the presence of TNF-α/GM-CSF and TNF-α/IL-3, respectively. CD1a⁺ DCs and CD14⁺ DCs were sorted according to their surface phenotype at day 8 and day 18. CD11c⁻ DCs were either freshly isolated from PBMCs at day 0 or matured for 5 days in the presence of IL-3 and CD40L. MoDCs were derived from monocytes by culturing with IL-4 and GM-CSF for 7 days and then cultured for 2 more days for maturation in monocyte-conditioned medium supplemented with TNF-α. Lin⁻ means TCR⁻CD14⁻CD16⁻CD19⁻CD56⁻. (B) Surface phenotype of each DC subset in immature and mature stages. Cells were stained with fluorescence-conjugated monoclonal antibodies (Becton Dickinson) shown above each image. Photographs of the DC subset on the right were taken on day 18 for CD1a⁺ DCs and CD14⁺ DCs, day 5 for CD11c⁻ DCs, and day 9 for MoDCs, respectively (magnification × 400).