DCs obtained by TNF-α or CD40LT exposure express characteristics of mature DCs.

(A) Monocytes were cultured for 4 days in AIM V containing 3% human AB serum, IL-4, and GM-CSF, as described in “Materials and methods.” TNF-α (●) was added at day 4, whereas PMA (▪) and CD40LT (▴) were added at day 6 as maturation stimuli. As a control, we used DCs to which no maturation signals were added (○). Cells were harvested at day 8, irradiated, and tested for CD4⁺ T-cell stimulatory capacity in a primary allogeneic mixed lymphocyte reaction at the indicated T/DC ratio. These results are the mean of round-bottom triplicate wells and are representative of 3 different experiments with different donors. (B) Flow cytometric analysis of immature DCs (■) or DCs matured by TNF-α (▧), CD40LT (▪), and PMA (░), as described in panel A. DCs were double-stained with a panel of phycoerythrin (PE)–conjugated monoclonal antibodies (mAbs) and with fluorescein isothiocyanate (FITC)–conjugated mAb HLA-DR. Cells were also stained with isotype controls for each PE- and FITC-conjugated mAb. Results are expressed as percentage of positive cells for each marker, in comparison with its isotype control. These results are representative of 3 different experiments with different donors.