At least 4 days of exposure to TNF-α are required for full maturation of DCs.

(A) Accumulation of DX-FITC in DCs. Immature DCs (top panel) cultured for 8 days in IL-4 and GM-CSF, or mature DCs (bottom panel) cultured for 4 days in IL-4 and GM-CSF plus 4 days in IL-4 and GM-CSF and TNF-α, were evaluated for their ability to endocytose DX-FITC as a function of time of exposure to TNF-α. For mature DCs, day 5 represents 1 day of exposure to TNF-α; day 6, 2 days of exposure to TNF-α; day 7, 3 days of exposure to TNF-α; and day 8, 4 days of exposure to TNF-α. The dark line represents DX-FITC uptake by samples incubated at 37°C, whereas the light line depicts DX-FITC uptake by samples incubated at 4°C as a negative control. These results are representative of 3 separate experiments with 3 different donors. (B) CD4⁺ T-cell stimulatory capacity of irradiated immature DCs (top) or mature DCs (bottom) in an allogeneic mixed lymphocyte reaction, when harvested at days 6, 7, and 8 of their culture as described in panel A. Frozen aliquots of responder CD4⁺ T cells from the same allogeneic donor were used for each time point. These results are the mean of triplicate wells and are representative of 2 different experiments.