In vitro function of DCs. (A) 5 × 10⁴ naive T cells were incubated with graded amounts of irradiated cells obtained after 4 weeks of culture with FLT3-L plus TPO (○). Naive T cells were also incubated with cells that were additionally cultured with GM-CSF plus IL-4 for 5 days (⧫) or with GM-CSF plus IL-4 for 2 days followed by 3 days with the latter cytokines plus TNF (▪). Measurements were done in triplicates, and the last significant value over background (horizontal line) was determined as the ³H-TdR incorporation of T cells measured in absence of DCs plus 3 SD. Mean ± SD of one representative experiment out of five are shown. (B) Internalization of FITC-dextran by CD1a⁺ cells. Cells from a 4-week FLT3-L plus TPO culture were incubated for 2 days in GM-CSF plus IL-4 (left histogram) or for 5 days with the same cytokines, with adjunction of TNF for the last 3 days (right histogram). Cells were then incubated with FITC-dextran for 1 hour at 4°C (thin lines) or 37°C (bold lines) before labeling with PE-conjugated anti-CD1a antibody. This result is from one experiment representative of four. Experiments performed with sorted cells obtained as in Fig 6 gave similar results, that is, incubation in TNF always decreased dextran internalization irrespective of the origin of the CD1a⁺ cells.