Inhibition of DNA synthesis and cell cycle progression by TRAIL. Splenocytes were prepared from 6–8-wk-old BALB/c mice (The Jackson Laboratory) and cultured in DMEM for 3 d in the presence of 10% FBS and 2.5 μg/ml of Con A. Live cells were then purified through a Ficoll gradient and cultured in 96-well plates at 3 × 105 cells per well in 200 μl of DMEM containing 10% FBS, with or without the following reagents: 100 ng/ml of TRAIL, 5 μg/ml of sDR5, 5 μg/ml of anti-CD95L mAb (MFL-3), and anti–mouse CD3 mAb (which was coated on the plate by preincubating the plate with 10 μg/ml of the antibody at 4°C for 16 h). For apoptosis and cell cycle analyses (A and B), cells were cultured for a total of 24 h, fixed in 70% ethanol, and stained with 50 μg/ml of propidium iodide. For thymidine incorporation assays (C and D), cells were cultured for 24 h, then pulsed with 10 μCi/ml of [3H]thymidine for an additional 16 h. Cells were then harvested, and radioactivity was determined using a Wallac β-plate counter. (A) Percentage of apoptotic cells as determined by flow cytometry. The spontaneous apoptotic rate in cultures containing no anti-CD3 mAb was 12%, which was subtracted from the data presented here. For cultures that contained TRAIL and anti-CD3 mAb, the percentage of anti-CD3–induced apoptosis was 16 ± 1%, which was comparable to that of the cultures treated with anti-CD3 mAb alone. The differences between anti-CD95L mAb–treated culture and all other cultures are statistically significant as determined by ANOVA (P < 0.0001). (B) Number of S-G2/M cells per well as determined by flow cytometry. For cultures that contained TRAIL and anti-CD3 mAb, the number of cells in the S-G2/M phases was 70–90% of that of the anti-CD3 mAb–treated culture. The total numbers of live cells per well recovered from each group were as follows: cultures with anti-CD3 mAb alone, 0.3 × 105; cultures with anti-CD3 mAb plus sDR5, 1.6 × 105; cultures with anti-CD3 mAb plus anti-CD95L mAb, 1.1 × 105; cultures with anti-CD3 mAb plus sDR5 plus TRAIL, 0.6 × 105. The differences between all four groups are statistically significant as determined by ANOVA (P < 0.01). (C and D) DNA synthesis as determined by [3H]thymidine incorporation. Data presented are means cpm ± SD of triplicate cultures. For cultures containing anti-CD3 mAb, the differences between the two groups are statistically significant as determined by ANOVA (P < 0.01). The experiments were repeated twice with similar results. The concentrations of TRAIL and sDR5 used in these experiments were selected based on the dose-dependency studies performed in our laboratory. When 0.2–1 μg/ml of sDR5 was used, less significant effects on cell cycle progression were observed. Similarly, when 50–150 ng/ml of TRAIL was tested, much less significant effects on cell cycle were detected (data not shown).