![Fig. 2. Mature DCs, but not immature DCs are resistant to death ligand–induced apoptosis. / (A) d3 cells (upper panel), d7 cells (middle panel), or d10 cells (lower panel) were incubated in the presence of 100 ng/mL LZ-CD95L, 1 μg/mL LZ-TRAIL, or the combination of both for 16 hours and survival of cells was determined by Annexin/propidium iodide staining and FACS. (B) Dose response curves of different populations of DCs (d3, d7, and d10). Cells were treated with the indicated concentrations of LZ-CD95L (square), LZ-TRAIL (triangle), or the combination of both death ligands (rhombs), and survival was determined 16 hours later by Annexin/propidum iodide staining by FACS (shown is a representative experiment [percentage of control survival] of a total of 4 independent experiments with similar results). (C) Cytokine cocktail-induced maturation of DCs is comparable to CD40L-induced maturation, but necessary to induce resistance to CD95L or TRAIL. The d7 DCs were cultured for 3 days either in GM-CSF and Il-4 alone, with addition of cytokine cocktail (see “Materials and methods”) or recombinant LZ-CD40L (1 μg/mL) and analyzed by surface analysis for CD1a, CD25, CD80, and CD83 (upper panel). Cells were subsequently treated with the LZ-CD95L (200 ng/mL), LZ-TRAIL (10 μg/mL), or the combination of both death ligands, and survival was determined 16 hours later by Annexin/propidum iodide staining by FACS as percentage of control (shown is a representative result of a total of 2 independent experiments with similar results). (D) cFLIP expression during maturation of DCs. Seventy-five micrograms of protein of monocyte-derived dendritic cells cultured for 4 days (d4) or 7 days (d7) in the presence of GM-CSF and IL-4 or subsequent 3 days in the presence of IL-1 α (500 IU/mL), IL-6, TNF-α, IL-1 β (all 1000 IU/mL), and PGE2 (10−8 mol/L) were analyzed by Western blotting with cFLIP specific antibodies. Low levels of cFLIPL are expressed in d4 and d7 immature DCs, whereas fully matured DCs (d10) show strong expression of cFLIPL. Reprobing of the membrane with tubulin Ab demonstrates comparable loading of protein.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/96/7/10.1182_blood.v96.7.2628/5/h81700080002.jpeg?Expires=1735555858&Signature=yJa2buBZKr8-tO0AZdn-UGSE~gYvUuIMZ-c7zwayOlAUSDndm1mvNPpuaY2l4b8ewKqLD1EiB8XG98Hq1gbZfVpi99ibuC1CJ51hdhHrEUUYhVBpbgg0fJ9SfZciigSDXD6gqMUblIgIFmVXfH~uQf2znADxe52yvKeXj4u3w8FRvWrY7vPPB0ZGAjnsobiE8UdXu38-X3-ehtf61AH39Ce9BBiSlA91uN1mcVENxq3TuJZz3iH~drft~gmvzA6LUAe364QYEVWOV4vDg6m-HxqzgpPUERmHt62~Ilt7WADu0XQiWlY8cvblqMaDR7E8fd0dCf8yAMd5iH2ZdXe-cA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Mature DCs, but not immature DCs are resistant to death ligand–induced apoptosis.
(A) d3 cells (upper panel), d7 cells (middle panel), or d10 cells (lower panel) were incubated in the presence of 100 ng/mL LZ-CD95L, 1 μg/mL LZ-TRAIL, or the combination of both for 16 hours and survival of cells was determined by Annexin/propidium iodide staining and FACS. (B) Dose response curves of different populations of DCs (d3, d7, and d10). Cells were treated with the indicated concentrations of LZ-CD95L (square), LZ-TRAIL (triangle), or the combination of both death ligands (rhombs), and survival was determined 16 hours later by Annexin/propidum iodide staining by FACS (shown is a representative experiment [percentage of control survival] of a total of 4 independent experiments with similar results). (C) Cytokine cocktail-induced maturation of DCs is comparable to CD40L-induced maturation, but necessary to induce resistance to CD95L or TRAIL. The d7 DCs were cultured for 3 days either in GM-CSF and Il-4 alone, with addition of cytokine cocktail (see “Materials and methods”) or recombinant LZ-CD40L (1 μg/mL) and analyzed by surface analysis for CD1a, CD25, CD80, and CD83 (upper panel). Cells were subsequently treated with the LZ-CD95L (200 ng/mL), LZ-TRAIL (10 μg/mL), or the combination of both death ligands, and survival was determined 16 hours later by Annexin/propidum iodide staining by FACS as percentage of control (shown is a representative result of a total of 2 independent experiments with similar results). (D) cFLIP expression during maturation of DCs. Seventy-five micrograms of protein of monocyte-derived dendritic cells cultured for 4 days (d4) or 7 days (d7) in the presence of GM-CSF and IL-4 or subsequent 3 days in the presence of IL-1 α (500 IU/mL), IL-6, TNF-α, IL-1 β (all 1000 IU/mL), and PGE2 (10−8 mol/L) were analyzed by Western blotting with cFLIP specific antibodies. Low levels of cFLIPL are expressed in d4 and d7 immature DCs, whereas fully matured DCs (d10) show strong expression of cFLIPL. Reprobing of the membrane with tubulin Ab demonstrates comparable loading of protein.
