Cytokine secretion of CD40L-activated CD1a− and CD1a+ monocyte-derived dendritic cells and cocultured activated allogeneic lymphocytes. Sorted CD1a− and CD1a+ immature DCs (2 × 105) were cultured on a monolayer of CD40L (CD40L)–expressing L929 fibroblasts (0.4 × 105) in 24-well plates as described in “Dendritic-cell cultures.” Culture supernatants of activated DCs were collected after 16 hours. Supernatants of cultures containing the parental L929 cells and sorted DCs were used as control to measure the cytokine secretion of immature DCs. (A-C) Mean values and SD of cytokine concentrations measured in the supernatant of nonactivated immature (IDC; □) and activated mature (MDC; ⊡) moDCs were calculated from data obtained in 2 independent experiments by the CBA assay (A-B) or with the cells of 7 individuals by ELISA (C). *P < .05; **P < .001. (D-E) Mean values and SD of cytokine concentrations measured by the CBA assay in the supernatants of CD1a− (□) and CD1a+ (▪) in moDC cultures derived from 7 individuals. (F) Comparison of intracellular IL-12p70 levels measured in CD1a+ (solid line) and CD1a− (thin line) in a typical experiment of 3 by flow cytometry. Dashed lines show fluorescence intensity of cells incubated with isotype-matched control antibody. (G) Mean and SD of biologically active TGF-β1 concentrations were calculated from data measured by ELISA in the acid-treated culture supernatants of 7 individuals. Sorted CD1a− and CD1a+ moDCs were activated by soluble or cell-bound CD40L and used for activating allogeneic lymphocytes. Typical experiments of 3 are documented. (H) Sorted CD1a− and CD1a+ DCs were seeded to flat-bottom 96-well plates at various numbers started at 5 × 104 cell/well, stimulated with 5 μg/mL soluble CD40L, and cocultured with 5 × 105/well allogeneic lymphocytes for 5 days. T-lymphocyte proliferation was measured by 3H-thymidine incorporation added for the last 16 hours of the culture. (I) In a similar assay, IFN-γ concentrations were measured in cell-culture supernatants taken at day 3 of cocultures. (J) CD1a+ and CD1a− moDCs were sorted, plated to 24-well plates, and activated by CD40L-expressing L929 cells for 24 hours. Activated moDCs were cocultured with allogeneic lymphocytes at 10:1 lymphocyte/DC ratios, and secreted IFN-γ was measured in the supernatant at day 3 by ELISA. (K) Cytokine secretion of primed T lymphocytes was also tested in a restimulation assay on day 14, when resting cells were collected, counted, and plated to anti-CD3–coated 96-well plates. In this experiment, IFN-γ secretion was measured 24 hours after anti-CD3 restimulation. (L) The same supernatants were subjected to IL-10 measurements by ELISA. Error bars represent SD of 3H-thymidine incorporation in triplicate cell cultures (H) or SD of cytokine concentration in triplicate samples (G, I-L).
