Figure 2. Maturation of CD137L-derived dendritic cells. (A–C) Monocytes were treated with immobilized CD137-Fc for 6 d and then exposed to the indicated maturation cocktails (see also Table 1) for 18 h, followed by a phenotypic and functional characterization. (A) Cells were immunostained for the CD83, CD86, and HLA-DR cell-surface expression and analyzed by fluorescence cytometry. Unshaded and gray histograms represent the unstained (control) and stained samples, respectively. Values associated with each histogram indicate the percentages of positive cells and mean fluorescence intensity (MFI). Bar charts on the right report the mean percentage of positive cells and mean MFI ± SD of data acquired from 2 different donors. (B) Upon 18 h of treatment with cocktails C0, C1, C3, or C4, CD137L-derived dendritic cells (CD137L-DCs) were co-cultured for additional 5 d with carboxyfluorescein succinimidyl ester (CFSE)-stained allogeneic T cells at a ratio of 1:10. T-cell proliferation was then quantified by fluorescence cytometry based on CFSE dilution. Numbers in the histograms and bar charts represent percentages of proliferating cells and CFSE MFI. (C) Supernatants from the co-cultures of mature CD137L-DCs and allogeneic T cells were harvested and the levels of interferon γ (IFNγ) were quantified by ELISA. Means ± SD of triplicate measurements are depicted. **P < 0.01 (two-tailed, unpaired Student t test). This experiment has been performed independently twice, yielding with comparable results. PHA, phytohemagglutinin; Rst, resting.