G-CSF–mobilized slanDCs retain their proinflammatory capacity. (A) Freshly purified PBMCs before and on day 5 of G-CSF treatment were stimulated as indicated. Intracellular cytokine staining was performed 18 hours after culture in the presence of brefeldin A.^12,13  One representative donor is shown. The mean fluorescence intensity (MFI) is indicated for each dot plot. (B) MFI results of 6 donors. Each symbol represents a single sample. Horizontal lines in each series represent median values (n = 6). (C) SlanDCs were isolated from PBSC donors before and after G-CSF treatment, using immunomagnetic beads. SlanDCs of each donor were incubated with identical naive CD4⁺CD45RA⁺ cord blood cells at both time points. Graded numbers of slanDCs were cocultured with 10⁵ naive T cells in triplicate for 5 days, and cultures were pulsed with ³H-thymidine for the last 16 hours. SlanDCs were added to the culture, either freshly isolated (immature) or after 6 hours of preculture (mature). In the left panel, results of 6 donors are shown. The right panel shows the T-cell stimulatory capacity of immature and mature slanDCs cultured at a ratio of 1:10 with cord blood T cells. Data are presented as mean counts per minute (cpm ± SEM). (D) Blood was drawn from 5 PBSC donors prior to and on day 5 of G-CSF therapy. SlanDCs were isolated and cocultured with allogeneic naive T cells again at a ratio of 1:10. Cells were incubated in the presence of LPS for 10 days. T cells were restimulated with PMA and ionomycin on day 10 of coculture. Supernatants were harvested after 24 hours and levels of secreted IFN-γ and IL-4 were measured by ELISA. Values are indicated for each individual donor before and after G-CSF (n = 5).