Function of in vivo FL-generated DCs.

(A) Comparison of alloreactive T-cell–stimulating capacity of the various PBMC populations depicted in Figure 1B. Sorted CD11c⁺ CD14⁺ monocytes (•), CD11c⁺ CD14⁻ DCs (○), and CD11c^lo CD14^lo neutrophils (▪) from day-15 PB samples were compared with control CD1a⁺ DCs generated in vitro from CD34⁺ BM progenitors (Δ). Data represent the mean ± SEM of triplicate wells. Results are representative of 4 separate experiments. (B) Comparison of Ag-induced T-cell proliferation by purified CD11c⁺ CD14⁻ DCs and CD11c⁺CD14⁺ monocytes from FL-treated individuals. CD11c⁺ CD14⁻ DCs or CD11c⁺CD14⁺ monocytes were isolated by flow cytometry and cultured with purified autologous T cells derived from cryopreserved PB samples taken before commencement of the study, in the presence of recall Ag (TT) or nominal Ags (Ova, KLH, or HBsAg peptide). Data represent the mean ± SEM of triplicate wells. Results are representative of 3 separate experiments. (C) Comparison of Ag uptake by PBMCs from FL-treated individuals. PBMCs were cultured with FITC-Ova at either 0°C or 37°C for 30 minutes and then incubated with anti-CD14–APC and anti-CD11c–PE. CD11c⁺CD14⁻ DCs, CD11c⁺ CD14⁺monocytes, and the enriched lymphocyte fraction (CD11c⁻ CD14⁻) were examined by flow cytometry for internalized FITC-Ova. Cells incubated at 0°C were used to discriminate between nonspecific cell surface staining and active uptake at 37°C. The data are presented as the mean fluorescence intensity (MFI) ± SEM from 3 FL-treated individuals. Results are representative of 3 separate experiments.