Ag-specific cytokine production and CD137 up-regulation are acDC-amplified in PBMCs and whole blood. (A) Intracellular IFN-γ/TNF-α staining performed on PBMCs in the absence (top row) or presence (bottom row) of the acDC cocktail. After 48 hours of incubation, nonadherent cells were stained for intracellular cytokines accumulated during a final 6 hours of incubation with brefeldin A. Percentages indicate cytokine-positive events among CD4⁺ (left) or CD8⁺ (right) T cells (absolute cell numbers are shown in brackets). (B) CD137 up-regulation detected on PBMCs with (bottom row) or without (top row) acDC amplification during a 48 hours of culture, as above. (C) Whole blood from the same draw was assayed in parallel by adding Ags with (bottom) or without (top) acDC cytokines. Dot plots were gated on CD4⁺ (left) or CD8⁺ (right) T cells to allow comparison between PBMCs and whole blood. Percentages indicate the CD137⁺ fraction among CD4⁺ or CD8⁺ T cells (absolute cell numbers are shown in brackets). (D) Whole blood (250 μL) was cultured for 48 hours with or without TTX in the presence (squares) or absence (circles) of acDC cocktail. Cytokines secreted in plasma supernatants were measured; only those significantly increased in response to Ag are shown. Results are shown as TTX-stimulated cytokine concentrations (filled symbols) and basal values in the absence of Ag (open symbols). Coefficients of variation among triplicate samples were 6.1%-16.2% (not shown). (E) Ag-specific IL-1β production by acDCs. After a 48-hour acDC stimulation, total PBMCs were collected and treated with brefeldin A for 6 hours. Intracellular IL-1β expression in CD11c⁺ cells (gated on CD14⁻CD19⁻CD8⁻CD4^lowHLA-DR⁺ events) and CD4⁺ cells (gated on CD14⁻CD19⁻CD8⁻CD11c^low events) is shown. Results refer to representative experiments of 10 or more performed using the anti-CD40/IFN-α maturation cocktail.