GM-CSF/IL-4 and maturation factors enhance Ag-specific T-cell stimulation and induce DCs in unfractionated PBMCs. (A-C) IFN-γ (A), IL-10 (B), and IL-4 (C) ELISPOT responses to TTX, KLH, or control Ag obtained by treating PBMCs with selected stimuli (see “Methods” and Figure 1 for details). Dotted lines indicate TTX-specific cytokine signals obtained in the absence of cytokines. *P < .04 for comparison with the “no cytokines” condition. One representative experiment of 3 is shown. (D) Phenotype comparison between acDCs (top) and moDCs (bottom). acDCs were obtained by culturing unfractionated PBMCs for 48 hours with GM-CSF/IL-4 alone (blue profiles) or in combination with TNF-α/PGE₂/IL-1β (added during the last 24 hours; red profiles). moDCs were generated by culturing purified monocytes for 7 days with the same cytokine cocktails (TNF-α/PGE₂/IL-1β added during the last 24 hours). Comparisons with cultures in the absence of cytokines (dotted profiles) and with isotype control staining (shaded profile) are shown (cells gated as CD19⁻CD8⁻CD4^lowCD11c^high). (E) Stimulatory potency in IFN-γ ELISPOT assays of acDCs and moDCs matured with TNF-α/PGE₂/IL-1β or left immature (GM-CSF/IL-4 only). For acDCs, whole PBMCs (1 × 10⁶/well) were cultured as before in the presence or absence of M tuberculosis PPD for 48 hours. To obtain moDCs, autologous monocytes were isolated by PBMC adherence (1 × 10⁶/well) and stimulated as above for 7 days. Fresh autologous PBMCs (1 × 10⁶/well) were then added onto moDCs with or without PPD for 48 hours. Nonadherent cells were subsequently recovered and subjected to IFN-γ ELISPOT. PPD-specific IFN-γ SFC frequencies were background-subtracted. For panels D and E, similar results were obtained in 3 separate experiments by maturing acDCs and moDCs with anti-CD40/IFN-α (not shown).