TLR stimulation inhibits GM-CSF– and IL-4–induced development of DCs from human CD14⁺ monocytes. Human CD14⁺ monocytes were cultivated for 6 days with GM-CSF and IL-4 (indicated as GI) in either the presence or absence of 10 ng/mL LPS (indicated as GI+LPS). CD14⁺ cells left unstimulated for 6 days are indicated as “none.” (A) Cell size and granula, and expression of CD1a and CD14, were measured by flow cytometry (representatives of at least 20 experiments). (B) Histograms of indicated surface molecules from either control DCs (open) or LPS-treated cells (filled). Data are from one representative experiment out of 4 experiments. (C) Expression of CD1a and CD14 was measured in cultures that were treated with graded amounts of LPS as indicated (1 out of 4 experiments). (D) Monocytes from 3 different donors (marked by circles, rectangles, and triangles) were cultivated either with GM-CSF+IL-4 alone (GI) or in the presence of an additional 10 μg/mL Pam₃ CysSK₄, 1 μg/mL FSL, 50 μg/mL poly[(I:C)], 50 μg/mL zymosan, or 10 ng/mL LPS. Cells were analyzed for expression of CD1a at day 6. (E) Cell numbers from LPS-treated cultures were determined as mean values of triplicate counts (n = 8). (F) Cells were analyzed for phagocytosis of FITC-labeled latex beads. Shown is ΔMFI (37°C-4°C) of 1 of 4 experiments with similar results. (G) Cells were restimulated at day 6 with 50 ng/mL LPS (▦) or left unstimulated (□) and analyzed for secretion of IL-6, TNF, IL-12p40, and IL-10 after overnight incubation (displayed as mean and SD from 1 of 3 donors with similar results). (H) DCs either treated with LPS or not during the differentiation period and completely untreated CD14⁺ cells were assayed for their capacity to induce proliferation of allogenic CD3-sorted T lymphocytes (triplicates of 1 of 3 similar experiments). (I) Similarly, cells were assayed in an autologous MLR with CD3-sorted T lymphocytes (triplicates of 1 of 3 similar experiments). To induce proliferation, 0.1 ng/mL superantigen SPEC was added to autologous cocultures.