DD-modified DCs show impaired STAT3 phosphorylation and induce T-cell anergy. (A) imDCs were incubated with maturation cocktail (MC) ± DD (100 ng/mL) for 48 hours. After washing, control maDC and DD maDC were cocultivated with allogenic CD4⁺ T cells (CD4:DC ratio: 66:1), referred to as MLR and DD-MLR. After 5 days, CD4⁺ T cells were collected from the MLR/DD-MLR cultures, and the number of live cells was determined (left bar diagram). T-cell aliquots were subsequently restimulated with anti-CD3 (OKT3; 0.1 µg/mL), PMA, or third-party allogeneic maDC (CD4:DC ratio: 66:1) for an additional 3 days, and cell proliferation was determined by overnight thymidine incorporation (Thy.incorp.) (right bar diagram). Data are shown as mean values ± SD of 4 independent experiments. P value was calculated with the Student t test (*P < .05); counts per minute (cpm). (B) Measurement of relative cytokine secretion from MLR/DD-MLR as described in panel A by cytometric bead array. Cytokine production for control MLR was set to 100%. Mean values ± SD are shown of 4 independent standardized experiments. (C) imDC were incubated with or without DD (100 ng/mL; 48 hours) (upper panels), maturation cocktail (MC) ± DD (100 ng/mL; 48 hours) (middle panels), or LPS ± DD (100 ng/mL; 48 hours) (lower panels). Subsequently, Stat3 and phosphorylated Stat3 (p-Stat3) were stained intracellularly and were analyzed by flow cytometry (FACScan). A single representative experiment of 3 is shown.