Inhibition on ATP-driven maturation of mouse DCs. (A) ATP-driven DC maturation. Immature bone marrow-derived mouse DCs were incubated for 24 hours without any activator (left panel) or with 10 ng/mL LPS or 200 μM ATP (right panels). Histograms are shown for the maturation marker CD86 of cells gated on CD11c; numbers indicate the percentage of CD86⁺ cells. (B) Prevention of ATP-driven maturation of mouse DCs by CD39⁺ Treg cells. LPS or ATP containing medium was used either directly (■) or after 20 minutes of pre-exposure to freshly isolated CD4⁺CD25⁻ cells (▒) or to activated CD4⁺CD25⁺ cells (▓) to incubate DC cultures. Bars represent the percentage of CD86⁺ cells (gated on CD11c); dashed line represents percentage of CD86⁺ cells obtained by spontaneous maturation. (C) Treg activation is required for inhibition of ATP-induced maturation. The experiment was carried out as in Figure 4B except that in addition to freshly isolated CD4⁺CD25⁻ cells and activated CD4⁺CD25⁺ cells, nonactivated CD4⁺CD25⁺ cells were also used. Left panel shows the ATP concentration remaining after 25 minutes of exposure of the ATP containing medium (200 μM) to the T cells; the right panel shows the inhibition of the ATP-driven DC maturation. Inhibition of maturation is expressed as a percentage and is determined after subtracting the spontaneous maturation in reference to the maximal increase of CD86⁺ cells after ATP exposure. Error bars indicate standard deviation of data points.