ATPase activity of mouse CD39⁺ Treg cells. (A) Expression of CD39 on activated and nonactivated Treg cells. Cell surface expression of CD39 is shown for CD4⁺CD25⁺ cells that were freshly isolated (left panel) or had been activated for 3d with α-CD3 and α-CD28 (right panel). Staining of CD39 is shown versus α-CD25, the mean fluorescence intensity (MFI) of CD39 is indicated. (B) ATP hydrolysis by activated and nonactivated CD39⁺ Treg cells. The indicated number of activated (●) or nonactivated (resting) CD25⁺CD4⁺ cells (○) were incubated for 10 minutes with 50 μM ATP (left panel). Numbers indicate the ATP consumption in fmol per second per cell and were calculated by determining the starting slope using a hyperbola regression. The experiment on the right panel was carried out in the same way except that 50 000 activated CD4⁺CD25⁺ T cells were incubated with ATP in the presence of indicated amounts of the ecto-ATPase inhibitor ARL67156. The ATP consumption was determined by a luminometric assay. Error bars indicate standard deviation of data points. (C) Abrogation of ATP-induced inhibition of proliferation. CD4⁺CD25⁺ T cells were labeled with CFSE and activated for 3d in the presence of 100 μM ATP, 250 μM ARL67156, 100 μM ATP/250 μM ARL67156, or without these reagents. Proliferation of the cells was determined by FACS analysis after gating on the PI-negative cells; numbers indicate percentage of total cells.