dAdo strongly inhibits ERKs activation and effector functions in ADA-deficient T cells activating A_2A AR signaling. (A) ERK activation was analyzed in CD4⁺ cells stimulated with cross-linked (1 μg/mL), anti-CD3 plus (10 μg/mL), anti-CD28 mAbs in the absence or presence of dAdo (500 μM). Samples of untreated and dAdo-treated stimulated cells were prepared in parallel, run on different gels but probed with the different Abs at the same time. (B) CFSE-labeled CD4⁺ cells were cultured in plain medium or stimulated with immobilized anti-CD3 mAb (1 μg/mL) in the absence or presence of dAdo (500 μM). After 64 hours, cells were analyzed by flow cytometry. TO-PRO-3 was added at the time of FACS analysis. Histograms depicting the CFSE content of TO-PRO-3⁻ viable cells are shown from one representative patient and one ND. The percentage of CFSE^dim cells is indicated in each plot. Background levels corresponding to unstimulated cells were subtracted. The total number of trypan blue–negative viable cells and the frequency of TO-PRO-3–positive cells from CFSE/TO-PRO-3 dot plots of total events are shown for 5 patients and 4 NDs. (C) CD4⁺ cells stimulated with 1 μg/mL coated anti-CD3 mAb in the absence or presence of dAdo (500 μM) or (D) CGS21680 (100 μM). The specified compounds (SCH58261 or dipyridamole) were included 1 hour before the addition of dAdo. After 48 hours, cell proliferation was assessed by [³H]thymidine incorporation. The result of a representative experiment including 5 patients (ADA^−/− and GT) and 5 NDs is reported. Percentages of inhibition with respect to the untreated condition are indicated. (E) Intracellular cAMP levels were determined in CD4⁺ T cells incubated for 15 minutes with culture medium containing 5 mM dAdo or 1 mM CGS21680. The inhibitor of adenylate cyclase (AC, 1 mM) was added 30 minutes before the other drugs. The data represent the mean plus or minus SD from 5 patients (ADA^−/− and GT) and 4 NDs in one of 3 independent experiments. Statistics: **P < .01; *P < .05.