Figure 4
dAdo strongly inhibits ERKs activation and effector functions in ADA-deficient T cells activating A2A 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 CFSEdim 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.

dAdo strongly inhibits ERKs activation and effector functions in ADA-deficient T cells activating A2A 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 CFSEdim 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.

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