Inhibition of TCR-induced up-regulation of T-cell activation marker CD69 in both A_2A receptor-expressing (+/+) and A_2A receptor-deficient (−/−) thymocytes by extracellular adenosine in the presence of ADA inhibitor.

Ex vivo thymocytes from A_2A receptor-expressing (+/+) and A_2A receptor-deficient (−/−) mice were incubated 16 hours at 37°C in 5% CO₂ incubator with immobilized anti-TCR/CD3 mAb (20 μg/mL) in the presence of extracellular adenosine (100 μmol/L) and adenosine deaminase inhibitor coformycin (cof, 10 μmol/L) as indicated on the graphs. Cells were harvested and stained with Annexin V, PI (to set gate for nonapoptotic cells), and anti-CD69-PE mAb to estimate the proportion of activated cells. The extent of up-regulation of CD69 was measured by flow cytometry as described in “Materials and methods.”