CD4⁺CD8⁺DP thymocytes are predominantly susceptible to extracellular adenosine-induced apoptosis.

Ex vivo thymocytes were incubated 16 hours at 37°C in 7% CO₂ incubator in the absence (control, A) or presence of 100 μmol/L adenosine (B). Cells were triple stained with anti-CD4, anti-CD8 mAb and PI, and the extent of cell death was measured by flow cytometry as described in “Materials and methods.” (C) Adenosine/A_2a receptor-mediated signaling targets CD4^interCD8^interCD4^lowHSA^lowCD5^highthymocytes. Thymocytes were incubated 16 hours with A_2AR selective and apoptosis-inducing agonist CGS21680 (1 μmol/L) in the presence or absence of A_2AR antagonist ZM241385 (2 μmol/L) or in the media alone. Cells were then stained with Annexin V, PI, and different mAb to important surface antigens and the proportion of cells with a particular surface marker was estimated by flow cytometry among live cells, which were gated on the basis of Annexin V and PI staining. (D) Effect of cAMP and cAMP-raising agents on apoptosis of thymocytes. Cells were incubated with different agents at concentrations that are indicated and, after 16 hours of incubation, the proportion of CD4⁺ versus CD8⁺ cells among surviving cells was estimated by flow cytometry. (E) cAMP-raising agents target for apoptosis mostly CD4⁺CD8⁺ DP thymocytes.