Extracellular adenosine inhibits TCR-triggered CD25 upregulation and blocks proliferation of antigenic peptide triggered TCR-transgenic T lymphocytes. Different concentrations of extracellular adenosine (0 to 200 μmol/L), 20 μmol/L of NBTI (inhibitor of nucleoside transporters), and 1 μmol/L of coformycin (ADA inhibitor) were present during 8 hours of incubation of B10A TCR-transgenic mouse splenocyte (5 × 10⁶ cells/mL) with 1 μmol/L of cytochrome c peptide. Expression of CD25 was evaluated by flow cytometry. Coformycin and NBTI were added to the assay in this experiment to prevent adenosine from entering the cell and from degradation by adenosine deaminase. These agents had no effect on their own when added alone (data not shown). (A) The effect of adenosine on CD25 upregulation. The shaded histograms represent profiles of CD25 expression in the presence of extracellular adenosine. The solid histograms represent the CD25 level in unstimulated cells. Flow cytometry analysis of CD25⁺ cells among TCRαβ⁺ cells was performed as in Fig 1. The coformycin and NBTI had no inhibitory effect on CD25 upregulation in the absence of adenosine. Graph a, control; graph b, 12.5 μmol/L adenosine; graph c, 25 μmol/L adenosine; graph d, 50 μmol/L adenosine; graph e, 100 μmol/L adenosine; and graph f, 200 μmol/L adenosine. (B) Extracellular adenosine inhibits TCR-triggered upregulation of CD25 on TCRαβ⁺ cells but does not have direct lymphotoxic effects. Flow cytometry data were used to calculate the effect of adenosine on percentage of CD25⁺ cells among live TCRαβ cells (•) and the percentage of PI⁺ (dead or apoptotic) cells (⋄) among TCRαβ⁺ cells. (C) Extracellular adenosine inhibits antigenic peptide/TCR-triggered proliferation of TCRαβ cells. Cells were incubated with medium alone (control) or with 1 μmol/L of cytochrome c peptide or with peptide in 20 μmol/L or with peptide and 50 μmol/L. Splenocytes were assayed for proliferation in parallel with the flow cytometry experiment in (B). Proliferation was tested as described in the Materials and Methods.