The poorly hydrolyzable adenosine analogs NECA, 2CADO, and CGS21680 inhibit the TCR-triggered CD25 upregulation on mouse splenocytes. B10A TCR-transgenic mouse splenocytes were incubated at 5 × 10⁶ cells/mL with 1 μmol/L of cytochrome c peptide for 8 hours. The cells were then labeled with (1) FITC antimouse CD25, (2) PE antimouse TCRαβ, and (3) PI. CD25 expression on the TCR⁺ cells was evaluated by flow cytometry by gating for PI⁻ (live) and PE-antimouse TCRαβ⁺ cells. 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. (A) Graph a, control, TCR-triggered CD25 upregulation in the absence of adenosine; graph b, effect of NECA (200 μmol/L); graph c, effect of 2CADO (10 μmol/L); graph d, effect of CGS21680 (20 μmol/L). (B) Comparison of effects of adenosine analogs on TCR-triggered CD25 upregulation. The changes in percentages of the CD25⁺ cells among TCRαβ⁺ cells were calculated from data in (A).