Altered CD39 and CD73 expression and decreased suppressive function of ADA^−/− Tregs. (A) TaqMan gene expression analysis for CD39 (ADA^+/+: n = 15, ADA^−/−: n = 16; **P = .0047) and CD73 (ADA^+/+: n = 9, ADA^−/−: n = 10. **P = .0045) in purified CD4⁺CD25⁺ Tregs; box and whiskers graphs. (B) Summarized MFI of CD39 and CD73 gated on CD4⁺CD25⁺FoxP3⁺ cells from ADA^+/+ (n = 5) and ADA^−/− (n = 5) mice; box and whiskers graphs. (C) Representative histogram plots for CD39 and CD73 on CD4⁺CD25⁺FoxP3⁺ splenocytes from untreated ADA^+/+ and ADA^−/− mice. Isotype-identical monoclonal antibodies served as controls (solid gray histogram). (D) AXP levels divided into ATP and AMP illustrate ectonucleotidase activity in serum samples from ADA^−/− (n = 7) and age-matched ADA^+/+ mice (n = 8) at 3 weeks of age. Data are mean ± SD. *P < .05. (E) Representative suppression assay using ADA^+/+ effector cells cocultured with Tregs from untreated ADA^+/+ (n = 3) or ADA^−/− mice (n = 5). E indicates unstimulated effector cells; and E*, effector cells stimulated with anti-CD3 and anti-CD28. Data are interexperimental mean ± SD. (F) Serum adenosine levels in ADA^−/− (n = 7) and age-matched ADA^+/+ mice (n = 8) at 3 weeks of age. Data are mean ± SD. ***P < .0005. (G) Serum hypoxanthine levels in ADA^−/− (n = 7) and age-matched ADA^+/+ mice (n = 8) at 3 weeks of age. Data are mean ± SD. **P < .005. (H) Representative histogram plots for CD39 and CD73 on CD4⁺CD25⁺FoxP3⁺ splenocytes from ADA^+/+ and ADA^−/− mice treated with 50 mg/kg of caffeine (nonselective Adora antagonist). Isotype-identical monoclonal antibodies served as controls (solid gray histogram). (I) Representative suppression assay using ADA^+/+ effector cells cocultured with Tregs from ADA^+/+ (n = 4) or ADA^−/− mice (n = 6) treated with 50 mg/kg of caffeine. Data are intraexperimental mean ± SD.