Inhibition of Treg function in the presence of PEG-ADA. (A) Percentage of CD25⁺FoxP3⁺ Tregs in ADA^−/− rescued with PEG-ADA (n = 20), lentiviral GT (n = 8), and BMT (n = 11) compared with ADA^+/+ mice (n = 23). Data are mean ± SEM. ***P = .0001. *P = .05. N.S. indicates not significant. (B) Absolute numbers of Tregs. Data are mean ± SEM. (C) Suppressive function versus ADA^+/+ effector cells of ADA^+/+ (n = 11) and ADA^−/− (n = 8) Tregs compared with Tregs from ADA^−/− mice treated with BMT (n = 3), GT (n = 3), and PEG-ADA after 10 (n = 3), 14 (n = 3), and 18 (n = 7) weeks. Data are interexperimental mean ± SD. ***P < .0005. **P < .005. *P < .05. (D) Summarized MFI of CD39 and CD73 gated on CD4⁺CD25⁺FoxP3⁺ cells from ADA^+/+ (n = 6) mice compared with ADA^−/− mice treated with PEG-ADA for 18 weeks (n = 4). (E) Representative histogram plots for CD39 and CD73 on CD4⁺CD25⁺FoxP3⁺ splenocytes from ADA^+/+ and ADA^−/− mice treated with PEG-ADA for 18 weeks. Isotype-identical monoclonal antibodies served as controls (solid gray histogram). (F) Measurement of ATP consumption and AMP formation (left panel) as well as adenosine formation (right panel) by purified CD4⁺CD25⁺ Treg cells from PEG-ADA–treated ADA^−/− (n = 4) and ADA^+/+ mice (n = 3). Data are interexperimental mean ± SD. *P < .05. (G) Serum adenosine levels in PEG-ADA–treated ADA^−/− (n = 4) and age-matched ADA^+/+ mice (n = 4) at 10, 14, and 18 weeks of follow-up. Data are interexperimental mean ± SD. *P < .05. (H) Serum hypoxanthine levels in PEG-ADA–treated ADA^−/− (n = 4) and age-matched ADA^+/+ mice (n = 4) at 10, 14, and 18 weeks of follow-up. Data are interexperimental mean ± SD. **P < .005.