LV-mediated ADA2 correction in patients’ macrophages restored ADA2 enzymatic activity and suppressed cytokine hyperproduction. (A) ADA2 protein expression was assessed in monocyte-derived macrophages from 6 patients with DADA2 and from HDs using immunoblot analysis. β-actin expression was included as a loading control. (B) ADA2 enzymatic activity was measured in cell-free supernatants from patients’ (n = 5) and HDs’ (n = 7) macrophages that were transduced or not with ADA2-LV. IL-6 and TNF expression and release were assessed in UT and ADA2-transduced M1 macrophages from patients and HDs using quantitative reverse transcription polymerase chain reaction (RT-PCR) (C) and enzyme-linked immunosorbent assay (ELISA) (D), respectively. HDs, n = 5; patients, n = 6, with the exception of RT-PCR for TNF expression (n = 5). (E) Immunoblot analysis of ADA2 expression and secretion ADA2^+/+ and ADA2^−/− U937 macrophages transduced with LV-ADA2 wild-type (ADA2^wt) or L188P mutant (ADA2^L188P). (F) ADA2 activity was measured in cell-free supernatants from ADA2^+/+ and ADA2^−/− U937 macrophages transduced with ADA2^WT and ADA2^L188P. Expression and secretion of IL-6 and TNF were measured in ADA2^−/− U937 macrophages expressing ADA2 wild-type or L188P mutant by quantitative RT-PCR (G) and ELISA (H). Data in (F-H) are mean ± standard deviation. *P < .05, **P < .01, ***P < .001. ns, not significant; PT, patient.