Characterization of the functional defect in CD4⁺ADA-deficient T cells. (A) Purified CD4⁺ T-cell lines from 5 patients and 5 NDs were stimulated with immobilized anti-CD3 mAb at the indicated concentrations, and [³H]thymidine incorporation was measured after 48 hours. One representative experiment of 3 is shown. (B) Cells were stimulated with 1 μg/mL anti-CD3, and culture supernatants were collected after 18 (IL-2) or 48 hours (all other cytokines). Cytokine concentrations were quantified by capture ELISA. Each value represents the mean of cytokine concentration measured in triplicate cultures for patients (n = 5; ADA^−/− and GT) and healthy controls (n = 5). Median values are also reported. Background levels were subtracted. One determination of 3 is shown. (C) Production of IFN-γ vs either IL-2, or IL-4, was analyzed by intracytoplasmic staining in CD4⁺ T-cell lines as described in “Cytokine detection.” The experiment shown is representative of 5 independent experiments. Two ADA-SCID patients, before and after GT, and 2 NDs are reported. Range values corresponding to 5 patients and 7 NDs are indicated in Table 1.