A defective transcription of cytokine genes in ADA-deficient T cells associates with reduced phosphorylation of CREB. (A) Cytokine gene expression after TCR/CD28-mediated stimulation of CD4⁺ T-cell lines from 5 patients (ADA^−/− and GT) and 4 NDs. Total RNA was prepared from either resting or stimulated cells (1 μg/mL anti-CD3 plus 10 μg/mL anti-CD28 mAbs) and gene expression were measured by semiquantitative RT-PCR. Values were normalized for the expression of HPRT and correspond to duplicate samples. Results are expressed as n-fold difference relative to resting condition. One representative experiment of 3 is shown. **P < .01; *P < .05. (B) Immunoblots depicting phosphorylated CREB, total CREB, and GAPDH. CD4⁺ T cells were stimulated with cross-linked (1 μg/mL), anti-CD3 plus (10 μg/mL), anti-CD28 mAbs for the indicated time points. The same membranes were sequentially probed for the different Abs. Upper panel: a representative experiment in Pt3 (ADA^−/− and GT) and one ND is shown. Lower panel: densitometric analysis and statistical significance (**P < .01) on 5 patients (ADA^−/− and GT) and 5 NDs are shown. Values corresponding to unstimulated cells were subtracted.