Perturbation of the PKA signaling node leads to release from inhibition of T-cell activation. (A) CD8 T-cell populations were TCR stimulated (OKT3, 1 μg/mL; CD28.2, 5 μg/mL) for 1 minute with or without PGE₂ (10μM; 10 minutes of preincubation) and with or without pretreatment with the PKA-RI antagonist Rp-8-Br-cAMPS (1mM; 30 minutes of preincubation). Cells were analyzed for CD3ζ phosphorylation at Tyr142. Data represent 4 independent experiments with individual blood donors expressed as mean ± SD; n = 4 of arcsinh median differences. (B) Effect of Rp-8-Br-cAMPS (1mM; 30 minutes of incubation) on basal CD3ζ phosphorylation at Tyr142. Warmer colors (yellow) in the heatmap representation (insert in B) indicate increases and cooler colors (blue) indicate decreases in Arcsinh median differences displayed on a scale of 1. Data represent 3 independent experiments with individual blood donors expressed as mean ± SD; n = 3 of arcsinh median differences. (C) CD8 T-cell populations were TCR stimulated (OKT3, 1 μg/mL; CD28, 5 μg/mL) for 1 minute with or without PGE₂ (10μM; 10 minutes of preincubation) and with or without pretreatment with the PKA-RI antagonist Rp-8-Br-cAMPS (1mM; 30 minutes of preincubation) and the myristoylated PKA inhibitor peptide PKI (45 minutes of preincubation). Cells were analyzed for CD3ζ phosphorylation at Tyr142. Data represent 3 independent experiments from 3 individual blood donors expressed as mean ± SD of arcsinh median differences. (D) As in panel C, but SLP-76 phosphorylation at Tyr128 was analyzed. Data represent 3 independent experiments from 3 independent blood donors expressed as mean ± SD of arcsinh median differences.