gp120-mediated regulatory T-cell activation depends on cAMP production and adenylate cyclase activity. (A) gp120 induces cytosolic cAMP production in human Tregs. Tregs were stimulated with gp120 or anti-CD3 mAbs for 10 hours, and cytosolic cAMP concentration was assessed by enzyme-linked immunosorbent assay (detection limit: 0.75 pmol/mL). Results of 5 individual donors are shown, represented by individual symbols. (B) Inhibition of the tyrosine kinase Lck by the inhibitor PP1 (15 μM) or inhibition of adenylate cyclase activity by the inhibitor MDL-12 (50 μM) prevent gp120-mediated cAMP production in Tregs. Each symbol represents 1 individual donor (**P < .005). (C) Blockade of adenylate cyclase activity holds off gp120-mediated Treg activation. Tregs were either preincubated with MDL-12 for 30 minutes or left untreated and subsequently washed and added to a coculture assay described in Figure 1. One representative result of 3 is shown. Ratios indicate numbers of Tregs to CD8⁺ T cells. (D) gp120-stimulated Tregs up-regulate intracellular CTLA-4 expression. Tregs were stimulated with gp120 or anti-CD3 mAbs for 10 hours, fixed, permealized, and stained for intracellular expression of CTLA-4.