CD4⁺ T cells activated in the presence of Env phosphorylate AKT, p38 MAPK, and STAT5a but fail to up-regulate the activation markers CD69, CD25, and HLA-DR. PBMCs were cocultured with Env or control RNA-expressing 293T cells for 24 hours. Phosphorylation of AKT or MAPK 10 minutes after anti-CD3 (1.0 μg/mL) stimulation was measured using flow cytometry with gating on CD4⁺ T cells. Purified CD4⁺ T cells were exposed to control RNA or Env-expressing 293T cells and activated using anti-CD3 in the absence of IL-2. Two days later, cells were stimulated with 20 U/mL IL-2 and analyzed for STAT5a phosphorylation 7 minutes later. CD4⁺ T cells exposed to IIIB Env-transfected 293T cells and stained with isotype control mAb (shaded gray) or specific phosphoprotein mAb (thin line) or specific phosphoprotein mAb-stained control-transfected 293T cells (thick line) are shown for each signaling molecule. Experiments shown are representative of 3 independent experiments.