WT Th2 cells require WASp signaling for IL-4 production. WT naive CD4⁺ T cells were primed under Th2 (A-C) or Th1 (D) conditions with peptide and WT APCs. Cells were retrovirally transduced with DN WASp (WASpΔVCA-IRES-GFP) or empty vector (-IRES-GFP) for 36 hours late during priming (day 4). On day 6, retrovirally transduced cells were sorted based on GFP expression and restimulated. (A) GFP⁻ and GFP⁺ cells were not activated or activated with anti-TCRβ mAb for 15 minutes. Cells were fixed and stained with phalloidin and analyzed by FACS. (B-C) Cells were restimulated with pOVA/APC for 24 hours. (B) The frequency of cytokine producers was determined using CSA (left panel) and cytokine produced in the supernatant by ELISA (right panel). Mean ± SEM; n = 3. *P ≤ .05 between GFP⁺ and GFP⁻ cells in 3 independent experiments; paired Student t test. (C) mRNA analysis 24 hours after restimulation. mRNA was normalized to CD3δ and expressed as fold change over naive unstimulated CD4⁺ T cells. (D) Th1-primed cells were restimulated with pOVA/APC for 24 hours. The frequency of cytokine producers was determined using CSA (left panel) and cytokine produced in the supernatant by ELISA (right panel). Mean ± SEM; n = 3. Data are from 1 of 3 comparable experiments.