Early events in the Th2 differentiation program are WASp-independent. (A) WT and WAS^−/− naive CD4⁺ T cells (CD4⁺CD62L^highCD44^low) were loaded with CFSE and primed under Th2 (anti-IFNγ mAb and rIL-4) conditions with plate-bound anti-TCRβ and anti-CD28 Ab for 5 days. Cells were harvested at given time points, and mRNA was extracted for qRT-PCR. mRNA was normalized to CD3δ and expressed as fold change over unstimulated naive CD4⁺ cells. mRNA from WT and WAS^−/− Th1 (anti–IL-4 mAb and rIL-12)–primed cells were collected at 48 hours as controls. Data from 1 representative experiment of 3. Results were not statistically different for all cytokine mRNAs tested over 3 independent experiments, by paired Student t test. (B) CFSE-labeled CD4⁺ T cells were harvested after 48 hours of primary Th2 stimulation and analyzed for phospho-STAT6 by FACS: dot plots are gated on CD4⁺ T cells, and quadrant numbers equal the percentage of CD4 T cells. Percentage of phospho-STAT6⁺ cells in negative control (Th1-primed) was less than 5%. (C) At 48 hours, IL-4 production was analyzed by the cytokine secretion assay (CSA) on gated live CD4⁺ T cells. Quadrant gates were set on “no catch” controls (< 0.5% IL-4⁺ for WT and WAS^−/−); numbers in quadrants equal the percentage of CD4⁺ T cells. (D) Kinetic analysis of IL-4 production by CD4⁺ T cells during Th2 differentiation by CSA. (E) IL-4 dose response during initial cell culture; IL-4 effects determined by the induction of IL-4 production by CD4⁺ T cells by CSA 48 hours after primary stimulation. Results represent one of at least 4 comparable experiments. (F) WT and WAS^−/− naive CD4⁺ T cells were primed under Th1 or Th2 conditions with pOVA/APC for 5 days. Th2 cells were primed a subsequent time under opposing Th1 conditions (anti–IL-4 mAb and rIL-12) with pOVA/APC. At 5 days later, cells were restimulated for 24 hours in the absence of exogenous cytokines with pOVA/APC. IFNγ ELISA analysis of collected supernatants 24 hours after restimulation, nonsignificant by Student t test. Error bars indicate mean and SEM.