Figure 4
Figure 4. IL-4 transcriptional enhancement but no IL-4 protein production in the absence of WASp. (A) Th2-primed WT and WAS−/− CD4+ T cells were restimulated with pOVA/APC (WT T-depleted splenocytes). Cells were collected at given times after stimulation, and mRNA was extracted for qRT-PCR. mRNA was normalized to CD3δ and expressed as fold change over Th2-primed unstimulated (resting) cells. Results were not statistically different for IL-4 mRNA tested over 3 independent experiments by paired Student t test. (B) Intracellular staining on Th2-primed cells stimulated with pOVA/APC. (C) ELISPOT analysis of Th2-primed cells for frequency of IL-4 secretors 24 hours after restimulation with pOVA/APC. Data are from 1 of 3 comparable experiments. *P ≤ .05; paired Student t test. (D) Fold change in mRNA versus protein in WAS−/− Th2 cells compared with WT Th2 cells. *P = .03, paired Student t test, for relative differences in IL-4 protein in WAS−/− and WT Th2 cells. Differences in absolute IL-4 mRNA between WAS−/− and WT Th2 cells was not significant. Average and SEM of data from 3 independent experiments. (E) Th2-primed WT and WAS−/− CD4+ T cells were restimulated with phorbol myristyl acetate (PMA) and ionomycin: IL-4 mRNA analysis (left) and protein secretion by ELISA (right) 12 hours after restimulation. Data are from 1 of 3 comparable experiments. *P ≤ .05; paired Student t test. Error bars indicate mean and SEM. (F) ERK phosphorylation after pOVA/APC restimulation of Th2-primed WT and WAS−/− CD4+ T cells using an anti–phospho-specific ERK Ab. Specificity of binding of the phospho-specific Ab was confirmed by treatment with the ERK inhibitor U0126 (bottom panel). Representative plots from 1 of 4 experiments. (G) qRT-PCR analysis of ERK transcriptional target gene c-FOS in Th2-primed WT and WAS−/− effectors restimulated with pOVA/APC. Representative data from 1 of 2 similar experiments. (H) Cytokine production after restimulation of WT Th1- and Th2-primed cells in the presence of the ERK inhibitor U0126. IFNγ and IL-4 were measured by ELISA and normalized to effector cell stimulation in the absence of the ERK inhibitor (DMSO vehicle control = 100%). Mean and standard error from 4 independent experiments. *P ≤ .05; Student t test. Error bars indicate mean and SEM.

IL-4 transcriptional enhancement but no IL-4 protein production in the absence of WASp. (A) Th2-primed WT and WAS−/− CD4+ T cells were restimulated with pOVA/APC (WT T-depleted splenocytes). Cells were collected at given times after stimulation, and mRNA was extracted for qRT-PCR. mRNA was normalized to CD3δ and expressed as fold change over Th2-primed unstimulated (resting) cells. Results were not statistically different for IL-4 mRNA tested over 3 independent experiments by paired Student t test. (B) Intracellular staining on Th2-primed cells stimulated with pOVA/APC. (C) ELISPOT analysis of Th2-primed cells for frequency of IL-4 secretors 24 hours after restimulation with pOVA/APC. Data are from 1 of 3 comparable experiments. *P ≤ .05; paired Student t test. (D) Fold change in mRNA versus protein in WAS−/− Th2 cells compared with WT Th2 cells. *P = .03, paired Student t test, for relative differences in IL-4 protein in WAS−/− and WT Th2 cells. Differences in absolute IL-4 mRNA between WAS−/− and WT Th2 cells was not significant. Average and SEM of data from 3 independent experiments. (E) Th2-primed WT and WAS−/− CD4+ T cells were restimulated with phorbol myristyl acetate (PMA) and ionomycin: IL-4 mRNA analysis (left) and protein secretion by ELISA (right) 12 hours after restimulation. Data are from 1 of 3 comparable experiments. *P ≤ .05; paired Student t test. Error bars indicate mean and SEM. (F) ERK phosphorylation after pOVA/APC restimulation of Th2-primed WT and WAS−/− CD4+ T cells using an anti–phospho-specific ERK Ab. Specificity of binding of the phospho-specific Ab was confirmed by treatment with the ERK inhibitor U0126 (bottom panel). Representative plots from 1 of 4 experiments. (G) qRT-PCR analysis of ERK transcriptional target gene c-FOS in Th2-primed WT and WAS−/− effectors restimulated with pOVA/APC. Representative data from 1 of 2 similar experiments. (H) Cytokine production after restimulation of WT Th1- and Th2-primed cells in the presence of the ERK inhibitor U0126. IFNγ and IL-4 were measured by ELISA and normalized to effector cell stimulation in the absence of the ERK inhibitor (DMSO vehicle control = 100%). Mean and standard error from 4 independent experiments. *P ≤ .05; Student t test. Error bars indicate mean and SEM.

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