FIGURE 3.

The expression of T-bet and GATA-3 is aberrant in lck^−/− Th2 skewed cells. The sequences used for qRT-PCR are listed in Table I. A, GATA-3 and T-bet mRNA expression from wt and lck^−/− Th2 cultures. CD4⁺CD62L⁺ T cells were stimulated under Th2 conditions for 4 d, and qRT-PCR was performed for T-bet and GATA-3. The data are graphed as the ratio of the expression of the target gene in lck^−/− Th2 cells relative to B6 Th2 skewed cells. Note that T-bet is overexpressed in these cultures. The graphs illustrate the average of a minimum of three experiments. B, GATA-3 and T-bet protein expression is altered in lck^−/− Th2 cultures. CD4⁺ cells were skewed under Th1 and Th2 conditions for 3 and 7 d, and protein expression was evaluated by immunoblotting. The data are representative of two (day 3) and three (day 7) experiments. C, GATA-3 overexpression normalizes IL-4 cytokine expression in lck^−/− Th2 cells. lck^−/− and B6 cells were infected with GATA-3 or empty vector 24 h after initiation of Th2 skewing and then skewed for an additional 6 d. Infected cells were identified by GFP expression and then analyzed for IL-4 and IFN-γ expression by ICCS. The flow plots show a representative experiment. The graph illustrates the average for the percentage of cells expressing IL-4 in GFP⁺ cells (four experiments). The expression of IL-4 in Lck Th2 vector versus Lck Th2 GATA-3–infected cells is statistically different. The asterisk indicates that p < 0.05 (Student paired t test). Note: IL-4 expression is similar in the wt Th2 and lck^−/− GATA-3–infected cells. D, c-Maf and Runx3 are elevated in lck^−/− Th2 cultures. lck^−/− and B6 CD4⁺CD62L⁺ cells were skewed under Th2 conditions for 7 d, and qRT-PCR was performed for c-Maf and Runx3. The data are graphed as the ratio of the expression of the target gene in lck^−/− Th2 cells relative to B6 Th2 skewed cells. The graph illustrates the average of three experiments.