The GATA-3 gene is expressed at similar levels in human Th1- and Th2-skewed cultures. (A) Quantitative analysis of gene expression in human and murine in vitro–polarized Th cells. Naive Th cells were isolated from human peripheral blood or BALB/c spleens and cultured under Th1- (□) or Th2-skewing conditions (▪) as specified in “Materials and methods.” Cells were restimulated (5 hours) with PMA and ionomycin to induce cytokine production. RNA was extracted in parallel stimulated and unstimulated cultures for quantitative real-time RT-PCR (see “Materials and methods”). Transcript levels are expressed as percent of the housekeeping control, PPIA, calculated as 2^−ΔCT. Values determined in human cells are plotted against the left y axes and those obtained in mouse against the right axes. Means ± SEM of 3 (mouse IL-4 and IFN-γ), 5 (mouse GATA-3 and T-bet; human IL-4 and IFN-γ), or 10 experiments (human GATA-3 and T-bet). *P < .05; **P < .005 relative to Th1; ns indicates nonsignificant. (B) Th1- and Th2-skewed cells were fixed and permeabilized and then stained with a GATA-3 mAb (see “Materials and methods”). The left histogram shows a typical experiment in which the level of staining for GATA-3 is compared in Th1 (dotted line) and Th2 cultures (solid line). The filled histogram is the Th1 population stained with a species- and isotype-matched control (a superimposable histogram was produced upon control staining of Th2 cells). The mean ± SEM frequency of GATA-3⁺ cells in 4 independent experiments is shown in the bar graph. The inset shows the electrophoretic mobility of the species recognized by the GATA-3 mAb (lower band) in whole-cell extracts from matching numbers of Th1 and Th2 cells. The upper band is generated upon staining for the housekeeping protein, α-tubulin. M indicates molecular weight markers.