Multiparameter flow cytometric analysis of GATA‐3 expression in T cell development and differentiation

Abstract

GATA‐3 is a key zinc‐finger transcription factor essential for T cell development and differentiation. In this study, a monoclonal antibody (mAb) was raised against mouse and human GATA‐3. This mAb was used to analyze GATA‐3 expression in intrathymic T cell progenitors and polarized Th1 and Th2 cells by Western blotting, and multicolor immunofluorescent staining, and flow cytometric analysis. Our results confirm that GATA‐3 is expressed at every stage of intrathymic mouse T‐cell development and that its expression levels vary among the T cell progenitor subsets. The highest levels of GATA‐3 are expressed by a subset of double negative CD4−CD8− cells and by the single positive CD4+CD8− cell population. In addition, we found that mature T cells express relatively low levels of GATA‐3 upon in vitro CD3 and CD28 costimulation. Moreover, their levels of GATA‐3 remain unchanged or rise significantly if these cells are skewed towards the Th1 or Th2 phenotypes in the presence of exogenous IL‐12 or IL‐4, respectively. In summary, we show that this antibody is compatible with both saponin and methanol permeabilization methods. It can be used in combination with fluorescent antibodies specific for staining various cell surface markers, as well as other intracellular transcription factors or cytokines to elucidate the T cell signaling pathways utilized during development and differentiation.