GATA-3 in human T cell helper type 2 development

Abstract

The delineation of the in vivo role of GATA-3 in human T cell differentiation is a critical step in the understanding of molecular mechanisms directing human immune responses. We examined T cell differentiation and T cell-mediated effector functions in individuals lacking one functional GATA-3 allele. CD4 T cells from GATA-3+/- individuals expressed significantly reduced levels of GATA-3, associated with markedly decreased T helper cell (Th)2 frequencies in vivo and in vitro. Moreover, Th2 cell-mediated effector functions, as assessed by serum levels of Th2-dependent immunoglobulins (Igs; IgG4, IgE), were dramatically decreased, whereas the Th1-dependent IgG1 was elevated compared with GATA-3+/+ controls. Concordant with these data, silencing of GATA-3 in GATA-3+/+ CD4 T cells with small interfering RNA significantly reduced Th2 cell differentiation. Moreover, GATA-3 mRNA levels increased under Th2-inducing conditions and decreased under Th1-inducing conditions. Taken together, the data strongly suggest that GATA-3 is an important transcription factor in regulating human Th2 cell differentiation in vivo.

Figure 1.

CD4 T cells from GATA-3^+/− individuals express reduced levels of GATA-3 associated with decreased Th2 cell differentiation and reduced Th2 effector functions. (A) Whole cell lysates of CD4 T cells from GATA-3^+/− (individual no. 1) and GATA-3^+/+ (control) individuals were analyzed by Western blotting with mAbs to GATA-3 and actin. One representative of three independent experiments with similar results is shown. (B) Freshly isolated memory CD4 T cells from GATA-3^+/− individuals were stimulated in vitro with PMA and ionomycin and analyzed for cytoplasmic IL-4 (Th2) and IFN-γ (Th1). Dots indicate Th2 and Th1 cell frequencies of the individual GATA-3^+/− individuals, whereas the shaded areas denote mean ± SD of 20 age-matched control individuals. (C) Freshly isolated naive T cells of GATA-3^+/− individuals were primed in vitro under Th2-inducing conditions, stimulated with PMA and ionomycin, and assessed for cytoplasmic IL-4 and IFN-γ. Dots indicate Th2 and Th1 cell frequencies of two GATA-3^+/− individuals. The third GATA-3^+/− individual was not analyzed because of limited numbers of available cells. Shaded areas denote the normal range of control individuals. (D and E) Serum Ig levels of GATA-3^+/− individuals. Shaded areas reflect the normal limits.

Figure 2.

Silencing of GATA-3 inhibits human Th2 cell differentiation. (A) Freshly isolated CD4 memory T cells from GATA-3^+/+ individuals were transfected with siRNA specific for GATA-3 or lamin (control), and GATA-3, actin, and STAT-6 protein levels were assessed 2 d later by Western blot analysis. GATA-3 protein levels were assessed by densitometric analysis after Western blot and normalized for actin. (B) After transfection, freshly isolated memory T cells from GATA-3^+/+ individuals were cultured under Th2-inducing conditions. For control, untransfected cells were analyzed after priming in neutral (non-Th1/Th2–inducing) conditions (medium). Cytoplasmic IL-4 was determined in the generated effector populations as described in Materials and Methods. Data from five independent experiments with cells from individual donors are indicated by dots and connected with lines.

Figure 3.

GATA-3 is regulated during human T cell differentiation in vitro. (A) Freshly isolated CD4 naive T cells from GATA-3^+/+ individuals were primed for 5 d in the presence of mAbs to CD3, CD28, and/or IL-4 and/or recombinant IL-4 or IL-12 as indicated. GATA-3 mRNA was assessed in the generated effector populations by real-time PCR (left). Th2 cell frequencies were determined in the same populations after an additional 2.5 d of rest by assessing cytoplasmic IL-4 and IFN-γ after stimulation with PMA and ionomycin (right). (B) Freshly isolated CD4 memory T cells from GATA-3^+/+ individuals were primed in the presence or absence of mAbs to CD28 and/or recombinant IL-4 as indicated and GATA-3 mRNA was assessed as in A (left). Th2 cell frequencies were determined as described in A in the same populations (right). (C) Th2 and Th1 effector memory T cells from GATA-3^+/+ individuals were sorted based upon their ability to produce IL-4 and IFN-γ, respectively, to purity. GATA-3 protein levels in the sorted populations were assessed by densitometric analysis after Western blot and normalized for actin.