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GATA-3 in human T cell helper type 2 development.

Skapenko A, Leipe J, Niesner U, Devriendt K, Beetz R, Radbruch A, Kalden JR, Lipsky PE, Schulze-Koops H - J. Exp. Med. (2004)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Nikolaus Fiebiger Center for Molecular Medicine, Clinical Research Group III, University of Erlangen-Nuremberg, 91054, Germany.

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.

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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.
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fig2: 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.

Mentions: To substantiate the important role of GATA-3 in human Th2 cell differentiation in greater detail, we blocked GATA-3 expression by transfecting freshly isolated memory CD4 T cells from GATA-3+/+ control individuals with siRNA specific for GATA-3. After 2 d of culture in the presence of nonmitogenic concentrations of IL-2, the GATA-3 protein level was assessed by Western blot analysis. Transfection of memory CD4 T cells with GATA-3 siRNA resulted in a markedly decreased level of GATA-3 protein when compared with control siRNA (Fig. 2 A, lamin). In contrast, the protein level of STAT-6, another transcription factor important in Th2 cell differentiation, was not affected. Priming of GATA-3 siRNA-transfected memory CD4 T cells under conditions that were optimal for Th2 cell differentiation from human memory cells (12, 17) resulted in significantly reduced Th2 differentiation (Fig. 2 B), indicating a dominant role of GATA-3 in initiating the Th2 cell differentiation program. Thus, the results are concordant with the results from GATA-3+/− CD4 T cells.


GATA-3 in human T cell helper type 2 development.

Skapenko A, Leipe J, Niesner U, Devriendt K, Beetz R, Radbruch A, Kalden JR, Lipsky PE, Schulze-Koops H - J. Exp. Med. (2004)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211796&req=5

fig2: 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.
Mentions: To substantiate the important role of GATA-3 in human Th2 cell differentiation in greater detail, we blocked GATA-3 expression by transfecting freshly isolated memory CD4 T cells from GATA-3+/+ control individuals with siRNA specific for GATA-3. After 2 d of culture in the presence of nonmitogenic concentrations of IL-2, the GATA-3 protein level was assessed by Western blot analysis. Transfection of memory CD4 T cells with GATA-3 siRNA resulted in a markedly decreased level of GATA-3 protein when compared with control siRNA (Fig. 2 A, lamin). In contrast, the protein level of STAT-6, another transcription factor important in Th2 cell differentiation, was not affected. Priming of GATA-3 siRNA-transfected memory CD4 T cells under conditions that were optimal for Th2 cell differentiation from human memory cells (12, 17) resulted in significantly reduced Th2 differentiation (Fig. 2 B), indicating a dominant role of GATA-3 in initiating the Th2 cell differentiation program. Thus, the results are concordant with the results from GATA-3+/− CD4 T cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Nikolaus Fiebiger Center for Molecular Medicine, Clinical Research Group III, University of Erlangen-Nuremberg, 91054, Germany.

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.

Show MeSH