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Control of the differentiation of regulatory T cells and T(H)17 cells by the DNA-binding inhibitor Id3.

Maruyama T, Li J, Vaque JP, Konkel JE, Wang W, Zhang B, Zhang P, Zamarron BF, Yu D, Wu Y, Zhuang Y, Gutkind JS, Chen W - Nat. Immunol. (2010)

Bottom Line: We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells).Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter.Therefore, a network of factors acts in a TGF-β-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.

View Article: PubMed Central - PubMed

Affiliation: Mucosal Immunology Unit, Oral Infection and Immunity Branch, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
The molecular mechanisms that direct transcription of the gene encoding the transcription factor Foxp3 in CD4(+) T cells remain ill-defined. We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells). We identify two transforming growth factor-β1 (TGF-β1)-dependent mechanisms that were vital for activation of Foxp3 transcription and were defective in Id3(-/-) CD4(+) T cells. Enhanced binding of the transcription factor E2A to the Foxp3 promoter promoted Foxp3 transcription. Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter. Furthermore, Id3(-/-) T cells showed greater differentiation into the T(H)17 subset of helper T cells in vitro and in a mouse asthma model. Therefore, a network of factors acts in a TGF-β-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.

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Id3 deficiency increases GATA-3a, GATA-3 mRNA expression relative to HPRT in naïve CD4+CD25− T cells at 12 h after activation with TCR and indicated reagents (mean ± s.d. of duplicate measurements in one experiment). b, Flow cytometry of CD4+CD25− T cells at 24 h after activation. Numbers in quadrants indicate percent Foxp3+GATA-3−(top left), Foxp3+GATA-3+(top right) or Foxp3−GATA3+ (bottom right) cells. c, IL-4 mRNA relative to HPRT in CD4+CD25− T cells at 2 h after activation (mean ± s.d. of duplicate measurements in one representative experiment). d, Flow cytometry of Tgfbr1f/f Cd4-cre+ and Tgfbr1f/+Cd4-cre+ CD4+CD25− T cells cultured for 24 h. Numbers in quadrants indicate same cells as in (b). e, Knockdown of E2a with SiRNA decreased TCR-driven IL-4 gene expression in WT naïve CD4+CD25− T cells. IL-4 mRNA expression relative to HPRT is shown (mean ± s.d. of triplicate measurements in one representative experiment). *P < 0.05. f, Relative GATA-3 binding (GATA-3/control IgG) at the Foxp3 promoter in CD4+CD25− T cells (cultured for 24 h). Data shown are mean ± s.d. of duplicate wells in one representative experiment . g, Elimination of GATA-3 with neutralization of IL-4 (αIL-4) restored enrichment of E2A binding to the Foxp3 promoter induced by TGF-β in Id3−/− CD4+ CD25− T cells (cultured for 24 h). Data shown are mean ± s.d. of duplicate wells in one representative experiment. Data shown in a,b,c,d are one experiment representative of at least three, and in e,f,g representative of two.
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Figure 5: Id3 deficiency increases GATA-3a, GATA-3 mRNA expression relative to HPRT in naïve CD4+CD25− T cells at 12 h after activation with TCR and indicated reagents (mean ± s.d. of duplicate measurements in one experiment). b, Flow cytometry of CD4+CD25− T cells at 24 h after activation. Numbers in quadrants indicate percent Foxp3+GATA-3−(top left), Foxp3+GATA-3+(top right) or Foxp3−GATA3+ (bottom right) cells. c, IL-4 mRNA relative to HPRT in CD4+CD25− T cells at 2 h after activation (mean ± s.d. of duplicate measurements in one representative experiment). d, Flow cytometry of Tgfbr1f/f Cd4-cre+ and Tgfbr1f/+Cd4-cre+ CD4+CD25− T cells cultured for 24 h. Numbers in quadrants indicate same cells as in (b). e, Knockdown of E2a with SiRNA decreased TCR-driven IL-4 gene expression in WT naïve CD4+CD25− T cells. IL-4 mRNA expression relative to HPRT is shown (mean ± s.d. of triplicate measurements in one representative experiment). *P < 0.05. f, Relative GATA-3 binding (GATA-3/control IgG) at the Foxp3 promoter in CD4+CD25− T cells (cultured for 24 h). Data shown are mean ± s.d. of duplicate wells in one representative experiment . g, Elimination of GATA-3 with neutralization of IL-4 (αIL-4) restored enrichment of E2A binding to the Foxp3 promoter induced by TGF-β in Id3−/− CD4+ CD25− T cells (cultured for 24 h). Data shown are mean ± s.d. of duplicate wells in one representative experiment. Data shown in a,b,c,d are one experiment representative of at least three, and in e,f,g representative of two.

Mentions: We then examined E2A binding to the Foxp3 promoter in Id3−/− CD4+CD25− T cells which exhibited a severely reduced frequencies of Foxp3+ Treg cells in response to TGF-β1 treatment (Fig. 2). The Id3−/− CD4+CD25− T cells had slightly but reproducibly higher basal levels of E2A binding to the Foxp3 promoter in response to TCR stimulation alone than did WT control T cells (data not shown and Fig. 5f), which could indicate an inhibitory function of Id3 for E2A protein binding to E boxes on target genes24. However, TGF-β1 treatment failed to enhance E2A binding to the Foxp3 promoter in Id3−/− T cells over the cells treated with TCR alone (Fig. 4 f). Freshly isolated CD4+CD25+ Treg cells from the spleens of Id3−/− mice showed no significant enrichment of E2A binding at the Foxp3 promoter compared to CD4+CD25− T cells in the same mice (Supplementary Fig. 6 c). The data were consistent with the positive function of E protein binding to E boxes at the Foxp3 promoter, but it raised an intriguing question as to why TGF-β1 treatment failed to upregulate E2A protein binding to the Foxp3 promoter and consequent Foxp3 expression in the Id3−/− T cells.


Control of the differentiation of regulatory T cells and T(H)17 cells by the DNA-binding inhibitor Id3.

Maruyama T, Li J, Vaque JP, Konkel JE, Wang W, Zhang B, Zhang P, Zamarron BF, Yu D, Wu Y, Zhuang Y, Gutkind JS, Chen W - Nat. Immunol. (2010)

Id3 deficiency increases GATA-3a, GATA-3 mRNA expression relative to HPRT in naïve CD4+CD25− T cells at 12 h after activation with TCR and indicated reagents (mean ± s.d. of duplicate measurements in one experiment). b, Flow cytometry of CD4+CD25− T cells at 24 h after activation. Numbers in quadrants indicate percent Foxp3+GATA-3−(top left), Foxp3+GATA-3+(top right) or Foxp3−GATA3+ (bottom right) cells. c, IL-4 mRNA relative to HPRT in CD4+CD25− T cells at 2 h after activation (mean ± s.d. of duplicate measurements in one representative experiment). d, Flow cytometry of Tgfbr1f/f Cd4-cre+ and Tgfbr1f/+Cd4-cre+ CD4+CD25− T cells cultured for 24 h. Numbers in quadrants indicate same cells as in (b). e, Knockdown of E2a with SiRNA decreased TCR-driven IL-4 gene expression in WT naïve CD4+CD25− T cells. IL-4 mRNA expression relative to HPRT is shown (mean ± s.d. of triplicate measurements in one representative experiment). *P < 0.05. f, Relative GATA-3 binding (GATA-3/control IgG) at the Foxp3 promoter in CD4+CD25− T cells (cultured for 24 h). Data shown are mean ± s.d. of duplicate wells in one representative experiment . g, Elimination of GATA-3 with neutralization of IL-4 (αIL-4) restored enrichment of E2A binding to the Foxp3 promoter induced by TGF-β in Id3−/− CD4+ CD25− T cells (cultured for 24 h). Data shown are mean ± s.d. of duplicate wells in one representative experiment. Data shown in a,b,c,d are one experiment representative of at least three, and in e,f,g representative of two.
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Figure 5: Id3 deficiency increases GATA-3a, GATA-3 mRNA expression relative to HPRT in naïve CD4+CD25− T cells at 12 h after activation with TCR and indicated reagents (mean ± s.d. of duplicate measurements in one experiment). b, Flow cytometry of CD4+CD25− T cells at 24 h after activation. Numbers in quadrants indicate percent Foxp3+GATA-3−(top left), Foxp3+GATA-3+(top right) or Foxp3−GATA3+ (bottom right) cells. c, IL-4 mRNA relative to HPRT in CD4+CD25− T cells at 2 h after activation (mean ± s.d. of duplicate measurements in one representative experiment). d, Flow cytometry of Tgfbr1f/f Cd4-cre+ and Tgfbr1f/+Cd4-cre+ CD4+CD25− T cells cultured for 24 h. Numbers in quadrants indicate same cells as in (b). e, Knockdown of E2a with SiRNA decreased TCR-driven IL-4 gene expression in WT naïve CD4+CD25− T cells. IL-4 mRNA expression relative to HPRT is shown (mean ± s.d. of triplicate measurements in one representative experiment). *P < 0.05. f, Relative GATA-3 binding (GATA-3/control IgG) at the Foxp3 promoter in CD4+CD25− T cells (cultured for 24 h). Data shown are mean ± s.d. of duplicate wells in one representative experiment . g, Elimination of GATA-3 with neutralization of IL-4 (αIL-4) restored enrichment of E2A binding to the Foxp3 promoter induced by TGF-β in Id3−/− CD4+ CD25− T cells (cultured for 24 h). Data shown are mean ± s.d. of duplicate wells in one representative experiment. Data shown in a,b,c,d are one experiment representative of at least three, and in e,f,g representative of two.
Mentions: We then examined E2A binding to the Foxp3 promoter in Id3−/− CD4+CD25− T cells which exhibited a severely reduced frequencies of Foxp3+ Treg cells in response to TGF-β1 treatment (Fig. 2). The Id3−/− CD4+CD25− T cells had slightly but reproducibly higher basal levels of E2A binding to the Foxp3 promoter in response to TCR stimulation alone than did WT control T cells (data not shown and Fig. 5f), which could indicate an inhibitory function of Id3 for E2A protein binding to E boxes on target genes24. However, TGF-β1 treatment failed to enhance E2A binding to the Foxp3 promoter in Id3−/− T cells over the cells treated with TCR alone (Fig. 4 f). Freshly isolated CD4+CD25+ Treg cells from the spleens of Id3−/− mice showed no significant enrichment of E2A binding at the Foxp3 promoter compared to CD4+CD25− T cells in the same mice (Supplementary Fig. 6 c). The data were consistent with the positive function of E protein binding to E boxes at the Foxp3 promoter, but it raised an intriguing question as to why TGF-β1 treatment failed to upregulate E2A protein binding to the Foxp3 promoter and consequent Foxp3 expression in the Id3−/− T cells.

Bottom Line: We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells).Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter.Therefore, a network of factors acts in a TGF-β-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.

View Article: PubMed Central - PubMed

Affiliation: Mucosal Immunology Unit, Oral Infection and Immunity Branch, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
The molecular mechanisms that direct transcription of the gene encoding the transcription factor Foxp3 in CD4(+) T cells remain ill-defined. We show here that deletion of the DNA-binding inhibitor Id3 resulted in the defective generation of Foxp3(+) regulatory T cells (T(reg) cells). We identify two transforming growth factor-β1 (TGF-β1)-dependent mechanisms that were vital for activation of Foxp3 transcription and were defective in Id3(-/-) CD4(+) T cells. Enhanced binding of the transcription factor E2A to the Foxp3 promoter promoted Foxp3 transcription. Id3 was required for relief of inhibition by the transcription factor GATA-3 at the Foxp3 promoter. Furthermore, Id3(-/-) T cells showed greater differentiation into the T(H)17 subset of helper T cells in vitro and in a mouse asthma model. Therefore, a network of factors acts in a TGF-β-dependent manner to control Foxp3 expression and inhibit the development of T(H)17 cells.

Show MeSH
Related in: MedlinePlus