STAT6-dependent regulation of Th9 development

Abstract

Th cell effector subsets develop in response to specific cytokine environments. The development of a particular cytokine-secreting pattern requires an integration of signals that may promote the development of opposing pathways. A recent example of this paradigm is the IL-9-secreting Th9 cell that develops in response to TGF-β and IL-4, cytokines that, in isolation, promote the development of inducible regulatory T cells and Th2 cells, respectively. To determine how the balance of these factors results in priming for IL-9 secretion, we examined the effects of each pathway on transcription factors that regulate Th cell differentiation. We demonstrated that TGF-β induces the PU.1-encoding Sfpi1 locus and that this is independent of IL-4-induced STAT6 activation. IL-4-activated STAT6 is required for repressing the expression of T-bet and Foxp3 in Th9 cells, transcription factors that inhibit IL-9 production, and STAT6 is required for the induction of IRF4, which promotes Th9 development. These data established a transcription factor network that regulates IL-9 and demonstrated how combinations of cytokine signals generate cytokine-secreting potential by altering the expression of a panel of transcription factors.

Figure 1. Differential requirement for STAT proteins in Th2 and Th9 cells

A, Naïve CD4+T cells from WT mice were activated with anti-CD3 and anti-CD28 and cultured with IL-4 and anti-IFN-γ (Th2 conditions) or with IL-4, TGF-β and anti-IFN-γ (Th9 conditions). Each day during differentiation, cells were stained for intracellular-phospho-STAT3 and phospho-STAT6. The bar graphs represent percentages of cells (top) and mean fluorescent intensity (MFI, bottom). Data are average ± S.E.M of 6 mice from 3 experiments.B, C. Naïve CD4+T cells from WT and Stat6^−/− (top), WT and Stat3^CD4−/− (middle), and WT and Parp14^−/− mice (bottom) were cultured under Th9 conditions. After 5d cells were harvested and stimulated with PMA and Ionomycin before intracellular staining for cytokines IL-9 and IL-4 was performed (B) or stimulated with anti-CD3 and 24h later cell-free supernatant was collected and IL-9 production was assessed by ELISA (C). Data are representative of 3 experiments with similar results.*, p<0.05. ***, p<0.001.

Figure 2. Th2 transcription factors are expressed in Th9 cells

WT naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and cultured under Th1, Th2, Th17, Th9, or Treg conditions or directly ex vivo (naïve) for 5d. Cell pellets were collected, RNA was isolated and quantitative PCR was performed for the indicated transcription factors. B, Naïve CD4+T cells from WT mice were differentiated under Th2 (left) or Th9 (right) cell conditions for 5d. Cells were then either kept unstimulated or stimulated for 6h before intracellular staining for GATA3 was performed. Data are representative of 3 experiments with similar results.C, Naïve CD4+T cells from WT mice were differentiated under Th2 or Th9 cell conditions. RNA was isolated from cells at each day during differentiation and examined for the expression of the indicated genes. D, WT and Stat6^−/− naïve CD4+T cells were cultured under Th9 cell conditions for 5d, RNA was isolated and quantitative PCR was performed for the indicated genes. Data are representative of 3 independent experiments with 2-3 mice in each group.E, Naïve CD4+T cells from WT and Stat6−/− mice were differentiated under Th2 (left) or Th9 (right) cell conditions for 5d. Cells were then stimulated for 6h before intracellular staining for GATA3 was performed.

Figure 3. Transcription factor binding to the Il9 locus

A, Schematic of the Il9 gene with conserved non-coding sequences used for ChIP analysis indicated. B, Naïve CD4+T cells from WT mice were activated with anti-CD3 and anti-CD28 and cultured under Th2 and Th9 cell conditions for 5d. Chromatin immunoprecipitation was performed for STAT6 before quantitative PCR for two Il9 CNS sites, or for the Irf4 or Maf promoters, were performed to determine the amount of Stat6 binding to the Il9 gene. Data are average ± S.D of 4 mice from 2 experiments. C, Naïve CD4+T cells from WT and Stat6^−/− mice were cultured under Th9 conditions for 5d. Chromatin immunoprecipitation was performed for PU.1 (top), and IRF4 (bottom) before quantitative PCR for two Il9 CNS sites were performed. Data are average ± S.D of 6-8 mice from 3-4 experiments.

Figure 4. Th2 transcription factors repress IL-9 in Th9 cultures

A-C. WT and Il4^−/− naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and cultured under Th9 cell conditions for 48h before being transduced with control or GATA3-expressing retroviruses (A), control or c-Maf expressing retroviruses (B), or control or JunB-expressing retroviruses (C). After 5d in culture cells were stimulated with PMA and Ionomycin for intracellular staining of IL-9 and IL-4. Data are representative of 2-3 independent experiments with similar results.*, p<0.05. **, p<0.01.

Figure 5. IL-9 is repressed by Th1 associated transcription factors

A, Naïve CD4+T cells from WT mice were analyzed directly ex vivo (naïve) or cultured under Th1, Th2, Th17, Th9, and Treg cell conditions for 5d. After RNA isolation, quantitative PCR was performed for the indicated genes. Data are representative of 2 experiments with similar results. B, Total cell lysates were prepared from Th1, Th2, and Th9 cells and immunoblot was performed for Runx3, β-actin was used as loading control. C, WT naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and cultured under Th9 cell conditions for 48h before being transduced with control or Runx3-expressing retroviruses and control or T-bet expressing retroviruses. Cells were harvested after 5d in culture and stimulated with PMA and ionomycin before intracellular IL-9 and IL-4 staining. Data are average ± S.D of 2 experiments with 4 mice. D, Naïve CD4+T cells from WT and Tbx21^−/− (bottom left) or WT and Runx3^fl/fl-CD4-Cre (bottom right) were cultured in Th9 cell conditions. After 5d cells were harvested and stimulated with PMA and Ionomycin before intracellular IL-9 and IL-4 staining. Data are representative of 2-3 experiments with similar results. E, WT and Stat6^−/−naïve CD4+T cells were cultured under Th9 cell conditions for 5d. Cells were harvested and quantitative PCR was performed for Runx3and Tbx21.*, p<0.05. **, p<0.01.

Figure 6. A balance of IL-4 and TGFβ signals are required for the development of IL-9-secreting T cells

A, WT naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and were cultured with increasing doses of TGF-β in the presence (Th9) or absence (Treg) of IL-4 for 5d. Cells were then stained for intracellular Foxp3 (top) and IL-9 (middle). Differentiated Th9 cells were stimulated with anti-CD3 and 24h later IL-9 production was assessed by ELISA (bottom). B, Naïve CD4+T cells were cultured under Th9 and Treg cell conditions as in A (left) or cultured with 2 ng/ml TGF-β and increasing doses of IL-4 (Th9, right) for 5d. After differentiation, RNA was isolated and Sfpi1 expression was measured by quantitative PCR. Data are representative of 2 experiments with similar results. C, WT and Stat6^−/− naïve CD4+T cells were cultured under Th9 cell conditions for 5d and Foxp3 expression was measured using qPCR. Data are average ± S.D of 4 mice from 2 experiments. D, Naïve CD4+T cells were activated with anti-CD3 and anti-CD28 and cultured in Th9 cell conditions for 48h before being transduced with control or Foxp3-expressing retroviruses. After 5d in culture cells were stimulated with PMA and Ionomycin and intracellularly stained for IL-9 and IL-17A. Data are representative of 2 experiments with similar results.E, Schematic showing a summary of a transcription factor network in Th9 cells.*, p<0.05.