The transcription factor STAT3 is required for T helper 2 cell development - PubMed (original) (raw)

The transcription factor STAT3 is required for T helper 2 cell development

Gretta L Stritesky et al. Immunity. 2011.

Abstract

Signal transducer and activator of transcription (STAT) family members direct the differentiation of T helper cells, with specific STAT proteins promoting distinct effector subsets. STAT6 is required for the development of T helper 2 (Th2) cells, whereas STAT3 promotes differentiation of Th17 and follicular helper T cell subsets. We demonstrated that STAT3 was also activated during Th2 cell development and was required for the expression of Th2 cell-associated cytokines and transcription factors. STAT3 bound directly to Th2 cell-associated gene loci and was required for the ability of STAT6 to bind target genes. In vivo, STAT3 deficiency in T cells eliminated the allergic inflammation in mice sensitized and challenged with ovalbumin or transgenic for constitutively active STAT6. Thus, STAT3 cooperates with STAT6 in promoting Th2 cell development. These results demonstrate that differentiating T helper cells integrate multiple STAT protein signals during Th2 cell development.

Copyright © 2011 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. STAT3 is Activated During Th2 cell Differentiation

(A) Wild type (WT) and _Stat3Cd4_−/ − naïve CD4+ T cells were activated with anti-CD3 and anti-CD28 and cultured in IL-4 and anti-IFN-γ (Th2 conditions). Each day during differentiation, cells were stained for intracellular phospho-STAT3 and phospho-STAT6. Numbers in flow cytometry dot plots indicate the percentages of cells in each quadrant. (B) Graphical representation of percent phospo-STAT-positive cells and mean fluorescent intensity (mean ± s.d.). Data are an average of two mice and are representative of 2–3 experiments. (C) Naïve DO11.10 TCR transgenic T cells were stimulated with anti-CD3 or ovalbumin (OVA) in the presence of antigen-presenting cells and cultured under Th2 conditions. Each day during differentiation, cells were stained for intracellular phospho-STAT3 and phospho-STAT6. Numbers in flow cytometry dot plots indicate the percentages of cells in each quadrant. (D) Graphical representation of average percent phospho-STAT-positive cells for DO11.10 TCR transgenic T cells stimulated with OVA or anti-CD3 (mean ± s.d.). Data are an average of two mice and are representative of 2 experiments.

Figure 2

Figure 2. Reduced Th2 Cytokine Production in the Absence of STAT3

(A) WT naïve CD4+ T cells were activated with anti-CD3 and anti-CD28 and cultured under Th2 conditions, in the presence or absence of antibodies to the indicated cytokines and receptors (20 μg/ml per antibody) for five days before stimulation with anti-CD3. Twenty-four hours after stimulation supernatants were tested for cytokine concentration using ELISA (mean ± s.d.). Results are representative of experiments with four mice. (B) Cells cultured as in (A) with control antibody or the combination of antibodies indicated were tested for amount of pSTAT by intracellular staining on day 4 of Th2 culture. Numbers represent mean fluorescence intensity. Results are representative of experiments with four mice. (C) WT and _Stat3Cd4_−/ − naïve CD4+ T cells were activated with anti-CD3 and anti-CD28 and cultured with IL-4 + anti-IFN-γ (Th2 conditions). After 5 days of differentiation cells were collected and counted. Differentiated cells (1x106) were then re-stimulated with anti-CD3 (4 μg/ml) for 24 hours. Cell-free supernatant was collected and tested for various cytokines using ELISA. Data are the mean ± s.d. of results from two mice and representative of more than 5 experiments. Students t test was performed to calculate p values. (D) Cells activated and cultured as in A were re-stimulated with anti-CD3 for 5 hours. Following stimulation cells were stained with anti-IL-4 and anti-IL-9. Numbers in flow cytometry dot plots indicate the percentages of cells in the quadrant. (E) After re-stimulation of differentiated Th2 cells with anti-CD3 and recovery of supernatants as described in (A), cell pellets were collected, RNA was isolated, and quantitative PCR was performed for the indicated cytokines (mean ± s.d.). Data are representative of two experiments.

Figure 3

Figure 3. Reduced Th2-Specific Transcription Factor Expression in the Absence of STAT3

(A) WT and _Stat3Cd4_−/− naïve CD4+ T cells were differentiated under Th2 conditions and cells were isolated each day during differentiation. RNA isolated from cells was analyzed for gene expression using qPCR (mean ± s.d.). Data are representative of experiments with 4 mice. (B) WT and _Stat3Cd4_−/ − naïve CD4+ T cells were used directly or differentiated as in (A) for five days. Chromatin immunoprecipitation was performed for the indicated histone modifications and qPCR was used to determine the amounts of each modification (mean ± s.d.). (C) Chromatin immunoprecipitation from WT naïve CD4+ T cells with control or STAT3 antibodies followed by qPCR for detection of the indicated loci (mean ± s.d.). (D) WT and _Stat3Cd4_−/ − naïve CD4+ T cells were differentiated as in (A) for five days. Nuclei were isolated and left untreated or treated with micrococcal nuclease for 10 minutes. Relative accessibility is defined as 2 to the power of the difference between the Ct value of qPCR of untreated and treated samples (mean ± s.d.). (E) WT and _Stat3Cd4_−/ − naïve CD4+ T cells were differentiated as in (A). On Day 2 cells were transduced with control, Batf or _Maf_-expressing retrovirus. Sorted transduced cells were re-stimulated with anti-CD3 for 24 hours. Supernatant was tested for Th2 cytokines using ELISA (mean ± s.d.). Data are representative of 2 independent experiments.

Figure 4

Figure 4. STAT3 and STAT6 Cooperate in Promoting Th2 Cytokine Production

(A) Naïve CD4+ T cells were activated for 48 hours in the absence of skewing cytokines before being transduced with control or STAT3C-expressing retrovirus. After 5 days in culture, cells were sorted and re-stimulated with anti-CD3 (4 μg/ml) for 24 hours before cell-free supernatant was tested for cytokines using ELISA (mean ± s.d.). (B) Naïve CD4+ T cells were activated for 48 hours under Th1 conditions (5 ng/ml IL–12 + 10 μg/ml anti-IL-4) before being transduced with control, STAT6VT-expressing, STAT3-expressing or both retroviruses. After 5 days in culture, cells were sorted and re-stimulated with anti-CD3 (4 μg/ml) for 24 hours before cell-free supernatant was tested for cytokines using ELISA (mean ± s.d.). (C) CD4+ cells were isolated from WT, STAT6VT and STAT6VT-_Stat3Cd4_−/ − mice. Cells were then re-stimulated with anti-CD3 for 24 hours. Cell-free supernatant was collected and tested for various cytokines using ELISA. Data are representative of 2 experiments (average of 2–6 mice ± s.d.). Students t test was performed to calculate p values.

Figure 5

Figure 5. STAT3 Binds Th2-Associated Gene Loci and Defines the STAT6 Binding Pattern

(A) Naïve CD4+ T cells activated with anti-CD3 and anti-CD28 and cultured under Th1, Th2 or Th17 conditions for 3 days were used for ChIP performed with normal rabbit IgG or anti-STAT3 before qPCR was performed for the indicated genes. Data are expressed as percent input ± s.d. and control Ig background values are subtracted from the values indicated. Data are representative of 3–4 experiments with similar results. (B) Naïve CD4+ T cells from WT and _Stat3Cd4_−/ − activated with anti-CD3 and anti-CD28 and cultured under Th2 conditions for 3 days were used for ChIP performed with normal rabbit IgG or anti-STAT6 and qPCR was performed for the indicated genes. Data are expressed as percent input ± s.d. and control Ig background values are subtracted from the values indicated. Data are representative of 3–4 experiments with similar results. ND, not detected. (C) CD4+ cells were isolated from WT, STAT6VT and STAT6VT-_Stat3Cd4_−/ − mice. Cells were then re-stimulated with anti-CD3 for 6 hrs before RNA was isolated and quantitative PCR was performed. Data are representative of 2 experiments (average of 2–6 mice ± s.d.). Students t test was performed to calculate p values.

Figure 6

Figure 6. STAT3 Promotes the Development of Allergic Inflammation

(A–C) WT and _Stat3Cd4_−/ − mice were immunized with OVA and Alum on days 0 and 7 and challenged as described in methods. After challenges, BAL cell numbers were determined by cell counting and flow cytometry (A), and cytokine levels were measured in BAL fluid and in culture supernatants from splenocytes stimulated with OVA for 72 h using ELISA. Data are average of 5–6 mice per group ± s.e.m (D) Incidence of blepharitis and atopic dermatitis of WT, STAT6VT and STAT6VT-_Stat3Cd4_−/ − mice are shown. Incidence was determined by visual examination of mice (n=25 per group). (E) Numbers of eosinophils (defined by flow cytometry) recovered in BAL. BAL data are representative of 2 independent experiments and shown as the average of 2 mice per group ± s.d. For A–C and E, Students t test was performed to calculate p values. (F) Ear tissue from WT, STAT6VT and STAT6VT-_Stat3Cd4_−/ − mice were fixed and paraffin-embedded sections were stained with hematoxylin-eosin. Magnification is indicated in the panel and photomicrographs are representative of 10 mice per group (G) Lungs from WT, STAT6VT and STAT6VT-_Stat3Cd4_−/ − mice were embedded in paraffin and stained with H & E. Magnification is indicated in the panel and photomicrographs are representative of 10 mice per group.

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