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The transcription factor musculin promotes the unidirectional development of peripheral Treg cells by suppressing the TH2 transcriptional program

Nature Immunology volume 18, pages 344–353 (2017)Cite this article

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Abstract

Although master transcription factors (TFs) are key to the development of specific T cell subsets, whether additional transcriptional regulators are induced by the same stimuli that dominantly repress the development of other, non-specific T cell lineages has not been fully elucidated. Through the use of regulatory T cells (Treg cells) induced by transforming growth factor-β (TGF-β), we identified the TF musculin (MSC) as being critical for the development of induced Treg cells (iTreg cells) by repression of the T helper type 2 (TH2) transcriptional program. Loss of MSC reduced expression of the Treg cell master TF Foxp3 and induced TH2 differentiation even under iTreg-cell-differentiation conditions. MSC interrupted binding of the TF GATA-3 to the locus encoding TH2-cell-related cytokines and diminished intrachromosomal interactions within that locus. MSC-deficient (Msc−/−) iTreg cells were unable to suppress TH2 responses, and Msc−/− mice spontaneously developed gut and lung inflammation with age. MSC therefore enforced Foxp3 expression and promoted the unidirectional induction of iTreg cells by repressing the TH2 developmental program.

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Figure 1: TGF-β-dependent early induction of MSC in CD4+ T cells.
Figure 2: Smad3 regulates MSC during iTreg cell differentiation.
Figure 3: MSC promotes Foxp3 expression during iTreg cell differentiation.
Figure 4: MSC-deficient iTreg cells exhibits enhanced TH2 response.
Figure 5: The TH2 response is the factor mainly responsible for the reduction in Foxp3 in the absence of MSC.
Figure 6: MSC suppresses GATA-3 activity in iTreg cells.
Figure 7: MSC suppresses the TH2 response in iTreg cells via regulation of the chromatin conformations of the TH2 locus.
Figure 8: MSC promotes the suppressive effect of iTreg cells on the TH2 response.

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Acknowledgements

We thank D. Kozoriz for cell sorting; T. Chatila for advice; M. Collins for advice and editing the manuscript; E.N. Olson (University of Texas Southwestern Medical Center at Dallas) for Msc−/− mice; L. Maggio-Price (University of Washington) for Smad3−/− mice; and A. Yoshimura (Keio University School of Medicine) for the plasmid pCMV5-Smad3. Supported by the National Multiple Sclerosis Society (Career Transition Award TA 3059-A-2 to C.W.) and the US National Institutes of Health (K99 NIH Pathway to Independence Award KAI110649A to C.W.; K01DK090105 to S.X.; and P01AI073748, P01AI056299, P01AI039671, R01NS045937 and R01 NS030843 to V.K.).

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  1. Chuan Wu, Valerie Dardalhon & Rafael F Franca

    Present address: Present addresses: Experimental Immunology Branch, National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA (C.W.), GMM-UMR5535-CNRS, Montpellier, France (V.D.), and Department of Pharmacology, University of São Paulo, São Paulo, Ribeirao Preto, Brazil (R.F.F.).,

  2. Chuan Wu and Zuojia Chen: These authors contributed equally to this work.

Authors and Affiliations

  1. Evergrande Center for Immunologic Diseases, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts, USA

    Chuan Wu, Zuojia Chen, Valerie Dardalhon, Sheng Xiao, Theresa Thalhamer, Asaf Madi, Rafael F Franca, Timothy Han & Vijay Kuchroo

  2. Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Mengyang Liao

  3. Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington, USA

    Mohammed Oukka

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Contributions

C.W. designed and performed experiments and wrote the manuscript; Z.C. performed experiments and wrote the manuscript; V.D., S.X., T.T., M.L., A.M., R.F.F., T.H. and M.O. performed experiments; A.M. analyzed the data; and V.K. supervised the study and edited the manuscript.

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Integrated supplementary information

Supplementary Figure 1 MSC is specifically expressed in iTreg cells.

(a) Naive CD4+ T cells from WT mice were stimulated under TH0 and iTreg conditions and harvested at 10 and 48 hours. Relative mRNA expression levels of indicated genes were shown by q-PCR; (b) Naive CD4+ T cells from WT mice were stimulated under TH0, TH1, TH2, TH17, iTreg conditions and harvested at 10 and 48 hours. MSC and Foxp3 protein expression under indicated conditions were determined by immunoblotting; (c) MSC and Foxp3 protein expression in in vivo isolated iTreg cells (CD4+Foxp3+Nrp1–) and tTreg cells (CD4+Foxp3+Nrp1+) from spleen were determined by immunoblotting. Foxp3 expression in CD4+ T cells from spleen, dLN and thymus of 8 weeks old WT and Msc–/– mice was determined by (d) flow cytometry and (e) quantification; (f) Ki76 level of Foxp3+ Treg cells from dLN and spleen of 8 weeks old WT and Msc–/– mice was determined by flow cytometry. Data are pooled from three independent experiments (a, e) or representative of three independent experiments (b, c, d, f) with n = 4 mice each group. NS: not statistically significant, (Student's t-test, error bars, SD).

Supplementary Figure 2 MSC deficiency induces compromised iTreg cell differentiation.

Flow cytometric analysis (a) and quantification (b) of Foxp3 expression in CD4+ T cells from the mediastina lymph nodes (lLN) and lung of 8 weeks old WT and Msc–/– mice; Flow cytometric analysis (c) and quantification (d) of Foxp3+Nrp1– iTreg cells or Foxp3+Nrp1+ tTreg cells from LP of WT and Msc–/– mice; Chimeric mice were generated by transferring WT or Msc–/– bone marrow into Rag2–/– mice. 8 weeks after reconstitution, flow cytometric analysis (e) and quantification (f) of Foxp3 expression in CD4+ cells in mLN and LP; Flow cytometric analysis (g) and quantification (h) of Foxp3+Nrp1– iTreg cells or Foxp3+Nrp1+ tTreg cells as in LP; (i) Naive CD4+ T cells from WT and Msc–/– mice were stimulated under TH0, TH1, TH2, TH17 conditions and harvested at 72 hours. Intracellular staining of indicated cytokines produced by different polarized T cell subsets cells from WT and Msc–/– mice was determined by flow cytometry; (j, k) CD45.1+ WT and CD45.2+ Msc–/– naive T cells were cultured (j) separately or (k) together in the presence of TGF-β. The frequency of Foxp3+ cells was then determined by flow cytometry. (l) Chimeric mice were generated by transferring WT and Msc–/– bone marrow at 1:1 ratio into Rag2–/– mice. 10 weeks after reconstitution, Foxp3 expression in CD4+ cells in LP were analyzed by flow cytometry. Data are pooled from three independent experiments (b, d, f, h) or are representative of three independent experiments (a, c, e, g, i, j, k, l) with n =5 mice each group. *P < 0.05, NS: not statistically significant (Student's t-test, error bars, SD).

Supplementary Figure 3 MSC-deficient iTreg cells exhibit enhanced expression of TH2-cell-related genes.

(a) The level of phosphorylated Smad3 was assessed by immunoblotting in naive T cells from WT and Msc–/– mice stimulated with TGF-β for the indicated time; (b) Naive CD4+ T cells from WT and Msc–/– mice were stimulated under iTreg conditions with TGF-β and harvested at 72 hours. Relative mRNA expression levels of indicated genes are shown by q-PCR; (c) TH2 RPKM values were than compared to all other T cell subsets (TH1, TH2, TH17, TH9 and Treg, GSE 39756). Differentially expressed genes (fold change >1.5) in each comparison were than overlapped with genes that were upregulated in the Msc–/– iTreg cells (fold change >1.5). Enrichment was calculated as the number of overlapping genes divided by the total number of genes upregulated in each T cell subset. Data are pooled from three independent experiments (b) or are representative of three independent experiments (a).

Supplementary Figure 4 MSC-deficient iTreg cells exhibit enhanced TH2 responses.

(a) Flow cytometry analysis of Foxp3 and GATA3 expression from CD4+ T cells in TH0 and iTregs from WT or Msc–/– mice; Flow cytometric analysis (b) and quantification (c) of IL-17, IFN-γ and IL-4 in CD4+ T cells from spleens of 10 months old WT and Msc–/– mice; Flow cytometric analysis of Foxp3 and IL-4 (d) and mRNA expression of Il4, Il5, Il13, Ifng and Il17 (e) in iTregs in the recipient mice transferred with OT-II Rag2–/– or OT-II Rag2–/–Msc–/– CD45.2+CD4+ T cells 5 days after OVA administration; (f) ELISA analysis of IL-4 production in iTregs in the recipient mice transferred with OT-II Rag2–/– or OT-II Rag2–/–Msc–/– CD45.2+CD4+ T cells after OVA administration. Data are pooled from three independent experiments (c, e, f) or are representative of three independent experiments (a, b, d) with n = 4 mice each group. *P < 0.05, **P < 0.01, NS: not statistically significant (Student's t-test, error bars, SD).

Supplementary Figure 5 MSC participates in epigenetic modification of the TH2 locus in iTreg cells.

ChIP-PCR analysis of the binding of AcH4 (a), H3K9Ac (b), H3K4me3 (c) and H3K27me3 (d) on the indicated sites on TH2 and Foxp3 locus in the WT or Msc–/– naïve T cells stimulated with TGF-β for 12 and 48 hours. Data representative of three independent experiments. *P < 0.05, **P < 0.01, (Student's t-test, error bars, SD).

Supplementary Figure 6 Depletion of IL-4 ‘rescues’ Foxp3 expression in MSC-deficient iTreg cells.

(a) Activated WT naive CD4+ T cells were stimulated with combinations of TGF-β with indicated dosage of anti-IL-4 antibody for 3 days. The frequency of Foxp3+ cells was determined by flow cytometry; (b) Quantification of the frequency of Foxp3+ iTregs as in (a); (c) Flow cytometry analysis and (d) Quantification of Foxp3+ Tregs in CD4+ T cells in the thymus from indicated mice; (e) Quantification of Foxp3+Nrp1– iTregs or Foxp3+Nrp1+ tTregs from dLN as in Fig 5d; (f) Quantification of Foxp3+Nrp1– iTregs or Foxp3+Nrp1+ tTregs from LP as in Fig 5d; The expression of Msc was analyzed by q-PCR. Data are representative of three independent experiments (a, c) with n = 4 mice each group or are pooled from three independent experiments (b, d-f). *P < 0.05, (Student's t-test, error bars, SD).

Supplementary Figure 7 MSC interacts with GATA3 in iTreg cells.

(a) GATA3 and MSC association was visualized in different T cell subsets with an in situ proximity ligation assay. Punctate staining (red) indicates a GATA3-MSC interaction as detected by the assay; (b) Co-immunoprecipitation of GATA3 and MSC from extracts of co-transfected 293T cells with or without ethidium bromide (EtBr). HEK293T cells were transfected with mouse Flag-tagged GATA3 and V5-tagged MSC. Anti-V5 immunoprecipitates (IP) and total lysates were immunoblotted with anti-V5 and anti-Flag antibodies; (c) Co-immunoprecipitation of GATA3 and MSC from extracts of in vitro differentiated iTregs with or without ethidium bromide (EtBr). Anti-GATA3 immunoprecipitates (IP) and total lysates were immunoblotted with anti-GATA3 and anti-MSC antibodies. Data are representative of three independent experiments.

Supplementary Figure 8 MSC promotes the suppressive effect of iTreg cells on the TH2 response.

(a) q-PCR analysis of indicated co-inhibitory molecules in GFP+ (Foxp3+) WT and Msc–/– iTregs; (b) Naive CD45.1+CD4+ T cells were cocultured with CD45.2+Foxp3+ WT and Msc–/– iTregs under differentiation conditions for TH1, TH2, and TH17 cells. Intracellular staining of indicated cytokines produced by CD45.1+ different polarized T cell subsets cells was determined by flow cytometry; (c) Flow cytometry analysis of intracellular cytokines in gated CD4+ T cells from the lung of HDM administrated mice with indicated treatments. Data are representative of three independent experiments (a, b, c) with n = 4 mice each group. *P < 0.05, **P < 0.01, (Student's t-test, error bars, SD).

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Wu, C., Chen, Z., Dardalhon, V. et al. The transcription factor musculin promotes the unidirectional development of peripheral Treg cells by suppressing the TH2 transcriptional program. Nat Immunol 18, 344–353 (2017). https://doi.org/10.1038/ni.3667

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