Abstract

Foxp3+ regulatory T cells (Treg cells) are crucial for the maintenance of immune homeostasis both in lymphoid tissues and in non-lymphoid tissues. Here we demonstrate that the ability of intestinal Treg cells to constrain microbiota-dependent interleukin (IL)-17–producing helper T cell (TH17 cell) and immunoglobulin A responses critically required expression of the transcription factor c-Maf. The terminal differentiation and function of several intestinal Treg cell populations, including RORγt+ Treg cells and follicular regulatory T cells, were c-Maf dependent. c-Maf controlled Treg cell–derived IL-10 production and prevented excessive signaling via the kinases PI(3)K (phosphatidylinositol-3-OH kinase) and Akt and the metabolic checkpoint kinase complex mTORC1 (mammalian target of rapamycin) and expression of inflammatory cytokines in intestinal Treg cells. c-Maf deficiency in Treg cells led to profound dysbiosis of the intestinal microbiota, which when transferred to germ-free mice was sufficient to induce exacerbated intestinal TH17 responses, even in a c-Maf-competent environment. Thus, c-Maf acts to preserve the identity and function of intestinal Treg cells, which is essential for the establishment of host–microbe symbiosis.

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Data availability

Sequencing data supporting the findings of this study have been deposited in the Sequence Read Archive (SRA) database under accession codes SRP123533 (ATAC-Seq) and PRJNA498200 (16S rRNA-Seq) and in the Gene Expression Omnibus (GEO) database under accession code GSE106396 (RNA-Seq).

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Acknowledgements

This research was supported by the Deutsche Forschungsgemeinschaft (DFG) under Germany’s Excellence Strategy—EXC 2167-390884018 (to A.S.), SFB650 (to A.S.) and CRC/TR 241 (to A.S. and C.R.), the German Federal Ministry of Education and Science (BMBF)—Project InfectControl 2020 Projekt DIAT FKZ: 03ZZ0827A (to A.S.), the state of Berlin and the ‘European Regional Development Fund’ ERDF 2014-2020, EFRE 1.8/11, Deutsches Rheuma-Forschungszentrum (to F.H., G.A.H, P.M. and M.F.M), the Helmholtz Association project grant VH-NG-933 (to T. Strowig), the National Health and Medical Research Council (NHMRC) project grants (1069075, 1106378 to A .Kallies), the Sylvia and Charles Viertel Foundation (fellowship to A. Kallies), a Walter and Eliza Institute Centenary Fellowship funded by the CSL (to W.S.), a fellowship by the Deutsche Forschungsgemeinschaft (to J.B.) and the Victorian State Government Operational Infrastructure Support and Australian Government NHMRC Independent Research Institute Infrastructure Support scheme. We thank the DRFZ Flow Cytometry Core Facility and the BCRT Flow Cytometry Lab for cell sorting. In addition, we thank Renee Gloury and Victoria von Götze for technical and experimental help.

Author information

Author notes

  1. These authors contributed equally: Sascha Rutz, Axel Kallies, Alexander Scheffold.

Affiliations

  1. Department of Cellular Immunology, Clinic for Rheumatology and Clinical Immunology, Charité—Universitätsmedizin Berlin, Berlin, Germany

    • Christian Neumann
  2. German Rheumatism Research Center (DRFZ) Berlin, Leibniz Association, Berlin, Germany

    • Christian Neumann
    • , Alexander Beller
    • , Frederik Heinrich
    • , Christina Stehle
    • , Gitta A. Heinz
    • , Patrick Maschmeyer
    • , Chiara Romagnani
    • , Hyun-Dong Chang
    • , Andrey Kruglov
    •  & Mir-Farzin Mashreghi
  3. Department of Microbiology and Immunology, University of Melbourne, The Peter Doherty Institute for Infection and Immunity, Melbourne, Australia

    • Jonas Blume
    • , Peggy P. Teh
    • , Ajithkumar Vasanthakumar
    • , Tom Sidwell
    •  & Axel Kallies
  4. Molecular Immunology Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia

    • Jonas Blume
    • , Peggy P. Teh
    • , Ajithkumar Vasanthakumar
    • , Tom Sidwell
    •  & Axel Kallies
  5. Research Group Microbial Immune Regulation, Helmholtz Centre for Infection Research, Braunschweig, Germany

    • Urmi Roy
    • , Eric J. C. Gálvez
    •  & Till Strowig
  6. Department of Medical Biology, University of Melbourne, Melbourne, Australia

    • Yang Liao
    • , Yifang Hu
    •  & Wei Shi
  7. Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia

    • Yang Liao
    • , Yifang Hu
    •  & Wei Shi
  8. Department of Cancer Immunology, Genentech, San Francisco, CA, USA

    • Teresita L. Arenzana
    •  & Sascha Rutz
  9. Department of Bioinformatics and Computational Biology, Genentech, San Francisco, CA, USA

    • Jason A. Hackney
  10. Department of Biochemical and Cellular Pharmacology, Genentech, San Francisco, CA, USA

    • Celine Eidenschenk
  11. Sanquin Research, Department of Hematopoiesis and Landsteiner Laboratory, Amsterdam Infection and Immunity Institute, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands

    • Derk Amsen
  12. Medical Department I, Charité—Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany

    • Chiara Romagnani
  13. Belozersky Institute of Physico-Chemical Biology and Biological Faculty, M.V. Lomonosov Moscow State University, Moscow, Russia

    • Andrey Kruglov
  14. Institute of Immunology, Christian-Albrechts−Universität zu Kiel & Universitätsklinik Schleswig Holstein, Kiel, Germany

    • Alexander Scheffold

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Contributions

C.N. and J.B. designed and performed most experiments, analyzed the data, generated the figures and wrote the manuscript. U.R. and T. Strowig performed and supervised microbiota studies. P.P.T., Y.L., C.S., P.M. and T. Sidwell performed experiments. A.V. provided discussion and proofread the manuscript. A.B. and H.C. performed and supervised IgA studies. F.H., T.L.A., J.A.H., E.J.C.G., Y.H. and W.S. performed computational analyses of 16 S rRNA-seq, RNA-seq, ChIP-seq and ATAC-seq data. C.E. performed studies with V463 mut-STAT3 mice. G.A.H. and M.F.M. supervised sequencing experiments and provided reagents and equipment for their execution. D.A. and C.R. provided expertise and Notch1floxNotch2floxCd4Cre mice and Il10GFP reporter mice, respectively. A. Kruglov contributed to data interpretation and discussions and assisted in DSS colitis experiments. S.R., A. Kallies and A.S. conceived the project and wrote the manuscript.

Competing interests

T.L.A., J.A.H. and S.R. are full-time employees of Genentech and shareholders of Roche.

Corresponding authors

Correspondence to Sascha Rutz or Axel Kallies or Alexander Scheffold.

Integrated supplementary information

  1. Supplementary Figure 1 Gating strategy.

    Flow cytometric profiles illustrating the gating strategy for phenotypic analysis of (a) splenic or (b) colonic LP CD4+ TCRβ+ T cells. The presented profiles are representative of at least five independent stainings.

  2. Supplementary Figure 2 Expression of eTreg cell and/or activation markers in c-Maf+ Treg cells versus that of c-Maf Treg cells.

    (a) Flow cytometric analysis shows expression of ICOS, CTLA4, TIGIT, CD69, Blimp-1 and CD25 in splenic c-Maf+ and c-Maf Foxp3+ Treg cells. Histograms are representative of three independent experiments. (b–c) Flow cytometric profiles of c-Maf versus (b) ICOS and (c) TIGIT expression by Foxp3+ Treg cells from different organs. Contour plots are representative of at least three independent experiments. (d) Expression of c-Maf by MACS-sorted CD4+ CD25+ splenic Treg cells after in vitro stimulation for 72 hours with anti-CD3/CD28 and rIL-2 in the presence or absence of the Notch ligand Dll-4. Histograms (left) show c-Maf expression (gated on Foxp3+ cells). Graph on the right shows frequency of c-Maf+ cells among Foxp3+ Treg cells (pooled data from three independent experiments, mean ± SEM, **P<0.0015). (e) Expression of c-Maf by mLNs Foxp3+ Treg cells from Notch1/2fl/flCd4Cre (N1/2∆CD4) and wild-type SPF (Ctrl) mice. Histograms (left) show c-Maf expression. Graph on the right shows quantification of c-Maf gMFI (n = 4, mean ± SEM). All statistical differences were tested using an unpaired Student’s t-test (two-tailed).

  3. Supplementary Figure 3 Phenotypic characterization of Maf∆Treg mice.

    (a) Expression of Il10:GFP by Foxp3+ and Foxp3 CD4+ TCRβ+ T cells isolated from indicated organs of naïve Il10GFP reporter mice as measured by flow cytometry. Representative flow cytometric profiles of Foxp3 versus Il10:GFP expression by splenic and colonic CD4+ TCRβ+ T cells are shown on top. The graph below shows quantification (n = 4, mean ± SEM, *P<0.05, ***P<0.001). (b) Flow cytometric profiles of c-Maf versus Foxp3 expression by CD4+ TCR+ T cells from different organs of Foxp3Cre (Ctrl) and Maffl/flFoxp3Cre (Maf∆Treg) mice. The presented profiles are representative of at least three independent stainings. (c) Frequency of Foxp3+ Treg cells among CD4+ TCRβ+ T cells from indicated organs of Maf∆Treg and control mice (n = 5, mean ± SEM). (d) Enumeration of total cells isolated from different organs of Maf∆Treg and control mice (n = 5, mean ± SEM, *P<0.05, **P<0.01). (e) Flow cytometric analysis shows expression of ICOS, TIGIT, CD69, CD25 in splenic CD62L Foxp3+ Treg cells of Maf∆Treg and control mice. Histograms are representative of three independent experiments. (f) Flow cytometric analysis of Blimp-1 expression in colonic Foxp3+ Treg cells from Maf∆Treg and control mice. On top, representative contour plots displaying Blimp-1 versus c-Maf expression are shown. Blimp-1-deficient Foxp3+ Treg cells from Prdm1fl/flCd4Cre (Prdm1∆CD4) mice served as a negative control for the Blimp-1 staining. Below, Blimp-1 expression is shown by representative histograms (left) and Blimp-1 gMFI is quantified (right, n = 3, mean ± SEM). (g) Flow cytometric analysis of IRF4 expression in colonic Foxp3+ Treg cells from Maf∆Treg and control mice. On the left, a representative contour plot displaying IRF4 versus c-Maf expression is shown. On the right, IRF4 expression in colonic Foxp3+ Treg cells is shown by representative histograms and IRF4 gMFI is quantified (n = 3, mean ± SEM). Foxp3 conventional T cells (Tcon) from control mice served as a negative control for the IRF4 staining. (h) Flow cytometric analysis of AhR expression in colonic Foxp3+ Treg cells from Maf∆Treg and control mice. On the left, representative contour plots displaying AhR versus c-Maf expression are shown. AhR-deficient Foxp3+ Treg cells from Ahr-/- mice served as a negative control for the AhR staining. On the right, AhR expression is shown by representative histograms and AhR gMFI is quantified (n = 3, mean ± SEM, **P = 0.0097). All statistical differences were tested using an unpaired Student’s t-test (two-tailed).

  4. Supplementary Figure 4 Rorc∆Treg mice have normal intestinal TH17 cell responses.

    (a) Flow cytometric analysis of c-Maf expression in different Treg cell subsets from PPs. Histograms are representative of three independent experiments. (b) Frequency of TFR cells (PD1+CXCR5+ cells among Foxp3+ Treg cells) in PPs of Maf∆Treg and control mice. Representative contour plots of PD1 versus CXCR5 expression by Foxp3+ Treg cells are shown on the left. The graph on the right shows quantification (n = 4, mean ± SEM, ***P < 0.0001). (c) Flow cytometric profiles of RORγt versus Foxp3:YFP expression by colonic Foxp3+ Treg cells of mosaic Foxp3Cre/+ or Maffl/flFoxp3Cre/+ female mice. The presented profiles are representative of at least two independent stainings. (d) RORγt (n = 4, mean ± SEM, *P = 0.0462) versus T-bet (n = 5, mean ± SEM, *P = 0.0212) expression by colonic Foxp3 conventional T cells of Maf∆Treg and control mice as measured by flow cytometry. Representative flow cytometric profiles are shown on top. Graphs below show quantification. (e) Contour plots show RORγt and c-Maf expression of Foxp3+ and Foxp3 CD4+TCRβ+ T cells from mLNs of Maf∆Treg and control mice as measured by flow cytometry. The presented plots are representative of at least three independent stainings. (f) Frequencies of IL-17A+ (*P = 0.0092) and IFN-γ+ cells among colonic Foxp3+ Treg cells of Foxp3Cre (Ctrl, n = 4, mean ± SEM) and Rorcfl/flFoxp3Cre (Rorc∆Treg, n = 3, mean ± SEM) mice after ex vivo PMA/ionomycin restimulation as measured by flow cytometry. (g) Frequency of IL-17A+ cells among colonic Foxp3 conventional T cells of Rorc∆Treg (n = 3, mean ± SEM) and control (n = 4, mean ± SEM) mice after ex vivo PMA/ionomycin restimulation as measured by flow cytometry. All statistical differences were tested using an unpaired Student’s t-test (two-tailed).

  5. Supplementary Figure 5 Tissue-specific gene regulation by c-Maf in gut-associated Treg cells.

    (a) Foxp3+ Treg cells were sorted from spleen of Maf∆Treg and control mice and subjected to RNA sequencing. MA plot showing comparison of gene expression between c-Maf-deficient and control Treg cells. Genes up-regulated in c-Maf-deficient Treg cells are highlighted in red; genes down-regulated are highlighted in blue (FDR < 0.1). DE analysis was performed using limma. P values were adjusted to control the global false discovery rate (FDR) across all comparisons with the ‘global’ option of the limma package. Data represent the combined analysis of three biologically independent samples. (b) Venn diagram displaying the overlap between genes differentially expressed (DE) between c-Maf-deficient and control Treg cells from LP, PPs and spleens. (c) Heatmap showing relative expression (z-score) of genes that were differentially expressed (FDR < 0.1) between c-Maf-deficient and control Treg cells from LP (n = 2), PPs (n = 3) and spleens (n = 3). DE analysis was performed using limma. P values were adjusted to control the global FDR across all comparisons with the ‘global’ option of the limma package. (d) Gene set enrichment plots showing significantly enriched (FDR < 0.1) hallmark gene sets in c-Maf-deficient versus control LP Treg cells. Data represent the combined analysis of two biologically independent samples. (e) MA plot showing comparison of gene expression between LP (n = 2) and splenic Treg cells (n = 3) from control mice. Genes up-regulated in LP Treg cells are highlighted in red; genes down-regulated are highlighted in blue (FDR < 0.05). DE analysis was performed using limma. P values were adjusted to control the global FDR across all comparisons with the ‘global’ option of the limma package. (f) Sorted c-Maf-deficient and control LP Treg cells (n = 3) were subjected to ‘Assay for Transposase-Accessible Chromatin’ sequencing (ATAC-seq). MA plot displaying regions of accessible chromatin. Regions over-represented in c-Maf-deficient Treg cells are highlighted in red; regions under-represented are highlighted in blue. Note that multiple peaks can be associated with individual genes. (g) Representative c-Maf chromatin immunoprecipitation sequencing (ChIP-seq) and ATAC sequencing tracks. Arrows indicate c-Maf occupancy in open chromatin regions of Treg cell gene loci. Data represent the combined analysis of three biologically independent samples. c-Maf ChIP-seq was published by Ciofani et al.34

  6. Supplementary Figure 6 Il10∆Treg mice are susceptible to DSS-induced colitis.

    Foxp3Cre (Ctrl) and Il10fl/flFoxp3Cre (Il10∆Treg) mice were orally challenged with 1.5% DSS (w/v) for 5 days. Colitis severity was assessed 3 days later (day 8). (a) Colon length (n = 4, mean ± SEM) and (b) colitis scores are shown (n = 4, mean ± SEM). *P = 0.03 as calculated by unpaired Student’s t-test (two-tailed). Data are representative of three independent experiments with four to five mice per genotype.

  7. Supplementary Figure 7 c-Maf is essential for Treg cell–mediated control of intestinal IgA responses.

    Analysis of faecal microbiota diversity of co-housed littermate Maf∆Treg (n = 10, mean ± SEM) and control mice (n = 11, mean ± SEM) using 16 S rRNA gene sequencing. (b) Quantification of IgA in serum of Maf∆Treg and control mice as measured by ELISA (n = 4, mean ± SEM, *P = 0.0129). (c) Frequency of IgA+ B cells in PP of naïve Maf∆Treg and control mice. Representative contour plots of IgA versus B220 expression by CD45+ cells are shown on the left. The graph on the right shows quantification (n = 4, mean ± SEM). (d) Quantification of IgA+ cells in the colonic LP of Maf∆Treg and control mice as measured by immunohistochemistry (n = 4, mean ± SEM, *P = 0.0045). (e) Differential representation of bacterial families between the IgA+ and IgA fractions of co-housed littermate Maf∆Treg and control mice shown in form of an IgA coating index (see Methods section). For a given taxon, the value of the IgA coating index can range from a maximum of 1.0 (taxon detected exclusively in the IgA+ fraction) to a minimum of −1.0 (present only in the IgA fraction). n = 4, *P = 0.0286 as calculated by Mann–Whitney U test (two-tailed). Whiskers represent the minimum and maximum of data. (f) Quantification of IgA in faeces of Il10∆Treg and control mice as measured by ELISA (n = 4, mean ± SEM). All statistical differences were tested using an unpaired Student’s t-test (two-tailed) unless stated otherwise.

  8. Supplementary Figure 8 Phenotypic analysis of intestinal Foxp3+ Treg cells in colonized germ-free mice.

    (a) Experimental design for colonization of germ-free (GF) mice with microbiota (Mb) from either Maf∆Treg or control mice. (b–d) GF + Ctrl Mb (n = 6, mean ± SEM) and GF + Maf∆Treg Mb (n = 8, mean ± SEM) mice were orally challenged with 1.5% DSS (w/v) for 5 days. Colonic Foxp3+ Treg cells were analyzed 2 days later. Graphs show pooled data of two independent experiments. (b) Frequency of Foxp3+ Treg cells among colonic CD4+ TCRβ+ T cells. (c) Frequency of c-Maf+ and RORγt+ cells among colonic Foxp3+ Treg cells. (d) Frequency of IL-10+ cells among colonic Foxp3+ Treg cells after ex vivo PMA/ionomycin restimulation. (e) Frequency of TFR cells (PD1+ CXCR5+) among Foxp3+ cells from PPs of GF + Ctrl Mb (n = 4, mean ± SEM) and GF + Maf∆Treg Mb (n = 3, mean ± SEM) mice after 2 weeks of co-housing with either Maf∆Treg or control mice. (f) Quantification of IgA in faeces of GF + Ctrl Mb (n = 4, mean ± SEM) and GF + Maf∆Treg Mb (n = 3, mean ± SEM) mice after 2 weeks of co-housing with either Maf∆Treg or control mice as measured by ELISA. (e–f) Data are representative of two independent experiments.

Supplementary information

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    Supplementary Figures 1–8, Supplementary Tables 1–5

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https://doi.org/10.1038/s41590-019-0316-2