Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The transcriptional regulators IRF4, BATF and IL-33 orchestrate development and maintenance of adipose tissue–resident regulatory T cells

An Erratum to this article was published on 21 April 2015

This article has been updated

Abstract

Foxp3+ regulatory T (Treg) cells in visceral adipose tissue (VAT-Treg cells) are functionally specialized tissue-resident cells that prevent obesity-associated inflammation and preserve insulin sensitivity and glucose tolerance. Their development depends on the transcription factor PPAR-γ; however, the environmental cues required for their differentiation are unknown. Here we show that interleukin 33 (IL-33) signaling through the IL-33 receptor ST2 and myeloid differentiation factor MyD88 is essential for development and maintenance of VAT-Treg cells and sustains their transcriptional signature. Furthermore, the transcriptional regulators BATF and IRF4 were necessary for VAT-Treg differentiation through direct regulation of ST2 and PPAR-γ expression. IL-33 administration induced vigorous population expansion of VAT-Treg cells, which tightly correlated with improvements in metabolic parameters in obese mice. Human omental adipose tissue Treg cells also showed high ST2 expression, suggesting an evolutionarily conserved requirement for IL-33 in VAT-Treg cell homeostasis.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Blimp-1+ eTreg cells have a distinct transcriptional profile and localize outside the T cell area.
Figure 2: ST2 is specifically expressed by eTreg cells, in particular in the VAT.
Figure 3: ST2 is required for the differentiation of VAT-Treg cells.
Figure 4: IL-33 induces proliferation and maintains identity of VAT-Treg cells.
Figure 5: TCR signals induce the VAT-Treg cell transcriptional program in a BATF- and IRF4-dependent manner.
Figure 6: IRF4 and BATF bind the Pparg and Il1rl1 loci, and continuous IRF4 expression is required for maintaining VAT-Treg cell identity.
Figure 7: IL-33 administration increases VAT-Treg cell numbers in genetically obese mice.

Accession codes

Primary accessions

Gene Expression Omnibus

Change history

  • 08 April 2015

    In the version of this article initially published, the Acknowledgments section was incomplete. The correct text should begin "We thank P. O'Brien, M. Mochizuki and N. Takeno for assistance with tissue collection...." The error has been corrected in the HTML and PDF versions of the article.

References

  1. Josefowicz, S.Z., Lu, L.F. & Rudensky, A.Y. Regulatory T cells: mechanisms of differentiation and function. Annu. Rev. Immunol. 30, 531–564 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Ohkura, N., Kitagawa, Y. & Sakaguchi, S. Development and maintenance of regulatory T cells. Immunity 38, 414–423 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Feuerer, M., Hill, J.A., Mathis, D. & Benoist, C. Foxp3+ regulatory T cells: differentiation, specification, subphenotypes. Nat. Immunol. 10, 689–695 (2009).

    Article  CAS  PubMed  Google Scholar 

  4. Campbell, D.J. & Koch, M.A. Phenotypical and functional specialization of FOXP3(+) regulatory T cells. Nat. Rev. Immunol. 11, 119–130 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Cretney, E., Kallies, A. & Nutt, S.L. Differentiation and function of Foxp3(+) effector regulatory T cells. Trends Immunol. 34, 74–80 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Burzyn, D., Benoist, C. & Mathis, D. Regulatory T cells in nonlymphoid tissues. Nat. Immunol. 14, 1007–1013 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chaudhry, A. & Rudensky, A.Y. Control of inflammation by integration of environmental cues by regulatory T cells. J. Clin. Invest. 123, 939–944 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Liston, A. & Gray, D.H. Homeostatic control of regulatory T cell diversity. Nat. Rev. Immunol. 14, 154–165 (2014).

    Article  CAS  PubMed  Google Scholar 

  9. Koch, M.A. et al. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nat. Immunol. 10, 595–602 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Linterman, M.A. et al. Foxp3+ follicular regulatory T cells control the germinal center response. Nat. Med. 17, 975–982 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Feuerer, M. et al. Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters. Nat. Med. 15, 930–939 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Feuerer, M. et al. Genomic definition of multiple ex vivo regulatory T cell subphenotypes. Proc. Natl. Acad. Sci. USA 107, 5919–5924 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Cipolletta, D. et al. PPAR-γ is a major driver of the accumulation and phenotype of adipose tissue Treg cells. Nature 486, 549–553 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Fontenot, J.D., Rasmussen, J.P., Gavin, M.A. & Rudensky, A.Y. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat. Immunol. 6, 1142–1151 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Pierson, W. et al. Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3(+) regulatory T cells. Nat. Immunol. 14, 959–965 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Smigiel, K.S. et al. CCR7 provides localized access to IL-2 and defines homeostatically distinct regulatory T cell subsets. J. Exp. Med. 211, 121–136 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Hall, A.O. et al. The cytokines interleukin 27 and interferon-γ promote distinct Treg cell populations required to limit infection-induced pathology. Immunity 37, 511–523 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Gratz, I.K. & Campbell, D.J. Organ-specific and memory Treg cells: specificity, development, function, and maintenance. Front. Immunol. 5, 333 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Cretney, E. et al. The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells. Nat. Immunol. 12, 304–311 (2011).

    Article  CAS  PubMed  Google Scholar 

  20. Weiss, J.M. et al. Neuropilin 1 is expressed on thymus-derived natural regulatory T cells, but not mucosa-generated induced Foxp3+ T reg cells. J. Exp. Med. 209, 1723–1742 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Schmitz, J. et al. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor–related protein ST2 and induces T helper type 2–associated cytokines. Immunity 23, 479–490 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Moro, K. et al. Innate production of TH2 cytokines by adipose tissue-associated c-Kit+Sca-1+ lymphoid cells. Nature 463, 540–544 (2010).

    Article  CAS  PubMed  Google Scholar 

  23. Molofsky, A.B. et al. Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages. J. Exp. Med. 210, 535–549 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Licona-Limon, P., Kim, L.K., Palm, N.W. & Flavell, R.A. TH2, allergy and group 2 innate lymphoid cells. Nat. Immunol. 14, 536–542 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Bonilla, W.V. et al. The alarmin interleukin-33 drives protective antiviral CD8+ T cell responses. Science 335, 984–989 (2012).

    Article  CAS  PubMed  Google Scholar 

  26. Miller, A.M. et al. Interleukin-33 induces protective effects in adipose tissue inflammation during obesity in mice. Circ. Res. 107, 650–658 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kakkar, R. & Lee, R.T. The IL-33/ST2 pathway: therapeutic target and novel biomarker. Nat. Rev. Drug Discov. 7, 827–840 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Boyman, O., Kovar, M., Rubinstein, M.P., Surh, C.D. & Sprent, J. Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science 311, 1924–1927 (2006).

    CAS  PubMed  Google Scholar 

  29. Turnquist, H.R. et al. IL-33 expands suppressive CD11b+ Gr-1int and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival. J. Immunol. 187, 4598–4610 (2011).

    Article  CAS  PubMed  Google Scholar 

  30. Martin, M.U. Special aspects of interleukin-33 and the IL-33 receptor complex. Semin. Immunol. 25, 449–457 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Murphy, T.L., Tussiwand, R. & Murphy, K.M. Specificity through cooperation: BATF-IRF interactions control immune-regulatory networks. Nat. Rev. Immunol. 13, 499–509 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Glasmacher, E. et al. A genomic regulatory element that directs assembly and function of immune-specific AP-1-IRF complexes. Science 338, 975–980 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ciofani, M. et al. A validated regulatory network for Th17 cell specification. Cell 151, 289–303 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Li, P. et al. BATF-JUN is critical for IRF4-mediated transcription in T cells. Nature 490, 543–546 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Man, K. et al. The transcription factor IRF4 is essential for TCR affinity-mediated metabolic programming and clonal expansion of T cells. Nat. Immunol. 14, 1155–1165 (2013).

    Article  CAS  PubMed  Google Scholar 

  36. Grusdat, M. et al. IRF4 and BATF are critical for CD8 T-cell function following infection with LCMV. Cell Death Differ. 7, 1050–1060 (2014).

    Article  CAS  Google Scholar 

  37. Brint, E.K. et al. Characterization of signaling pathways activated by the interleukin 1 (IL-1) receptor homologue T1/ST2. A role for Jun N-terminal kinase in IL-4 induction. J. Biol. Chem. 277, 49205–49211 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Zheng, Y. et al. Regulatory T-cell suppressor program co-opts transcription factor IRF4 to control TH2 responses. Nature 458, 351–356 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kluge, R., Scherneck, S., Schurmann, A. & Joost, H.G. Pathophysiology and genetics of obesity and diabetes in the New Zealand obese mouse: a model of the human metabolic syndrome. Methods Mol. Biol. 933, 59–73 (2012).

    CAS  PubMed  Google Scholar 

  40. Wood, I.S., Wang, B. & Trayhurn, P. IL-33, a recently identified interleukin-1 gene family member, is expressed in human adipocytes. Biochem. Biophys. Res. Commun. 384, 105–109 (2009).

    Article  CAS  PubMed  Google Scholar 

  41. Zeyda, M. et al. Severe obesity increases adipose tissue expression of interleukin-33 and its receptor ST2, both predominantly detectable in endothelial cells of human adipose tissue. Int. J. Obes. 37, 658–665 (2013).

    Article  CAS  Google Scholar 

  42. Boraschi, D. & Tagliabue, A. The interleukin-1 receptor family. Semin. Immunol. 25, 394–407 (2013).

    Article  CAS  PubMed  Google Scholar 

  43. Makrinioti, H., Toussaint, M., Jackson, D.J., Walton, R.P. & Johnston, S.L. Role of interleukin 33 in respiratory allergy and asthma. Lancet. Respir. Med. 2, 226–237 (2014).

    Article  CAS  PubMed  Google Scholar 

  44. Han, J.M. & Levings, M.K. Immune regulation in obesity-associated adipose inflammation. J. Immunol. 191, 527–532 (2013).

    Article  CAS  PubMed  Google Scholar 

  45. Williams, L.M. & Rudensky, A.Y. Maintenance of the Foxp3-dependent developmental program in mature regulatory T cells requires continued expression of Foxp3. Nat. Immunol. 8, 277–284 (2007).

    Article  CAS  PubMed  Google Scholar 

  46. Sakaguchi, S., Vignali, D.A., Rudensky, A.Y., Niec, R.E. & Waldmann, H. The plasticity and stability of regulatory T cells. Nat. Rev. Immunol. 13, 461–467 (2013).

    Article  CAS  PubMed  Google Scholar 

  47. Wang, Y., Su, M.A. & Wan, Y.Y. An essential role of the transcription factor GATA-3 for the function of regulatory T cells. Immunity 35, 337–348 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Wohlfert, E.A. et al. GATA3 controls Foxp3+ regulatory T cell fate during inflammation in mice. J. Clin. Invest. 121, 4503–4515 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Guo, L. et al. IL-1 family members and STAT activators induce cytokine production by Th2, Th17, and Th1 cells. Proc. Natl. Acad. Sci. USA 106, 13463–13468 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Kwon, H. et al. Analysis of interleukin-21-induced Prdm1 gene regulation reveals functional cooperation of STAT3 and IRF4 transcription factors. Immunity 31, 941–952 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Oboki, K. et al. IL-33 is a crucial amplifier of innate rather than acquired immunity. Proc. Natl. Acad. Sci. USA 107, 18581–18586 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Adachi, O. et al. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9, 143–150 (1998).

    Article  CAS  PubMed  Google Scholar 

  53. Jacob, J. & Baltimore, D. Modelling T-cell memory by genetic marking of memory T cells in vivo. Nature 399, 593–597 (1999).

    Article  CAS  PubMed  Google Scholar 

  54. Schraml, B.U. et al. The AP-1 transcription factor Batf controls TH17 differentiation. Nature 460, 405–409 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Kamanaka, M. et al. Expression of interleukin-10 in intestinal lymphocytes detected by an interleukin-10 reporter knockin tiger mouse. Immunity 25, 941–952 (2006).

    Article  CAS  PubMed  Google Scholar 

  56. Wan, Y.Y. & Flavell, R.A. Identifying Foxp3-expressing suppressor T cells with a bicistronic reporter. Proc. Natl. Acad. Sci. USA 102, 5126–5131 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Mittrucker, H.W. et al. Requirement for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function. Science 275, 540–543 (1997).

    Article  CAS  PubMed  Google Scholar 

  58. Klein, U. et al. Transcription factor IRF4 controls plasma cell differentiation and class-switch recombination. Nat. Immunol. 7, 773–782 (2006).

    Article  CAS  PubMed  Google Scholar 

  59. Hoshino, K. et al. The absence of interleukin 1 receptor-related T1/ST2 does not affect T helper cell type 2 development and its effector function. J. Exp. Med. 190, 1541–1548 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Kallies, A. et al. Plasma cell ontogeny defined by quantitative changes in blimp-1 expression. J. Exp. Med. 200, 967–977 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Kallies, A. et al. Transcriptional repressor Blimp-1 is essential for T cell homeostasis and self-tolerance. Nat. Immunol. 7, 466–474 (2006).

    Article  CAS  PubMed  Google Scholar 

  62. Kawamoto, S. et al. The inhibitory receptor PD-1 regulates IgA selection and bacterial composition in the gut. Science 336, 485–489 (2012).

    Article  CAS  PubMed  Google Scholar 

  63. Liao, Y., Smyth, G.K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    Article  CAS  PubMed  Google Scholar 

  64. Gentleman, R.C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5 (2004).

  65. Lee, H.Y. et al. Blockade of IL-33/ST2 ameliorates airway inflammation in a murine model of allergic asthma. Exp. Lung Res. 40, 66–76 (2014).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank P. O'Brien, M. Mochizuki and N. Takeno for assistance with tissue collection, S. Wada for animal care, M. Febbraio and A. Lew for critical advice, E. Cretney for mice and E. Bandala-Sanchez, V. Bryant and J. Brady for reagents. We are grateful to K. Nakanishi (Hyogo College of Medicine), T. Mak (The Campbell Family Institute for Breast Cancer Research), and U. Klein (Columbia University) for mice. Supported by the National Health and Medical Research Council of Australia (A.K., S.L.N. and G.K.S.), the Sylvia and Charles Viertel Foundation (A.K.), the Australian Research Council (A.K. and S.L.N.), the Diabetes Australia Research Trust (J.M.W.), PRESTO from the Japan Science and Technology Agency (K.M.), and a Grant-in Aid for Scientific Research (B) (26293110 to K.M.) and a Grant-in-Aid for Scientific Research (S) (22229004 to S. Koyasu) from the Japan Society for the Promotion of Science. W.L., P.L. and W.J.L. are supported by the Division of Intramural Research, National Heart, Blood, and Lung Institute, US National Institutes of Health. This study was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC Independent Research Institute Infrastructure Support scheme.

Author information

Authors and Affiliations

Authors

Contributions

A.V. planned and performed most experiments; K.M. performed experiments related to the IL-33 and ST2-deficient mice; A.X., S.A.-S., Y.L., P.L., W.L., W.S., W.J.L. and G.K.S. did or analyzed the RNA and ChIP sequencing experiments; S. Kawamoto and S.F. did the immunofluorescence; L.A.M. and R.G. performed additional experiments; S.N. and H.S. contributed reagents; S.L.M. and J.M.W. contributed to the scientific planning and organization of experiments; S.L.N. and S. Koyasu designed experiments; A.K. oversaw and designed the study; A.K. and A.V. wrote the manuscript.

Corresponding author

Correspondence to Axel Kallies.

Ethics declarations

Competing interests

S.K. is a consultant for Medical and Biological Laboratories (MBL).

Integrated supplementary information

Supplementary Figure 1 eTreg cells are a transcriptionally distinct Treg cell population.

(a) Gating strategy used to purify Blimp1- cTreg cells (blue gate) and Blimp1+ eTreg cell (red gate) from pooled spleen and lymph nodes (LNs) of Blimp1GFP mice. Representative of 6 experiments. (b) Heat map showing top 100 differentially expressed genes between cTreg cells and eTreg cells determined using likelihood ratio test. (c) RNAseq tracks showing the expression of Foxp3 in cTreg and eTreg cells. (d) Heat maps showing expression of transcriptional regulators (left) and genes related to migration and adhesion (right) that are differentially expressed between cTreg cells and eTreg cells. RNAseq for the indicated Treg cell populations performed in triplicate.

Supplementary Figure 2 ST2 expression correlates with other VAT-Treg cell markers.

(a) Expression of ST2 against other surface molecules Ly6C, Ccr2, KLRG1, PD-1, CD69 and Tigit on Treg cells from spleen and VAT. Flow cytometric plots displaying CD4+Foxp3+ cells from a 35-week-old wild-type mouse, representative of 7 individual mice from two experiments.

Supplementary Figure 3 IL-33 is specifically required for VAT-Treg cells but dispensable for other Treg cell populations.

(a-c) Treg proportions and phenotype in selected lymphoid (a-b) and non-lymphoid (c) organs of wild-type (WT) and Il1rl1–/– mice as indicated. Values are mean ± S.D. of 9 individual mice from 3 experiments. LPL - Lamina propria lymphocytes of the small intestine. (d) Treg cell proportions in WT and Il33–/– mice and expression of KLRG1 in WT and Il33–/– Treg cells. Values are mean ± S.D. of 5 individual mice from two experiments. Numbers indicate percentages of cells. (e) Weight of VAT from 35-week old Il1rl1–/–, Il33–/– and WT mice. Values are mean ± S.D. from 8 individual mice from 3 experiments. (f-g) Glucose tolerance tests for Il1rl1–/– (f), Il33–/– (g) and corresponding WT control mice. The graphs are representative of at least two independent experiments with 3-5 mice per experiment. Two-way ANOVA test (P<0.0001), error bars denote S.E.M. (h) HOMA-IR calculated for Il33–/– and WT mice. (i) Flow cytometric analysis of adipose tissue from Il33–/– and WT mice. Graphs show VAT macrophages (TCRβ-, CD11b+, F4/80+ and CD11c+), pro-inflammatory monocytes (TCRβ-, CD11b+ Ly6C+) and CD8+ T cells. Panels representative of more than 5 mice analyzed in two independent experiments. (j) Serum leptin levels in Il33–/– mice. Values are mean ± S.D. *P<0.04; NS – not significant (unpaired, two tailed t-test).

Supplementary Figure 4 IL-33 drives proliferation of VAT-Treg cells.

(a-b) In vitro proliferation of VAT-Treg cells. Equal number of purified VAT lymphocytes from wild-type (WT) mice were CTV labeled and cultured for 3.5 days with (a) or without (b) plate bound CD3 and soluble CD28 antibodies, cytokines, and with or without IL-2 blocking antibodies as indicated. Bar graph shows relative numbers of Treg cells at the end of the culture. Figure representative of three experiments. (c) Relative Il33 mRNA expression in the VAT of young (8 weeks) versus old (35 weeks) mice. (d) IL-33 protein expression analyzed by immunoblotting of adipose tissue from young and old mice that were on a normal diet. Il33–/– mice were used as specificity control. Actin was used as loading control. Representative of 5 experiments. (e-f) ST2 expression on VAT-Treg cells isolated from WT mice of different ages as indicated. (e) and correlation of age and VAT-Treg prevalence (f). One way ANOVA for both panels, P<0.0001. (g) Frequency of Foxp3+ cells of CD4+ T cells in selected lymphoid and non-lymphoid organs from PBS, IL-33 and IL-2/anti-IL-2 Ab complex (IL-2c) treated mice. LPL - Lamina propria lymphocytes of the small intestine. (h) ST2 expression on KLRG1+ and KLRG1- Treg cells. (i) Flow cytometric analysis of splenic Foxp3+ cells showing expression of KLRG1 and ST2 in PBS and IL-33 treated mice (left). Graph showing proportion of KLRG1+ cells ± S.D. of total Treg cells in the spleen in control and IL-33 treated mice (right). (j) Proportion of Foxp3+ cells within CD4+ T cells in the VAT at different time points post IL-33 injection. One way ANOVA, P=0.0047. Symbols indicate data points for individual mice, values are mean ± S.D. Values in (a-c) are means ± S.D. from 3 experiments. Values in (e-g) are means ± S.D. from 5 individual mice from 2 experiments. *P=0.017; **P<0.008; ***P=0.0001; ****P<0.0001 (unpaired, two tailed t-test).

Supplementary Figure 5 IL-33 signaling through MyD88 is required for VAT-Treg cell differentiation.

(a-b) Proportion of Treg cells in selected lymphoid (a) and non-lymphoid (b) organs of wild-type (WT) and Myd88–/– mice. LPL - lamina propria lymphocytes of the small intestine. (c) Flow cytometric analysis of Treg cells from the lymph nodes (LN) of wild-type and Myd88–/– mice assessed for eTreg cell markers ICOS and KLRG1 (left), frequency of KLRG1+ cells of lymph node Treg cells from WT and Myd88–/–mice (right). Numbers indicate percentages of cells. (d) VAT weight from WT and Myd88–/– mice. (e) Treg cells enriched from spleens of wild-type (WT) mice and cultured in the presence of plate bound CD3 and soluble CD28 antibodies, and cytokines for 3 days. Expression of ST2 and Foxp3 is shown in the flow cytometric plots. (f) Treg cells enriched from spleen of WT and Myd88–/– mice, CTV labeled and cultured as in (e). Expression of ST2 (dot plots, left) and proliferation measured by CTV dilution. Values in (a-d) are means ± S.D. from 8-9 mice. **P=0.003; NS – Not significant (unpaired, two tailed t-test).

Supplementary Figure 6 BATF and IRF4 are required for VAT-Treg cell development.

(a-b) Proportion of Treg cells in the spleen and VAT of wild-type (WT) mice compared to Batf–/– (b) and Irf4–/– (b) mice. Values are means ± S.D. from 5-7 mice per group. (c-d) VAT mass (c) and body weight (d) of WT, Irf4–/– and Batf–/– mice. Values are the means from each 6-8 mice per group (one way ANOVA). (e) Bar graph showing proportions of WT and knock-out Foxp3+ cells as indicated from the spleens and VAT of Ly5.1 (WT) / Batf–/– (left) and Ly5.1 (WT) / Irf4–/– peripheral chimeric mice. (f) Flow cytometric analysis of ST2 expression on Treg cells from the VAT of mice of the indicated genotype. (g-h) MACS enriched CD4+CD25+ cells from WT (Ly5.1), Batf–/– (Ly5.2) and Irf4–/– (Ly5.2) mice as indicated were mixed as indicated, CTV labeled and cultured in conditions that induce ST2. Flow cytometric analysis of total Foxp3+ cells. Numbers indicate percentages of cells. Bar graphs show the proportion of Foxp3+ cells of the indicated genotype that express ST2. Histograms (gated on Foxp3+ cells) show CTV dilution profiles. Values are mean ± S.D. from 5 male 30-week-old mice per group. (i) RNAseq track showing expression of GzmB by cTreg cells and eTreg cells. (j) Weight of VAT from Irf4fl/flGzmBCre+ and control mice. Values are mean from 4 mice per group. *P<0.01; **P<0.002; ***P=0.0001; ****P<0.0001 (unpaired, two tailed t-test)..

Supplementary Figure 7 IL-33 administration can rescue VAT-Treg defects in genetically obese and HFD mice.

(a) Percentages of VAT and spleen Treg cells within the CD4+ compartments of C57BL/6 and NZO mice. Values are mean ± S.D. from 5 mice of each genotype. (b) Intraperitoneal glucose tolerance test (GTT) for NZO mice treated with PBS and IL-33. (c) Area under curve (AUC) for GTT performed on HFD and NZO mice as indicated. Values are mean from 4 and 5 mice per group. (d) Proportion of CD8+ T cells and VAT macrophages in the VAT of NZO mice treated with PBS or IL-33 as indicated. (e) Representative flow cytometric analysis of HFD mice. Plots show VAT macrophages (TCRβ-, CD11b+, F4/80+ and CD11c+) and pro-inflammatory monocytes (TCRβ-, CD11b+ Ly6C+). Numbers in boxes indicate percentages of cells. (f) Expression of Ccl2 (Mcp1), Ccl3 (Mip1α), Ccl5 (RANTES) and Il1β in the VAT of HFD and NZO mice treated with PBS or IL-33 analyzed by qPCR. Values are means ± S.D. For the GTT experiments in (b) a two way ANOVA test was performed (P<0.0001) and error bars denote S.E.M. P values for other graphs *P<0.05; **P=0.002; NS – not significant (unpaired, two tailed t-test).

Supplementary Figure 8 IL-33 treatment increases Treg cell numbers and improves metabolic parameters in NZO and HFD mice.

(a) Weight of VAT isolated from NZO and HFD mice treated with PBS or IL-33. Values are mean from 4 and 5 mice per group. (b) Hematoxylin and eosin staining of VAT sections. Numbers of adipocytes per field and adipocyte sizes from PBS and IL-33 treated NZO (upper panels) and HFD mice (lower panels) as indicated. Values are means ± S.E.M. of three sections each from 4-5 mice analyzed. (c) HOMA-IR calculated from PBS or IL-33 treated NZO and HFD mice as indicated. (d) Immunoblot showing Akt phosphorylation in VAT of PBS and IL-33 treated NZO or HFD mice after intravenous insulin injection. Representative of three experimets. (e) Analysis of ST2 expression on human Treg cells from peripheral blood mononuclear cells or omental fat as indicated; representative of three samples. * P<0.01 (a); ***P=0.0001 (b); ****P<0.0001 (b).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, and Supplementary Tables 4 and 5 (PDF 5066 kb)

Supplementary Table 1

Top 200 DE genes_cTregs Vs eTregs (CSV 13 kb)

Supplementary Table 2

DE genes encoding transcription factors_cTregs Vs eTregs (CSV 2 kb)

Supplementary Table 3

DE genes involved in migration_cTregs Vs eTregs (CSV 1 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Vasanthakumar, A., Moro, K., Xin, A. et al. The transcriptional regulators IRF4, BATF and IL-33 orchestrate development and maintenance of adipose tissue–resident regulatory T cells. Nat Immunol 16, 276–285 (2015). https://doi.org/10.1038/ni.3085

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.3085

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing