Regulatory iNKT cells lack expression of the transcription factor PLZF and control the homeostasis of Treg cells and macrophages in adipose tissue

Abstract

Invariant natural killer T cells (iNKT cells) are lipid-sensing innate T cells that are restricted by the antigen-presenting molecule CD1d and express the transcription factor PLZF. iNKT cells accumulate in adipose tissue, where they are anti-inflammatory, but the factors that contribute to their anti-inflammatory nature, as well as their targets in adipose tissue, are unknown. Here we found that iNKT cells in adipose tissue had a unique transcriptional program and produced interleukin 2 (IL-2) and IL-10. Unlike other iNKT cells, they lacked PLZF but expressed the transcription factor E4BP4, which controlled their IL-10 production. The adipose iNKT cells were a tissue-resident population that induced an anti-inflammatory phenotype in macrophages and, through the production of IL-2, controlled the number, proliferation and suppressor function of regulatory T cells (Treg cells) in adipose tissue. Thus, iNKT cells in adipose tissue are unique regulators of immunological homeostasis in this tissue.

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Figure 1: iNKT cells are tissue resident in adipose tissue and do not rely on ICAM or LFA-1 for retention.
Figure 2: Adipose iNKT cells lack PLZF and are present in Plzf−/− mice.
Figure 3: Adipose iNKT cells have characteristics similar to those of PLZF iNKT cells.
Figure 4: Adipose iNKT cells express E4BP4, which induces IL-10 production.
Figure 5: Adipose iNKT cells interact with macrophages in vivo and induce M2 macrophages via IL-10.
Figure 6: Adipose iNKT cells control adipose Treg cells through IL-2 production.
Figure 7: Adipose iNKT cells enhance the suppressive ability of Treg cells and have functions similar to those of Treg cells.

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References

  1. 1

    Cohen, N.R., Garg, S. & Brenner, M.B. Antigen presentation by CD1 lipids, T cells, and NKT cells in microbial immunity. Adv. Immunol. 102, 1–94 (2009).

    CAS  Article  Google Scholar 

  2. 2

    Savage, A.K. et al. The transcription factor PLZF directs the effector program of the NKT cell lineage. Immunity 29, 391–403 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Kovalovsky, D. et al. The BTB-zinc finger transcriptional regulator PLZF controls the development of invariant natural killer T cell effector functions. Nat. Immunol. 9, 1055–1064 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Martin, E. et al. Stepwise development of MAIT cells in mouse and human. PLoS Biol. 7, e54 (2009).

    Google Scholar 

  5. 5

    Kreslavsky, T. et al. TCR-inducible PLZF transcription factor required for innate phenotype of a subset of γδ T cells with restricted TCR diversity. Proc. Natl. Acad. Sci. USA 106, 12453–12458 (2009).

    CAS  PubMed  Google Scholar 

  6. 6

    Watarai, H. et al. Development and function of invariant natural killer T cells producing TH2- and TH17-cytokines. PLoS Biol. 10, e1001255 (2012).

    CAS  Article  Google Scholar 

  7. 7

    Brennan, P.J., Brigl, M. & Brenner, M.B. Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions. Nat. Rev. Immunol. 13, 101–117 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Berzins, S.P., Smyth, M.J. & Baxter, A.G. Presumed guilty: natural killer T cell defects and human disease. Nat. Rev. Immunol. 11, 131–142 (2011).

    CAS  PubMed  Google Scholar 

  9. 9

    Godfrey, D.I. & Kronenberg, M. Going both ways: immune regulation via CD1d-dependent NKT cells. J. Clin. Invest. 114, 1379–1388 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Novak, J., Griseri, T., Beaudoin, L. & Lehuen, A. Regulation of type 1 diabetes by NKT cells. Int. Rev. Immunol. 26, 49–72 (2007).

    CAS  PubMed  Google Scholar 

  11. 11

    Araki, M. et al. Th2 bias of CD4+ NKT cells derived from multiple sclerosis in remission. Int. Immunol. 15, 279–288 (2003).

    CAS  PubMed  Google Scholar 

  12. 12

    van der Vliet, H.J. et al. Circulating V(α24+) Vβ11+ NKT cell numbers are decreased in a wide variety of diseases that are characterized by autoreactive tissue damage. Clin. Immunol. 100, 144–148 (2001).

    CAS  PubMed  Google Scholar 

  13. 13

    Bosma, A., Abdel-Gadir, A., Isenberg, D.A., Jury, E.C. & Mauri, C. Lipid-antigen presentation by CD1d+ B cells is essential for the maintenance of invariant natural killer T cells. Immunity 36, 477–490 (2012).

    CAS  PubMed  Google Scholar 

  14. 14

    Novak, J. & Lehuen, A. Mechanism of regulation of autoimmunity by iNKT cells. Cytokine 53, 263–270 (2011).

    CAS  PubMed  Google Scholar 

  15. 15

    Lynch, L. et al. Invariant NKT cells and CD1d+ cells amass in human omentum and are depleted in patients with cancer and obesity. Eur. J. Immunol. 39, 1893–1901 (2009).

    CAS  PubMed  Google Scholar 

  16. 16

    Lynch, L. et al. Adipose tissue invariant NKT cells protect against diet-induced obesity and metabolic disorder through regulatory cytokine production. Immunity 37, 574–587 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Lumeng, C.N., DelProposto, J.B., Westcott, D.J. & Saltiel, A.R. Phenotypic switching of adipose tissue macrophages with obesity is generated by spatiotemporal differences in macrophage subtypes. Diabetes 57, 3239–3246 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    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 

  20. 20

    Ji, Y. et al. Activation of natural killer T cells promotes M2 macrophage polarization in adipose tissue and improves systemic glucose tolerance via interleukin-4 (IL-4)/STAT6 protein signaling axis in obesity. J. Biol. Chem. 287, 13561–13571 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Schipper, H.S. et al. Natural killer T cells in adipose tissue prevent insulin resistance. J. Clin. Invest. 122, 3343–3354 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Huh, J.Y. et al. A novel function of adipocytes in lipid antigen presentation to iNKT cells. Mol. Cell. Biol. 33, 328–339 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Weisberg, S.P. et al. Obesity is associated with macrophage accumulation in adipose tissue. J. Clin. Invest. 112, 1796–1808 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Lumeng, C.N., Bodzin, J.L. & Saltiel, A.R. Obesity induces a phenotypic switch in adipose tissue macrophage polarization. J. Clin. Invest. 117, 175–184 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Schipper, H.S. et al. Natural killer T cells in adipose tissue prevent insulin resistance. J. Clin. Invest. 122, 3343–3354 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Ji, Y. et al. Short term high fat diet challenge promotes alternative macrophage polarization in adipose tissue via natural killer T cells and interleukin-4. J. Biol. Chem. 287, 24378–24386 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Hams, E., Locksley, R.M., McKenzie, A.N. & Fallon, P.G. Cutting edge: IL-25 elicits innate lymphoid type 2 and type II NKT cells that regulate obesity in Mice. J. Immunol. 191, 5349–5353 (2013).

    CAS  PubMed  Google Scholar 

  28. 28

    Wright, D.E., Wagers, A.J., Gulati, A.P., Johnson, F.L. & Weissman, I.L. Physiological migration of hematopoietic stem and progenitor cells. Science 294, 1933–1936 (2001).

    CAS  PubMed  Google Scholar 

  29. 29

    Ohteki, T., Maki, C., Koyasu, S., Mak, T.W. & Ohashi, P.S. Cutting edge: LFA-1 is required for liver NK1.1+TCRαβ+ cell development: evidence that liver NK1.1+TCRαβ+ cells originate from multiple pathways. J. Immunol. 162, 3753–3756 (1999).

    CAS  PubMed  Google Scholar 

  30. 30

    Geissmann, F. et al. Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids. PLoS Biol. 3, e113 (2005).

    PubMed  PubMed Central  Google Scholar 

  31. 31

    Thomas, S.Y. et al. PLZF induces an intravascular surveillance program mediated by long-lived LFA-1-ICAM-1 interactions. J. Exp. Med. 208, 1179–1188 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Cohen, N.R. et al. Shared and distinct transcriptional programs underlie the hybrid nature of iNKT cells. Nat. Immunol. 14, 90–99 (2013).

    CAS  Google Scholar 

  33. 33

    Kronenberg, M. Toward an understanding of NKT cell biology: progress and paradoxes. Annu. Rev. Immunol. 23, 877–900 (2005).

    CAS  PubMed  Google Scholar 

  34. 34

    Bendelac, A., Savage, P.B. & Teyton, L. The biology of NKT cells. Annu. Rev. Immunol. 25, 297–336 (2007).

    CAS  Google Scholar 

  35. 35

    Brennan, P.J., Brigl, M. & Brenner, M.B. Invariant natural killer T cells: an innate activation scheme linked to diverse effector functions. Nat. Rev. Immunol. 13, 101–117 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Constantinides, M.G. & Bendelac, A. Transcriptional regulation of the NKT cell lineage. Curr. Opin. Immunol. 25, 161–167 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Lee, Y.J., Holzapfel, K.L., Zhu, J., Jameson, S.C. & Hogquist, K.A. Steady-state production of IL-4 modulates immunity in mouse strains and is determined by lineage diversity of iNKT cells. Nat. Immunol. 14, 1146–1154 (2013).

    CAS  Google Scholar 

  38. 38

    Chang, P.P. et al. Identification of Bcl-6-dependent follicular helper NKT cells that provide cognate help for B cell responses. Nat. Immunol. 13, 35–43 (2012).

    CAS  Google Scholar 

  39. 39

    Motomura, Y. et al. The transcription factor E4BP4 regulates the production of IL-10 and IL-13 in CD4+ T cells. Nat. Immunol. 12, 450–459 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Sakaguchi, S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 22, 531–562 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Cheng, G. et al. IL-2 receptor signaling is essential for the development of Klrg1+ terminally differentiated T regulatory cells. J. Immunol. 189, 1780–1791 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Monteiro, M. et al. Identification of regulatory Foxp3+ invariant NKT cells induced by TGF-β. J. Immunol. 185, 2157–2163 (2010).

    CAS  PubMed  Google Scholar 

  43. 43

    Moran, A.E. et al. T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse. J. Exp. Med. 208, 1279–1289 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Constantinides, M.G., Picard, D., Savage, A.K. & Bendelac, A. A naive-like population of human CD1d-restricted T cells expressing intermediate levels of promyelocytic leukemia zinc finger. J. Immunol. 187, 309–315 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Sag, D., Krause, P., Hedrick, C.C., Kronenberg, M. & Wingender, G. IL-10-producing NKT10 cells are a distinct regulatory invariant NKT cell subset. J. Clin. Invest. 124, 3725–3740 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Xu, X. et al. Obesity activates a program of lysosomal-dependent lipid metabolism in adipose tissue macrophages independently of classic activation. Cell Metab. 18, 816–830 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Wu, L. & Van Kaer, L. Contribution of lipid-reactive natural killer T cells to obesity-associated inflammation and insulin resistance. Adipocyte 2, 12–16 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Lynch, L. Adipose iNKT cells. Immunology 142, 337–346 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Swann, J., Crowe, N.Y., Hayakawa, Y., Godfrey, D.I. & Smyth, M.J. Regulation of antitumour immunity by CD1d-restricted NKT cells. Immunol. Cell Biol. 82, 323–331 (2004).

    CAS  PubMed  Google Scholar 

  50. 50

    Fontenot, J.D. et al. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22, 329–341 (2005).

    CAS  Google Scholar 

  51. 51

    Dash, P. et al. Paired analysis of TCRα and TCRβ chains at the single-cell level in mice. J. Clin. Invest. 121, 288–295 (2011).

    CAS  PubMed  Google Scholar 

  52. 52

    Uldrich, A.P. et al. CD1d-lipid antigen recognition by the γδ TCR. Nat. Immunol. 14, 1137–1145 (2013).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank M. Exley (Harvard Medical School) for Cd1d−/− mice; P.P. Pandolfi (Harvard Medical School) for the use of Plzf−/− mice; A. Rudensky (Memorial Sloan-Kettering Cancer Center) for Foxp3-GFP mice; E. Pamer (Memorial Sloan-Kettering Cancer Center) for the pLD53 SC-AB vector system; and K. Rothamel (Harvard Medical School) and the US National Institutes of Health Tetramer Core for mouse CD1d-PBS57 tetramers; and E. Lynch for assistance with imaging. This work benefited from public data generated by the Immunological Genome Project. Supported by Marie Curie Actions (L.L.), the US National Institutes of Health (AI063428, AI028973 and DK057521 to M.B.B.; and T32 A1049823 to E.E.V.-D.), the American Diabetes Association (7-12-IN-07 to M.B.B.), the American Academy of Allergy, Asthma and Immunology ARTrust (P.J.B.), the Trudeau Institute (M.T., E.E.V.-D. and E.A.L.), The National Health and Medical Research Council of Australia (1013667 to D.I.G. and H.-F.K.; and a1020770 to D.I.G.), the National Institute of Allergy and Infectious Diseases of the US National Institutes of Health (R01 AI083988 and AI059739 to D.B.S.) and the Robert Wood Johnson Foundation (67038 to the Child Health Institute of New Jersey, support for D.B.S.).

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Contributions

L.L. designed and performed experiments, analyzed data and wrote the paper; X.M., S.Z., A.M., C.L. and H.-F.K. performed experiments; P.J.B. contributed to the microarray analysis and other analysis; G.B. synthesized α-GalCer; E.E.V.-D., M.T. and E.A.L. developed analytical tools; D.I.G., D.B.S. and U.v.A. contributed to the design of experiments and provided materials and tools; and M.B.B. designed experiments and wrote the paper.

Corresponding authors

Correspondence to Lydia Lynch or Michael B Brenner.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Adipose iNKT cells are not present in athymic mice.

iNKT levels were measured by flow with aGalCer-loaded CD1d tetramers in the thymus and adipose tissue of nude mice (nu-/-) and littermate controls (nu+/-).

Supplementary Figure 2 Fate mapping of PLZF reveals that adipose iNKT cells previously expressed PLZF.

BAC transgene that has cre knocked into the PLZF gene directly behind the ATG (the Pcre mouse). The cre gene has a stop codon so that PLZF is not expressed from the transgene. PLZF-Cre mice were crossed to a floxed stop, tdtomato ROSA26 allele. Any cell that expresses PLZF (and therefore cre) will be permanently marked with the red color. Gating on iNKT cells in spleen and adipose tissue revealed they were all tdtomato positive, however, PLZF mRNA is transiently expressed in HSCs, with no apparent functional consequence. Therefore 50% of all splenocytes are also tdTomato positive

Supplementary Figure 3 Use and sequences of genes encoding Vα and Vβ TCRs in thymic and adipose iNKT cells.

(a) Cells derived from thymus and adipose tissue were single-cell sorted by aGalCer loaded-CD1d tetramer and TCRb+ expression. (b) RNA was extracted from single cells and amplified by PCR, paired TCR alpha/beta chain sequences. (c) A PCR was performed with TRAV11 (Va14) primers to determine whether adipose iNKT cells expressed the canonical invariant chain. (d) TCR sequences for 5 thymic iNKT cells and 10 adipose iNKT cells. This data is representative of 16 individual thymic iNKT cells and 22 adipose iNKT cells.

Supplementary Figure 4 Expression of transcription factors and cytokine production by adipose iNKT cells.

(a) Intranuclear staining for Gata3, Tbet and Rorγt in splenic, hepatic and adipose iNKT cells (n=10 mice). (b&c) Intracellular cytokine staining of iNKT cells in spleen, liver and adipose tissue, for IFNγ, TNF, IL-17A, and IL-10 after aGalCer stimulation (b), or PMA and Ianomycin (c).

Supplementary Figure 5 Macrophage switching after co-culture with adipose or splenic Treg cells or iNKT cells.

Peritoneal macrophages were co-cultured with Tregs or iNKT cells from wither spleen or adipose tissue overnight, and M2/M2 like surface markers were examine on macrophages by flow cytometry.

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Lynch, L., Michelet, X., Zhang, S. et al. Regulatory iNKT cells lack expression of the transcription factor PLZF and control the homeostasis of Treg cells and macrophages in adipose tissue. Nat Immunol 16, 85–95 (2015). https://doi.org/10.1038/ni.3047

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