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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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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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