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Insulin signaling establishes a developmental trajectory of adipose regulatory T cells

Nature Immunology volume 22, pages 1175–1185 (2021)Cite this article

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Abstract

Systematic characterizations of adipose regulatory T (Treg) cell subsets and their phenotypes remain uncommon. Using single-cell ATAC-sequencing and paired single-cell RNA and T cell receptor (TCR) sequencing to map mouse adipose Treg cells, we identified CD73hiST2lo and CD73loST2hi subsets with distinct clonal expansion patterns. Analysis of TCR-sharing data implied a state transition between CD73hiST2lo and CD73loST2hi subsets. Mechanistically, we revealed that insulin signaling occurs through a HIF-1α–Med23–PPAR-γ axis to drive the transition of CD73hiST2lo into a CD73loST2hi adipose Treg cell subset. Treg cells deficient in insulin receptor, HIF-1α or Med23 have decreased PPAR-γ expression that in turn promotes accumulation of CD73hiST2lo adipose Treg cells and physiological adenosine production to activate beige fat biogenesis. We therefore unveiled a developmental trajectory of adipose Treg cells and its dependence on insulin signaling. Our findings have implications for understanding the dynamics of adipose Treg cell subsets in aged and obese contexts.

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Fig. 1: Integrated single-cell data reveal two adipose Treg cell subsets with distinct clonal expansion patterns.
Fig. 2: Tracing of adipose Treg cell subsets in aged and obese contexts.
Fig. 3: Insulin signals induce PPAR-γ and drive transition of CD73hiST2lo into CD73loST2hi adipose Treg cells.
Fig. 4: Insulin signals occur through the HIF-1α–Med23 complex to induce PPAR-γ expression.
Fig. 5: Hif1afl/flFoxp3Cre and Med23fl/flFoxp3Cre mice have enriched CD73hiST2lo Treg cells and increased IL-33 in fat.
Fig. 6: In vivo enriched CD73hiST2lo adipose Treg cells primarily activate beige fat biogenesis.

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Data availability

Gene Expression Omnibus accession numbers for the CUT&RUN DNA-seq, bulk RNA-seq, paired scRNA-seq and TCR-seq and scATAC-seq data reported in this paper are GSE164256, GSE152082, GSE124721, GSE137493 and GSE147768, respectively. Other data generated or analyzed during this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Code availability

No custom code or algorithms were used in this study.

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Acknowledgements

We thank X. Feng for cell sorting and members of SII family and our reviewers for constructive comments and suggestions. We thank for the support from Core Facility of Basic Medical Sciences and the sequencing core at Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine. Our research is supported by the National Natural Science Foundation of China (NSFC)-National Institutes of Health (NIH) collaborative grant (81161120417 to B.L.), National Natural Science Foundation of China (3213000541, 31525008, 81830051 and 31961133011 to B.L.), National Key R&D Program of China (2019YFA09006100 to B.L.), innovative research team of high-level local universities in Shanghai (SSMU-ZDCX20180101 to B.L.), Program of Shanghai Academic Research Leader (16XD1403800 to B.L.); Shanghai Jiao Tong University, The Chinese University of Hong Kong Joint Research Collaboration Fund and the Fundamental Research Funds for Central Universities (to B.L.). The work was supported by the Shanghai Municipal Science and Technology Major Project (19JC1410200 to X.T.) and the National Natural Science Foundation of China (81972210 and 92057117 to X.T. and 82003012 to Y. Lu). This work was supported in part by the Ministry of Science and Technology of China (2017YFA0102700 to G.W.) and by the National Natural Science Foundation of China (32030028 to G.W.). This work was also supported by the National Natural Science Foundation of China (31700775 to Y. Li), National Postdoctoral Program for Innovative Talents (BX201700159 to Y. Li) and China Postdoctoral Science Foundation (2017M621497 to Y. Li).

Author information

Author notes

  1. These authors contributed equally: Yangyang Li, Ying Lu.

Authors and Affiliations

  1. Department of Immunology and Microbiology, Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Yangyang Li, Ning Li, Yichao Han, Qianru Huang, Yi Zhao, Feng Xie, Yixian Guo, Biaolong Deng, Andy Tsun, Dan Li & Bin Li

  2. Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Ying Lu, Shu-Hai Lin & Xuemei Tong

  3. Biotheus Inc, Zhuhai, China

    Andy Tsun & Joanne Sun

  4. Department of Pulmonary and Critical Care Medicine, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Juan Du, Guochao Shi & Bin Li

  5. Department of Immunology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Fang Zheng

  6. Unit of Respiratory Infection and Immunity, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China

    Xiao Su

  7. Laboratory of Oral Microbiota and Systemic Diseases, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Shengzhong Duan

  8. Department of Clinical Immunology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China

    Song Guo Zheng

  9. State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, China

    Gang Wang

  10. Henan Key Laboratory for Digestive Organ Transplantation, Department of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China

    Bin Li

  11. Institute of Arthritis Research, Guanghua Integrative Medicine Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China

    Bin Li

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  1. Yangyang Li
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Contributions

Y. Li and Y. Lu designed and performed mice experiments; Y. Li wrote the manuscript; Y. Lu, N.L., B.D., Y.G. and Y.H. contributed to mouse work, flow cytometry, immunoblotting, RT–qPCR and so on; Y.Z., F.X. and Q.H. helped to analyze RNA-seq, ATAC-seq and DNA-seq data; D.L., A.T., J.S., S.-H.L., J.D., G.S., F.Z., X.S., S.D. and S.G.Z. contributed to scientific discussion; G.W., X.T. and B.L. supervised the overall project and revised the manuscript.

Corresponding authors

Correspondence to Gang Wang, Xuemei Tong or Bin Li.

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Competing interests

B.L. is a co-founder of Biotheus Inc. and Chairman of its scientific advisory board. The remaining authors declare no competing interests.

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Peer review information Zoltan Fehervari was the primary editor(s) on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Increased adipose inflammation in aged and obese contexts.

a, Expression of IFNγ, IL-17A and IL-4 by adipose CD4+YFP− Teff and CD8+ T cells from 4-month old WT, 4-month old HFD-treated WT and 9-month old WT mice (n = 6 per group). b and c, Percentages of IFNγ+, IL-17A+ and IL-4+ adipose Teff (b) and CD8+ T (c) cells from a. Data are mean ± s.d., P values were determined by unpaired two-tailed Student’s t-test. ns, not significant.

Source data

Extended Data Fig. 2 Insulin signals induce PPAR-γ expression and CD73loST2hi Treg cell generation in vitro.

a, Expression of insulin receptor β (InsRβ) in CD4+Foxp3+ Treg cells in the thymus, spleen, peripheral lymphoid nodes (pLNs) and fat of 4-month old WT (n = 6) mice. b, MFI of InsRβ among CD4+Foxp3+ Treg cells from a. c, Gating strategy showing CD62L and InsRβ expression by CD73hiST2lo and CD73loST2hi adipose Treg cells from 4-month old WT (n = 6) mice. d, MFI of InsRβ among CD73hiST2lo and CD73loST2hi adipose Treg cells from c. e, Relative mRNA levels of Sell, CD44, Insr, Hif1a and Med23 genes in CD73hiST2lo and CD73loST2hi adipose Treg cells (n = 3 over three independent experiments). f, Expression of CD73 and ST2 in CD4+Foxp3+ Treg cells and CD4+Foxp3− Teff cells from the thymus, spleen, pLNs and fat of 4-month old WT (n = 6) mice. g and h, Frequencies of CD73hiST2lo and CD73loST2hi Treg subsets (g) and Teff subsets (h) from f. i, CD4+CD25−YFP− Teff cells and CD4+CD25hiYFP+ Treg cells were sorted from lymphoid tissues of Foxp3Cre (WT) and Insrfl/flFoxp3Cre (IKO) mice. Expression of Insr in each population was further analyzed by qRT-PCR (n = 4 per group). j, Lymphoid tissue-derived CD73hiST2lo Treg cells were primed with anti-CD3/CD28 DynaBeads (1 cell to 1 bead) in the presence of mIL-2 (50 U/ml) for indicated time. Cells were harvested for Western Blotting. Results are representative of two independent experiments. k, TCR-primed lymphoid-tissue-derived CD73hiST2lo Treg cells from WT and IKO mice were rested and further stimulated with or without 5 ng/ml Insulin for 48 hours. Cells were harvested for Western Blotting. Results are representative of two independent experiments. l, TCR-primed splenic CD73hiST2lo Treg cells from 6-week old WT and IKO mice were rested and further stimulated with or without 5 ng/ml Insulin for 48 hours. Cells were next analyzed for CD73 and ST2 expression. m, Percentages of CD73loST2hi Treg cells as indicated in l (n = 5 per group). Data are mean ± s.d., P values were determined by unpaired two-tailed Student’s t-test. ns, not significant.

Source data

Extended Data Fig. 3 Hif1afl/flFoxp3Cre and Med23fl/flFoxp3Cre mice show no signs of systemic inflammation.

a and b, CD4+CD25−YFP− Teff cells and CD4+CD25hiYFP+ Treg cells were sorted from lymphoid tissues of Foxp3Cre (WT), Hif1afl/flFoxp3Cre (HKO) and Med23fl/flFoxp3Cre (MKO) mice. Expression of Hif1a (a) or Med23 (b) in each population was further analyzed by qRT-PCR (n = 3 per group). c, Image of thymus, spleen and pLNs from 4-month-old WT, HKO and MKO mice. Results are representative of three independent experiments. d, Weight of spleens isolated from 4-month old WT (n = 9), HKO (n = 8) and MKO (n = 9) mice. e, Total number of cells in thymus, spleen and pLNs from 4-month-old WT, HKO and MKO mice (n = 6 per group). f, Distribution of CD4+CD8− (CD4-SP), CD4−CD8+ (CD8-SP), CD4−CD8− (DN) and CD4+CD8+ (DP) thymocytes in 4-month old WT, HKO and MKO mice (n = 6 per group). g, Percentages of CD4-SP, CD8-SP, DN and DP thymocytes in mice from f. h, Expression of CD62L and CD44 by splenic CD4+YFP− Teff and CD8+ T cells from 4-month old WT (n = 8), HKO (n = 7) and MKO (n = 7) mice. i, Percentages of splenic CD62LloCD44hi effector memory T cells from h. j and k, Expression of IFNγ (j), IL-17A (j), IL-2 (k) and IL-4 (k) by splenic CD4+YFP− Teff and CD8+ T cells from 4-month old WT (n = 7), HKO (n = 7 or 6) and MKO (n = 7) mice. l and m, Percentages of IFNγ+, IL-17A+, IL-2+ and IL-4+ splenic Teff cells (l) and CD8+ T cells (m) as indicated in j and k. Data are mean ± s.d. (a and b), box plots (d, e, g, i, l and m) include center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. P values were determined by unpaired two-tailed Student’s t-test. ns, not significant.

Source data

Extended Data Fig. 4

The HIF-1α-Med23 complex specifically regulates the homeostasis of adipose Treg cells a, Expression of Foxp3 in CD4+CD8− T cells from thymus and CD4+ T cells from spleen, LNs, liver, fat, muscle and lung of 4-month old Foxp3Cre (WT, n = 6) and Hif1afl/flFoxp3Cre (HKO, n = 6) mice. b, Expression of Foxp3 in CD4+CD8− T cells from thymus and CD4+ T cells from spleen, LNs, liver, fat, muscle and lung of 4-month old WT (n = 6) and Med23fl/flFoxp3Cre (MKO, n = 6) mice. c, Percentages of Foxp3+ Treg cells as indicated in a. d, Percentages of Foxp3+ Treg cells as indicated in b. e, Bone marrow (BM) cells from WT (CD45.1) and HKO (CD45.2) mice were adoptively co-transferred into irradiated Rag1−/− mice. Eight weeks after adoptive transfer, expression of CD45.1 (WT) and CD45.2 (HKO) by CD4+TCRβ+Foxp3+ T cells was determined in the thymus, spleen, LNs and fat. f, Percentages of CD45.1+ (WT) and CD45.2+ (HKO) Treg cells as indicated in e (n = 6 per group). g, Bone marrow (BM) cells from WT (CD45.1) and MKO (CD45.2) mice were adoptively co-transferred into irradiated Rag1−/− mice. Eight weeks after adoptive transfer, expression of CD45.1 (WT) and CD45.2 (MKO) by CD4+TCRβ+Foxp3+ T cells was determined in the thymus, spleen, LNs and fat. h, Percentages of CD45.1+ (WT) and CD45.2+ (MKO) Treg cells as indicated in g (n = 6 per group). Box plots (c, d, f and h) include center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. P values were determined by unpaired two-tailed Student’s t-test. ns, not significant.

Source data

Extended Data Fig. 5 WT, HKO and MKO adipose Treg cells are capable of sensing insulin signals.

a, Serum insulin levels of 4-month-old male Foxp3Cre (WT), Hif1afl/flFoxp3Cre (HKO) and Med23fl/flFoxp3Cre (MKO) mice (n = 8 per group). b, Expression of InsRβ in CD4+Foxp3+ adipose Treg cells of 4-month-old male WT (n = 8), HKO (n = 8) and MKO (n = 8) mice. c, MFI of InsRβ among Foxp3+ adipose Treg cells as indicated in b. d, WT (n = 7), HKO (n = 7) and MKO (n = 7) mice were fasted overnight. SVF pellets from mouse fat were further treated with or without insulin (5 ng/ml) for 30 min. pAKT (Ser473) levels in adipose Treg cells from SVF pellets were further analyzed by flow cytometry. e, MFI of pAKT (Ser473) among Foxp3+ adipose Treg cells as indicated in d. Data are mean ± s.d., P values were determined by unpaired two-tailed Student’s t-test. ns, not significant.

Source data

Extended Data Fig. 6 The HIF-1α-Med23 complex establishes the identity of CD73loST2hi adipose Treg cells, while IL-33 regulates ST2hi cell expansion.

a, Expression of CD73 and ST2 in CD4+Foxp3+ adipose Treg cells from 4-month old Il33+/+ (WT, n = 5) and Il33−/− (n = 5) mice. b, Frequencies of CD73hiST2lo and CD73loST2hi adipose Treg subsets as indicated in a. Exact P values: CD73hiST2lo, 4.86 × 10−5; CD73loST2hi, 0.000999. c, Expression of CD73 and ST2 in CD4+Foxp3+ adipose Treg cells from 4-month old PBS-treated or IL-33-administered Foxp3Cre (WT), Hif1afl/flFoxp3Cre (HKO) and Med23fl/flFoxp3Cre (MKO) mice (n = 4 per group). d, Frequencies of CD73hiST2lo and CD73loST2hi adipose Treg subsets as indicated in c. Exact P values (WT): CD73hiST2lo, 9.13 × 10−5; CD73loST2hi, 0.0055. Exact P values (HKO): CD73hiST2lo, 0.747; CD73loST2hi, 0.196. Exact P values (MKO): CD73hiST2lo, 0.491; CD73loST2hi, 0.488. Data are mean ± s.d., **P≤0.01, ***P≤0.001, as determined by unpaired two-tailed Student’s t-test. ns, not significant.

Source data

Extended Data Fig. 7 PPAR-γ represses Nt5e gene transcription in adipose Treg cells.

a, tSNE plots of adipose Treg cells in scATAC-seq and scRNA-seq datasets, highlighting the scaled gene accessibility or expression of Pparg and Nt5e genes in different clusters. Cluster 1 and 2 were separated by a red dashed line. b, Upper, aggregated single cell profiles showed significant Nt5e gene accessibility differences in different clusters and a Pparg motif around Nt5e gene promoter. Lower, mouse adipose Treg cells were subjected to Cut&Run assay with anti-PPAR-γ antibody. Enriched DNA was further sequenced and showed binding of PPAR-γ to the Nt5e gene promoter. c, The enriched DNA from Cut&Run assay (as indicated in b) was quantified by real-time PCR using Nt5e promoter primers (n = 6 per group). d, WT Nt5e promoter and the corresponding promoter depleted of the Pparg motif, were cloned into the luciferase reporter gene and transfected into HEK293T cells together with increasing doses of PPAR-γ. Luciferase activity was further analyzed 48 hours later (n = 3 per group). Data are mean ± s.d., P values were determined by unpaired two-tailed Student’s t-test. ns, not significant.

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Extended Data Fig. 8 Genetic ablation of Nt5e in Treg cells from Nt5efl/flFoxp3Cre, Nt5efl/flHif1afl/flFoxp3Cre and Nt5efl/flMed23fl/flFoxp3Cre mice.

a and b, CD4+CD25−YFP− Teff cells and CD4+CD25hiYFP+ Treg cells were sorted from lymphoid tissues of Foxp3Cre (WT), Nt5efl/flHif1afl/flFoxp3Cre (NHKO) and Nt5efl/flMed23fl/flFoxp3Cre (NMKO) mice. Expression of Nt5e (a and b), Hif1a (a) and Med23 (b) in each population was further analyzed by qRT-PCR (n = 3 per group). c, Expression of CD73 and ST2 by CD4+Foxp3+ adipose Treg cells from 4-month old Foxp3Cre (WT, n = 12), Hif1afl/flFoxp3Cre (HKO, n = 5), Med23fl/flFoxp3Cre (MKO, n = 5), Nt5efl/flHif1afl/flFoxp3Cre (NHKO, n = 5), Nt5efl/flMed23fl/flFoxp3Cre (NMKO, n = 5) and Nt5efl/flFoxp3Cre (NKO, n = 5) mice. d and e, MFI of CD73 (d) and ST2 (e) among Foxp3+ adipose Treg cells from mice as indicated in c. f, Percentages of CD73loST2lo, CD73hiST2lo, CD73hiST2hi and CD73loST2hi adipose Treg subsets from mice as indicated in c. Data are mean ± s.d., P values were determined by unpaired two-tailed Student’s t-test. ns, not significant.

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Extended Data Fig. 9 Hif1afl/flFoxp3Cre and Med23fl/flFoxp3Cre mice are protected against age-related metabolic disorders.

a, Representative image of fat from 10-month-old male Foxp3Cre (WT), Hif1afl/flFoxp3Cre (HKO) and Med23fl/flFoxp3Cre (MKO) mice. Results are representative of three independent experiments. b, Weight of total body, lean and fat of 10-month-old male WT (n = 7), HKO (n = 6) and MKO (n = 8) mice. c, Mass of SAT and VAT in 10-month-old male WT, HKO and MKO mice (n = 8 per group). d, H&E staining of adipose tissues from 10-month-old WT, HKO and MKO mice. Scale bars, 100 μm. Results are representative of three independent experiments. e, Adipocyte size in mice as indicated in d. Results are representative of three independent experiments. f, Food consumption of 10-month-old male WT, HKO and MKO mice (n=8 per group). g-i, Average respiratory exchange ratio (RER) (g), average oxygen consumption rate (VO2) (h) and average CO2 release rate (VCO2) (i) of 10-month-old male WT (n = 8), HKO (n = 6) and MKO (n = 6) mice. j, Serum insulin levels in 10-month-old fasted WT (n = 8), HKO (n = 7) and MKO (n = 9) mice. k and l, Glucose (k) and insulin (l) tolerance tests of 10-month-old WT (n = 7), HKO (n = 5) and MKO (n = 5) mice. m and n, Glucose (m) and insulin (n) tolerance tests of 10-month-old WT and Insrfl/flFoxp3Cre (IKO) mice (n = 6 per group). o and p, Glucose (o) and insulin (p) tolerance tests of 10-month-old WT and Ppargfl/flFoxp3Cre (PKO) mice (n = 6 per group). Box plots (b, c, e, f, g, h, i and j) include center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. Data (k to p) are mean ± s.d.. P values were determined by unpaired two-tailed Student’s t-test. ns, not significant.

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Extended Data Fig. 10 Delayed onset of diet-induced obesity in Hif1afl/flFoxp3Cre and Med23fl/flFoxp3Cre mice.

a and b, 6-week-old Foxp3Cre (WT, n = 11), Hif1afl/flFoxp3Cre (HKO, n = 10) and Med23fl/flFoxp3Cre (MKO, n = 5) mice were placed on HFD and body weight changes were monitored for 12 weeks. Exact P values (HKO, 8 to 18 week): 0.006, 0.009, 0.018, 0.012, 0.015, 0.012, 0.008, 0.005, 0.005, 0.007 and 0.039. Exact P values (MKO, 8 to 18 week): 0.0022, 0.0006, 0.0006, 0.0004, 4.14 × 10−5, 0.0002, 0.0009, 0.0014, 0.024, 0.011 and 0.038. c, Weight of total body, lean and fat of 18-week old WT (n = 6), HKO (n = 5) and MKO (n = 5) mice after a 12-week long HFD. d, Mass of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) in 18-week old WT (n = 6), HKO (n = 5) and MKO (n = 5) mice after a 12-week long HFD. e, Food consumption of 18-week old WT (n = 8), HKO (n = 8) and MKO (n = 8) mice after a 12-week long HFD. f-h, Average RER (f), average oxygen consumption rate (VO2) (g) and average CO2 release rate (VCO2) (h) of 18-week old WT (n = 6), HKO (n = 6) and MKO (n = 5) mice after a 12-week long HFD. i, H&E staining of liver from 18-week old WT, HKO and MKO mice after a 12-week long HFD. Scale bars, 100 μm. Results are representative of three independent experiments. j, Serum insulin levels in fasted 18-week old WT (n = 6), HKO (n = 6) and MKO (n = 5) mice after a 12-week long HFD. k and l, Glucose (k) and insulin (l) tolerance tests of 18-week old WT (n = 6), HKO (n = 6) and MKO (n = 5) mice after a 12-week long HFD. Data (b, k and l) are mean ± s.d., box plots (c, d, e, f, g, h and j) include center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. *P≤0.05, **P≤0.01, ***P≤0.001, as determined by unpaired two-tailed Student’s t-test. ns, not significant.

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Supplementary information

Supplementary Information

Supplementary gating strategy and supplementary work model.

Reporting Summary

Supplementary Table 1

Antibody information is provided in Excel format as Supplementary Table 1 (Methods). Sequences of RT–qPCR primers are provided in Excel format as Supplementary Tables 2–4 (Methods).

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Li, Y., Lu, Y., Lin, SH. et al. Insulin signaling establishes a developmental trajectory of adipose regulatory T cells. Nat Immunol 22, 1175–1185 (2021). https://doi.org/10.1038/s41590-021-01010-3

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