Innate lymphoid cells support regulatory T cells in the intestine through interleukin-2

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Abstract

Interleukin (IL)-2 is a pleiotropic cytokine that is necessary to prevent chronic inflammation in the gastrointestinal tract1,2,3,4. The protective effects of IL-2 involve the generation, maintenance and function of regulatory T (Treg) cells4,5,6,7,8, and the use of low doses of IL-2 has emerged as a potential therapeutic strategy for patients with inflammatory bowel disease9. However, the cellular and molecular pathways that control the production of IL-2 in the context of intestinal health are undefined. Here we show, in a mouse model, that IL-2 is acutely required to maintain Treg cells and immunological homeostasis throughout the gastrointestinal tract. Notably, lineage-specific deletion of IL-2 in T cells did not reduce Treg cells in the small intestine. Unbiased analyses revealed that, in the small intestine, group-3 innate lymphoid cells (ILC3s) are the dominant cellular source of IL-2, which is induced selectively by IL-1β. Macrophages in the small intestine produce IL-1β, and activation of this pathway involves MYD88- and NOD2-dependent sensing of the microbiota. Our loss-of-function studies show that ILC3-derived IL-2 is essential for maintaining Treg cells, immunological homeostasis and oral tolerance to dietary antigens in the small intestine. Furthermore, production of IL-2 by ILC3s was significantly reduced in the small intestine of patients with Crohn’s disease, and this correlated with lower frequencies of Treg cells. Our results reveal a previously unappreciated pathway in which a microbiota- and IL-1β-dependent axis promotes the production of IL-2 by ILC3s to orchestrate immune regulation in the intestine.

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Fig. 1: IL-2 is dominantly produced by ILC3s in the small intestine.
Fig. 2: Sensing of the microbiota and production of IL-1β induces ILC3-derived IL-2.
Fig. 3: ILC3-derived IL-2 is essential for Treg cell maintenance, immunological homeostasis and oral tolerance to dietary antigen in the small intestine.
Fig. 4: Production of IL-2 by ILC3s is impaired in the intestine of patients with Crohn’s disease.

Data availability

RNA sequencing data are available at Gene Expression Omnibus under accession number GSE126580. All datasets generated and/or analysed during the current study are presented in this published article, the accompanying Source Data or Supplementary Information, or are available from the corresponding author upon reasonable request.

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  • 23 April 2019

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Acknowledgements

We thank members of the Sonnenberg Laboratory for discussions and critical reading of the manuscript, and T. Shima and Y. Umesaki from Yakult Central Institute for providing segmented filamentous bacteria and advice. Research in the Sonnenberg Laboratory is supported by the National Institutes of Health (R01AI143842, R01AI123368, R01AI145989, R21DK110262 and U01AI095608), the NIAID Mucosal Immunology Studies Team (MIST), the Crohn’s and Colitis Foundation, the Searle Scholars Program, the American Asthma Foundation Scholar Award, Pilot Project Funding from the Center for Advanced Digestive Care (CADC), an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund, a Wade F. B. Thompson/Cancer Research Institute CLIP Investigator grant, the Meyer Cancer Center Collaborative Research Initiative and the Jill Roberts Institute (JRI) for Research in IBD. L.Z. and J.G. are supported by fellowships from the Crohn’s and Colitis Foundation (608975 and 519428). N.J.B. is supported by a fellowship from the NIH (F32AI124517). We thank the Epigenomics Core of Weill Cornell Medicine, and all contributing members of the JRI IBD Live Cell Bank, which is supported by the JRI, Jill Roberts Center for IBD, Cure for IBD, the Rosanne H. Silbermann Foundation and Weill Cornell Medicine Division of Pediatric Gastroenterology and Nutrition. Research in the Vivier laboratory is supported by funding form the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (TILC, grant agreement no. 694502); the Agence Nationale de la Recherche; Equipe Labellisée ‘La Ligue’, Ligue Nationale contre le Cancer, MSDAvenir, Innate Pharma and institutional grants to the CIML (INSERM, CNRS and Aix-Marseille University) and to Marseille Immunopôle. Research reported in this publication was supported by the National Center for Advancing Translational Science of the National Institute of Health, under award number UL1TR002384.

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Nature thanks Koji Hase, Scott Snapper and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Contributions

L.Z. and G.F.S. conceived the project. L.Z. performed most experiments and analysed the data. F.T., C.C., N.J.B., J.G., H.K. and E.K.S. helped with experiments. G.G.P. performed bioinformatics analyses. J.R.K., R.N.B., M.A.S. and R.E.S. provided human samples, scientific advice and expertise. E.V., G.E. and K.A.S. provided essential mouse models, scientific advice and expertise. L.Z. and G.F.S. wrote the manuscript, with input from all the authors.

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Correspondence to Gregory F. Sonnenberg.

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Extended data figures and tables

Extended Data Fig. 1 IL-2 blockade results in disrupted T cell homeostasis throughout the intestinal tract and associated lymphoid tissues.

ac, Sex- and age- matched C57BL/6 mice were treated with anti-IL-2 monoclonal antibodies every other day for two weeks, and examined for the size of the spleen and mesenteric lymph nodes (mLN) (a), the frequency of Treg cells (b) and Ki-67+CD4+ T cells (c) of mesenteric lymph nodes by flow cytometry (n = 10). dg, Mice from ac were also analysed for the frequencies of Treg cells (d) and Ki-67+CD4+ T cells (e) in large-intestinal lamina propria cells, and for the frequencies and numbers of Treg cells (f) and Ki-67+CD4+ T cells (g) in small-intestinal lamina propria cells by flow cytometry (n = 10). hk, Mice from ac were analysed for the frequency of TH1 cells (h) and TH17 cells (i) in large-intestinal lamina propria cells, and the frequencies and numbers of TH1 cells (j) and TH17 cells (k) in small-intestinal lamina propria cells by flow cytometry (n = 10). Data in a are representative of two independent experiments with similar results. Data in bk are pooled from two independent experiments. Results are shown as mean ± s.e.m. All statistics are calculated by unpaired two-tailed Student’s t-test. LI, large intestine; SI, small intestine. Source data

Extended Data Fig. 2 T-cell-derived IL-2 is essential for maintaining immunological homeostasis in the mesenteric lymph node and large intestine.

a, Sex- and age- matched Il2f/f and LckcreIl2f/f mice were examined for the deletion efficiency of IL-2 in CD4+ T cells in the large and small intestines. bc, Mice in a were examined for the frequency of Treg cells (b) and Ki-67+ CD4+ T cells (c) from the mesenteric lymph nodes by flow cytometry (n = 6). dg, Mice in a were analysed for the frequencies and numbers of Treg cells (d), Ki-67+CD4+ T cells (e), TH1 cells (f) and TH17 cells (g) of large-intestinal lamina propria cells by flow cytometry (n = 6). hk, Mice in a were analysed for the frequencies and numbers of Treg cells (h), Ki-67+CD4+ T cells (i), TH1 cells (j) and TH17 cells (k) of small-intestinal lamina propria cells by flow cytometry (n = 6). Data in a are representative of two independent experiments with similar results. Data in bk are pooled from two independent experiments. Results are shown as mean ± s.e.m. All statistics are calculated by unpaired two-tailed Student’s t-test. Source data

Extended Data Fig. 3 Gating strategy to analyse subsets of innate lymphoid cells and CD4+ T cells in the small intestine.

Gating strategy for flow cytometry analysis of innate lymphoid cells and CD4+ T cells in small-intestinal lamina propria cells. Lineage 1, CD11b, CD11c and B220; lineage 2, CD3ε, CD5 and CD8α. CD4+ T cells were identified as CD45+Lineage 2+CD4+; group-1 innate lymphoid cells were identified as CD45+LineageCD127+CD90.2+T-bet+RORγt; group-2 innate lymphoid cells were identified as CD45+LineageCD127+CD90.2+GATA3+; ILC3s were identified as CD45+LineageCD127+CD90.2+RORγt+; and subsets of ILC3s further identified as CCR6+T-bet ILC3s or CCR6T-bet+ ILC3s.

Extended Data Fig 4 IL-2+ cells in the large intestine of wild-type mice and in the small and large intestines of Rag1−/− mice.

a, Flow cytometry plots with graph of frequency and numbers of IL-2 in ILC3s and effector and memory (E/M) CD4+ T cells (CD3+CD4+FOXP3CD44highCD62Llow) in small-intestinal lamina propria cells of wild-type mice (n = 8). b, Flow cytometry plots show IL-2+ cells in large-intestinal lamina propria cells of C57BL/6 mice. Lineage 1, CD11b, CD11c and B220; lineage 2, CD3ε, CD5 and CD8α. c, d, Flow cytometry plots with graph of frequency (c) and absolute numbers (d) of IL-2+ cells in large-intestinal lamina propria cells of C57BL/6 mice (n = 6). e. The frequency and number of IL-2+ subsets of ILC3s in small-intestinal lamina propria cells of C57BL/6 mice (n = 8). f, Flow cytometry plots show IL-2+ cells in small-intestinal lamina propria cells of Rag1−/− mice. g, h, Flow cytometry plots with graph of frequency (g) and absolute numbers (h) of IL-2+ cells in small-intestinal lamina propria cells of Rag1−/− mice (n = 5). i, j, Flow cytometry plots with graph of frequency (i) and absolute numbers (j) of IL-2+ subsets of ILC3s in small-intestinal lamina propria cells of Rag1−/− mice (n = 5). k, Flow cytometry plots show IL-2+ cells in large-intestinal lamina propria cells of Rag1−/− mice. Lineage 1, CD11b, CD11c and B220; lineage 2, CD3ε, CD5 and CD8α. Data in b and fk are representative of two independent experiments with similar results. Data in a and ce are pooled from two independent experiments. Results are shown as mean ± s.e.m. Statistics are calculated by paired or unpaired two-tailed Student’s t-test. Source data

Extended Data Fig. 5 Natural killer cells and group-1 innate lymphoid cells are dispensable for maintenance of Treg cells in the small intestine.

a, IL-2 was assessed in T-bet+ ILC3s, total ILC3s, CD4+ T cells, natural killer cells, group-1 innate lymphoid cells and dendritic cells in small-intestinal lamina propria cells of Il2f/f and Ncr1creIl2f/f mice. b, The number of IL-2+ cells was quantified in small-intestinal lamina propria cells of Il2f/f and Ncr1creIl2f/f mice (n = 4). c, d, Sex- and age- matched C57BL/6 mice were treated with anti-NK1.1 monoclonal antibody every three days for two weeks and examined for efficiency of depletion of natural killer cells (c), and for the frequency and number of Treg cells in small-intestinal lamina propria cells (d) (n = 7). Data in ac are representative of two independent experiments with similar results. Data in d are pooled from two independent experiments. Results are shown as mean ± s.e.m. Statistics are calculated by unpaired two-tailed Student’s t-test. Source data

Extended Data Fig. 6 ILC3-derived IL-2 is dispensable for the maintenance of small-intestinal TH17 cells, ILC3 homeostasis and large-intestinal Treg cells.

ad, Il2f/f and Ncr1creIl2f/f mice were analysed for the percentage of Treg cells (a), TH1 cells (b), Ki-67+CD4+ T cells (c) and the frequency and cell number of TH17 cells (d), in small-intestinal lamina propria cells at steady state by flow cytometry (n = 8). e, Il2f/f and Ncr1creIl2f/f mice were examined for the frequency and number of Treg cells in large-intestinal lamina propria cells by flow cytometry (n = 8). f, Il2f/f and Ncr1creIl2f/f mice were examined for the frequency and number of ILC3s in small-intestinal lamina propria cells by flow cytometry (n = 8). g, IL-22 was assessed in ILC3s from small-intestinal lamina propria cells of Il2f/f or Ncr1creIl2f/f mice. h, Representative histograms and bar graph examination of CD25 staining on Treg cells and IL-2+ ILC3s. i, Representative histograms that demonstrate IL-2 binding capacity and quantification of bound IL-2 mean fluorescence intensity in Treg cells and ILC3s. j, Experimental design of the delayed-type hypersensitivity model. Data in fh are representative of two independent experiments with similar results (at least three mice per group). Data in ae and i are pooled from two independent experiments. Results are shown as mean ± s.e.m. Statistics are calculated by paired or unpaired two-tailed Student’s t-test. Source data

Extended Data Fig. 7 Deletion of ILC3-intrinsic IL-2 affects the population size of peripherally induced Treg cells but not their suppressive capacity.

a, b, The frequency of peripheral Treg cells (labelled ‘Nrp-1lo pTregs’) and thymic Treg cells (labelled ‘Nrp-1hi tTregs’) were characterized in small-intestinal lamina propria cells of Il1rf/f and Ncr1creIl1rf/f mice (a) or Il2f/f and Ncr1creIl2f/f mice (b) (n = 5). c, d, The frequency of subsets of Treg cells were analysed in small-intestinal lamina propria cells of Il2f/f and Ncr1creIl2f/f mice (n = 5). e, Small-intestinal Treg cells were examined for expression of Lag3, Tgfb1, Ctla4, Ebi3 and Il10 in Il2f/f and Ncr1creIl2f/f mice (n = 7). f, g, Sort-purified small-intestinal CD45+CD3+CD4+CD25+ Treg cells were co-cultured with sort-purified CFSE-labelled splenic effector T cells (CD3+CD4+CD25CD45RBhigh) in the presence of purified splenic dendritic cells and soluble anti-CD3 for three days. CFSE dilution was analysed and quantified (n = 6). Data in ad and f are representative of two independent experiments with similar results. Data in e and g are pooled from two independent experiments. Results are shown as mean ± s.e.m. Statistics are calculated by unpaired two-tailed Student’s t-test. Source data

Extended Data Fig. 8 ILC3-derived IL-2 does not exhibit functional redundancy or hierarchies with ILC3-specific GM-CSF or MHCII.

a, Flow cytometry plots with graph of frequency and quantification of cell numbers of IL-2+ ILC3s in small-intestinal lamina propria cells of wild-type and Csf2−/− mice (n = 8). b, c, Flow cytometry plots with graph of frequency and quantification of cell numbers of Treg cells (b) and IL-2+ ILC3s (c) in small-intestinal lamina propria cells of H2-Ab1f/f littermate controls and mice lacking ILC3-specific MHCII (MHCIIΔILC3 mice) (n = 7). d, e, Flow cytometry plots with graph of frequency and quantification of cell numbers of MHCII+ ILC3s (d) and GM-CSF+ ILC3s (e) in small-intestinal lamina propria cells of Il2f/f and Ncr1creIl2f/f mice (n = 7). Data are pooled from two independent experiments. Results are shown as mean ± s.e.m. Statistics are calculated by unpaired two-tailed Student’s t-test. Source data

Extended Data Fig. 9 ILC3-derived IL-2 promotes essential immune regulation in the intestine.

ah, CD4+ T cells were adoptively transferred into Il2f/fRag1−/− or RorccreIl2f/fRag1−/− recipient mice. ad, Recipient mice were examined for changes in weight (a), colon length (b), histological haematoxylin and eosin staining in the terminal colon (c) and lipocalin-2 presence in faecal samples (d) (n = 8). e, Flow cytometry plots with graph of percentage and absolute cell number of Treg cells in large-intestinal lamina propria cells in defined recipient mice. f, Absolute cell number of Treg cells in small-intestinal lamina propria cells in defined recipient mice. g, Flow cytometry plots and graph of frequency and absolute number of IFNγIL-17A+ and IFNγ+IL-17A+ cells in large-intestinal lamina propria cells in defined recipient mice. h, Cell number of TH1 and TH17 cells in small-intestinal lamina propria cells in defined recipient mice. n = 7 mice, Il2f/fRag1−/− group; n = 8 mice, RorccreIl2f/fRag1−/− (eh). Data in ah are pooled from two independent experiments. Results are shown as mean ± s.e.m. Statistics are calculated by unpaired two-tailed Student’s t-test. Source data

Extended Data Fig. 10 A IL-1β–ILC3–IL-2 circuit is essential for the maintenance of Treg cells and immunological homeostasis uniquely within the small intestine.

Here we define a pathway of immune regulation in the small intestine. This pathway is continuously required, and involves MYD88- and NOD2-dependent microbial sensing by macrophages, production of IL-1β and induction of ILC3-derived IL-2 to support the maintenance of peripherally induced intestinal Treg cells. Consequently, this is essential to maintain immunological homeostasis and oral tolerance, and becomes dysregulated in inflammatory bowel disease in humans.

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Zhou, L., Chu, C., Teng, F. et al. Innate lymphoid cells support regulatory T cells in the intestine through interleukin-2. Nature 568, 405–409 (2019). https://doi.org/10.1038/s41586-019-1082-x

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