Abstract
Interleukin (IL-)23 is a major mediator and therapeutic target in chronic inflammatory diseases that also elicits tissue protection in the intestine at homeostasis or following acute infection1,2,3,4. However, the mechanisms that shape these beneficial versus pathological outcomes remain poorly understood. To address this gap in knowledge, we performed single-cell RNA sequencing on all IL-23 receptor-expressing cells in the intestine and their acute response to IL-23, revealing a dominance of T cells and group 3 innate lymphoid cells (ILC3s). Unexpectedly, we identified potent upregulation of the immunoregulatory checkpoint molecule cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) on ILC3s. This pathway was activated by gut microbes and IL-23 in a FOXO1- and STAT3-dependent manner. Mice lacking CTLA-4 on ILC3s exhibited reduced regulatory T cells, elevated inflammatory T cells and more-severe intestinal inflammation. IL-23 induction of CTLA-4+ ILC3s was necessary and sufficient to reduce co-stimulatory molecules and increase PD-L1 bioavailability on intestinal myeloid cells. Finally, human ILC3s upregulated CTLA-4 in response to IL-23 or gut inflammation and correlated with immunoregulation in inflammatory bowel disease. These results reveal ILC3-intrinsic CTLA-4 as an essential checkpoint that restrains the pathological outcomes of IL-23, suggesting that disruption of these lymphocytes, which occurs in inflammatory bowel disease5,6,7, contributes to chronic inflammation.
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Data availability
All data necessary to understand and evaluate the conclusions of this paper are provided. Single-cell RNA sequencing data have been deposited in the Gene Expression Omnibus database under the accession number GSE229976. Bulk RNA sequencing data have been deposited under the accession number GSE247742. Source data are provided with this paper.
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Acknowledgements
We thank members of the Sonnenberg Laboratory for discussions and critical reading of the manuscript. Research in the Sonnenberg Laboratory is supported by the US National Institutes of Health (grant nos. R01AI143842, R01AI123368, R01AI145989, U01AI095608, R01AI162936, R01CA274534 and R37AI174468), an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund, the Meyer Cancer Center Collaborative Research Initiative, the Dalton Family Foundation and Linda and Glenn Greenberg. A.A. and M.L. are supported by fellowships from the Crohn’s and Colitis Foundation (grant nos. 902451 and 935259). A.M.J. and J.U. are supported by US National Institutes of Health grant no. T32DK116970. J.Z. is supported by grant no. F32DK136248. V.H. is supported by a DFG Walter Benjamin Fellowship (grant no. HO 7399/1-1). G.F.S. is a CRI Lloyd J. Old STAR. We thank the Epigenomics Cores of Weill Cornell Medicine, N. Zahan for administrative support and D. Garone and A. Brcic-Susak for technical support. The Jill Roberts Institute (JRI) Live Cell Bank is supported by the JRI, the Jill Roberts Center for IBD, Cure for IBD, the Rosanne H. Silbermann Foundation, the Sanders Family and Weill Cornell Medicine Division of Pediatric Gastroenterology and Nutrition.
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A.A. and G.F.S. conceived the project. A.A. performed most experiments and analysed the data. A.M.J., J.Z., V.H., J.U., M.L. and J.G. helped with experiments and data analyses. JRI Live Cell Bank and R.E.S. contributed to clinical sample acquisition, annotation, processing and evaluation. J.B.W., E.V. and S.S. provided essential mouse models, scientific advice and expertise. A.A. and G.F.S. wrote the manuscript, with input from all of the authors.
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Extended data figures and tables
Extended Data Fig. 1 Single cell analysis of all IL-23R+ cells in small intestine of healthy mice.
a, Gating strategy and experimental design to sort IL-23R+ immune cells from small intestine lamina propria of IL-23R-eGFP mice for scRNA-seq. Violin plot confirming Il23r expression among all the identified IL-23R+ cell clusters. b, Feature plots showing expression of indicated genes in different clusters. c, Violin plot showing Mki67 expression among all identified IL-23R+ cell clusters.
Extended Data Fig. 2 Characterization of IL-23R+ immune cells in the intestine.
a, Flow cytometry plots with donut plot of final frequencies of different IL-23R+ immune cells in small intestine lamina propria of IL-23R-eGFP mice (n = 4 mice). Lineage 1, CD11b, CD11c, B220, CD3ε, CD5, CD8α, NK1.1 and TCRγδ. b, Flow cytometry plots showing eGFP expression (IL-23R) in different immune cells in small intestine lamina propria of IL-23R-eGFP mice (n = 4 mice). c, Donut plots of final frequencies of different IL-23R+ immune cells in large intestine lamina propria of naïve and C. rodentium infected IL-23R-eGFP mice at day 14 post infection (n = 4 mice). Data in a and c are representative of two or three independent experiments with similar results.
Extended Data Fig. 3 Acute IL-23 driven responses in IL-23R+ intestinal cells.
a, Volcano plots of differentially expressed genes in scRNA-seq dataset from IL-23R+ immune cells from the small intestine of IL-23R-eGFP mice before and after IL-23 stimulation in annotated cell types. b, UMAP plots of scRNA-seq data showing indicated gene expression in different clusters before and after IL-23 stimulation (1-NKp46+ ILC3s, 2-CCR6+ ILC3s, 3-DN ILC3s, 4-γδ T cells, 5-TH17 cells, 6-Proliferative cells). The statistics was obtained by the Wilcoxon test as implemented by Seurat; red dots are significantly different.
Extended Data Fig. 4 Flow cytometry gating for ILCs and CTLA-4.
a, Gating strategy for flow cytometry analysis of different immune cells in intestinal lamina propria of mice. Lineage 1, CD11b, CD11c, F4/80 and B220; lineage 2, CD3ε, CD5, CD8α and TCRγδ; Group 1 innate lymphoid cells (ILC1s) were identified as live CD45+ Lineage− CD127+ CD90.2+ T-bet+ RORγ− NK1.1+ Eomes−; group 2 innate lymphoid cells (ILC2s) were identified as live CD45+ Lineage− CD127+ CD90.2+ GATA3+; group 3 innate lymphoid cells (ILC3s) were identified as live CD45+ Lineage− CD127+ CD90.2+ RORγ+; and subsets of ILC3s were further identified as CCR6+ T-bet− ILC3s or CCR6− T-bet+ ILC3s. For T cell analysis Tregs were identified as CD45+CD4+FoxP3+ T cells and TH 17 as CD45+CD4+ RORγ+FoxP3− T cells. b, Representative flow cytometry plots of CTLA-4 staining in different immune cells from small intestine lamina propria of C57BL/6 mice (n = 4 mice). c, Flow cytometry plots of CTLA-4+ cells in small intestine lamina propria of Rag1−/− mice (n = 4 mice). Data in b and c are representative of two independent experiments with similar results.
Extended Data Fig. 5 Microbiota dependent regulation of CTLA-4.
a, Flow cytometry plots with graph of CTLA-4+ ILC1s and CTLA-4+ NK cells frequencies in large intestine lamina propria of conventional SPF, germ-free (GF) and SPF Rag1−/− mice treated with antibiotics (ABX) (n = 8 mice). b, Graph of CTLA-4+ ILC1s and CTLA-4+ NK cells frequencies in small intestine lamina propria of conventional SPF, germ-free and SPF mice treated with antibiotics on Rag1−/− background (n = 8 mice). c, Graph of CTLA-4+ ILC1s, CTLA-4+ NK cells and CTLA-4+ γδ T cells frequencies in small intestine lamina propria of SPF mice ex vivo stimulated with indicated cytokines (n = 4 mice). Data in a-b were pooled from two independent experiments and shown as Mean ± s.d. Data in c are representative of two or three independent experiments with similar results and shown as Mean ± s.d. The statistics in a-c were calculated by one-way ANOVA with Dunnett’s multiple comparisons.
Extended Data Fig. 6 Steady state analysis of Ncr1cre Ctla4f/f mice.
a, Flow cytometry plots of NKp46+ cells in large intestine lamina propria of naïve and C. rodentium infected C57BL/6 mice (n = 4 mice). Lineage 1, CD11b, CD11c and B220; lineage 2, CD3ε, CD5, CD8α, and TCRγδ. b, UMAP plot of scRNA-seq data of IL-23R+ immune cells from the small intestine of IL-23R-eGFP mice showing Ctla4 expression in different clusters. The large intestine of Ncr1cre Ctla4f/f and Ctla4f/f were analyzed at steady state and graph displaying the frequency of (c) ILC3s (percentage of Lin- CD90+ CD127+) and (d) IL-22+ ILC3s (percentage of total ILC3s). e, Graph of TH17 cells frequency (percentage of CD4+ T cells). Flow plots with graph displaying the frequency of (f) Treg cells (percentage of CD4+ T cells) and (g) neutrophils gated as CD11b+Ly6G+ cells (percentage of CD45+ cells). Data in a,c,d,e,f and g are representative of two or three independent experiments with similar results (n = 5 mice) and shown as Mean ± s.d. The statistics in c-g were determined by Mann–Whitney U-test (unpaired, two-tailed).
Extended Data Fig. 7 NK cells and ILC1s do not impact intestinal inflammation following enteric infection with C. rodentium.
C57BL/6 mice were orally infected with C. rodentium and treated with IgG or anti-IL-23 monoclonal antibody. a, Flow cytometry plots with graph displaying the frequency of CTLA-4 expression on ILC1s, NK cells and γδ T cells (n = 5 mice). C57BL/6 mice were orally infected with C. rodentium and treated with IgG or anti-NK1.1 monoclonal antibody. Mice were analyzed at day 14 post infection and graph displaying the (b) depletion efficiency of natural killer cells and ILC1s, and the frequency of (c) ILC3s (percentage of Lin− CD90+ CD127+); (d) Treg cells (percentage of CD4+ T cells); (e) TH17 cells (percentage of CD4+ T cells); (f) Neutrophils gated as CD11b+Ly6G+ cells (percentage of CD45+ cells) in large intestinal lamina propria immune cells. Data in b-f are representative of two or three independent experiments with similar results (n = 4 mice) and shown as Mean ± s.d. The statistics in a-f were determined by Mann–Whitney U-test (unpaired, two-tailed).
Extended Data Fig. 8 Innate immune responses are intact in mice lacking ILC3-specific CTLA-4.
Ncr1wt/wt and Ncr1cre/wt mice were orally infected with C. rodentium and at day 14 post infection large intestinal lamina propria immune cells were analyzed. a, Flow cytometry plots with graph of Tregs frequency (percentage of CD4+ T cells) and graph displaying the frequency of (b) TH17 cells (percentage of CD4+ T cells) and (c) neutrophils gated as CD11b+Ly6G+ cells (percentage of CD45+ cells) with (d) colon length. e, Flow cytometry plots and graph of CTLA-4+ ILC3s frequency (percentage of total ILC3s) in Rag1−/− Ctla4f/f and Rag1−/− RORγtcre Ctla4f/f mice. Rag1−/− Ctla4f/f and Rag1−/− RORγtcre Ctla4f/f mice were treated with 100 µg of anti-CD40 antibody, and graph displaying the frequency of (f) ILC3s (percentage of Lin− CD90+ CD127+); (g) IL-22+ ILC3s (percentage of total ILC3s) and (h) neutrophils gated as CD11b+Ly6G+ cells (percentage of CD45+ cells) in the large intestines with (i) colon length at day 7 post treatment. Data in a-i are representative of two independent experiments with similar results (n = 4 mice) and shown as Mean ± s.d. The statistics in a-i were determined by Mann–Whitney U-test (unpaired, two-tailed).
Extended Data Fig. 9 CTLA-4+ ILC3s reduce co-stimulatory molecules and enhance PD-L1 on myeloid cells in response to IL-23.
Sorted ILC3s and myeloid cells from the large intestine of C57BL/6 mice were co-cultured with recombinant IL-23 in presence or absence of indicated blocking antibodies. Flow cytometry plots with graph of MFI of (a) CD80 and (b) CD86 expression on myeloid cells gated as CD11c+ MHCIIhi. c, Flow cytometry plots with graph of free PD-L1 frequency on myeloid cells gated as CD11c+ MHCIIhi. d, Gating strategy to analyze T cells, myeloid cells and ILC3s in human samples. Data in a-c are representative of two independent experiments with similar results (n = 4 mice) and shown as Mean ± s.d. The statistics were calculated by one-way ANOVA with Dunnett’s multiple comparisons.
Extended Data Fig. 10 ILC3s restrain IL-23 mediated inflammation through CTLA-4.
Here we define a pathway of immune regulation in the large intestine. This pathway is activated by gut microbes and IL-23 in a FOXO1- and STAT3-dependent manner. ILC3-intrinsic CTLA-4 shapes the levels of co-inhibitory and co-stimulatory molecules on intestinal myeloid cells to support Tregs and restrict T effector (Teff) cells. Consequently, ILC3-intrinsic CTLA-4 function as a checkpoint to restrain the pathologic functions of IL-23, suggesting that disruption of these lymphocytes, which occurs in IBD, contributes to chronic inflammation.
Supplementary information
Supplementary Table 1
Clinical metadata on human samples for bulk RNA sequencing.
Supplementary Table 2
Clinical metadata on paediatric patients with IBD and matched healthy controls.
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Ahmed, A., Joseph, A.M., Zhou, J. et al. CTLA-4-expressing ILC3s restrain interleukin-23-mediated inflammation. Nature 630, 976–983 (2024). https://doi.org/10.1038/s41586-024-07537-3
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DOI: https://doi.org/10.1038/s41586-024-07537-3
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