Abstract
Regulatory T cells (Treg cells) are instrumental in establishing immunological tolerance. However, the precise effector mechanisms by which Treg cells control a specific type of immune response in a given tissue remains unresolved. By simultaneously studying Treg cells from different tissue origins under systemic autoimmunity, in the present study we show that interleukin (IL)-27 is specifically produced by intestinal Treg cells to regulate helper T17 cell (TH17 cell) immunity. Selectively increased intestinal TH17 cell responses in mice with Treg cell-specific IL-27 ablation led to exacerbated intestinal inflammation and colitis-associated cancer, but also helped protect against enteric bacterial infection. Furthermore, single-cell transcriptomic analysis has identified a CD83+CD62Llo Treg cell subset that is distinct from previously characterized intestinal Treg cell populations as the main IL-27 producers. Collectively, our study uncovers a new Treg cell suppression mechanism crucial for controlling a specific type of immune response in a particular tissue and provides further mechanistic insights into tissue-specific Treg cell-mediated immune regulation.
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Data availability
All data are present in the article and supplementary information files are available from the corresponding authors upon reasonable request. RNA-seq data underlying Figs. 1 and 2 and Extended Data Fig. 1, as well as scRNA-seq data underlying Fig. 7 and Extended Data Fig. 7, are available from the National Center for Biotechnology Information under accession no. GSE217949. Source data are provided with this paper.
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Acknowledgements
The present study was supported by the NIH (grant nos. AI108651, AI127751 and AI163813 to L.-F.L., DK110534 and DK120515 to H.C., AI126277, AI145325 and AI154644 to M.R. and AI132122 and BX005106 to J.T.C.). Work in M.R.’s laboratory is also supported by the University of California, San Diego, Center for Mucosal Immunology, Allergy, and Vaccines, Chiba University. M.R. holds an Investigator in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund. R.P. is a BioLegend fellow. R.R.G. is partly supported by a fellowship from the Crohn’s and Colitis Foundation. We thank all members of our laboratory for discussions.
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C.-H.L. and L.-F.L. conceived and designed the project. C.-H.L., C.-J.W., S.C., R.P., W.J.H., R.R.G. and C.-Y.H. performed the experiments. C.-H.L., R.P., E.I., J.B., M.N., M.C., R.A.M., S.A.P., H.G.D. and L.-F.L. analyzed the data. L.-L.L., M.-C.C., H.C., M.R. and J.T.C. contributed critical reagents, materials and analytical tools. C.-H.L. and L.-F.L. wrote the manuscript.
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L.-F.L. is a scientific advisor for Elixiron Immunotherapeutics and receives research grants from AstraZeneca, Avidity Biosciences and Molecular Axiom. E.I., J.B., M.N., M.C. and R.A.M are or were employees of AstraZeneca and may own stock or stock options. As such, they declare that they are bound by confidentiality agreements that prevent them from disclosing their competing interests in this work. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Establishing an in vivo experimental model to simultaneously study active suppressor program in different tissue Treg cell subsets.
a, Schematic of the experimental model for studying Treg cell-mediated control of systemic autoimmunity. FACS analysis and frequencies of b, Ki67+ and c, CD25+ cells within the Tconv cells gated on the live CD4+Foxp3− population (or Treg cells gated on the live CD4+Foxp3+ population) in spleens of control PBS-treated or DT-treated Foxp3DTR mice with or without transfer of Foxp3Thy1.1+ Treg cells. Each symbol represents an individual mouse (n = 6). Data are presented as mean values +/- SD. In b, ****P < 0.0001 (up); ****P < 0.0001 (bottom left); ****P < 0.0001 (bottom right). In c, *P = 0.0352 (up); ****P < 0.0001 (bottom left); ****P < 0.0001 (bottom right). Statistical significance was determined by two-tailed unpaired t test. d, Heatmap of selected genes characteristic of activated T cells as well as Th1, Th2 and Th17 subsets that were expressed in Tconv cells isolated from indicated tissues in control PBS-treated or DT-treated Foxp3DTR mice 10 days after Treg cell transfer. Heatmaps of top 10% of most variable genes in Treg cells isolated from indicated tissues in e, control PBS-treated or f, DT-treated Foxp3DTR mice 10 days after Treg cell transfer. g, PCA of gene expression by different Treg and Tconv cell subsets. Different cell samples were grouped by treatment (top) or anatomical location (bottom).
Extended Data Fig. 2 Tissue Treg cells consistently produced high levels of IL-10 regardless of the presence or absence of inflammatory conditions.
a, qPCR analyses for the expressions of Il10 in Tconv and Treg cells in different tissues from control PBS- or DT-treated Foxp3DTR mice. Each symbol represents an individual mouse (n = 5). b, ELISA analyses of the production of IL-10 by Tconv and Treg cells in different tissues from control PBS- or DT-treated Foxp3DTR mice. Each symbol represents FACS-isolated cell sample pooled from two to three mice (n = 4). Dotted line represents the minimum detection limit of the cytokine. Data are presented as mean values +/- SD. In a, **P = 0.0021 (up); **P = 0.0084 (middle); n.s. = 0.4266 (bottom). In b, **P = 0.0081 (up), *P = 0.0385 (middle); n.s. = 0.3009 (bottom). Statistical significance was determined by two-tailed unpaired t test.
Extended Data Fig. 3 Loss of IL-27 produced by Treg cells did not lead to any obvious immune phenotype except for increased Th17 responses in the intestine.
Frequencies and numbers of Foxp3+ Treg cells and frequencies of Ki67+, CD44hiCD62Llo, IL-17+, and IFNγ+ Tconv cells gated on the live CD4+Foxp3− population in a, spleen and b, LI LP of Foxp3CreIl27fl/fl mice and WT littermates (~8-12 weeks). c, FACS analysis and d, frequencies of RORgt+ in Tconv cells gated on the live CD4+Foxp3− population in spleen and SI LP of Foxp3CreIl27fl/fl mice and WT littermates (~8-12 weeks). e, Frequencies of IL-17+ in RORgt+ Tconv cells gated on the live CD4+Foxp3− population in spleen and SI LP of Foxp3CreIl27fl/fl mice and WT littermates (~8-12 weeks). f, Frequencies of IL-17+ and IFNγ+ Ly5.1+ Teff cells (isolated from Foxp3KO mice) gated on the live CD4+Foxp3− population in spleens of RAG-deficient mice three weeks after co-transferred with Treg cells isolated from either Foxp3CreIl27fl/fl mice or WT littermates. Each symbol represents an individual mouse. Data are presented as mean values +/- SD. In a, from right to left: n.s. = 0.2762 (n = 10 for Foxp3CreIl27+/+; 8 for Foxp3CreIl27fll/fl); n.s. = 0.2290 (n = 9 for Foxp3CreIl27+/+; 7 for Foxp3CreIl27fll/fl); n.s. = 0.9168 (n = 8 for Foxp3CreIl27+/+; 7 for Foxp3CreIl27fll/fl); n.s. = 0.9197 (n = 8 for Foxp3CreIl27+/+; 7 for Foxp3CreIl27fll/fl); n.s. = 0.2547 (n = 9 for Foxp3CreIl27+/+; 7 for Foxp3CreIl27fll/fl); n.s. = 0.3885 (n = 10 for Foxp3CreIl27+/+; 8 for Foxp3CreIl27fll/fl). In b, from right to left: n.s. = 0.7752; n.s. = 0.5144; n.s. = 0.5537; n.s. = 0.8933; *P = 0.0155; n.s. = 0.0577 (n = 8). In d, Spl: n.s. = 0.9907; SI: **P = 0.0025 (n = 7). In e, Spl: n.s. = 0.7866; SI: n.s. = 0.4099 (n = 7). In f, IL-17: n.s. = 0.7980; IFNγ: n.s. = 0.7652 (n = 12). Statistical significance was determined by two-tailed unpaired t test.
Extended Data Fig. 4 Treg cell-derived IL-27 likely limits Th17 responses through directly acting on T cells.
FACS analysis and frequencies of a, IL-17+ and b, IFNγ+ cells in Tconv cells gated on the live CD4+Foxp3− population cultured in the presence or absence of IL-27 (100 ng/ml) under Th17 and Th1 polarizing conditions, respectively. Each symbol represents an individual experiment (n = 3). qPCR analyses for the expressions of c, Il1b, d, Il6, e, Tgfb, f, ll23p19, g, Il12p40, and h, Il12p35 in DCs isolated from SI LP of either Foxp3CreIl27fl/fl mice or WT littermates. Each symbol represents FACS-isolated cell sample pooled from two to three mice (n = 2). Data are presented as mean values +/- SD. In a, ***P = 0.0009. In b, n.s. = 0.8920. Statistical significance was determined by two-tailed unpaired t test.
Extended Data Fig. 5 IL-27 produced by other non-Treg intestinal resident cell types is not required for IL-27-mediated regulation of Th17 responses.
FACS analysis of IL-17+ Tconv cells gated on the live CD4+Foxp3− population in SI LP of a, CD4CreIl27rafl/fl mice, b, LysMCreIl27fl/fl, c, CD11cCreIl27fl/fl, d, Vil1CreIl27fl/fl, and their corresponding WT littermates ( ~ 8-12 weeks). e, n-fold changes (on the basis of corresponding WT controls) of IL-17+ Tconv cell frequencies in indicated mouse lines. FACS analysis of IL-17+ Tconv cells gated on the live CD4+Foxp3− population in SI LP of f, CD4CreIl27rafl/fl mice g, LysMCreIl27fl/fl, h, CD11cCreIl27fl/fl, i, Vil1CreIl27fl/fl, and their corresponding WT littermates 4 days after initial aCD3 mAb injection. j, n-fold changes (on the basis of corresponding WT controls) of IL-17+ Tconv cell frequencies in indicated mouse lines. Each symbol represents an individual mouse. Data are presented as mean values +/- SD. In e, from left to right: **P = 0.0065 (n = 7); **P = 0.006 (n = 8); n.s. = 0.7795 (n = 8); n.s. = 0.7158 (n = 11); n.s. = 0.5244 (n = 12). In j, from left to right: ***P = 0.001 (n = 6); ***P = 0.001 (n = 5); n.s. = 0.1755 (n = 9); n.s. = 0.6306 (n = 7); n.s. = 0.4163 (n = 7). Statistical significance was determined by two-tailed unpaired t test.
Extended Data Fig. 6 Elevated IL-27 production by intestinal Treg cells could be observed in other autoimmune- and infection-driven inflammatory settings.
ELISA analyses of the production of IL-27 or IL-35 by Tconv and Treg cells in a, spleen and b, SI LP from PBS or aCD3 mAb treated Foxp3CreIl27fl/fl mice and WT littermates. Each symbol represents FACS-isolated cell sample pooled from two to three mice (n = 4 for Foxp3CreIl27+/+; 2 for Foxp3CreIl27fll/fl). ELISA analyses of the production of IL-27 or IL-35 by Tconv and Treg cells in c, spleen and d, LI LP from Foxp3CreIl27fl/fl mice and WT littermates at day 10 post C. rodentium infection. Each symbol represents FACS-isolated cell sample pooled from two to three mice (n = 4). Dotted line represents the minimum detection limit of the indicated cytokine. Data are presented as mean values +/- SD. In a, n.s. = 0.4560 (top); ***P = 0.0003 (bottom left); **P = 0.0090 (bottom right). In b, **P = 0.0021 (top); n.s. = 0.2264 (bottom left); **P = 0.0074 (bottom right). In c, ***P = 0.0005 (top), ***P = 0.0010 (bottom left), n.s. = 0.2535 (bottom right). In d, ***P = 0.0005 (top), ***P = 0.0003 (bottom left), ****P < 0.0001 (bottom right). Statistical significance was determined by two-tailed unpaired t test.
Extended Data Fig. 7 Expression of known intestinal Treg cell markers in different Treg cell clusters.
Violin plots of a, Il10 and Gzmb, b, Sell and Bach2, c, Il1rl1, and d, Rorc and Gata3 in different intestinal Treg cell clusters from C. rodentium-infected mice.
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Lin, CH., Wu, CJ., Cho, S. et al. Selective IL-27 production by intestinal regulatory T cells permits gut-specific regulation of TH17 cell immunity. Nat Immunol 24, 2108–2120 (2023). https://doi.org/10.1038/s41590-023-01667-y
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DOI: https://doi.org/10.1038/s41590-023-01667-y
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