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Interleukin-33 activates regulatory T cells to suppress innate γδ T cell responses in the lung

Nature Immunology volume 21, pages 1371–1383 (2020)Cite this article

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Abstract

Foxp3+ regulatory T (Treg) cells expressing the interleukin (IL)-33 receptor ST2 mediate tissue repair in response to IL-33. Whether Treg cells also respond to the alarmin IL-33 to regulate specific aspects of the immune response is not known. Here we describe an unexpected function of ST2+ Treg cells in suppressing the innate immune response in the lung to environmental allergens without altering the adaptive immune response. Following allergen exposure, ST2+ Treg cells were activated by IL-33 to suppress IL-17-producing γδ T cells. ST2 signaling in Treg cells induced Ebi3, a component of the heterodimeric cytokine IL-35 that was required for Treg cell-mediated suppression of γδ T cells. This response resulted in fewer eosinophil-attracting chemokines and reduced eosinophil recruitment into the lung, which was beneficial to the host in reducing allergen-induced inflammation. Thus, we define a fundamental role for ST2+ Treg cells in the lung as a negative regulator of the early innate γδ T cell response to mucosal injury.

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Fig. 1: ST2+ Treg cells expand rapidly in the lung and airways of mice and humans after aeroallergen exposure.
Fig. 2: ST2+ Treg cells respond to IL-33 but not directly to allergen peptide.
Fig. 3: Treg cell-specific deletion of ST2 increases γδ T cells in the lung in response to HDM.
Fig. 4: IL-33–ST2 signaling in Treg cells restrains γδ T cell activation and function.
Fig. 5: γδ T cells mediate exacerbated allergic pulmonary inflammation in mice lacking ST2 in Treg cells.
Fig. 6: IL-17 blockade decreases eosinophil-attracting chemokine production and eosinophil recruitment into the lung.
Fig. 7: ST2+ Treg cell-derived Ebi3 is required to suppress γδ T cell IL-17 responses in the lung.
Fig. 8: IL-35 suppresses γδ T cell IL-17 responses in the lung induced by HDM.

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Data availability

The data generated or analyzed to support the findings of this study are available from the corresponding author upon request without restrictions. Source data are provided with this paper.

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Acknowledgements

We thank R. Lee for providing Il1rl1fl/fl mice and L. Wu for providing Il33–/– mice. This work was supported by grants from the National Institutes of Health to A.D.L. (R01AI040618, U19AI095261 and T32HL116275), J.W.G. (K08AI125816), R.A.R. (K08 HL140173) and D.A.A.V. (R01CA203689) and from the National Council of Scientific Development and Technology to L.D.F. (CNPq; 237062/2012-7).

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Authors and Affiliations

  1. Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    Lucas D. Faustino, Jason W. Griffith, Rod A. Rahimi, Keshav Nepal, Daniel L. Hamilos, Josalyn L. Cho, Benjamin D. Medoff, James J. Moon & Andrew D. Luster

  2. Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    Jason W. Griffith, Rod A. Rahimi, Josalyn L. Cho, Benjamin D. Medoff & James J. Moon

  3. Department of Immunology, University of Pittsburgh School of Medicine and Tumor Microenvironment Center, UPMC Hillman Cancer Center, Pittsburgh, PA, USA

    Dario A. A. Vignali

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Contributions

L.D.F. conceived the study, performed the experiments in the HDM and A. alternata model, analyzed the human samples and wrote the manuscript; J.W.G. performed the experiments in the influenza model and assisted in the interpretation of the experiments in the HDM model; R.A.R. performed the parabiosis experiments; K.N. assisted in the execution of the experiments; D.L.H. recruited the human participants; J.L.C. and B.D.M. performed the SAC studies; J.J.M. provided reagents and expertise for experiments using the HDM-specific tetramer; D.A.A.V. provided the reagents and expertise for experiments studying IL-35; and A.D.L. conceived the study, reviewed and assisted in interpretation of the data and writing the manuscript.

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Correspondence to Andrew D. Luster.

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Extended data

Extended Data Fig. 1 Profiling ST2-expressing Treg and TH cells in the lungs of HDM-treated mice.

a–d, Foxp3YFPcre mice were treated with HDM i.n. on days 0 and 7-11 and analyzed on day 14. a, Schematic of experimental design. b, NanoString analysis of gene expression comparison between flow sort-purified CD4+Foxp3– T helper (TH) cells expressing ST2 (TH2) or not (ST2– TH) from lung parenchyma. Data are presented as volcano plot from three independent experiments of pooled mice (n = 7 mice per experiment). Unpaired two-tailed t test with Holm-Sidak correction for multiple comparisons was used to obtain the P values. c, Representative flow cytometry plots for IL-13 and IL-5 (top), and for IL-17A and IFN-γ (bottom) in TH2 and ST2– TH cells from lung parenchyma. d, Representative histograms showing the expression of the indicated transcription factors in ST2+ Treg cells (red) or ST2– Treg cells (blue) from lung parenchyma. Isotype control is shown in gray.

Source data

Extended Data Fig. 2 Subject characteristics.

Aeroallergen-allergic human subjects screened for eligibility with a full medical history, baseline spirometry, methacholine challenge, and allergen skin testing to confirm allergy to either cat dander or Dermatophagoides pteronyssinus (DP). The threshold level of allergen sensitivity was determined by skin prick test titration using serial threefold dilutions of allergen extract.

Extended Data Fig. 3 IL-33 activates ST2+ Treg cells in the lung after HDM exposure.

a–c, Wild-type (WT) and Il33–/– mice were treated with HDM i.n. on days 0 and 7-11 and analyzed on day 10. a, Representative flow cytometry for Foxp3 and ST2 in CD4+ T cells from lung parenchyma. b, Percentage of ST2+ Treg cells, ST2– Treg cells, and TH2 (Foxp3–ST2+) cells in the lung. c, Representative histograms showing the expression of the indicated surface markers in ST2+ Treg and TH2 cells from the lung parenchyma of WT (black) and Il33–/– (gray) mice. Data represent one experiment with n = 6 mice per group of two independent experiments in panels (a–c). Unpaired two-tailed t test was used for statistical analysis in panel (b). Error bars denote mean ± s.d. P values are indicated in the figure.

Extended Data Fig. 4 Treg cell-specific deletion of ST2 does not result in systemic alterations in the number or activation of T cells.

Thymus, spleen, cervical lymph nodes (cLN) and mesenteric lymph nodes (MLN) were harvested from naive Il1rl1fl/flFoxp3YFPcre mice, Il1rl1fl/+Foxp3YFPcre mice, and Il1rl1+/+Foxp3YFPcre (Foxp3YFPcre) littermate control mice for flow cytometric analysis. a, Representative flow cytometry for Foxp3 and CD4 in CD4+ T cells (top) and percentage of Foxp3+ Treg cells (bottom) from the indicated tissues. b, Representative flow cytometry for CD8 and CD4 in CD3+ T cells (top) and percentage of CD8+ T cells and CD4+ T cells (bottom) from the indicated tissues. c, Representative flow cytometry for CD44 and CD62L in CD4+ T cells (top) and percentage of CD44+CD4+ T cells (bottom) from the indicated tissues. Data represent one experiment (Foxp3YFPcre n = 5; Il1rl1fl/+Foxp3YFPcre n = 3; Il1rl1fl/flFoxp3YFPcre n = 4 mice per group) of two independent experiments in panels (a–c). Unpaired two-tailed t test with Holm-Sidak correction for multiple comparisons was used for statistical analysis in panels (a–c bottom). No statistical difference was found. Error bars denote mean ± s.d.

Extended Data Fig. 5 Immune response in the lung to HDM in mice with a Treg cell-specific deletion of ST2.

a–c, Il1rl1fl/flFoxp3YFPcre mice and Foxp3YFPcre littermate controls were left untreated (Naive) or were treated with HDM i.n. on days 0 and 7-11 and analyzed on day 14. a, b, Representative flow cytometry for IL-13 and IL-5 (a) and for IFN-γ and IL-17A (b) in CD4+Foxp3– (TH) cells from lung parenchyma. Data represent one experiment with n = 5 mice per group of two independent experiments in panels (a, b). c, Lung explants from Naive and HDM-treated mice were re-stimulated ex vivo with HDM and the indicated cytokines measured in culture supernatants 72 h later by ELISA. Data represent one experiment with n = 4 mice per group of two independent experiments. Unpaired two-tailed t test with Holm-Sidak correction for multiple comparisons was used for statistical analysis. Error bars denote mean ± s.d. P values are indicated in the figure.

Extended Data Fig. 6 Innate lymphocyte response in the lung to HDM.

a–g, Il1rl1fl/flFoxp3YFPcre mice and Foxp3YFPcre littermate controls were left untreated (Naive) or were treated with HDM i.n. on days 0 and 7-11 and analyzed on day 14. a, Gating strategy for flow cytometric analysis of innate lymphocytes in lung parenchyma. b–d, Percentage of ILC1 (T-bet+), ILC2 (ST2+), and ILC3 (ST2–T-bet–) of CD3–Lin–CD127+ cells (b), percentage of natural killer (NK) cells of CD3–TCRαβ– cells (c), and percentage of NKT cells of CD3+TCRγδ– cells (d) in the lung parenchyma. e–g, Percentage of lung ILCs (e), NK (f), and NKT cells (g) expressing IL-13 and IL-5, IFN-γ, or IL-17A. Data represent one experiment with n = 5 mice per group of three independent experiments in panels (b–g). Unpaired two-tailed t test with Holm-Sidak correction for multiple comparisons was used for statistical analysis in panels (b–g). No statistical difference was found. Error bars denote mean ± s.d.

Extended Data Fig. 7 Lung and airway inflammatory response to HDM and A. alternata in mice with a Treg cell-specific deletion of ST2.

a, b, Il1rl1fl/flFoxp3YFPcre mice and Foxp3YFPcre littermate controls were left untreated (Naive) or were treated with HDM i.n. on days 0 and 7-11 and analyzed on day 14. a, Gating strategy for flow cytometric analysis of eosinophils (CD11c–Siglec-F+), neutrophils (Siglec-F–CD11b+Ly6G+), and Ly6C+ inflammatory monocytes (Ly6C+ iMo, Siglec-F–CD11b+Ly6C+) in the lung parenchyma of HDM-treated mice. b, Quantification of cell differential counts in BAL. Mn - Mononuclear cells; Ne - Neutrophils; Eo - Eosinophils. Data represent one experiment (Foxp3YFPcre Naive n = 3; Foxp3YFPcre HDM n = 5; Il1rl1fl/flFoxp3YFPcre Naive n = 3; Il1rl1fl/flFoxp3YFPcre HDM n = 5 mice per group) of two independent experiments. Unpaired two-tailed t test with Holm-Sidak correction for multiple comparisons was used for statistical analysis. c, Il1rl1fl/flFoxp3YFPcre mice and Foxp3YFPcre littermate controls were treated with A. alternata i.n. on days 0, 1 and 17-19 and analyzed on day 20. Gating strategy for flow cytometric analysis of eosinophils and neutrophils in the lung parenchyma of A. alternata-treated mice. Error bars denote mean ± s.d. P values are indicated in the figure.

Extended Data Fig. 8 Decreased lung CCL11 and CCL24 and allergic pulmonary inflammation in γδ T cell-deficient mice.

a–c, TCRγδ-deficient (Tcrd–/–) and WT mice were treated with HDM i.n. on days 0 and 7-11 and analyzed on day 14. a, Lung Ccl11 and Ccl24 mRNA levels relative to β2M determined by RT-qPCR. Data represent one experiment with n = 4 mice per group of two independent experiments. b, Number of eosinophils (CD11c–Siglec-F+) and neutrophils (Siglec-F–CD11b+Ly6G+) in lung parenchyma. c, Quantification of cell differential counts in BAL. Mn - Mononuclear cells; Ne - Neutrophils; Eo - Eosinophils. Data represent one experiment (WT n = 5; Tcrd–/– n = 6 mice per group) of two independent experiments in panels (b, c). Unpaired two-tailed t test was used for statistical analysis in panels (a, b) and unpaired two-tailed t test with Holm-Sidak correction for multiple comparisons was used for statistical analysis in panel (c). Error bars denote mean ± s.d. P values are indicated in the figure.

Extended Data Fig. 9 ST2+ Treg cell-derived Ebi3 suppresses γδ T cell responses in the lung to influenza infection.

a–d, Foxp3YFPcre, Il1rl1fl/flFoxp3YFPcre and Ebi3fl/flFoxp3YFPcre mice were infected with PR8 influenza and lungs were harvested for flow cytometry analysis in the indicated time points. a, Representative flow cytometry for IL-17A and IFN-γ in γδ T cells from lung parenchyma on day 11 post-infection. b, Number of IL-17A+ γδ T cells in the lung over the course of infection. Data pooled from two independent experiments (day 0: Foxp3YFPcre n = 5 and Il1rl1fl/flFoxp3YFPcre n = 4; day 4: Foxp3YFPcre n = 18 and Il1rl1fl/flFoxp3YFPcre n = 16; day 7: Foxp3YFPcre n = 10 and Il1rl1fl/flFoxp3YFPcre n = 12; day 11: Foxp3YFPcre n = 9 and Il1rl1fl/flFoxp3YFPcre n = 9 mice from the two experiments in each time point). Unpaired two-tailed t test with Holm-Sidak correction for multiple comparisons was used for statistical analysis in panel (b). c, Representative flow cytometry for TCRαβ and TCRγδ in CD3+ T cells (top) and for IL-17A and IFN-γ in γδ T cells (bottom) from lung parenchyma on day 7 post-infection. d, Number of lung IL-17A+ γδ T cells. Data pooled from two independent experiments (Foxp3YFPcre n = 10; Il1rl1fl/flFoxp3YFPcre n = 8; Ebi3fl/flFoxp3YFPcre n = 10 mice from the two experiments) in panel (d). Ordinary one-way ANOVA with Tukey’s multiple comparisons test was used for statistical analysis in panel (d). Error bars denote mean ± s.d. P values are indicated in the figure.

Supplementary information

Source data

Source Data Fig. 7

Raw NanoString dataset.

Source Data Extended Data Fig. 1

Raw NanoString dataset.

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Faustino, L.D., Griffith, J.W., Rahimi, R.A. et al. Interleukin-33 activates regulatory T cells to suppress innate γδ T cell responses in the lung. Nat Immunol 21, 1371–1383 (2020). https://doi.org/10.1038/s41590-020-0785-3

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