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Regulatory T cells function in established systemic inflammation and reverse fatal autoimmunity

Nature Immunology volume 22, pages 1163–1174 (2021)Cite this article

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Abstract

The immunosuppressive function of regulatory T (Treg) cells is dependent on continuous expression of the transcription factor Foxp3. Foxp3 loss of function or induced ablation of Treg cells results in a fatal autoimmune disease featuring all known types of inflammatory responses with every manifestation stemming from Treg cell paucity, highlighting a vital function of Treg cells in preventing fatal autoimmune inflammation. However, a major question remains whether Treg cells can persist and effectively exert their function in a disease state, where a broad spectrum of inflammatory mediators can either inactivate Treg cells or render innate and adaptive pro-inflammatory effector cells insensitive to suppression. By reinstating Foxp3 protein expression and suppressor function in cells expressing a reversible Foxp3 null allele in severely diseased mice, we found that the resulting single pool of rescued Treg cells normalized immune activation, quelled severe tissue inflammation, reversed fatal autoimmune disease and provided long-term protection against them. Thus, Treg cells are functional in settings of established broad-spectrum systemic inflammation and are capable of affording sustained reset of immune homeostasis.

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Fig. 1: Restoration of Foxp3 expression in Treg cell ‘wannabes’ cures fulminant autoimmunity in male Foxp3LSL mice.
Fig. 2: Restoration of Foxp3 expression in Treg cell ‘wannabes’ in mosaic adult female Foxp3LSL/DTR–GFP mice suppresses immune activation caused by DT-mediated Treg cell ablation.
Fig. 3: Rescued Treg cells in inflamed mice are activated and potently suppressive in inflammatory settings.
Fig. 4: Rescued Treg cells in male Foxp3LSL mice provide long-term protection from autoimmune inflammatory disease.
Fig. 5: Single-cell transcriptomic analysis of control and long-lived rescued Treg cells.
Fig. 6: Identification and analysis of γREG+ Treg cells from the single-cell RNA-sequencing data.

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Data availability

All sequencing data generated in this study can be accessed at the Gene Expression Omnibus (GEO) under accession GSE179710. The custom mouse genome used for scRNA-seq analysis, which was generated by adding the tdTomato sequence to GRCm38 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/), is also available at the GEO. The following gene-set enrichment analysis gene sets were used for data analysis: GO_CELL_CYCLE_G1_S_PHASE_TRANSITION, GO_CELL_CYCLE_G2_M_PHASE_TRANSITION, GO_DNA_REPLICATION, HALLMARK_APOPTOSIS, HALLMARK_FATTY_ACID_METABOLISM, HALLMARK_G2M_CHECKPOINT, HALLMARK_GLYCOLYSIS, HALLMARK_IL2_STAT5_SIGNALING, HALLMARK_OXIDATIVE_PHOSPHORYLATION, HALLMARK_PI3K_AKT_MTOR_SIGNALING, HALLMARK_REACTIVE_OXYGEN_SPECIES_PATHWAY, HALLMARK_TGF_BETA_SIGNALING, HALLMARK_WNT_BETA_CATENIN_SIGNALING, KEGG_CITRATE_CYCLE_TCA_CYCLE, KEGG_PURINE_METABOLISM and REACTOME_EUKARYOTIC_TRANSLATION_INITIATION. Source data are provided with this paper.

Code availability

All custom scripts are available upon request to the corresponding authors.

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Acknowledgements

We thank E. Azizi for advice on scRNA-seq data analysis and members of the laboratory of A.Y.R. for technical support and discussion, Memorial Sloan Kettering Cancer Center (MSKCC) Single Cell Research Initiative and Integrated Genomics Operation Core facility for single-cell and bulk RNA-seq. This work was supported by the National Institutes of Health (grant R01 AI034206 to A.Y.R.), an Irvington-Cancer Research Institute Postdoctoral Fellowship (to W.H.), the Ludwig Center at Memorial Sloan Kettering, Parker Institute for Cancer Immunotherapy (PICI) (to A.Y.R.) and the National Cancer Institute Cancer Center (grant P30 CA08748). A.Y.R is an investigator with the Howard Hughes Medical Institute. Z.-M.W. is supported by a Bruce Charles Forbes Fellowship.

Author information

Author notes

  1. Yongqiang Feng

    Present address: Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, USA

  2. Joris van der Veeken

    Present address: Research Institute of Molecular Pathology, Vienna BioCenter, Vienna, Austria

  3. These authors contributed equally: Wei Hu, Zhong-Min Wang.

Authors and Affiliations

  1. Howard Hughes Medical Institute, Immunology Program, and Ludwig Center, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Wei Hu, Zhong-Min Wang, Yongqiang Feng, Michail Schizas, Beatrice E. Hoyos, Joris van der Veeken, Jacob G. Verter, Regina Bou-Puerto & Alexander Y. Rudensky

  2. Gerstner Sloan Kettering Graduate School of Biomedical Sciences, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Zhong-Min Wang

  3. Immunology and Microbial Pathogenesis Program, Weill Cornell Graduate School of Medical Sciences, New York, NY, USA

    Regina Bou-Puerto

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Contributions

W.H. and A.Y.R. conceived the study and designed experiments. W.H., Z.-M.W. and A.Y.R. interpreted the data and wrote the manuscript. W.H. and Z.-M.W. performed the experiments. Y.F. designed the targeting construct and generated the Foxp3LSL mouse. B.E.H. performed the antibody ELISA assays. J.G.V., J.v.d.V. and R.B.-P. provided assistance with some experiments. J.G.V. assisted with mouse generation and animal colony maintenance. Z.-M.W. and M.S. performed analysis of the bulk RNA-seq experiment. M.S. performed analysis of scRNA-seq and TCR-sequencing datasets. W.H. and A.Y.R. supervised the study. Correspondence and requests for materials should be addressed to the corresponding authors.

Corresponding authors

Correspondence to Wei Hu or Alexander Y. Rudensky.

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Competing interests

A.Y.R. is a co-founder and Science Advisory Board member of and holds stock options in Vedanta Bioscience and Sonoma Biotherapeutics. The other authors declare no competing interests.

Additional information

Peer review information Nature Immunology thanks Masahiro Ono and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Generation and characterization of Foxp3LSL mice.

a, Schematics of the targeting construct and the Foxp3LSL allele before and after Flp recombinase-mediated removal of the neo cassette. Homologous regions between the targeting construct and the WT allele are demarcated with dashed gray lines. Gene map is based on RefSeq record NM_001199347.1. pA, bovine growth hormone polyadenylation signal; neo, neomycin resistance gene; PGK, mouse phosphoglycerate kinase 1 promoter; 3xSV40pA, triple-tandem SV40 early polyadenylation signals (STOP cassette); DTA, diphtheria toxin A. b, Genotyping PCR showing the WT (275 bp) and knock-in-specific (350 bp) bands using primers labeled in a. Gel picture is reproduced for and representative of all animals analyzed in this study. c, d, Flow cytometric analysis of T cells and TCRβhi single positive thymocytes from 3-week-old male Foxp3LSL/y (c) and 8–10-week-old female Foxp3LSL/WT (d) mice. LN, lymph nodes. e, Expression of molecules associated with T cell activation in splenic Foxp3+ Treg and Thy1.1+ Treg ‘wannabes’, and naïve (CD44loCD62Lhi) and activated (CD44hiCD62Llo) conventional CD4 T cells shown in d. f, Percentages of TCRVβ5+, Vβ6+ and Vβ8+ cells among the indicated splenic CD4 T cell populations from 8–10-week-old female Foxp3LSL/DTR–GFP mice. Cells are gated on the CD44loCD62Lhi subset to avoid potential clonal expansion during activation. One-way ANOVA. g, 2×106 CD45.1+Foxp3– conventional CD4 T cells were co-transferred with 2×105 CD45.2+ Treg ‘wannabes’ from 2–3-week-old Foxp3LSL/y mice or Treg cells from 6–8-week-old Foxp3GFP mice into Tcrb–/–Tcrd–/– mice. Recipients were orally gavaged with tamoxifen and injected i.p. with diphtheria toxin (DT) at the indicated time points (top). DT was administered to deplete the few contaminating DTR-expressing Treg cells in the transferred FACS-purified conventional T cells or those induced to express Foxp3 after transfer. Cellularity and proliferation of CD45.1+ responder cells from lymph nodes were analyzed using flow cytometry (bottom). Data are representative of two independent experiments. All error bars denote mean ± s.e.m. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Source data

Extended Data Fig. 2 Restoration of Foxp3 expression reverses spontaneous lymphocyte expansion, myelo-proliferation, and cytokine production, and normalizes circulatory Ig levels in male Foxp3LSL mice.

a, 4-OHT mediated recombination efficiency in lymphoid and non-lymphoid organs. Experimental scheme (left) and recombination efficiency of Rosa26Tom in Treg cells from indicated organs (right). One-way ANOVA. LN, lymph nodes. b-g, Male Foxp3LSL mice were treated with 4-OHT on postnatal day 14 and analyzed throughout the following 4 weeks. b, Representative histograms showing expression of proliferation and activation markers by splenic conventional CD4 and CD8 T cells at the indicated time points. c, Representative contour plots of flow cytometric analyses of activated and cytokine-producing splenic conventional CD4 and CD8 T cell populations. d, e, frequencies (d) and numbers (e) of activated, proliferating and cytokine-producing conventional CD4 and CD8 T cells from lymph nodes of mice of indicated genotypes at designated time points after 4-OHT treatment. Two-way ANOVA with Tukey’s multiple comparison correction. f, Numbers of splenic myeloid cell populations at indicated time points after 4-OHT treatment. Two-way ANOVA with Tukey’s multiple comparison correction. g, Serum antibody levels as in Fig. 1g. One-way ANOVA with Tukey’s multiple comparison test. All error bars denote mean ± s.e.m. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Source data

Extended Data Fig. 3 Restoration of Foxp3 expression in peripheral Treg ‘wannabes’ rescues immune activation in male Foxp3LSL mice.

a, Experimental design. 2-week-old mice were treated with 4-hydroxytamoxifen (4-OHT) while being continuously treated with FTY720 to block thymic output. A Rosa26loxP−STOP−loxP−tdTomato (R26LSL−tdTomato) recombination reporter allele was introduced to the mice to enable labeling of all CD4 T cells undergoing Cre-mediated recombination induced by 4-OHT. b, Number of Treg cells in the thymus (left), and percentage of tdTomato+ cells among Foxp3–Thy1.1– CD4SP thymocytes (right), from FTY720-treated mice and untreated controls on day 14. Two-tailed unpaired t-tests with multiple hypothesis testing correction using the Holm-Sidak method. c, d, Numbers of activated, proliferating, and cytokine-producing conventional CD4 and CD8 T cells in the spleen (c) and lymph nodes (d). One-way ANOVA with Tukey’s multiple comparison test. All error bars denote mean ± s.e.m. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

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Extended Data Fig. 4 Restoration of Foxp3 expression in Treg ‘wannabes’ rescues tissue damage in the skin and liver of male Foxp3LSL mice.

Mice were treated with 4-OHT on postnatal day 14 and examined at the indicated time points post-treatment. a, b, H&E staining of skin sections showing epidermal hyperplasia and formation of serocellular crust (arrow heads). Dashed lines demarcate the boundary between epidermis and dermis. Images are representative of 4 Foxp3LSL/yCd4wt, 4 Foxp3LSL/yCd4creERT2 and 2 Foxp3DTR−GFP/yCd4creERT2 mice. c, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay followed by immunohistochemistry to visualize apoptotic cells (arrow heads) in H&E counter-stained liver sections from mice of indicated genotypes. d, Quantification of TUNEL+ cells. e, Measurement of serum albumin levels. Two-way ANOVA with Tukey’s multiple comparison. All error bars denote mean ± s.e.m. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

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Extended Data Fig. 5 Restoration of Foxp3 expression in Treg ‘wannabes’ in mosaic adult female Foxp3LSL/DTR−GFP mice suppresses immune activation caused by diphtheria toxin-mediated Treg cell ablation.

Experimental scheme shown in Fig. 2a. a, Representative histograms showing expression of activation and proliferation markers in splenic conventional T cell populations. b, Representative contour plots of splenic conventional CD4 and CD8 T cells showing cytokine production. c, Percentages (left) and numbers (right) of Treg and Treg ‘wannabes’ from indicated tissues 5 weeks post 4-OHT administration. Two-way ANOVA with Tukey’s multiple comparison test. All error bars denote mean ± s.e.m. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. pLN, peripheral (brachial, axillary, and inguinal) lymph nodes; mLN, mesenteric lymph nodes; LP, lamina propria.

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Extended Data Fig. 6 Analysis of gene expression changes in Treg ‘wannabes’ induced upon activation.

a, Experimental scheme. 8–10-week-old heterozygous female Foxp3LSL/DTR−GFPCd4wt mice were treated with diphtheria toxin (DT) on designated days to deplete Foxp3DTR−GFP-expressing Treg cells and induce activation of Foxp3LSL-expressing Treg ‘wannabes’ which were sorted and analyzed by RNA-seq. b, FC-FC plot of activation-induced gene expression changes in Treg cells and ‘wannabes’. Genes with mean normalized counts of >100 are shown. Differentially expressed genes (p < 0.05) are colored based on the direction of the change in either or both cell types. c, Venn diagram showing the numbers of genes with larger than a 2-fold change in activated Treg cells and ‘wannabes’. d, eCDF plots showing expression changes in activated Treg ‘wannabes’ for all genes (black) and Treg activation signature genes that are up- (red) or down- (blue) regulated. Two-sided Kolmogorov-Smirnov test. e, FC-FC plots showing gene expression changes in Treg cells vs. ‘wannabes’ isolated from sick and healthy mice. Signature genes that are up- or down-regulated in activated Treg and conventional CD4 T cells are highlighted in different colors.

Extended Data Fig. 7 Rescued Treg cells in male Foxp3LSL mice exert long-term control of adaptive and innate immune cells.

Mice were treated with a single dose of 4-OHT at 2 weeks of age and analyzed 4 months later. a, Body weight of rescued Foxp3LSLCd4creERT2 and control Foxp3DTR−GFPCd4creERT2 mice over a 4-month time course. Two-way ANOVA with Sidak’s multiple comparison test. b, Analysis of 4-OHT ‘functional’ pharmacokinetics in 2-week-old male mice. Mice were injected with 4-OHT 3–72 hours before transfer of congenically marked recombination-proficient CD4 T cells from Cd4creERT2R26Tom mice. 4-OHT-induced recombination was assayed 7 days later by tdTomato expression among donor CD4 T cells as a readout for 4-OHT activity. Data are pooled from two independent experiments with 2 to 4 mice per time point each. Ctrl, no 4-OHT injection. c, Suppression of in vitro proliferation of conventional CD4 T cells induced by α-CD3 antibody and antigen-presenting cells by control Treg cells from Foxp3DTR−GFP/y or rescued Treg cells from Foxp3LSL/y mice. Two-way ANOVA with Tukey’s multiple comparison test. d-f, Numbers of activated, proliferating, and cytokine-producing conventional CD4 T cells (d), CD8 T cells (e) and myeloid populations (f) in indicated tissues. pLN, peripheral (brachial, axillary, and inguinal) lymph nodes; mLN, mesenteric lymph nodes; LP, lamina propria. Data are pooled from two independent experiments. Two-tailed t-tests with multiple hypothesis testing correction using the Holm-Sidak method. All error bars denote mean ± s.e.m. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

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Extended Data Fig. 8 Rescued Treg cell population persisting for 7 months in male Foxp3LSL mice prevents relapse of rampant autoimmunity.

Mice were treated with 4-OHT on postnatal day 14 and analyzed 7 months later. a-c, Frequencies of Treg cells (a) and proliferating, activated, and cytokine-producing conventional CD4 (b) and CD8 (c) T cells. Two-tailed unpaired t-tests with multiple hypothesis testing correction using the Holm-Sidak method. pLN, peripheral (brachial, axillary, and inguinal) lymph nodes; mLN, mesenteric lymph nodes; LP, lamina propria. d, Serum antibody levels. Scales for IgM, IgG1, and IgE were kept the same as in Fig. 1g. Two-tailed unpaired t-test. e, Representative images of hematoxylin and eosin-stained sections of the indicated organs. Images are representative of 5 Foxp3DTR−GFP/y and 5 Foxp3LSL/y mice. f, Clonal diversity of the TCRα repertoire of the long-lived rescued Treg cells from Foxp3LSL/yCd4creERT2 mice at indicated time points after restoring Foxp3 expression upon 4-OHT treatment. The inverse Simpson Index was calculated based on the clone size distribution using clonotypes defined by full nucleotide sequence (left) or CDR3 amino acid sequence (right). g, Total number of unique clones (left) and Gini coefficient (right) of the TCRα repertoire of the long-lived rescued Treg cells. Clonotypes were defined by using the full nucleotide sequence. One-way ANOVA with Dunnett’s multiple hypothesis test. All error bars denote mean ± s.e.m. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

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Extended Data Fig. 9 Analysis of long-lived Treg cells in Foxp3LSL and control Foxp3DTR−GFP mice.

Mice were treated with 4-OHT on postnatal day 14 and analyzed 7 months later. a, UMAP visualization of the single-cell transcriptomes colored by imputed expression levels of representative genes. b, Frequencies of CD62Lhi and CD103+ cells among Treg cells in rescued Foxp3LSLCd4creERT2 and control Foxp3DTR−GFPCd4creERT2 mice. Two-tailed multiple t-tests with Holm-Sidak’s correction. pLN, peripheral (brachial, axillary, and inguinal) lymph nodes; mLN, mesenteric lymph nodes; LP, lamina propria. c, Foxp3WTCd4creERT2R26Tom mice were treated with 4-OHT on postnatal day 14 and analyzed 4 months later. Representative contour plots show expression of activation markers by tdTomato+ and tdTomato– Foxp3+ CD4 T cells. d, Histogram depicting the density of Foxp3LSL and tdTomato+ or tdTomato– Foxp3DTR−GFP Treg cells along the average expression values for the indicated gene sets. All error bars denote mean ± s.e.m. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

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Extended Data Fig. 10 Flow cytometric analysis of γREG+ Treg cells.

a, Gating strategy for γREG+ Treg cells in adult mice. b, c, Representative histogram (b) and quantification (c) of IL-4Rα expression in γREG+ and other Treg cell populations identified in a. Two-way ANOVA with Tukey’s multiple comparison correction. d, e, Representative histogram (d) and quantification (e) of CD25 expression in γREG+ and other Treg cell populations identified in a. Two-way ANOVA with Tukey’s multiple comparison correction. f, Percentages of γREG+ Treg cells in lymphoid organs and non-lymphoid tissues of CD45 i.v.-labeled 8–10-week-old Foxp3GFP mice. One-way ANOVA with Sidak’s multiple comparison test. g, Percentages (left) and numbers (right) of γREG+ Treg cells in different organs of 2- or 8-week-old Foxp3GFP−BirA−AVI−TEV mice. Two-sided unpaired t-tests with correction for multiple hypothesis testing using the Holm-Sidak method. All error bars denote mean ± s.e.m. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. pLN, peripheral (brachial, axillary, and inguinal) lymph nodes; mLN, mesenteric lymph nodes; cLP, colonic lamina propria.

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Supplementary information

Supplementary Information

Supplementary Fig. 1

Reporting Summary

Supplementary Table 1

Differentially expressed genes for clusters identified in the scRNA-seq analysis as shown in Fig. 6d.

Supplementary Table 2

Sample sizes for all summary data.

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Hu, W., Wang, ZM., Feng, Y. et al. Regulatory T cells function in established systemic inflammation and reverse fatal autoimmunity. Nat Immunol 22, 1163–1174 (2021). https://doi.org/10.1038/s41590-021-01001-4

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