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Th17 cells transdifferentiate into regulatory T cells during resolution of inflammation

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Abstract

Inflammation is a beneficial host response to infection but can contribute to inflammatory disease if unregulated. The Th17 lineage of T helper (Th) cells can cause severe human inflammatory diseases. These cells exhibit both instability (they can cease to express their signature cytokine, IL-17A)1 and plasticity (they can start expressing cytokines typical of other lineages)1,2 upon in vitro re-stimulation. However, technical limitations have prevented the transcriptional profiling of pre- and post-conversion Th17 cells ex vivo during immune responses. Thus, it is unknown whether Th17 cell plasticity merely reflects change in expression of a few cytokines, or if Th17 cells physiologically undergo global genetic reprogramming driving their conversion from one T helper cell type to another, a process known as transdifferentiation3,4. Furthermore, although Th17 cell instability/plasticity has been associated with pathogenicity1,2,5, it is unknown whether this could present a therapeutic opportunity, whereby formerly pathogenic Th17 cells could adopt an anti-inflammatory fate. Here we used two new fate-mapping mouse models to track Th17 cells during immune responses to show that CD4+ T cells that formerly expressed IL-17A go on to acquire an anti-inflammatory phenotype. The transdifferentiation of Th17 into regulatory T cells was illustrated by a change in their signature transcriptional profile and the acquisition of potent regulatory capacity. Comparisons of the transcriptional profiles of pre- and post-conversion Th17 cells also revealed a role for canonical TGF-β signalling and consequently for the aryl hydrocarbon receptor (AhR) in conversion. Thus, Th17 cells transdifferentiate into regulatory cells, and contribute to the resolution of inflammation. Our data suggest that Th17 cell instability and plasticity is a therapeutic opportunity for inflammatory diseases.

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Figure 1: Th17 cells lose IL‐17A and acquire IL‐10 in vivo.
Figure 2: Tr1exTh17 cells have a similar gene expression profile and function compare with Tr1 cells.
Figure 3: Tr1exTh17 cell development in EAE and helminth infection.
Figure 4: TGF‐β1 via Smad3, and AhR support the conversion of Th17 to Tr1.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The RNA sequencing data have been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE68242.

Change history

  • 08 July 2015

    Minor changes were made to the Acknowledgements, and the GEO accession code was added.

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Acknowledgements

The authors would like to thank C. Lieber, P. Musco, E. Hughes-Picard and J. Alderman for expert administrative assistance. J. Stein, L. Evangelisti and C. Hughes for generating the IL-17A IRES-eGFP-CRE-ERT2 constructs, embryonic stem cells and chimaeric mice, respectively. We thank E. Baiocchi for remote key support. LXG is supported by grants K01ES025434 from NIH/BD2K and P20 COBRE GM103457 from NIH/NIGMS. E.E. was supported by the DFG (EXC 257 NeuroCure and SFB633) and by the Crohn’s & Colitis Foundation of America (#311143). N.G. is supported by the Dr. Keith Landesman Memorial Fellowship of the Cancer Research Institute. S.H. is supported by the DFG (HU1714/3) and by Ernst Jung-Stiftung Hamburg and has an endowed Hofschneider-Professorship from the Stiftung Experimentelle Biomedizin. This work was supported, by the Howard Hughes Medical Institute, by Cariplo foundation (2013-0937 to J.G. and R.A.F.), by the AbbVie-Yale Collaboration (R.A.F.) and by the Francis Crick Institute (B.S.).

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Authors

Contributions

N.G. designed and performed the experiments, analysed the data and wrote the manuscript. M.C.A.V., A.I., H.X. and L.B. performed the experiments and analysed the data. N.W.P. and M.R.d.Z. optimized the isolation of intestinal cells. P.L-L. provided the expertise and supervised the experiments with N. brasiliensis. T.C. performed bioinformatics analysis and L.X.G. supervised the bioinformatics analysis. C.W. provided the IL-10Thy1.1 mice. X.Z., X.P., R.F. performed and supervised the extraction, amplification and library preparation for RNA sequencing. M.J.C., Y.D. and B.B. assisted with expression profiling and data analysis. M.R.d.Z., P.L.-L., J.G., R.S.P. and E.E. discussed and interpreted the results. N.W.P. discussed and interpreted the results and helped in writing the paper. B.S. provided the IL-17A Cre mice and the protocol for the intracellular staining. R.A.F. and S.H. wrote the manuscript and supervised the project.

Corresponding authors

Correspondence to Samuel Huber or Richard A. Flavell.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Description of Fate+ mice and characterization of exTh17 IL‐10eGFP+ cells under steady state condition.

a, Constructs contained in the Fate+ mice. b, During anti‐CD3 mAb induced transient inflammation in the S.I., a sufficient number of exTh17 IL‐10eGFP+was generated to test whether Fate+ mice faithfully report IL17A and IL‐10 expression. In particular, Tr1 (CD4+ IL‐17AKatushka– YFP IL‐10eGFP+ Foxp3RFP–), Th17 (CD4+ IL‐17AKatushka+ YFP+ IL‐10eGFP– Foxp3RFP–), exTh17 IL‐10eGFP+ (CD4+ IL‐17AKatushka– YFP+ IL‐10eGFP+ Foxp3RFP–) and exTh17 (CD4+ IL‐17AKatushka– YFP+ IL‐10eGFP– Foxp3RFP–) were FACS sorted from the small intestine of anti‐CD3 monoclonal antibody treated Fate+ mice and mRNA expression relative to Th17 cells for Il17a and relative to Tr1 for Il10 is reported. ExTh17 IL‐10eGFP+express Il10dim/high and Il17alow. Data are cumulative of three independent experiments. In each experiment we pooled cells from at least 7 mice. Mean and s.e.m., ***P ≤ 0.0005 by ANOVA (Tukey’s multiple comparison test). ce, Under steady state conditions, intestinal lymphocytes were isolated and re‐stimulated in vitro for 3 h with PMA/ionomycin for the intracellular staining of IFN‐γ and IL‐4, while they were freshly analysed for the expression of CCR6 and RORγt. c, Frequencies of IFN‐γ and IL‐4 among the exTh17 cells is shown. Pie chart reports the frequencies of the indicated cytokine among the exTh17 IL‐10eGFP+. One biological replicates out of five is shown. d, e, Frequencies and MFI of CCR6 (d) and MFI of RORγt are reported for Tr1 (CD4+ IL‐17AKatushka– YFP IL‐10eGFP+), Th17 (CD4+ IL‐17AKatushka+ YFP+/– IL‐10eGFP–), exTh17 IL‐10+ (CD4+ IL‐17AKatushka– YFP+ IL‐10eGFP+) (e). Each dot represents one biological replicate. Mean and s.e.m., **P ≤ 0.005, ***P ≤ 0.0005 by ANOVA (Dunnett’s multiple comparison test, comparison all columns vs control (Th17 cells)).

Extended Data Figure 2 Characterization of Tr1exTh17 cells.

Tr1 (CD4+ IL‐17AKatushka– YFP IL‐10eGFP+), Th17 (CD4+ IL‐17AKatushka+ YFP+/– IL‐10eGFP–), Tr1exTh17 (CD4+ IL‐17AKatushka– YFP+ IL‐10eGFP+) were isolated from the small intestine of anti‐CD3 monoclonal antibody treated‐Fate+ mice and analysed by FACS. ad, Frequencies of LAG‐3 (a), CCR6 (b), CD49b (c) and MFI of RORγt (d) are reported. Each dot represents one biological replicate. Mean and s.e.m., **P ≤ 0.005, ***P ≤ 0.0005 by ANOVA (Dunnett’s multiple comparison test, Comparison all columns vs control (Th17 cells)). e, Th1 (CD4+ IFN‐γKatushka+ Foxp3RFP–), Th2 (CD4+ IL‐4GFP+ Foxp3RFP–), Tr1 (CD4+ IL‐17AKatushka– IL‐10eGFP+ Foxp3RFP–), Th17 (CD4+ IL‐17AKatushka+ Foxp3RFP–), Tr1exTh17 (CD4+ IL‐17AKatushka– YFP+ IL‐10eGFP+ Foxp3RFP–) and exTh17 (CD4+ IL‐17AKatushka– YFP+ IL‐10eGFP– Foxp3RFP–) were FACS sorted from the small intestine of anti‐CD3 monoclonal antibody treated mice. mRNA expression relative to HPRT of Ifng, Il2, Tbx21, Il4 and Gata3 of the indicated populations is reported. Mean and s.e.m., *P ≤ 0.05, by Mann–Whitney U‐-test, two tailed.

Extended Data Figure 3 Th17 fate in DNIL‐10R Fate mice.

Fate (WT) and Fate dominant negative IL‐10R (DNIL‐10R) were injected with anti‐CD3 monoclonal antibody and the fate of small intestinal Th17 cells analysed. a, Frequencies of Th17 and exTh17 gated on CD4+ T cells are reported. b, Representative flow cytometric analysis of the IL‐10 and IFN‐γ expression in exTh17 cells. c, Ratio between Tr1exTh17 (CD4+ IL‐17A YFP+ IL‐10eGFP+ IFN‐γ) and Th1exTh17 (CD4+ IL‐17A YFP+ IL‐10eGFP– IFN‐γ+) in WT and DNIL‐10R mice. Mean ± s.e.m. Each dot represents one biological replicate. *P ≤ 0.05; **P ≤ 0.005 by Mann–Whitney U‐-test, two tailed.

Extended Data Figure 4 Constructs and validation of iFate mice.

a, Targeting strategy and constructs of IL‐17AeGFP‐CRE‐ERT2 mice (iFate). Black circles represent Flp recombinase target (FRT) sites. b, Schematic of Th17 cell development in iFate showing that Th17 cell plasticity can be tested only after tamoxifen treatment. c, iFate mice were injected with tamoxifen and anti‐CD3 monoclonal antibody and non Th17 cells (IL‐17AGFP–YFP), Th17 YFP+ cells (IL‐17AeGFP+ YFP), Th17 cells (IL‐17AeGFP+ YFP+) and exTh17 cells (IL‐17AeGFP– YFP+) were FACS sorted. Il17a mRNA expression in the indicated populations is reported. The mRNA expression is normalized to HPRT. One representative experiment out of two is shown. d, Representative flow cytometric analysis of intestinal Th17 cells under steady state condition or after anti‐CD3 monoclonal antibody (aCD3) in the absence of tamoxifen treatment. The efficiency of CRE‐mediated recombination after tamoxifen is reported as frequencies of YFP+ cells (YFP) and is the result of the following calculation: YFP+ cells (gate 2 + 3) / (IL‐17AeGFP+ YFP cells (gate 1) + YFP+ cells (gate 2 + 3).

Extended Data Figure 5 Relative expression of Il17a, Il10 and Foxp3 and FPKM values of signature genes of bona fide Th17 and Tr1 cells.

a, Tr1 (CD4+ IL‐17AKatushka– YFP IL‐10eGFP+ Foxp3RFP–) Th17 (CD4+ IL‐17AKatushka+ YFP+/− IL‐10eGFP+/– Foxp3RFP–), Tr1exTh17 (CD4+ IL‐17AKatushka– YFP+ IL‐10eGFP+ Foxp3RFP–), exTh17 (CD4+ IL‐17AKatushka– YFP+ IL‐10eGFP– Foxp3RFP–) Foxp3+ TReg (CD4+ IL‐17AKatushka– YFP IL‐10eGFP– Foxp3RFP+) and Foxp3+ TReg IL‐10+ (CD4+ IL‐17AKatushka– YFP IL‐10eGFP+ Foxp3RFP+) cells were isolated from the small intestine of Fate+ mice after anti‐CD3 monoclonal anttibody injections. The transcriptome of these populations was sequenced and the relative FPKM expressions of Il10, Il17a and Foxp3 compared to Tr1, Th17 and Foxp3+ TReg cells are reported. b, FPKM values of the indicated populations of the reported genes are shown.

Extended Data Figure 6 Tr1exTh17 cell development in EAE and during helminth infection using iFate mice.

a, Schematic of the experiment, showing iFate+ mice immunized with MOG, treated for 3 times with tamoxifen and then injected with anti‐CD3 monoclonal antibody 70 and 72 days after MOG immunization. The intestinal lymphocytes were analysed 4 h after the second injection of anti‐CD3 monclonal antibody. b, Representative flow cytometric analysis of Th17 and exTh17 (gated on YFP+ cells) and Tr1exTh17 cells (gated on exTh17). The YFP+ percentages (YFP) shown on the dot plots report the efficiency of tamoxifen‐induced CRE‐recombination. Three representative biological replicates out of six are shown. c, Schematic of the experiment, showing iFate mice infected with N. brasiliensis and injected i.p. with tamoxifen at the indicated time points. d, Representative flow cytometric analysis of CD4+ T cells isolated from the lung of iFate mice before (steady state) and after the second infection ± tamoxifen (control N. brasiliensis (no tamoxifen) and N. brasiliensis (+ tamoxifen) respectively)). Cumulative dot plots of 3 biological replicates are shown. One representative experiments out of 3 is shown. The YFP+ percentages (YFP) shown on the dot plots report the efficiency of tamoxifen‐induced CRE‐recombination. The frequencies within the cumulative dot/density plot report the percentage of Th17 and exTh17 among the YFP+ cells, and the frequency of IL‐10+ cells among the exTh17 cells. e, Frequencies of Tr1exTh17 cells (gated on exTh17). Each dot represents a biological replicates. Results are cumulative from three independent experiments.

Extended Data Figure 7 Conversion of Th17 cells into Tr1 over the course of S. aureus infection using Fate and iFate mice.

a, Fate+ mice were left untreated (control) or injected i.v. with S. aureus (S. aureus). Representative flow cytometric analysis of intestinal Th17 and exTh17 (gated on CD4+ Foxp3RFP–) and Tr1exTh17 cells (CD4+ IL‐17AKatushka– YFP+ IL‐10eGFP+; gated on exTh17) are shown. One representative experiment out of three is shown. b, Frequencies and numbers of the indicated population in the small intestine of untreated (control) and infected mice (S. aureus) are reported. Results are cumulative from three independent experiments. Mean and s.e.m., **P ≤ 0.005, ***P ≤ 0.0005 by Mann–Whitney U‐-test, two tailed. c, IFN‐γ and IL‐10eGFP expression of exTh17 cells. Pie chart reports the frequencies of the indicated cytokine among the Tr1exTh17 cells. ac, One representative biological replicate out of three is shown. One representative experiment out of two is shown. d, Representative flow cytometric analysis of intestinal lymphocytes isolated from iFate mice 4 days after S. aureus infection. One representative biological replicate out of 5 is shown. The YFP+ percentages (YFP) shown on the dot plots report the efficiency of tamoxifen‐induced CRE‐recombination. e, Frequencies of Tr1exTh17 cells (CD4+ IL‐17AeGFP– YFP+ IL‐10CD90.1+; gated on exTh17) are shown. Results are cumulative from two independent experiments.

Extended Data Figure 8 Gene expression of Tr1, Tr1exTh17 and Th17 cells.

a, Heat map of genes selectively expressed in both Tr1exTh17 and Tr1 compared to Th17 cells. The bioinformatics analysis is based on the genes listed in Supplementary Table 1. Red squares highlight genes linked to TGF‐β1 signalling. b, Relative mRNA expression of the indicated genes in Tr1, Tr1exTh17 and Th17 cells FACS sorted form the intestine of Fate+ mice treated with anti‐CD3 monoclonal antibody is shown. Values shown are relative to Th17 cell gene expression. Mean and s.e.m. of biological independent experiments (IRF8 n = 2; SMAD3 n = 4; FOXO1 n = 2; STAT5a n = 3; SMAD4 n = 4) except for IL‐23 and Runx1 (n = 2 technical replicates) are shown. In each experiment we pooled intestinal lymphocytes isolated from 7 treated mice before FACS sorting.

Extended Data Figure 9 Characterization of in vitro generated Tr1exTh17 cells.

a, IL‐1β counteracted Th17 plasticity. IL‐17A MFI in Th17 (CD4+ Foxp3RFP– IL‐17AKatushka+ YFP+/– IL‐10eGFP+/–) differentiated in the presence of TGF‐β1, IL‐6, IL‐23 or IL‐1β, IL‐6, IL‐23. One experiment out of five is shown. Two technical replicates are reported. b, Dose‐response effect of TGF‐β1 on the induction of Tr1exTh17 cells cultured in the presence of IL‐6, IL‐23, IL‐1β. In the last conditions we added anti‐TGF‐β1 monoclonal antibody. TGF‐β1 was diluted 1:2 starting from the concentration of 4 ng ml−1. One experiment out of five is shown. Two technical replicates are reported. c, In line with the literature29, Smad3 chemical inhibition also favours Th17 cell development. Frequency of Th17 cells cultured in the presence or in the absence Smad3 inhibitor at the indicated different concentrations of TGF‐β1 (4–0.25 ng ml−1). One experiment out of five is shown. Three technical replicates are reported. d, mRNA expression of Ahr in CD4 T cells cultured in the presence of either TGF‐β1, IL‐6, IL‐23 or IL‐1β, IL‐6, IL‐23. The expression is normalized to HPRT. One experiment out of two is shown. Two technical replicates are reported. e, Tr1exTh17 cells were polarized in vitro in the presence of TGF‐β1+IL‐6+IL‐23+FICZ and transferred into Rag1−/− mice ± (p)TH17 cells. Endoscopic colitis score and percentage of initial body weight in the indicated groups are shown. Each dot represents one mouse. Results are cumulative from three independent experiments. Mean and s.e.m., *P ≤ 0.05 by Mann–Whitney U‐-test, two‐tailed. f, Th17 cells were isolated from the intestine of anti‐CD3 monoclonal antibody and then restimulated in vitro in the presence of either anti‐TGF‐β+IL‐6 or IL‐6+TGF‐β1+FICZ for 5 days. Frequencies of Tr1exTh17 cells among total cells (left) and among exTh17 cells (right) are reported. Results are cumulative from three independent experiments. Each dot represents a pool of Th17 cells isolated from five mice treated with anti‐CD3. Mean and s.e.m., *P ≤ 0.05, by Mann–Whitney U‐-test, two tailed.

Supplementary information

Supplementary Table 1

This table contains the list of Th17 related genes. This list was used to generate the cluster analysis shown in Figure 3a. (XLSX 36 kb)

Supplementary Table 2

This table contains the list of cytokine genes used to generate the cluster analysis shown in Figure 3b. (XLSX 38 kb)

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Gagliani, N., Vesely, M., Iseppon, A. et al. Th17 cells transdifferentiate into regulatory T cells during resolution of inflammation. Nature 523, 221–225 (2015). https://doi.org/10.1038/nature14452

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