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. 2015 Jul 9;523(7559):221-5.
doi: 10.1038/nature14452. Epub 2015 Apr 29.

Th17 Cells Transdifferentiate Into Regulatory T Cells During Resolution of Inflammation

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Free PMC article

Th17 Cells Transdifferentiate Into Regulatory T Cells During Resolution of Inflammation

Nicola Gagliani et al. Nature. .
Free PMC article

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) and plasticity (they can start expressing cytokines typical of other lineages) 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 transdifferentiation. Furthermore, although Th17 cell instability/plasticity has been associated with pathogenicity, 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.

Conflict of interest statement

Author Information The authors declare no competing financial interests. Readers are welcome to comment on the online versionof the paper.

Figures

Extended Data Figure 1
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). c–e, 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
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. a–d, 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-17AKatushk− IL-10eGFP+ Foxp3RFP−), TH17 (CD4+ IL-17AKatushka+ Foxp3RFP−), TR1exTH17 (CD4+ IL-17AKatushka− YFP+ IL-10eGFP+ Foxp3RFP−) and exTH17 (CD4+ IL-17AKatushk− 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
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
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
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
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
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 TrlexTH17 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 populationin 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. a–c, 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
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
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-β1onthe inductionofTR1exTH17 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 literature, 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 (4–0.25ng 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 byMann–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.
Figure 1
Figure 1. TH17 cells lose IL-17A and acquire IL-10 in vivo
a, Flow cytometric analysis of small intestinal CD4+ T cells. Steady state, vancomycin-treated, or treated with anti-CD3 monoclonal antibody (inflammation) depicted. b, Number and frequencies of exTH17 cells (gated on CD4+ T cells) and TR1exTH17 cells (gated on exTH17 cells) are cumulative of two and three independent experiments respectively. IL-10 mean fluorescence intensity (MFI) data (n = 3 biological replicates) of one representative experiment out of three are shown. Mean ± s.e.m.; *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 by ANOVA (Bonferroni’s multiple comparison test) or by t-test for percentage (MannWhitney U-test, two tailed) and MFI (paired t-test, two-tailed) of TR1exTH17 cells. c, Hypotheses: first model, expansion of pre-existing TR1exTH17; second model, conversion of TH17 cells expanded/induced over the course of immune response. d, Flow cytometric analysis of small intestinal CD4+ T cells isolated from iFate mice in steady state, and upon anti-CD3 monoclonal antibody and tamoxifen (Inflammation + tamoxifen) treatment. Frequencies of YFP+ cells, TH17 and exTH17 (gated on YFP+ cells), and of IL-10+ cells (gated on exTH17) are representative of two experiments (n = 6 biological replicates).
Figure 2
Figure 2. TR1exTH17 cells have a similar gene expression profile and function compare with TR1 cells
a, b, Correlative heatmaps based on the expression of TH17 related genes (n = 97) (a) and cytokine genes (n = 191) (b). The indicated cell populations were isolated from the small intestine of 10 anti-CD3 treated Fate+ mice from two independent experiments. c, Pathogenic (p)TH17 were differentiated in vitro and then injected alone or in combination with the depicted populations into Rag1−/− mice. TH17, exTH17, TR1exTH17 and TR1 cells (YFP) were isolated from the small intestine of Fate+ mice treated with anti-CD3 mAb. d, Endoscopic and histological pictures. Scale bars: 200 μm. Endoscopy pictures show stool inconsistency (s), increased mucosal granularity (g), lack of translucency (t) and bleeding (arrow). The histological pictures show oedema (**), inflammation (*) and crypt loss (C). e, f, Endoscopic colitis score (e) and percentage of initial body weight (f). Each dot represents one mouse. Mean and s.e.m. are indicated. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 by ANOVA (Tukey’s multiple comparison test).
Figure 3
Figure 3. TR1exTH17 cell development in EAE and helminth infection
a, Clinical EAE-score.Anti-CD3 was injected 35 days after MOG-immunization. b, Flow cytometric analysis of TH17 and exTH17 (gated on YFP+ cells) cells in dLNs. c, d, Flow cytometric analysis (c)and percentages of TR1exTH17 cells (gated on exTH17)in dLNs (d). e, IFN-γ/IL-10eGFP expression of exTH17 cells. a–e, Representative of three independent experiments. f, Flow cytometric analysis of MOG-tetramer staining of intestinal CD4 cells of EAE mice left untreated (control) or treated with anti-CD3. g, h, Representative flow cytometric analysis (g) and frequencies of TR1exTH17 (gated on MOG ± cells) (h). Each dot represents one mouse. IL-10 MFIs (average of three mice ± s.e.m.) are reported. **P ≤ 0.05 by Mann–Whitney U-test, two tailed. i, IFN-γ/IL-10eGFP expression of exTH17 isolated from small intestine of EAE mice. f–i, Representative of two independent experiments. j, Schematic of the experiment. k, l, Flow cytometric analysis and frequencies of exTH17 and TR1exTH17 from the lung (upper panel: gatedonCD4+ T cells; lower panel: gated on exTH17 cells). One experiment of three is shown. Mean ± s.e.m., *P ≤ 0.05, **P ≤ 0.005 by ANOVA (Tukey’s multiple comparison test).
Figure 4
Figure 4. TGF-β1 via Smad3, and AhR support the conversion of TH17 to TR1
TH17 cells were differentiated in vitro in the presence of IL-6, IL-23 with TGF-β1 or with IL-1β and anti-TGF-β monoclonal antibody. TGF-β1 was diluted 1:2 starting from 4 ng ml−1. a, Flow cytometric analysis of TH17, exTH17 and TR1exTH17 (gated on exTH17). b, Percentages and IL-10 MFI of TR1exTH17 cells. Technical replicates (n = 2) of one experiment out of seven. c, d, Endoscopic pictures and score of mice injected with TH17 or TR1exTH17 cells polarized with TGF-β1. Stool inconsistency (s), increased mucosal granularity (g) and a lack of translucency (t). Each dot denotes one biological replicate. Mean and s.e.m., ***P ≤ 0.0005 by Mann Whitney U-test, two tailed. e, Flow cytometric analysis of TH17, exTH17 and TR1exTH17 cells cultured in the presence of TGF-β1 (diluted as above), IL-6, IL-23 ± Smad3 inhibitor. f, Percentages and IL-10 MFI of TR1exTH17 cells. Technical replicates (n = 3) of one experiment out of five are shown. Mean and s.e.m., *P ≤ 0.05, **P ≤ 0.005 by paired t-test. g, Flow cytometric analysis of TR1exTH17 (gated on exTH17) cultured in the presence of TGF-β1 (diluted as above), IL-6, IL-23 ± AhR ligand (FICZ) or AhR antagonist. h, Percentages and IL-10 MFI of TR1exTH17 (gated on exTH17). Technical replicates (n = 3) of one experiment out of five are shown. Mean and s.e.m.; *P ≤ 0.005, **P ≤ 0.005, ***P ≤ 0.0005 by ANOVA (Tukey’s multiple comparison test). NS, non-significant. i, Flow cytometric analysis of TR1exTH17 cells cultured in the presence of TGF-β1, IL-6, IL-23 ± FICZ in the indicated medias. One experiment of two is shown.

Comment in

  • T CELLS: Role reversal.
    Bird L. Bird L. Nat Rev Immunol. 2015 Jun;15(6):332. doi: 10.1038/nri3864. Nat Rev Immunol. 2015. PMID: 25998962 No abstract available.

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