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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Hirota, K. et al. Fate mapping of IL-17-producing T cells in inflammatory responses. Nature Immunol. 12, 255–263 (2011)
Annunziato, F. et al. Phenotypic and functional features of human Th17 cells. J. Exp. Med. 204, 1849–1861 (2007)
Graf, T. & Enver, T. Forcing cells to change lineages. Nature 462, 587–594 (2009)
Komatsu, N. et al. Heterogeneity of natural Foxp3+ T cells: a committed regulatory T-cell lineage and an uncommitted minor population retaining plasticity. Proc. Natl Acad. Sci. USA 106, 1903–1908 (2009)
Nylander, A. & Hafler, D. A. Multiple sclerosis. J. Clin. Invest. 122, 1180–1188 (2012)
Huber, S., Gagliani, N. & Flavell, R. A. Life, death, and miracles: Th17 cells in the intestine. Eur. J. Immunol. 42, 2238–2245 (2012)
Roncarolo, M. G. & Battaglia, M. Regulatory T-cell immunotherapy for tolerance to self antigens and alloantigens in humans. Nature Rev. Immunol. 7, 585–598 (2007)
Huber, S. et al. Th17 cells express interleukin-10 receptor and are controlled by Foxp3− and Foxp3+ regulatory CD4+ T cells in an interleukin-10-dependent manner. Immunity 34, 554–565 (2011)
Gagliani, N. et al. Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells. Nature Med. 19, 739–746 (2013)
Heinemann, C. et al. IL-27 and IL-12 oppose pro-inflammatory IL-23 in CD4+ T cells by inducing Blimp1. Nature Commun. 5, 3770 (2014)
Okamura, T. et al. TGF-β3-expressing CD4+CD25−LAG3+ regulatory T cells control humoral immune responses. Nature Commun. 6, 6329 (2015)
Littman, D. R. & Rudensky, A. Y. Th17 and regulatory T cells in mediating and restraining inflammation. Cell 140, 845–858 (2010)
Apetoh, L. et al. The aryl hydrocarbon receptor interacts with c-Maf to promote the differentiation of type 1 regulatory T cells induced by IL-27. Nature Immunol. 11, 854–861 (2010)
Zhou, L. et al. TGF-β-induced Foxp3 inhibits Th17 cell differentiation by antagonizing RORγt function. Nature 453, 236–240 (2008)
Beriou, G. et al. IL-17-producing human peripheral regulatory T cells retain suppressive function. Blood 113, 4240–4249 (2009)
McGeachy, M. J. et al. TGF-β and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain Th-17 cell-mediated pathology. Nature Immunol. 8, 1390–1397 (2007)
Esplugues, E. et al. Control of Th17 cells occurs in the small intestine. Nature 475, 514–518 (2011)
Zielinski, C. E. et al. Pathogen-induced human Th17 cells produce IFN-γ or IL-10 and are regulated by IL-1β. Nature 484, 514–518 (2012)
Kamanaka, M. et al. Expression of interleukin-10 in intestinal lymphocytes detected by an interleukin-10 reporter knockin tiger mouse. Immunity 25, 941–952 (2006)
Ciofani, M. et al. A validated regulatory network for Th17 cell specification. Cell 151, 289–303 (2014)
Yosef, N. et al. Dynamic regulatory network controlling TH17 cell differentiation. Nature 496, 461–468 (2014)
Cua, D. J. et al. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421, 744–748 (2003)
Gause, W. C., Wynn, T. A. & Allen, J. E. Type 2 immunity and wound healing: evolutionary refinement of adaptive immunity by helminths. Nature Rev. Immunol. 13, 607–614 (2013)
Chen, F. et al. An essential role for Th2-type responses in limiting acute tissue damage during experimental helminth infection. Nature Med. 18, 260–266 (2012)
Panzer, M. et al. Rapid in vivo conversion of effector T cells into Th2 cells during helminth infection. J. Immunol. 188, 615–623 (2011)
Milner, J. D. et al. Impaired Th17 cell differentiation in subjects with autosomal dominant hyper-IgE syndrome. Nature 452, 773–776 (2008)
Lee, Y. et al. Induction and molecular signature of pathogenic Th17 cells. Nature Immunol. 13, 991–999 (2012)
Ghoreschi, K. et al. Generation of pathogenic Th17 cells in the absence of TGF-beta signalling. Nature 467, 967–971 (2010)
Martinez, G. J. et al. Smad3 differentially regulates the induction of regulatory and inflammatory T cell differentiation. J. Biol. Chem. 284, 35283–35286 (2009)
Stockinger, B., Di Meglio, P., Gialitakis, M. & Duarte, J. H. The aryl hydrocarbon receptor: multitasking in the immune system. Annu. Rev. Immunol. 32, 403–432 (2014)
Maynard, C. L. et al. Regulatory T cells expressing interleukin 10 develop from Foxp3+ and Foxp3− precursor cells in the absence of interleukin 10. Nature Immunol. 8, 931–941 (2007)
Jinnin, M., Ihn, H. & Tamaki, K. Characterization of SIS3, a novel specific inhibitor of Smad3, and its effect on transforming growth factor-beta1-induced extracellular matrix expression. Mol. Pharmacol. 69, 597–607 (2006)
Guo, S. et al. Nonstochastic reprogramming from a privileged somatic cell state. Cell 156, 649–662 (2014)
Pan, X. et al. Two methods for full-length RNA sequencing for low quantities of cells and single cells. Proc. Natl Acad. Sci. USA 110, 594–599 (2013)
Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25,. 2078–2079 (2009)
Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnol. 28, 511–515 (2010)
Anders, S., Pyl, P. T. & Huber, W. HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2014)
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.).
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). 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)).
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‐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.
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.
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. 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.
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.
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.
About this article
Cite this article
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
Interleukin 17A promotes cell migration, enhances anoikis resistance, and creates a microenvironment suitable for triple negative breast cancer tumor metastasis
Cancer Immunology, Immunotherapy (2021)
The FEBS Journal (2021)
Journal of Internal Medicine (2021)
Aryl Hydrocarbon Receptor Plasma Agonist Activity Correlates With Disease Activity in Progressive MS
Neurology - Neuroimmunology Neuroinflammation (2021)
Journal of Bone and Mineral Metabolism (2021)