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Thymus-derived Foxp3+ regulatory T cells upregulate RORγt expression under inflammatory conditions

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Abstract

Foxp3+ regulatory T cells (Tregs) co-expressing the Th17-lineage specification factor RORγt represent a unique Treg subpopulation that has been reported to be induced upon response to gut microbiota within the intestinal immune system. Hence, RORγt+ Tregs are considered to solely consist of peripherally induced Foxp3+ Tregs (pTregs), and the possibility that also thymus-derived Treg (tTregs) can upregulate RORγt expression and contribute to the pool of RORγt+ Tregs was largely ignored. Here, we expand our knowledge on the origin of RORγt+ Tregs by demonstrating that also tTregs can attain RORγt expression. In transgenic Foxp3∆CNS1-Cre reporter mice, a substantial fraction of CNS1-independent Tregs, predominantly consisting of tTregs, was found to co-express RORγt. In addition, genuine tTregs isolated from thymi of Foxp3hCD2RAGGFP reporter mice initiated RORγt expression both in vitro and in vivo, particularly under inflammatory conditions. In conclusion, our data demonstrate that tTregs can upregulate RORγt expression under inflammatory conditions and that hence RORγt+ Tregs can be regarded as a heterogeneous population consisting of both pTregs and tTregs.

Key messages

  • RORγt cannot be considered as a marker for pTregs.

  • CNS1-independent tTregs within the colon display RORγt expression.

  • RORγt can be induced in genuine tTregs, particularly under inflammatory conditions.

  • RORγt+ Tregs are a heterogeneous population consisting of both pTregs and tTregs.

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Abbreviations

BAC:

Bacterial artificial chromosome

mLN:

Mesenteric lymph node

Treg:

Regulatory T cell

pTreg:

Peripherally induced Treg

tTreg:

Thymus-derived Treg

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Acknowledgments

We thank all members of the animal facility, the flow cytometry facility, and the Department Experimental Immunology of the Helmholtz Centre for Infection Research (HZI), especially Dr. Lothar Gröbe, Yassin Elfaki, and Ahmed Elfiky for the technical assistance, and Dr. Shohei Hori (University of Tokyo, Japan) for providing Foxp3hCD2RAGGFP mice.

Funding

This work was supported by the German Research Foundation (SFB738-C7) and the Alexander von Humboldt Foundation.

Author information

Authors and Affiliations

  1. Department Experimental Immunology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124, Braunschweig, Germany

    Juhao Yang, Mangge Zou, Joern Pezoldt & Jochen Huehn

  2. Key Laboratory of Pathogenic Microbiology and Immunology, University of the Chinese Academy of Sciences, Beijing, 100080, China

    Mangge Zou & Xuyu Zhou

Authors
  1. Juhao Yang
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  2. Mangge Zou
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  3. Joern Pezoldt
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  4. Xuyu Zhou
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  5. Jochen Huehn
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Corresponding author

Correspondence to Jochen Huehn.

Ethics declarations

All animal experiments were approved by the Lower Saxony Committee on the Ethics of Animal Experiments as well as the responsible state office (Lower Saxony State Office of Consumer Protection and Food Safety) under the permit numbers 33.9-42502-04-13-1170 and 33.9-42502-04-12/1012. Foxp3∆CNS1-CreRosa26YFP mice (C57BL/6 background) were bred in-house (Institute of Microbiology, Chinese Academy of Sciences). All experiments were conducted according to the guidelines of the Institute of Microbiology, Chinese Academy of Sciences Institutional Animal Care and Use Committee (permit no. PZIMCAS2011005).

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Supplementary Figure 1.

Genuine tTregs virtually lack RORγt expression and can be identified upon adoptive transfer into immunocompetent hosts. Genuine tTregs (CD25+Foxp3hCD2+RAGGFP+ CD4SP thymocytes) isolated from thymi of Foxp3hCD2RAGGFP reporter mice (CD45.1) were adoptively transferred into three-week-old immunocompetent recipient mice (CD45.2). PBS-injected mice served as controls. (a) Scatterplot summarizes RORγt expression among sorted CD25+Foxp3hCD2+RAGGFP+ CD4SP thymocytes before transfer. Data pooled from nine independent experiments are shown as mean ± SD. (b) Representative dotplot confirms lack of false-positive CD45.1+ cells among CD4+ cells from mLN of PBS-injected control mice four weeks post transfer. Number indicates frequency of CD45.1+ cells. (c) Scatterplot summarizes frequencies of CD45.1+ cells among CD4+ cells in mLN of recipient mice four weeks after adoptive transfer of genuine tTregs. (d) Scatterplot summarizes absolute number of CD45.1+CD4+ cells recovered from mLN of recipient mice four weeks after adoptive transfer of genuine tTregs. Each dot represents one mouse. Data pooled from three independent experiments are shown as mean ± SD (n = 6-9). (EPS 671 kb)

Supplementary Figure 2.

Impact of IL-6 on phenotype, survival and proliferation of in vitro cultured tTregs. Genuine tTregs (CD25+Foxp3hCD2+RAGGFP+ CD4SP thymocytes) isolated from thymi of Foxp3hCD2RAGGFP reporter mice were cultured for four days with or without IL-6, followed by flow cytometric analysis. (a) Scatterplots summarize mean fluorescence intensity (MFI) of RORγt (left) and Foxp3 expression (right) in indicated groups normalized to medium control. Each dot represents data from one independent experiment (n=5). (b) Representative dotplots determine LIVE/DEAD (LD) fixable Blue expression (left), and scatterplot (right) summarizes frequencies of LD+ cells among RORγt+ or RORγt- tTregs at the end of the culture. Each dot represents one independent experiment. Data are shown as mean ± SD. (c) Representative histograms depicts CellTrace Violet (CTV) signal of RORγt+ or RORγt- tTregs at the end of the culture (left), and scatterplot summarizes MFI of CTV signal among RORγt+ or RORγt- tTregs at the end of the culture. Unstimulated naïve T cells served as control. Each dot represents one independent experiment. Data are shown as mean ± SD. (EPS 735 kb)

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Yang, J., Zou, M., Pezoldt, J. et al. Thymus-derived Foxp3+ regulatory T cells upregulate RORγt expression under inflammatory conditions. J Mol Med 96, 1387–1394 (2018). https://doi.org/10.1007/s00109-018-1706-x

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  • DOI: https://doi.org/10.1007/s00109-018-1706-x

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