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Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors

Nature Immunology volume 16, pages 628–634 (2015)Cite this article

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Abstract

Most T lymphocytes, including regulatory T cells (Treg cells), differentiate in the thymus. The age-dependent involution of this organ leads to decreasing production of T cells. Here we found that the output of new Treg cells from the thymus decreased substantially more than that of conventional T cells. Peripheral mouse and human Treg cells recirculated back to the thymus, where they constituted a large proportion of the pool of Treg cells and displayed an activated and differentiated phenotype. In the thymus, the recirculating cells exerted their regulatory function by inhibiting interleukin 2 (IL-2)-dependent de novo differentiation of Treg cells. Thus, Treg cell development is controlled by a negative feedback loop in which mature progeny cells return to the thymus and restrain development of precursors of Treg cells.

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Figure 1: Thymic production of Treg cells decreases substantially with age.
Figure 2: Peripheral Treg cells are able to recirculate to the thymus.
Figure 3: Recirculating Treg cells in the thymus have an activated and differentiated phenotype.
Figure 4: Mature peripheral Treg cells that reenter the thymus inhibit the differentiation of Treg cells.
Figure 5: Activated and differentiated peripheral Treg cells also recirculate to the thymus in humans.

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Acknowledgements

We thank A. Lew (Walter & Eliza Hall Institute of Medical Research) and A. Egle (Salzburg Cancer Research Institute) for GK-transgenic mice; P. Fink (University of Washington, Seattle) for B6 Rag-GFP mice; A. Rudensky (Memorial Sloan-Kettering Cancer Center) and A. Liston (University of Leuven) for Foxp3-Thy-1.1 mice; S. Guerder and J.-C. Guéry for critical reading of the manuscript; F. Auriol and A. Garnier for help in obtaining human thymuses; F.-E. L'Faqihi-Olive and V. Duplan-Eche for technical assistance at the flow-cytometry facility; S. Allart and A. Canivet for technical assistance at the cellular imaging facility of Inserm U1043, Toulouse; the personnel of the Inserm US006 ANEXPLO/CREFRE animal facility for expert animal care; and the GeT-PlaGe and GenoToul bioinformatics platforms Toulouse Midi-Pyrénées for sequencing, computing and storage resources. Supported by the Fondation pour la Recherche Médicale (O.P.J. and J.P.M.v.M.), the IdEx Toulouse (E.A.R. and P.R.), the Région Midi Pyrénées (O.P.J. and J.P.M.v.M.) and the Agence Nationale pour la Recherche (O.P.J.).

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  1. Nicolas Thiault, Julie Darrigues and Véronique Adoue: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut National de la Santé et de la Recherche Médicale U1043, Toulouse, France

    Nicolas Thiault, Julie Darrigues, Véronique Adoue, Marine Gros, Bénédicte Binet, Corine Perals, Nicolas Fazilleau, Olivier P Joffre, Joost P M van Meerwijk & Paola Romagnoli

  2. Centre National de la Recherche Scientifique U5282, Toulouse, France

    Nicolas Thiault, Julie Darrigues, Véronique Adoue, Marine Gros, Bénédicte Binet, Corine Perals, Nicolas Fazilleau, Olivier P Joffre, Joost P M van Meerwijk & Paola Romagnoli

  3. Université de Toulouse, Université Paul Sabatier, Centre de Physiopathologie de Toulouse Purpan, Toulouse, France

    Nicolas Thiault, Julie Darrigues, Véronique Adoue, Marine Gros, Bénédicte Binet, Corine Perals, Nicolas Fazilleau, Olivier P Joffre, Joost P M van Meerwijk & Paola Romagnoli

  4. Department of Biology, Ecole Normale Supérieure de Lyon, 69007 Lyon, France.,

    Marine Gros

  5. Department of Pediatric Cardiology and Cardiovascular surgery, Children Hospital, University Hospital of Toulouse, Toulouse, France

    Bertrand Leobon

  6. Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, California, USA

    Ellen A Robey

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  1. Nicolas Thiault
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Contributions

N.T., J.D. and V.A. performed experiments, analyzed data and contributed to writing the manuscript; M.G. and B.B. performed experiments and analyzed data; C.P. performed experiments; B.L. provided clinical samples; N.F., O.P.J. and E.A.R. designed experiments and analyzed data; J.P.M.v.M. and P.R. designed experiments, analyzed the data and wrote the manuscript; and all authors critically reviewed the manuscript.

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Correspondence to Joost P M van Meerwijk or Paola Romagnoli.

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Integrated supplementary information

Supplementary Figure 1 Gating strategy used to calculate the proportions of GFP− and GFP+ Tconv cells and Treg cells in thymus and spleen from Rag‑GFPFoxp3-Thy-1.1 mice.

(a) Thymocytes and (b) splenocytes were stained with fluorescent antibodies against the indicated markers and analyzed by flow cytometry. CD4 and CD8 expression on electronically gated live cells (FSC/SSC gate, not shown) is depicted (upper left). CD4+CD8− (CD4SP and CD4+) cells were electronically gated as depicted and Thy1.1 expression was analyzed (upper right). GFP-expression was then analyzed on further gated Thy1.1− Tconv cells (lower left) and Thy1.1+ Treg cells (lower right). Numbers indicate percentages of cells within the indicated electronic gates.

Supplementary Figure 2 Longitudinal analysis of the proportion of splenic Treg cells.

Splenocytes from Rag‑GFPFoxp3-Thy-1.1 mice of indicated ages were stained with fluorescent antibodies against CD4, CD8, and Thy1.1 and analyzed by flow cytometry. Live cells were gated electronically (FSC/SSC gate, not shown) and the proportion of Thy1.1+ Treg cells among CD4+CD8− splenocytes was calculated. Values for all the individual mice analyzed are shown.

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Supplementary Figure 3 Analysis of the TCR Vβ repertoire of GFP+ and GFP− Treg cells in the thymus and spleen from Rag-GFPFoxp3-Thy-1.1 mice.

(a) Thymocytes and splenocytes were analyzed as in supplementary Fig. 1 but with the addition of antibodies to the indicated TCR Vβ segments. Expression of Vβ5, Vβ6, and Vβ11 on the indicated, electronically gated CD4+CD8− Thy1.1+ Treg populations is shown. Numbers indicate percentages of cells within indicated electronic gates. Fig. 2c shows the quantitative analysis of thus performed experiments. (b) Percentages of Vβ5-expressing cells in the indicated populations (left). Percentage of peripheral deletion of Vβ5-expressing cells calculated from the data in the left panel (right). The data shown are means ± SD, n=9 from three independent experiments. *p<0.01; Wilcoxon matched pairs signed rank test. (c) Newly developing GFP+ and recirculating GFP− thymic CD4+CD8−Thy-1.1+ Treg cells were analyzed by immunoscope. The peaks within each distribution represent sequences that differ by three bases (one amino acid). The Gaussian-like Vβ-Cβ distribution profiles obtained indicate that the populations of Vβ6- or Vβ11-expressing Treg cells are polyclonal (Vβ5 plots are shown as a control). Shown are representative results from three experiments performed. (d) The percentages of cells expressing the indicated control TCR Vβ segments among the electronically gated indicated CD4+CD8− Thy1.1+ Treg cell populations were analyzed by flow cytometry. The data shown are means ± SD, n=9 from three independent experiments. NS, not significant as determined by the Wilcoxon matched pairs signed rank test.

Source data

Supplementary Figure 4 Peripheral depletion of CD4+ T cells and reduced proportions of thymic GFP− Treg cells in GK-transgenic Rag-GFPFoxp3-Thy-1.1 mice.

(a) Flow cytometry of CD4 versus CD8 expression on electronically gated TCRhi splenocytes from control (“Wt”) and GK-transgenic Rag-Gfp/Foxp3-Thy1.1 mice. Numbers indicate percentages of cells within indicated gates. A representative experiment out of three performed is shown. (b) Flow cytometry of CD4 versus CD8 expression by thymocytes from indicated mice. The numbers above the boxed populations indicate the percentages of mature CD4+CD8− cells among total thymocytes. A representative experiment out of three performed is shown. (c) Analysis of Thy-1.1 expression by electronically gated CD4+CD8− thymocytes from indicated mice. Numbers indicate percentage of cells within indicated gates. Quantitative analysis of thus performed experiments is shown in Fig. 2d, left. (d) GFP expression on electronically gated CD4+CD8−Thy-1.1+ thymic Treg cells. Numbers indicate percentage of cells within indicated gates. Quantitative analysis of thus performed experiments is shown in Fig. 2d, right. (e) Cell cycle analysis of GFP+ and GFP− thymic Treg cells in non-transgenic and GK-transgenic mice. Cell cycle status was analyzed by flow-cytometry. Percentages of cycling cells (S, G2, and M phase) for all studied populations are indicated (means ± SD, n=4 control and 4 GK mice). NS, not significant, Mann-Whitney test.

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Supplementary Figure 5 Peripheral Treg cells recirculate to the thymus.

(a) In vitro expanded and cell trace violet (CTV)-labeled Treg cells (8–10×106 cells/mouse) were injected i.v. into wt mice. One day later, the thymus of the adoptive host was analyzed by flow-cytometry using antibodies to CD4, CD8, CD25, and Foxp3. In the left plot, expression of Foxp3 and CD25 on electronically gated CD4+CD8− thymocytes is shown. The right plot shows CTV-marked cells in the Treg population, electronically gated as shown. The number indicates the percentage of cells within the indicated gate. Quantitative analysis of thus performed analyses is shown in Fig. 2e. One day after their i.v. injection into wild-type mice, in vitro activated Tconv cells (b) and in vitro generated iTreg cells (c) were found in the spleen and thymus of host mice. Shown are the percentages of indicated cells among Tconv (b) and Treg (c) cells in spleen and thymus (individual mice are shown, horizontal lines indicate mean values, 3 independent experiments, *p<0.01; Wilcoxon matched pairs signed rank test). (d) CXCR4 versus GFP expression by electronically gated CD4+CD8−Foxp3+ thymic Treg cells from Rag-GFP/Foxp3-Thy-1.1 mice. A representative experiment out of three performed is shown. (e) Percentages of Foxp3+ Treg cells among electronically gated CD4+CD8−TCRhiH-2Kb-negative cells from fetal thymus cultures performed in the absence or presence of added in vitro activated H-2Kb-positive Tconv cells. The data from two independent experiments are shown. NS, not significant; Mann Whitney test.

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Thiault, N., Darrigues, J., Adoue, V. et al. Peripheral regulatory T lymphocytes recirculating to the thymus suppress the development of their precursors. Nat Immunol 16, 628–634 (2015). https://doi.org/10.1038/ni.3150

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