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Thymic regulatory T cell niche size is dictated by limiting IL-2 from antigen-bearing dendritic cells and feedback competition

Nature Immunology volume 16, pages 635–641 (2015)Cite this article

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Abstract

The thymic production of regulatory T cells (Treg cells) requires interleukin 2 (IL-2) and agonist T cell antigen receptor (TCR) ligands and is controlled by competition for a limited developmental niche, but the thymic sources of IL-2 and the factors that limit access to the niche are poorly understood. Here we found that IL-2 produced by antigen-bearing dendritic cells (DCs) had a key role in Treg cell development and that existing Treg cells limited new development of Treg cells by competing for IL-2. Our data suggest that antigen-presenting cells (APCs) that can provide both IL-2 and a TCR ligand constitute the thymic niche and that competition by existing Treg cells for a limited supply of IL-2 provides negative feedback for new production of Treg cells.

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Figure 1: Limited Treg cell development in thymic slices regardless of the route of antigen delivery.
Figure 2: Existing thymic Treg cells reduce the size of the Treg developmental niche.
Figure 3: Existing thymic Treg cells do not prevent TCR signaling.
Figure 4: Availability of IL-2 from the thymic environment influences the Treg cell niche.
Figure 5: Existing Treg cells limit available IL-2 and/or IL-15 within the thymus.
Figure 6: Antigen-bearing DCs provide a local source of IL-2 to developing thymic Treg cells.

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Acknowledgements

We thank all the members of the Robey Lab for critical reading of the manuscript; H. Chu, H. Melichar and K. Taylor for technical assistance and suggestions; H. Nolla and A. Valeros for help with cell sorting; and N. Shastri (University of California, Berkeley) for the CTLL2 cell line. Supported by US National Institutes of Health (R01AI064227 to E.A.R. and T32AI100829 to B.M.W.).

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  1. Jeremy Boussier

    Present address: Present address: Centre d'immunologie humaine, Institut Pasteur, Paris, France.,

Authors and Affiliations

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

    Brian M Weist, Nadia Kurd, Shiao Wei Chan & Ellen A Robey

  2. Ecole Normale Supérieure, Paris, France

    Jeremy Boussier

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Contributions

B.M.W. designed, performed and analyzed experiments and wrote the manuscript; N.K., J.B. and S.W.C. performed experiments; and E.A.R. supervised the study and wrote the manuscript.

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Correspondence to Ellen A Robey.

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Integrated supplementary information

Supplementary Figure 1 Time course of OT-II Treg cell development in thymic slices.

Time course of Treg cell development following addition of OT-II TCR transgenic Rag2 -/- mice (OT-II) thymocytes to thymic slices from mice that were previously injected with 2mg OVA protein. Data are representative of 4 independent experiments, gated on OT-II CD4SP.

Supplementary Figure 2 Treg cell development in thymic slices recapitulates normal thymic development rather than in vitro–induced Treg cell development.

(a) OT-II thymocytes or mature OT-II T cells were added to thymic slices from wild type mice that were injected with OVA protein 1hr prior to sacrifice. TGF-β (5ng/ml) was added along with mature OT-II T cells in some samples as indicated. (b) Neuropilin-1 expression levels on gated OT-II Foxp3+ CD4SP from thymocytes, splenocytes, or Foxp3 negative thymic CD4SP. (c) OT-II thymocytes were depleted of CD4SP prior to introduction into thymic slices. Flow cytometric analysis of samples before and after depletion are shown. (d) OT-II CD4SP depleted thymocytes overlaid onto WT thymic slices along with 1μM OVA peptide. Flow cytometry of gated OT-II CD4SP after days 3 or 4 of culture are shown. Data representative of 2 (a,b) or 3 (c,d) independent experiments.

Supplementary Figure 3 Relationship between the frequency of precursors of Treg cells and Treg cell development.

The % Treg cells (CD25+Foxp3+) amongst OT-II donor thymocytes plotted versus the % of donor OT-II thymocytes within a thymic slice. Each dot represents the values for an individual thymic slice and data are compiled from 3-4 independent experiments. Ovalbumin was introduced via i.v. injection of mice prior to sacrifice and preparation of thymic slices (a), use of thymic slices from mice expressing a RIPmOVA transgene (b), or addition of OVA loaded bone marrow derived DCs to thymic slices containing OT-II thymocytes (c). Data compiled from multiple experiments, (a) n=15 (4 exp.); (b) n=16 (3 exp.); (c) n=19 (4 exp.).

Supplementary Figure 4 Treg cell development is enhanced in the absence of existing thymic Treg cells.

(a) Slices from WT or AND (Treg deficient) thymi, overlaid with 2D2 TCR transgenic thymocytes in the presence of 3.8μM MOG 35-55 peptide. Treg cell development was analyzed on day 3 by flow cytometry. (b) Number of 2D2 Treg cells recovered per thymic slice after 3d. *p<0.01 by student’s t-test, n=6 and 3 slices respectively, representative of 2 independent experiments. (c) Slices from WT or F5 TCR transgenic Rag2-/-(Treg deficient) thymi, overlaid with OT-II thymocytes in the presence of the indicated concentrations of OVA peptide. Treg cell development analyzed on day 3 by flow cytometry. (d) Number of OT-II Treg cells recovered per thymic slice after 3d. *p<0.01 and **p<0.001 one-way ANOVA using Tukey’s post test analysis. Data pooled from 3 independent experiments, n=9 slices each.

Supplementary Figure 5 DCs derived from IL-2-deficient bone marrow have normal antigen-presentation capacity.

DCs were generated in culture from WT and Il2−/− bone marrow, incubated with 1mg/ml OVA protein, and introduced into thymic slices along with OT-II thymocytes. Flow cytometric analysis of activation markers on OT-II thymocytes was performed after 16h of culture. (a) Representative flow cytometry plots and (b) expression levels of activation markers on gated CD4SP OT-II thymocytes. Error bars represent +/- SEM, n=6 slices per condition, ns= not significant by unpaired student’s t-test, data representative of 3 independent experiments.

Supplementary Figure 6 Localized IL-2 production by the DC population presenting antigen.

Il2−/− thymic slices were overlaid with OT-II thymocytes followed by the addition of WT or Il2−/− DCs loaded with 1mg/ml OVA protein. OT-II Treg cell development was measured on d3. (a) Representative flow cytometry plots. (b) Quantification of the number of OT-II Treg cells recovered per thymic slice. *p<0.001 by student’s t-test. Data representative of 3 independent experiments, n=8 slices per condition. (c) Treg precursors (orange) interact with niche-defining APCs that bear agonist self-peptide (orange rectangles) and also supply IL2 (black dots). As precursors upregulate CD25 and differentiate into Treg cells (blue) they limit IL2 availability from the APC, providing negative feedback and preventing future Treg cell development within the niche. Shaded stars indicate cells in the surrounding tissue that are not part of the niche.

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Weist, B., Kurd, N., Boussier, J. et al. Thymic regulatory T cell niche size is dictated by limiting IL-2 from antigen-bearing dendritic cells and feedback competition. Nat Immunol 16, 635–641 (2015). https://doi.org/10.1038/ni.3171

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