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A regulatory role for TGF-β signaling in the establishment and function of the thymic medulla

Nature Immunology volume 15, pages 554–561 (2014)Cite this article

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Abstract

Medullary thymic epithelial cells (mTECs) are critical in establishing and maintaining the appropriate microenvironment for negative selection and maturation of immunocompetent T cells with a self-tolerant T cell antigen receptor repertoire. Cues that direct proliferation and maturation of mTECs are provided by members of the tumor necrosis factor (TNF) superfamily expressed on developing thymocytes. Here we demonstrate a negative role of the morphogen TGF-β in tempering these signals under physiological conditions, limiting both growth and function of the thymic medulla. Eliminating TGF-β signaling specifically in TECs or by pharmacological means increased the size of the mTEC compartment, enhanced negative selection and functional maturation of medullary thymocytes as well as the production of regulatory T cells, thus reducing the autoreactive potential of peripheral T cells.

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Figure 1: Enhanced mTEC expansion in the absence of TEC-specific TGF-β signaling.
Figure 2: Altered TGF-β signaling in TEC affects the maturation of mTECs.
Figure 3: TGF-β dampens the thymocyte-derived signals required for mTEC differentiation.
Figure 4: TGF-β and its target gene Myc affect proliferation of mTECs.
Figure 5: Consequences of the altered medulla in Tgfbr2fl/flFN1-Cre mice on thymocyte development and export.
Figure 6: Increased generation of Treg cells in the thymus of Tgfbr2fl/flFN1-Cre mice.
Figure 7: Reduced autoimmunity in Tgfbr2fl/flFN1-Cre mice upon chronic in vivo ablation of Foxp3+ regulatory T cells.

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Acknowledgements

We thank D.J. Campbell and J.A. Hamerman for helpful discussions about these data, and S. McCarty for help with preparation of the manuscript. This work was partially supported by grants from the Swiss National Science Foundation (M.H.-H. and G.A.H.).

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Author notes

  1. Georg A Holländer and Steven F Ziegler: These authors contributed equally to this work.

Authors and Affiliations

  1. Immunology Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington, USA

    Mathias Hauri-Hohl & Steven F Ziegler

  2. Department of Biomedicine, Laboratory of Pediatric Immunology, University of Basel, Basel, Switzerland

    Saulius Zuklys & Georg A Holländer

  3. Basel University's Hospital, Basel, Switzerland

    Saulius Zuklys & Georg A Holländer

  4. Department of Pediatrics, Developmental Immunology, University of Oxford, Oxford, UK

    Georg A Holländer

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Contributions

M.H.-H. performed all of the experiments in the manuscript, with help from S.Z.; M.H.-H., G.A.H. and S.F.Z. designed the experiments, analyzed data and contributed to writing the manuscript.

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Correspondence to Steven F Ziegler.

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Integrated supplementary information

Supplementary Figure 1 FN1-Cre–mediated deletion of TGFβRII does not affect the phenotype of cTECs.

(a) Assessment of FN1-Cre-mediated genomic deletion of the floxed sequence in total TEC sorted from E14 thymic stroma, derived from either TβRIIlox/lox/FN1-Cre (denoted ‘Crepos’) or TβRIIlox/lox (‘Creneg’) embryos. (b) Gating strategy to identify TEC subpopulations from total thymic tissue, digested with Collagenase and DNAse. (c) Characterization of the cTEC compartment in 4-week-old TβRIIlox/lox/FN1-Cre mice or littermate controls for the expression of MHCII, β5t and DEC205.

Supplementary Figure 2 Altered expression of TRA with preserved thymic architecture in thymi with altered TGF-β signaling.

(a) Thymic section derived from 4 week old TβRIIlox/lox/FN1Cre or littermates stained for H&E, cytokeratin-5, cytokeratin-14 or UEA1 respectively. (b) Expression of indicated TRAs within total thymus of TβRIIlox/lox/FN1Cre (white bars, N=4), relative to expression found in littermate controls (black bars, N=5). (c) Absolute numbers of thymocytes and (d) relative distribution of thymocyte subpopulations in 6-8 week-old C57Bl/6 mice treated daily with SB431542 (white bars, N=8) or vehicle only (black bars, N=8) for 6 consecutive days, analysed on day 12 after the first injection. For statistical analysis, a two-tailed, unpaired t-test was used; NS denotes not statistically different whereas asterisks indicate a p-value of ≤0.05 (*),≤0.01 (**) or ≤0.001(***).

Supplementary Figure 3 TGF-β limits RANKL-induced mTEC differentiation.

Lymphocyte-depleted fetal thymi were cultured in the presence of the indicated stimuli for 5 days and assessed for the up-regulation of MHCII and CD80 within the mTEC compartment. Note the reduction in MHCIIhigh and CD80pos cells in the presence of RANKL/TGFβ vs RANKL alone, and a corresponding increase in the MHCIIlow population.

Supplementary Figure 4 Reduced thymic cellularity but normal thymocyte development and maturation in mice with c-myc–deficient TECs.

(a) Thymocyte cellularity and (b) relative distribution of thymocyte subpopulations with respect to CD4 and CD8 (upper panel) or CD69 and TCRβ (lower panel) expression in 4 week old c-myclox/lox/FN1-Cre mice (white bar) and littermate controls (black bar).

Supplementary Figure 5 Thymocyte maturation, export and functional changes of peripheral T cells in Tgfbr2fl/flFN1-Cre mice.

(a) Absolute numbers of DP and DN thymocytes in 4 week-old TβRIIlox/lox/FN1-Cre mice (white bars, N=5) and controls (black bars, N=5). (b) Flow cytometry profiles (left panel) of thymocytes for CD69 and TCR□ expression derived from 4-week-old mice and (right panel) statistical summary of the indicated subpopulations (R3: TCRintCD69pos, R4: TCRhighCD69pos, R5: TCRhighCD69neg). White bars denote TβRIIlox/lox/FN1-Cre mice (N=5), black bars littermate controls (N=5). (c) Absolute numbers of immature (i.e. CD24highCD62Llow) CD4 or CD8 TCRhigh SP thymocytes from TβRIIlox/lox/FN1-Cre mice (white bars, N=5) and controls (black bars, N=7). (d) GFP expression in immature (upper panel) and mature (lower panel) TCRhigh SP4 thymocytes derived from either controls (shaded histogram) or TβRIIlox/lox/FN1-Cre mice (open histogram), which were lethally irradiated and reconstituted with T-cell-depleted bone marrow cells from congenically marked mice that express GFP under the control of the Rag2-promoter (representative of 3 (TβRIIlox/lox/FN1-Cre mice) and 4 (littermate controls) mice from two independent experiments are shown). (e) Percentage of splenic CD4 or CD8 T cells in TβRIIlox/lox/FN1-Cre mice and littermate controls at the age of 3 days (left panel) and 6 days (right panel). White bars denote TβRIIlox/lox/FN1-Cre mice, black bars littermate controls. Two individual litters per time point with a minimum of total 3 mice per genotype are shown. (f) Absolute numbers of FITCpos recent thymic emigrants in peripheral lymphoid organs recovered 24 hours after intrathymic injection of 10μl FITC. White bars denote TβRIIlox/lox/FN1-Cre mice (N=16), black bars littermate controls (N=14). (g) Frequency of CD69pos cells within sorted peripheral naïve CD4 T cells from TβRIIlox/lox/FN1-Cre mice (white bars) or littermate controls (black bars) 20 hours after stimulation with the indicated stimuli. (h) Proliferation assessed by CFSE-dilution in sorted peripheral naïve CD4 T cells derived from TβRIIlox/loxFN1-Cre mice (upper histogram) and littermate controls (lower histogram) after a 4-day stimulation culture with 1μl/ml α-CD3 and 5μg/ml α-CD28. For statistical analysis, a two-tailed, unpaired t-test was used; NS denotes not statistically different whereas asterisks indicate a p-value of ≤0.05 (*), ≤0.01 (**) or ≤0.001(***).

Supplementary Figure 6 Reduced frequency of natural TH17 cells in thymus and spleen of Tgfbr2fl/flFN1-Cre mice.

Flow cytometry profiles (left panel) and corresponding bar graphs and statistics (right panel) of CD4SP TCRhigh thymocytes (a) and splenic CD4pos T cells (b) derived from 4-week-old mice after a 4 hour culture with PMA, Ionomycin and Brefeledin A. Data shown from 2 experiments with a total of 9 mice (asterisks indicate a p-value of ≤0.05 (*) or ≤0.01 (**)). (a, b) White bars denote TβRIIlox/lox/FN1-Cre mice, black bars littermate controls.

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Hauri-Hohl, M., Zuklys, S., Holländer, G. et al. A regulatory role for TGF-β signaling in the establishment and function of the thymic medulla. Nat Immunol 15, 554–561 (2014). https://doi.org/10.1038/ni.2869

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