Nr4a receptors are essential for thymic regulatory T cell development and immune homeostasis

Journal name:
Nature Immunology
Volume:
14,
Pages:
230–237
Year published:
DOI:
doi:10.1038/ni.2520
Received
Accepted
Published online

Abstract

Regulatory T cells (Treg cells) develop from progenitor thymocytes after the engagement of T cell antigen receptors (TCRs) with high-affinity ligands, but the underlying molecular mechanisms are still unclear. Here we show that the Nr4a nuclear receptors, which are encoded by immediate-early genes upregulated by TCR stimulation in thymocytes, have essential roles in Treg cell development. Mice that lacked all Nr4a factors could not produce Treg cells and died early owing to systemic autoimmunity. Nr4a receptors directly activated the promoter of the gene encoding the transcription factor Foxp3, and forced activation of Nr4a receptors bypassed low-strength TCR signaling to drive the Treg cell developmental program. Our results suggest that Nr4a receptors have key roles in determining CD4+ T cell fates in the thymus and thus contribute to immune homeostasis.

At a glance

Figures

  1. Compound deletion of all Nr4a receptors resulted in loss of Treg cells and systemic lethal autoimmunity.
    Figure 1: Compound deletion of all Nr4a receptors resulted in loss of Treg cells and systemic lethal autoimmunity.

    (a,b) Flow cytometry profiles of the thymus (a) and pooled spleens and lymph nodes (b) from 18-day-old wild-type (WT) and Nr4a-TKO mice (n = 5 mice per group). CD4SP thymocyte fraction in a and CD4+ T cell fraction in b were further analyzed for CD25 and Foxp3 expression (bottom). Numbers adjacent to outlined areas or in quadrants indicate percent cells in each. (c) Flow cytometry profiles showing the expression of CD44 and CD62L in CD4+ T cells pooled from the spleen and lymph node of 18-day-old wild-type and Nr4a-TKO mice (n = 5 mice per group). (d) Spleens and lymph nodes from 18-day-old wild-type and Nr4a-TKO mice (left), and total cellularity of spleens in 18- to 20-day-old wild-type and Nr4a-TKO mice (right; n = 5 per group). *P = 0.0007 (unpaired two-tailed Student's t-test). (e) Titers of IgG, IgM and IgA antibodies to double-stranded DNA (Anti-dsDNA) in serum from 18- to 20-day-old wild-type and Nr4a-TKO mice (n = 5 per group). *P = 0.006 (unpaired two-tailed Student's t-test). (f) Survival of wild-type and Nr4a-TKO mice, and for mice doubly deficient in Nr4a1 and Nr4a2 (a1a2-DKO), Nr4a1 and Nr4a3 (a1a3-DKO), or Nr4a2 and Nr4a3 (a2a3-DKO), monitored for 36 d (n = 7–10 mice per group). (g) Hematoxylin and eosin staining of liver, lung, colon and kidney sections from 19-day-old wild-type and Nr4a-TKO mice: red dashed line indicates venous endotheliitis; blue dashed line indicates portal endotheliitis; green dashed line indicates lupus-like thickening of the glomeruli. Scale bars, 100 μm. Each symbol (d, right, and e) represents an individual mouse; small horizontal lines indicate the mean. Data represent three independent experiments.

  2. Nr4a-TKO mice developed a TH2-type inflammation.
    Figure 2: Nr4a-TKO mice developed a TH2-type inflammation.

    (a) Flow cytometry profiles of pooled spleens and lymph nodes from 18-day-old wild-type and Nr4a-TKO mice (n = 5 per group), showing expression of IL-4 and IFN-γ (top) and of IL-10 and IL-17 (bottom) in the CD4+ T cell fraction of cells restimulated for 5 h with PMA plus ionomycin in the presence of brefeldin A. Numbers in quadrants indicate percent cells in each. (b) Flow cytometry profiles of liver and lung lymphocytes in 19-day-old wild-type and Nr4a-TKO mice (n = 3 per group) showing expression of IL-4, IFN-γ, IL-10, IL-17 and Foxp3 in the CD4+ T cells restimulated for 5 h with PMA plus ionomycin in the presence of brefeldin A. (c) Titers of IgG1, IgE and IgG2b in serum from 18- to 20-day-old wild-type and Nr4a-TKO mice (n = 5 per genotype). Each symbol represents an individual mouse; small horizontal lines indicate the mean. *P = 0.01, **P = 6.0 × 10−5 and ***P = 1.2 ×10−7 (unpaired two-tailed Student's t-test). (d) Flow cytometry profiles of Foxp3 expression by naive wild-type and Cd4-Cre+Nr4a1fl/flNr4a2fl/flNr4a3+/ (Nr4a1−/−a2−/−a3+/) CD4+ T cells transduced with shRNA targeting Nr4a3 (sh-Nr4a3) or control shRNA (sh-control) and cultured for 72 h under iTreg cell–skewing conditions (with TGF-β). Numbers above bracketed lines (b, right, and d) indicate percent Foxp3+ cells. Data are representative of three independent experiments.

  3. Mice deficient in both Nr4a1-Nr4a3 mice have fewer Treg cells and develop autoimmunity.
    Figure 3: Mice deficient in both Nr4a1-Nr4a3 mice have fewer Treg cells and develop autoimmunity.

    (a) Flow cytometry profiles of the thymus and pooled spleens and lymph nodes (Peripheral) from 3-week-old wild-type and mice deficient in Nr4a1-Nr4a2, Nr4a2-Nr4a3 or Nr4a1-Nr4a3 (n = 4 mice per group), showing the expression of CD25 and Foxp3 in the CD4SP thymocyte fraction in the thymus and CD4+ T cell fraction in the periphery and of CD44 and CD62L in peripheral CD4+ T cells. Numbers adjacent to outlined areas indicate percent cells in each. (b,c) Flow cytometry analysis of Foxp3 expression (left; measured as mean fluorescence intensity (MFI) at right) in Treg cells in the thymus (b) or in pooled spleens and lymph nodes (c) from wild-type and mutant mice as in a. *P = 0.04, **P = 0.03, ***P = 0.02 (unpaired two-tailed Student's t-test, n = 4 per group). (d) Total cellularity of spleens in 18- to 20-day-old wild-type and mutant mice as in a (n = 3–5 per group). *P = 0.003 (unpaired two-tailed Student's t-test). (e) Frequency of Foxp3+ Treg cells in the CD4SP thymocyte fraction in the thymus and the CD4+ T cell fraction in pooled spleens and lymph nodes from 18- to 20-day-old wild-type and mutant mice as in a and Nr4a-TKO mice (n = 4 per group). *P = 0.01 and **P = 0.005 (unpaired two-tailed Student's t-test). Data are pooled from four independent experiments with four mice per group (mean and s.d.)

  4. Redundancy among Nr4a factors in Foxp3 induction.
    Figure 4: Redundancy among Nr4a factors in Foxp3 induction.

    (a) Chromatin-immunoprecipitation analysis of Nr4a receptors at the Foxp3 promoter in naive CD4+ T cells stimulated for 2 h with antibody to CD3 and to CD28, assessed with IgG (control) or antibody (α-) to each Nr4a receptor (horizontal axis). (b) Structure (top) of Nr4a1, Nr4a2 and the chimera with the DNA−binding domain of Nr4a2 replaced with that of Nr4a1 (Nr4a2-a1-DBD). Below, induction of Foxp3 expression (assessed as coexpression of human CD2 (hCD2)) by Nr4a1, Nr4a2 and Nr4a2-a1-DBD in CD4+ T cells transduced with a control retrovirus or Nr4a-expressing retroviruses (assessed as GFP expression) and cultured for 72 h under TH0 conditions. Numbers in quadrants indicate percent cells in each. (c) Frequency of Foxp3+ cells in b separated into four fractions (negative (Neg), low (Lo), medium (Med), and high (Hi)) according to Nr4a1 expression (GFP fluorescence strength). *P = 8.0 × 10−4 (unpaired two-tailed Student's t-test). (d,e) Quantitative PCR analysis of mRNA encoding each Nr4a protein in Treg cells (CD4+CD25+hCD2+ cells in Foxp3-hCD2-hCD52-KI mice; d), and in unstimulated (–) naive CD4+ T cells or naive CD4+ T cells stimulated (+) for 1 h with antibody to CD3 and to CD28 (TCR stim; e); results are presented relative to expression of the control gene Hprt. Data represent at least three independent experiments (a,c,d,e; mean and s.d.) or four independent experiments (b).

  5. CD4+ T cell-intrinsic defect in the Treg cell development of Nr4a-TKO cells.
    Figure 5: CD4+ T cell–intrinsic defect in the Treg cell development of Nr4a-TKO cells.

    (a) Flow cytometry profiles of mixed–bone marrow chimeras (n = 3 per group) given wild-type (Ly5.1+) and either wild-type or Nr4a-TKO (Ly5.2+) cells, showing expression of Foxp3 and IL-4 in the Ly5.1+ and Ly5.2+ fractions of CD4SP thymocytes and CD4+ T cells in pooled spleens and lymph nodes, for cells stimulated for 5 h with PMA plus ionomycin in the presence of brefeldin A. Numbers in quadrants indicate percent cells in each. (b) Quantification of the results in a. *P = 0.01 and **P = 0.002 (unpaired two-tailed Student's t-test. Data are representative of three independent experiments (mean and s.d. of nine mice per group in b).

  6. Activation of Nr4a compensates for suboptimal TCR signaling strength in thymic Treg cell development.
    Figure 6: Activation of Nr4a compensates for suboptimal TCR signaling strength in thymic Treg cell development.

    (a) Flow cytometry profiles of thymocytes from bone marrow–chimeras (n = 3 per group), showing expression of Flag peptide (control) or Nr4a2-ΔLBD-ERT2 (assessed as Thy-1.1 expression) and Foxp3 (intracellular staining). Numbers adjacent to outlined areas or in quadrants indicate percent cells in each. (b) Quantification of results in a (n = 6 mice per group), showing the frequency of Foxp3+ CD4SP cells in the Thy-1.1+ fraction (left) and the ratio of the Thy-1.1+ fraction to total CD4SP cells (right), with ratio for tamoxifen-untreated samples set as 1. *P = 0.02 (unpaired two-tailed Student's t-test). Data are representative of two independent experiments (a) or are pooled from two independent experiments with six mice per group (b; mean and s.d.).

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Author information

Affiliations

  1. Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan.

    • Takashi Sekiya,
    • Ikkou Kashiwagi,
    • Rei Yoshida,
    • Tomohiro Fukaya,
    • Rimpei Morita,
    • Akihiro Kimura &
    • Akihiko Yoshimura
  2. Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.

    • Hiroshi Ichinose
  3. Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Illkirch, France.

    • Daniel Metzger &
    • Pierre Chambon
  4. Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Tokyo, Japan.

    • Akihiko Yoshimura

Contributions

T.S. and A.Y. designed the research and analyzed data; T.S., I.K., R.Y., T.F., A.K. and R.M. did the experiments; H.I., D.M. and P.C. provided the Nr4a2fl and Nr4a1fl mouse strain, plasmids and feedback on the manuscript; and T.S. and A.Y. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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