The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4+ T cells.
Regulatory T cells (Tregs) have a central role in maintaining immune homoeostasis through various mechanisms.Nr4a2 binds to regulatory regions of Foxp3, where it mediates permissive histone modifications.Nr4a2-deficeint Tregs are prone to lose Foxp3 expression and have attenuated suppressive ability both in vitro and in vivo.
Affiliation: Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo 160-8582, Japan.
- Cell Differentiation*
- Forkhead Transcription Factors/genetics/immunology*
- Gene Expression Regulation*
- Nuclear Receptor Subfamily 4, Group A, Member 2/genetics/immunology*
- T-Lymphocytes, Regulatory/cytology*/immunology
- Cells, Cultured
- Mice, Transgenic
- Protein Binding
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f4: Nr4a2 induces suppression activity in vitro by upregulating CD25 and repressing IL-2.(a) qPCR analysis of the effects of Nr4a2 expression on the expression of Treg effector genes. mRNA was prepared from naïve T cells (columns 1 and 2), Tregs (columns 3 and 4), the GFP+ fraction of eMIGR1 retrovirus-transduced cells (columns 5, 6, 11 and 12), and the GFP+Foxp3– (columns 7, 8, 13 and 14) or GFP+Foxp3+ (columns 9, 10, 15 and 16) fraction of eMIGR1-Nr4a2 retrovirus-transduced cells, which were incubated with (even-numbered columns) or without (odd-numbered columns) PMA/ionomycine for 2 h. Retrovirus-transduced cells were sampled at 48 h (blue bars, columns 5 to 10) and 84 h (green bars, columns 11 to 16) after infection. Expression levels of the indicated mRNA, are presented relative to the Hprt expression. Data are representative of three independent experiments (mean and s.d. of triplicate). (b) In vitro suppression assay. Top: suppression of CFSE-labeled naïve CD4+ T cells (responder, Tresp) by Tregs (open squares), the GFP+ fraction of eMIGR1 retrovirus-transduced cells (open circles), the GFP+Foxp3+ fraction of eMIGR1-Nr4a2 retrovirus-transduced cells (filled triangles), or the GFP+Foxp3+ fraction of eMIGR1-Nr4a2 retrovirus-transduced cells supplemented with IL-2 (5 ng ml−1, crosses). Cells were cultured for 96 h with anti-CD3/anti-CD28-coated beads. The ratio of responder cells with more than three divisions are presented. Bottom: results in the top panel at the 1:1 ratio of regulatory cells/Tresp are presented. Numbers in FACS plots represent percentages of CD4+ T cells in the gated area. Data are representative of three independent experiments (mean and s.d. of triplicate). **P<0.01 (two-tailed Student′s t-test).
Next, we characterized Nr4a2-induced Foxp3+ cells by analysing the expression profile of Treg effectors and suppression activity. Expression levels of Foxp3 mRNA in Nr4a2-induced Foxp3+ cells were one-quarter to one-third of that in restimulated Tregs (Fig. 4a). IL-2 was completely repressed, and CD25, a subunit of the high affinity Il-2 receptor complex, was higher than that in Tregs, suggesting that Nr4a2-induced Foxp3+ cells can consume IL-2 by ligand–receptor interactions, similar to Tregs. Although TGF-β1 and GITR were highly expressed in the Nr4a2-induced Foxp3+ cells, IL-10 was repressed, and CTLA-4 required a long interval for induction. These data indicate that Nr4a2-induced Foxp3+ cells expressed similar but distinctive set of Treg effectors.