Resistance of Foxp3+ Regulatory T Cells to Nur77-Induced Apoptosis Promotes Allograft Survival

Ran Tao,Wayne W Hancock,Derya Unutmaz

doi:10.1371/journal.pone.0002321

Ran Tao, Wayne W Hancock + Show 1 more

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https://doi.org/10.1371/journal.pone.0002321

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Abstract

The NR4A nuclear receptor family member Nur77 (NR4A1) promotes thymocyte apoptosis during negative selection of autoreactive thymocytes, but may also function in mature extrathymic T cells. We studied the effects of over-expression of Nur77 on the apoptosis of murine peripheral T cells, including thymic-derived Foxp3+ regulatory (Treg) cells. Overexpression of Nur77 in the T cell lineage decreased numbers of peripheral CD4 and CD8 T cells by ∼80% compared to wild-type (WT) mice. However, the proportions of Treg cells were markedly increased in the thymus (61% of CD4+Foxp3+ singly positive thymocytes vs. 8% in WT) and secondary lymphoid organs (40–50% of CD4+Foxp3+ T cells vs. 7–8% in WT) of Nur77 transgenic (Nur77Tg) mice, and immunoprecipitation studies showed Nur77 was associated with a recently identified HDAC7/Foxp3 transcriptional complex. Upon activation through the T cell receptor in vitro or in vivo, Nur77Tg T cells showed only marginally decreased proliferation but significantly increased apoptosis. Fully allogeneic cardiac grafts transplanted to Nur77Tg mice survived long-term with well-preserved structure, and recipient splenocytes showed markedly enhanced apoptosis and greatly reduced anti-donor recall responses. Allografts in Nur77Tg recipients had significantly increased expression of multiple Treg-associated genes, including Foxp3, Foxp1, Tip60 and HDAC9. Allograft rejection was restored by CD25 monoclonal antibody therapy, indicating that allograft acceptance was dependent upon Treg function in Nur77Tg recipients. These data show that compared to conventional CD4 and CD8 T cells, Foxp3+ Tregs are relatively resistant to Nur77-mediated apoptosis, and that tipping the balance between the numbers of Tregs and responder T cells in the early period post-transplantation can determine the fate of the allograft. Hence, induced expression of Nur77 might be a novel means to achieve long-term allograft survival.

Highlights

Nur77 is an inducible orphan nuclear receptor comprised of an N-terminal AF1 transactivation domain, a DNA binding domain containing two zinc fingers, and a C-terminal ligand binding domain
We found by quantitative real-time PCR, that expression of the inducible immediate early gene, Nur77, was upregulated within 4 h of in vitro stimulation by PMA/ionomycin (Figure 1a) or CD3 monoclonal antibody (Figure 1b), and returned to basal levels by 24 h in both non-thymic-derived Foxp3+ regulatory (Treg) and Tregs (p.0.05)
We found that Nur77 formed a complex with HDAC7 and Foxp3 in resting Tregs (Figure 1c), to that of HDAC7 and Foxp3 [19], and intranuclear staining for Nur77 protein showed that Nur77 was expressed by a small population of naive CD4+ T cells, especially CD4+ Foxp3+ T cells, but not by naive CD8+ T cells (Figure 1d)

Summary

Introduction

Nur ( known as NR4A1, TR3, NGFI-B or NAK1) is an inducible orphan nuclear receptor comprised of an N-terminal AF1 transactivation domain, a DNA binding domain containing two zinc fingers, and a C-terminal ligand binding domain. Transgenic mice expressing dominant-negative Nur are defective in the process of negative selection whereas positive selection proceeds normally [3,5]. Nur77-deficient mice are phenotypically normal, suggesting functional redundancy between Nur and related proteins [6]. Aspects of the mechanism by which Nur induces cell apoptosis remain unclear. T cell receptor (TCR) activation induces HDAC7 phosphorylation by the serine/ threonine kinase PKD1, and phosphorylated HDAC7 binds to 143-3 protein and is exported from the nucleus, leading to depression of Nur and other gene targets [7,8,9,10]. Nur downstream target genes responsible for regulating T cell apoptosis remain to be identified.

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