Decidual CD4+CD25+CD127dim/− regulatory T cells in patients with unexplained recurrent spontaneous miscarriage

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Abstract

Objectives

To investigate the frequency and function of CD4+CD25+CD127dim/− regulatory T (Treg) cells in decidua of patients with unexplained recurrent spontaneous miscarriage (URSM).

Study design

The decidual lymphocytes from patients who experienced URSM and normal pregnant women (controls) were collected by Ficoll density gradient centrifugation. CD4+CD25+CD127dim/− Treg cells were isolated by magnetic cell sorting. The proportion of Treg cells and IL-10, TGF-β in Treg cells were determined by flow cytometry. Inhibitory effects of Treg cells on effecter T cells were detected with or without the presentation of anti-IL-10 antibodies and anti-TGF-β antibodies.

Results

The frequency of CD4+CD25+CD127dim/− Treg cells was decreased in URSM decidua compared to controls (2.09% ± 0.86% vs. 2.97% ± 1.19%, p = 0.005), and the expression of IL-10 and TGF-β in Treg cells was lower in the URSM group than in the control group (p = 0.04 and p = 0.01, respectively). Furthermore, the suppressive effect of CD4+CD25+CD127dim/− Treg cells on the proliferation of effector T cells was decreased in URSM decidua (p < 0.05). Suppression was mediated predominantly through IL-10 and TGF-β in decidua.

Conclusions

Decreased frequency and immunosuppressive capacity of CD4+CD25+CD127dim/− Treg cells was found in URSM decidua. Treg cells inhibit proliferation of effector T cells mainly via IL-10 and TGF-β in URSM decidua.

Introduction

Recurrent spontaneous miscarriage, defined as two or more consecutive pregnancy losses, occurs in 1–5% of reproductive age women, and has a negative effect on human reproductive health [1], [2]. Although the causes of recurrent spontaneous miscarriage involve genetic, anatomic, endocrinologic, infectious, and autoimmunologic abnormalities, in some cases no cause can be identified. Recent research has shown that unexplained recurrent spontaneous miscarriage (URSM) is primarily associated with the failure of feto-maternal immunologic tolerance [3], and that regulatory T (Treg) cells are necessary for the maternal immune system to tolerate paternal alloantigen expressed by the fetus [4]. The CD4+CD25+ Treg cell subset plays an important role in the induction of tolerance, and shows potential regulatory properties in both the induction and maintenance phases of tolerance to alloantigen [5].

Foxp3 has been described as a specific marker for CD4+CD25+ Treg cells [6], but the intracellular staining procedure required for its determination renders this marker unsuitable for the routine analysis of CD4+CD25+ Treg cells in patients. Until recently, the determination of human Treg cells was based solely on the combined expression of CD4 and CD25 markers. Among CD4+CD25+ Treg cells, however, only cells expressing the highest level of CD25 (CD25high cells) are thought to possess regulatory properties in humans [7]. Due to lack of a reliable, generally accepted method for the detection of CD25high Treg cells, the reported percentages of CD25high Treg cells measured in healthy individuals have been highly variable [8].

Recent reports have shown that low levels of the IL-7 receptor-chain (CD127) are expressed on Treg cell surfaces. The expression of CD127 is inversely correlated with Foxp3 expression and the suppressive function of CD25high Treg cells [9], [10]. These findings indicate that CD127 could be used as a new biomarker in human Treg cell determinations. Detection of the combination of CD4, CD25, and CD127 may enhance the purification of Treg cells and improve the previous isolation method based on other cell surface markers.

In our previous study on CD4+CD25+ Treg cells in URSM, we found that the proportions of CD4+CD25bright T cells in URSM patients were statistically significantly lower than those in control women [11]. The present study is the first to characterize CD4+CD25+CD127dim/− as a biomarker for the identification of Treg cells, and to explore its frequency and influence on effector T cell function in URSM.

Section snippets

Patients

Twenty-one patients who had at least three successive spontaneous early miscarriages of unexplained etiology were recruited to participate in the study. The diagnosis of “unexplained” miscarriage was made as in a previous report [2]. For a relevant control group, we randomly selected 30 healthy women who were undergoing elective termination during the first trimester. In all 30 cases, fetal heart activity had been identified. There were no significant differences in the age and pregnancy

Proportion of Treg cells in decidual lymphocytes

By flow cytometry analysis, the proportion of CD4+CD25+CD127dim/− Treg cells was 2.09% ± 0.86% in total decidual lymphocytes in URSM and 2.97% ± 1.19% in controls. The proportion of this subset of Treg cells was significantly reduced in URSM (p = 0.005). Foxp3 was introduced as another cell surface marker in Treg detection to compare the proportion of Foxp3 that was highly expressed (Foxp3+) in CD4+CD25+ Treg cells in the decidua of the two groups. The proportion of decidual CD4+CD25+Foxp3+ Treg

Comments

With their immunosuppressive effect, the CD4+CD25+ Treg cells are recognized as a critical subset of Treg cells in the establishment of tolerance to alloantigen. In animal experiments, it was reported that CD4+CD25+ T cells increased from an early stage of pregnancy [4]. Such an increase was also observed in syngeneic pregnant mice. It was suggested that the increase in CD4+CD25+ Treg cells might be alloantigen-independent. Zenclussen and colleagues reported that adoptive transfer of CD4+CD25+

Acknowledgement

This work was supported by a grant of the National Natural Science Foundation of China (Shanghai) (30530740/C030304).

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