Keywords: regulatory T cells, indoleamine 2, 3-dioxygenase, human, decidua
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Copyright © 2004 Blackwell Publishing Ltd Phenotypic characterization of regulatory T cells in the human decidua Correspondence: Dr Jenni Heikkinen, Turku Graduate School of Biomedical Sciences Department of Medical Microbiology, Turku University, Kiinamyllynkatu 13, FIN-20520 Turku, Finland. E-mail: jenni.heikkinen/at/utu.fi Accepted February 10, 2004.  This article has been cited by other articles in PMC. | |||||||||||
Abstract Pregnancy is a unique situation for the maternal immune system. We have studied and identified a CD4+CD25+ regulatory T (Treg) cell population isolated from the human decidua. This mucosal surface in the uterus is in direct contact with semiallogenic fetal cells. We observed that about 14% of the decidual CD4+ T cells have the CD4+CD25+ phenotype. The decidual CD4+CD25+ T cells expressed high frequency of intracellular CTLA-4 (CTLA-4i). The majority of CD4+CD25+CTLA-4i+ cells were also positive for GITR and OX40, typical markers for human Treg cells. The frequency of CD4+CD25+ T cells in the peripheral blood from pregnant women was found to be increased during the first and second trimester of gestation when compared to nonpregnant controls. Being an important molecule for Treg cells, the role of CTLA-4 in the regulation of indoleamine 2,3-dioxygenase (IDO) expression was also examined. The stimulation with CTLA-4Ig did not increase IDO mRNA expression in CD14+ cells from pregnant women, while IFN-γwas observed to up-regulate IDO expression. The presence of Treg cells in the human decidua suggests that these cells are important in protecting the fetus from alloreactive immune responses at the maternal–fetal interface. Keywords: regulatory T cells, indoleamine 2, 3-dioxygenase, human, decidua | |||||||||||
INTRODUCTION During pregnancy fetal semiallogeneic trophoblast cells migrate through the decidua into the myometrium and invade the walls of maternal spiral arteries. Thereby, decidua has an important role in the regulation of immunological responses between the mother and the fetus. Many mechanisms have been demonstrated for the protection of the fetus from the maternal immune system. These mechanisms include the deviation of the maternal immune system towards Th2 type of responses, the expression of HLA-G and Fas ligand in the placenta and the production of interleukin (IL)-10 and tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) at the maternal–fetal interface [1, 2, 3, 4, 5, 6]. Recently, we showed that the major antigen presenting cell (APC) population in the human decidua, CD14+ macrophages, are phenotypically immunoinhibitory [7]. These cells produce spontaneously IL-10 and express IDO transcripts. Decidual CD14+ cells express HLA-DR, but low levels of the costimulatory molecules CD80/CD86 suggesting that they could induce regulatory T (Treg) cells. Human decidua has also recently been shown to contain a small population of immature myeloid DCs [8]. Based on the presence of immunoinhibitory CD14+ mononuclear cells and immature DCs in the human decidua led us to explore the presence of Treg cells and to further study the mechanisms how immunoinhibitory CD14+ macrophages or immature DCs may function to prevent maternal T cell activation against fetal cells. CD4+ T cells expressing constitutively IL-2 receptor alpha chain (CD25) are recognized to be involved in the regulation of immune responses and as an important component of peripheral tolerance [9–11]. Treg cells are included in the CD4+CD25+ T cell population and can be distinguished from the recently activated T cells by the expression of intracellular cytotoxic lymphocyte-associated antigen 4 (CTLA-4). These CD4+CD25+ Treg cells have the ability to inhibit the IL-2 production and the proliferation of both CD4+ and CD8+ T cells. The specific mechanism of the suppressive function of these cells is, still unclear, but it seems to require direct T cell-T cell contact. It has also been shown the Treg cells are able to inhibit the function of APCs [12]. Recently, it was proposed that Treg cells could also modify the function of APCs through CTLA-4-B7 (CD80/CDC86) signalling [13]. The CTLA-4-immunoglobulin (CTLA-4Ig) fusion protein was shown to induce IFN-γ production in dendritic cells (DCs), which led to the expression of IDO in DCs. The resulting tryptophan catabolism was able to protect pancreatic islet allografts in mice from T cell mediated rejection [14]. Many studies have demonstrated the important role of IDO during pregnancy, as blocking the function of this enzyme causes allogenic fetal loss [6,15]. More recently, it has also been shown that DCs producing IDO inhibit T cell proliferation [16]. As the important events in the maintenance of pregnancy are most likely to occur at the maternal–fetal interface we investigated whether Treg cells are present in the maternal decidua, in the close proximity to the semiallogenic fetal tissues, the placenta and the fetal membranes. We have identified a population of CD4+CD25+ T cells, which express intracellular CTLA-4 (CTLA-4i) in the human decidua. These cells also expressed Treg markers glucocorticoid-induced tumour necrosis factor receptor (TNFR) family related gene (GITR) and OX40. Decidual Treg cells are most likely important in the regulation of local maternal tolerance towards the fetus. | |||||||||||
MATERIALS AND METHODS Peripheral blood and decidual cells This study was approved by the joint ethical committee of Turku University Hospital and Turku University and informed consent was obtained from the women participating in this study. The peripheral blood (PB) samples were obtained from the pregnant women visiting Turku University Hospital antenatal care unit and from healthy female controls. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. Fetal membranes were obtained from women delivered by elective caesarean section at term (> 37 weeks of gestation) in Turku University Hospital. In brief, the decidual cells were isolated by scraping the decidual tissue from the maternal surface of the fetal membranes [7,17]. The tissue was digested with DNase I (50 µg/ml), collagenase (300 U/ml) and hyaluronidase (2 mg/ml) in RPMI 1640 culture medium. Ten ml of this enzyme cocktail was used per 1 g wet weight of tissue, with pulsed digestion of 3 × 20 min at 37°C with stirring. After each incubation the tissue was allowed to settle and the supernatant containing released cells was removed. Finally, the dispersed cells were filtered through metal sieve and silk, and washed twice with Hanks’ buffered solution (HBS). Mononuclear cells were then separated with Ficoll-Hypaque gradient centrifugation.Reagents and antibodies In the isolation of decidual cells RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS; Bioproducts for Science, Indianapolis, IN, USA), penicillin and streptomycin (Biological Industries, Kibbutz beit Haemek, Israel) was used. The human decidual cells were cultured in Isocove's modified Dulbecco's medium (Gibco BRL) supplemented with 10% FCS, 1 mmol/l HEPES (Gibco BRL), 0·1 mm 2-mercaptoethanol and 100 µg/ml gentamycin (Biological Industries, Kibbutz beit Haemek, Israel). Antibodies used in the flow cytometry are described in the Table 1. Fix & Perm® cell permeabilization kit was obtained from Caltag Laboratories, Inc. (Burlingame, CA, USA). Anti-CD14 conjugated microbeads for magnetic cell sorting (MACS) was from Miltenyi Biotec (Auburn, CA, USA). The purified recombinant human (rh) CTLA-4Ig chimera and M-CSF were from R & D Systems (Minneapolis, MN, USA) and interferon gamma (IFN-γ) was obtained from DNAX Research Institute (Palo Alto, CA, USA). Enzymes DNase I, hyaluronidase and collagenase used for decidual cell isolation were obtained from Sigma (St. Louis, MO, USA).
Flow cytometry and cell sorting The isolated cells were incubated with FITC- or PE-conjugated mAb for 30 min at 4°C followed by two washes. As negative controls FITC- or PE-conjugated nonspecific mouse IgG was used. The intracellular staining was performed using the Fix & Perm® cell permeabilization kit from Caltag Laboratories, Inc. according to the manufacturer's instructions. The cells were analysed using FACSCalibur flowcytometer and Cellquest software (Becton Dickinson, San Jose, CA, USA). In the PB and decidual cell samples the CD4+ cells were gated and analysed for the expression of surface and intracellular antigens. Mean fluorescence intensity ratio (MFIR) (mean fluorescence intensity with mAb of interest/mean fluorescence intensity with control mAb) was used to compare the intensity of CD25 on CD4+ T cells from the decidua and PB. The cell sorting was done using MACS and CD14 microbeads (Miltenyi Biotec).Cell culture and cytokine measurement The PB CD14+ cells (0·4–0·8 × 106/well) were cultured in flat-bottom 48-well plates (Costar, Cambridge, MA, USA) in medium (IMDM, 10% FCS), with IFN-γ (100 ng/ml) or with rhCTLA-4Ig chimera (10 µg/ml) from R & D Systems for 24 h. Production of IL-10 was measured from the cell culture supernatants by specific ELISA (CLB, Netherlands) with a detection limit of 2 pg/ml.IDO mRNA expression by real-time-PCR Cultured PB CD14+ cells (purity of positive cells ranged between 72 and 90%) were resuspended in TRIzol® (Invitrogen, Life Technologies) total RNA isolation reagent (n = 4). As a positive control for the IDO mRNA expression PB CD14+ cells (0·25 × 106/well) were cultured in the presence of M-CSF (200 U/ml) for five days and on the fifth day IFN-γ (10 ng/ml) was added for 24 h [18]. After the culture the cells were resuspended in Ultraspec (Biotecx, Houston, TX, USA) lysis buffer. RNA isolation was performed as described by the manufacturer. Oligo-p(dT)-primed cDNA were synthesized using avian myeloblastosis virus (AMV) reverse transcriptase in a 20 µl reaction volume (1st Strand cDNA Synthesis Kit for RT-PCR, Roche Diagnostics, Mannheim, Germany). To exclude possible contamination of genomic DNA, AMV reverse transcriptase was omitted from the control RT reactions. Each reaction mixture was incubated at 42°C for 1 h using a DNA thermal cycler (Perkin-Elmer, Norwalk, CT, USA). A heat inactivation step at 99°C for 5 min was performed to denaturate reverse transcriptase, and this was followed by a cooling step to +4°C for 5 min. The reagents and capillaries for Real-Time quantitative PCR (LightCycler™, Roche Molecular Diagnostics, Mannheim, Germany) were purchased from the Roche Molecular Diagnostics. Real-time RT-PCR for the quantification of IDO expression was conducted using Light Cycler – FastStart DNA Master SYBR Green I kit (Roche) according to the manufacturer's instructions. Additional MgCl2 to achieve the optimal final concentration 4 mm for IDO and 2 mm for β-actin, each primer at 10 pmol/µl and 2 µl of 1 : 10 and 1 : 100 diluted template cDNA was used. The specific primers used for IDO were: sense (5′-AACTCCTGGACAATCAG TAAAG-3′) and antisense (5′-ATATATGCGAAGAACACT GAAAAA-3′). As a control constitutively expressed β-actin gene was amplified from the same pool of cDNA to normalize the concentration of cDNA; sense primer (5′-AGCCTCGCCTTTGC CGA- 3′) and antisense (5′-CTGGTGCCTGGGGCG-3′) [19]. At the beginning of thermal cycler program activation of the FastStart Taq DNA Polymerase at 95°C for 10 min was done. PCR amplification steps for IDO (15 s at 95°C, 5 s at 60°C, 28 s at 72°C) and for β-actin (15 s at 95°C, 5 s at 67°C, 9 s at 72°C) were repeated 50 times. Fluorescence was measured at channel F1 after each elongation. Melting curves were done by lowering the temperature to 65°C, then raising the temperature by 0·1°C/ s to 95°C for IDO and to 99°C for β-actin and measuring the fluorescence continuously. The standard curve for IDO was constructed using IDO positive cDNA in four concentrations (1 : 10, 1 : 100, 1 : 1000, 1 : 10 000) and was used for calculations. Finally, the products were analysed by electrophoretic separation on a SeaKem 1·5% agarose gel (FMC Bioproducts, Rockland, ME, USA).Statistical analysis Independent samples student's t-test was used in the statistical analysis of the phenotypic differences. Cytokine concentrations were analysed using Mann–Whitney U-test. | |||||||||||
RESULTS Regulatory T cells are present in the maternal decidua The decidual mononuclear cells were analysed using flow cytometry and the cells expressing CD4 were observed to constitute 2–9% of the total mononuclear cells. As Treg cells are characterized by the expression of IL-2 receptor α-chain, CD25, we next analysed the expression of CD25 on the CD4+ cells from the decidua (Fig. 1); 14 ± 5% of decidual CD4+ cells expressed CD25. The intensity of CD25 expression was higher in the decidual CD4+CD25+ cells when compared to peripheral blood (PB) of nonpregnant controls (mean fluorescence intensity ratio, 1·9 ± 0·3 versus 1·5 ± 0·2, P = 0·050). Intracellular and surface staining was used to assess the CTLA-4 expression. The frequency of cells expressing intracellular and cell surface CTLA-4 was significantly increased in the decidual CD4+CD25+ cell population compared to PB controls from pregnant and nonpregnant women (Table 2).
To further analyse the phenotype of human decidual Treg cells the four-colour staining was used. Cell surface expression of CD40 ligand (CD40L), CD69, OX40 and GITR were analysed in following populations: CD4+CD25+, CD4+CD25-, CD4+CD25+ CTLA-4i+. The expression of TNFR superfamily members GITR and OX40 have been found among cell surface receptors which demarks the CD4+CD25+ Treg cells from the CD4+CD25− fraction [20]. In the human decidual cells the expression of GITR was clearly associated with the CD4+CD25+ (48%) population compared to CD4+CD25− (27%) population and even more in the triple positive CD4+CD25+CTLA-4i+ population (86%). Similar association was observed in the expression of OX40. These results show clearly the presence of Treg cells in the human decidua. The expression CD40L was slightly increased in decidual CD4+CD25− cell fraction when compared to CD4+CD25+ population. The expression of CD69 was increased in decidual Treg cells compared to the corresponding populations in PB. The frequency of CD4+CD25+ T cells was significantly increased in PB CD4+ population during the first and second trimester of pregnancy when compared to nonpregnant controls (25 ± 5·5%, 18 ± 2·9%versus 9·7 ± 6·1%, P > 0·01, Fig. 2). Also the intensity of CD25 expression was increased in PB CD4+CD25+ cells during the first and second trimester of pregnancy when compared to nonpregnant controls (mean fluorescence intensity ratio, 1·9 ± 0·2 P < 0·01, 1·7 ± 0·1 P < 0·05, versus 1·5 ± 0·2).
The role of CTLA-4 and IFN-γ in the regulation of IDO expression in PB CD14+ monocytes from pregnant women We also wanted to study the effects of recombinant CTLA-4Ig, whether it induces an increase in IDO expression through the activation of CD80/CD86 molecules on the PB CD14+ cells from pregnant women. CTLA-4Ig was not observed to increase the IDO mRNA expression in PB monocytes whereas stimulation with IFN-γ clearly up-regulated the IDO mRNA expression (Table 3). However, we observed that although CD14+ cells from PB of pregnant women produced spontaneously high levels of IL-10 (2216 pg/ml, mean, n = 4, Fig. 3), the stimulation with CTLA-4Ig could slightly up-regulate the production of IL-10 (2522 pg/ml). In contrast IFN-γ clearly inhibited IL-10 production (1630 pg/ml).
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Discussion The maternal immune system tolerates the semiallogenic fetus in the uterus, where the fetal derived tissues are in direct contact with the maternal decidua. It is known that maternal immune system is capable of reacting against paternal cells during pregnancy while the fetus is tolerated. Most likely, it is the local immunological events that play a key role in creating and maintaining the maternal tolerance towards the fetus. The CD4+CD25+ T cell population comprise about 14% of the CD4+ T cell population in the term decidua. During first trimester the frequency of CD4+CD25+ cells is estimated to be around 10%[21]. Chao et al. [21] proposed that these cells represent activated T cells. Based on our detailed characterization of these cells, we suggest that the identified CD4+CD25+ T cell subset in the human decidua display the phenotype of Treg cells. High frequency of decidual CD4+CD25+ cells express intracellular CTLA-4. The surface expression of both GITR and OX40 was found in these decidual CD4+CD25+ T cells and was enriched in CD4+CD25+CTLA-4i+ cells. Recently GITR has been proposed to have also a functional role in the suppression mechanisms by Treg cells, as stimulation through GITR on CD4+CD25+ cells has been shown to abrogate the suppressive function of these cells [20,22]. Contradictory evidence has also been presented suggesting that the GITR antigen would be purely a cell surface marker for suppressive Treg cells [23]. Nevertheless, the intensity of the expression of GITR and CTLA-4 has been shown to correlate with the suppressive capacity of Treg cells [23]. OX40 is known to be expressed on activated T cells. OX40L on APCs can provide costimulation to CD4+ T cells inducing proliferation, cytokine production and promote effective memory [24,25]. OX40 signalling has also been suggested to be involved in Th2 differentiation although this is still controversial [26]. Recently, OX40 signals were shown to break an established state of tolerance, which could indicate a negative regulatory role for OX40 signalling in Treg cells [25]. The expression of CD69, an early activation marker of T cells, was also expressed on the decidual CD4+CD25+CTLA-4+ population. This suggests that the decidual Treg cells are activated, as the expression of CD69 has been shown to be increased after activation of Treg cells [27,28]. The clear phenotypic differences between the peripheral blood and decidual CD4+CD25+ cells suggest that decidual Treg cells are activated whereas the cells in PB are rather resting Treg cells. Despite of this association of CD69 with the phenotype of activated Treg cells, the inhibitory function of CD4+CD25+ T cells has been shown to be independent of CD69 expression [29]. Interestingly, the frequency of CD4+CD25+ cells was found to be increased in the PB of pregnant women in the first trimester suggesting that Treg cells function already during early pregnancy. We observed that the frequency of CD4+CD25+ cells was highest during the first trimester but decreased towards term suggesting dynamic behaviour of Treg cells during pregnancy. In addition to the suppressive effects on both CD4+ and CD8+ cells, Treg cells have been shown to inhibit the function of APCs by down-regulating the expression of costimulatory molecules [12]. Furthermore, it was shown that soluble CTLA-4Ig was able to induce expression of IDO in DCs. The ability of Treg cells to induce expression of IDO in APCs through the CTLA-4–CD80/CD86 interaction was suggested as one of the mechanisms of the peripheral tolerance [13,14]. However, CTLA-4Ig was not observed to induce IDO mRNA expression in PB CD14+ cells from pregnant women, it is possible that only a certain DC subset is able to respond to CTLA-4 by producing IDO. Furthermore, also placental cells has been shown to express CTLA-4, which support a role for CTLA-4 in the maternal-fetal immuneregulation [30]. In recent in vivo experiment by Mellor et al. [31] CTLA-4Ig treatment induced IDO expression selectively in specific DC subsets. In the human decidua, four subtypes of APCs have been characterized. We have characterized decidual CD14+ macrophages and shown that these cells display a unique phenotype resembling immunoregulatory immature DCs [7]. In addition, small population of immature myeloid DCs, mature CD83+ DCs as well as CD14+ DC-SIGN+ cells have been shown to be present in early human decidua [8, 32, 33]. The IL-10 producing CD14+ decidual cells [7] and immature DCs [8] could induce differentiation of Treg cells locally in the decidua. The presence of Treg cells and tolerance inducing APCs (either immunoinhibitory CD14+ macrophages or immature DCs) are most likely connected in the human decidua. However, it remains open how decidual Treg cells and APCs interact in the maintenance of maternal tolerance. Human decidua can be compared to the other mucosal surfaces, such as lung and intestine, where DCs producing IL-10 and TGF-β are able to induce the differentiation of Treg cells [34]. We propose that the decidual APCs, being either macrophages or immature DCs, are able to induce Treg cells locally in the uterus. In conclusion, this is the first report to demonstrate the presence of a population of CD4+CD25+CTLA-4i+ Treg cells in the human decidua. As a small number of Treg cells is able to prevent the rejection of transplants [35], it is possible that the number of Treg cells present in the human decidua is sufficient to prevent the maternal alloreactivity and maintain tolerance. Our results also provide new insight into the immunoregulatory events in maternal–fetal interface and further evidence for the special immunosuppressive environment in the human decidua regulated by both APCs and Treg cells. | |||||||||||
Acknowledgments We thank Anna Karvonen and Jasperiina Mattsson for technical assistance. Dr Elli Veistinen is acknowledged for the help and advice concerning the quantification assays using the real-time PCR. This study was supported by the Academy of Finland, the special funds for Turku University Hospital, Turku University Foundation and The Finnish Cultural Foundation of South-western Finland. Dr Jenni Heikkinen is a recipient of a training grant from Turku Graduate School of Biomedical Sciences. | |||||||||||
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