Interface During Early Human Pregnancy Regulatory T Cells Into The

Human Chorionic Gonadotropin Attracts Regulatory T Cells into the Fetal-Maternal Interface during Early Human Pregnancy

This information is current as Anne Schumacher, Nadja Brachwitz, Sindy Sohr, Kurt of September 24, 2021. Engeland, Stefanie Langwisch, Maria Dolaptchieva, Tobias Alexander, Andrei Taran, Sara Fill Malfertheiner, Serban-Dan Costa, Gerolf Zimmermann, Cindy Nitschke, Hans-Dieter Volk, Henry Alexander, Matthias Gunzer and Ana Claudia Zenclussen Downloaded from J Immunol 2009; 182:5488-5497; ; doi: 10.4049/jimmunol.0803177 http://www.jimmunol.org/content/182/9/5488 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2009/04/20/182.9.5488.DC1 Material References This article cites 92 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/182/9/5488.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Human Chorionic Gonadotropin Attracts Regulatory T Cells into the Fetal-Maternal Interface during Early Human Pregnancy1

Anne Schumacher,* Nadja Brachwitz,* Sindy Sohr,† Kurt Engeland,† Stefanie Langwisch,* Maria Dolaptchieva,‡ Tobias Alexander,§ Andrei Taran,*¶ Sara Fill Malfertheiner,*¶ Serban-Dan Costa,¶ Gerolf Zimmermann,† Cindy Nitschke,ʈ Hans-Dieter Volk,‡ Henry Alexander,† Matthias Gunzer,ʈ and Ana Claudia Zenclussen2*

Regulatory T cells (Treg) expand during pregnancy and are present at the fetal-maternal interface at very early stages in pregnancy. The migration mechanisms of Treg to the pregnant uterus are still unclear. Human chorionic gonadotropin (hCG) is secreted by the blastocyst immediately after fertilization and has chemoattractant properties. Therefore, we sought

to analyze whether hCG secreted by early trophoblasts attracts Treg to the uterus and hence contributes to maternal Downloaded from tolerance toward the fetus. Decidua and placenta tissue samples from patients having spontaneous abortions or ectopic pregnancies were employed to evaluate Treg and hCG levels. Age-matched samples from normal pregnant women served as controls. We further performed in vitro studies with primary first trimester trophoblast cells and a choriocarcinoma cell line (JEG-3) aiming to evaluate the ability of secreted hCG to attract Treg. Patients having miscarriages or ectopic pregnancy presented significantly decreased hCG mRNA and protein levels associated with decreased Foxp3, neuropilin-1, IL-10, and ␤ TGF- mRNA levels as compared with normal pregnant women. Using migration assays we demonstrated that Treg were http://www.jimmunol.org/ attracted by hCG-producing trophoblasts or choriocarcinoma cells. Treg migration toward cells transfected with hCG expression vectors confirmed the chemoattractant ability of hCG. Our data clearly show that hCG produced by trophoblasts attracts Treg to the fetal-maternal interface. High hCG levels at very early pregnancy stages ensure Treg to migrate to the site of contact between paternal Ags and maternal immune cells and to orchestrate immune tolerance toward the fetus. The Journal of Immunology, 2009, 182: 5488–5497.

regnancy is a fascinating phenomenon if one keeps in mind creases very early after conception, peaking during the second trimes- that it comprises the tolerance of the fetus, bearing foreign ter to a decline postpartum (9). Treg have been shown to suppress P Ags of paternal origin, by the maternal immune system. The proliferative responses of autologous CD4ϩCD25Ϫ T cells to alloge- by guest on September 24, 2021 transient tolerance during gestation is thought to be achieved, at least neic dendritic cells and therefore to facilitate fetal survival (9, 10). The ϩ ϩ ϩ partially, via the presence of CD4 CD25 Foxp3 regulatory T cells suppressive function of Treg is mediated either via cell-cell contact 3 (Treg). This unique subpopulation, which plays a central role in tol- (11) or by secretion of immunosuppressive cytokines such as IL-10 erance and prevention of autoimmunity (1–3), is known to play an and TGF-␤ (12–16). Additionally, it has been demonstrated that the important role in the survival of allogeneic organ grafts (4–8). Re- proportion of decidual Treg was signiﬁcantly lower in specimens garding pregnancy, it has been found that the number of Treg in- from spontaneous abortion compared with those from induced abortions (10). Furthermore, infertile women have lower expression of

*Department of Experimental Obstetrics and Gynecology, Medical Faculty, Otto- Foxp3 in their endometrium when compared with fertile women (17). von-Guericke University, Magdeburg, Germany; †Department of Obstetrics and Human chorionic gonadotropin (hCG) is a heterodimeric pla- ‡ Gynecology, University of Leipzig, Leipzig, Germany; Institute of Medical Im- cental glycoprotein hormone required to maintain pregnancy. The munology and §Department of Rheumatology and Clinical Immunology, Charite´, Universita¨tsmedizin, Berlin, Germany; and ¶Department of Obstetrics and Gyne- ␣-subunit is identical to that of other glycoprotein hormones such ʈ cology and Institute of Molecular Immunology, Medical Faculty, Otto-von-Gu- as thyroid-stimulating hormone, follicle-stimulating hormone, and ericke University, Magdeburg, Germany luteinizing hormone, whereas the ␤-subunit is unique to hCG (18). Received for publication September 23, 2008. Accepted for publication February 23, ␤ 2009. -hCG is encoded by six different but very similar genes located ␤ The costs of publication of this article were defrayed in part by the payment of page in a gene cluster on chromosome 19 together with the -luteinizing charges. This article must therefore be hereby marked advertisement in accordance hormone gene (19). ␤-hCG1 and ␤-hCG2 were described as pseu- with 18 U.S.C. Section 1734 solely to indicate this fact. dogenes (20, 21). In pregnancy and in germ cell tumors mainly 1 This work was supported by grants from the Deutsche Forschungsgemeinschaft ␤-hCG3, ␤-hCG5, and ␤-hCG8 are expressed, which code for an (Ze526/04-2) to A.C.Z. and the Interdisciplinary Center for Clinical Research (IZKF) ␤ Leipzig (Projekt D02) to K.E. identical protein. In contrast, -hCG7 encodes a protein differing 2 Address correspondence and reprint requests to Dr. Ana Claudia Zenclussen, in three amino acids and is expressed at low levels in normal Experimental Obstetrics and Gynecology, Medical Faculty, Otto-von-Guericke- nonmalignant tissues (20, 22, 23). During pregnancy hCG is ini- University Magdeburg, Gerhart-Hauptmann-Strasse 35, 39108 Magdeburg, Ger- tially produced by the blastocyst 6–8 days after fertilization (24, many. E-mail address: [email protected] 25) and later by the syncytiotrophoblast (26). The level of hCG 3 Abbreviations used in this paper: Treg, regulatory T cells; hCG, human chorionic gonadotropin; HCT116, human colon carcinoma 116; RT, room temperature; Nrp-1, increases during the ﬁrst trimester of pregnancy and decreases to neuropilin-1; LH/CG, luteinizing hormone/chorionic gonadotropin; IVF, in vitro 10% of the peak value during the second and third trimesters (27). fertilization. It is widely known that hCG has immunoregulatory properties, for Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 example, it is able to suppress mitogen-induced responses of T (28, www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803177 The Journal of Immunology 5489

Table I. Numbers and characteristics (mean Ϯ SEM) of normal Foxp3, IL-10, and TGF-␤, real-time PCR was conducted using the ABI pregnant (NP), spontaneous abortion (SA), and extrauterine pregnant PRISM 7700 sequence detection system (PerkinElmer/Applied Biosys- patients (EU) tems) with primers and fluorescent probes, whereas for hCG and neuropilin-1 (Nrp-1) real-time PCR analysis was performed on the iCycler (Bio- Rad) using SYBR Green (Applied Biosystems) for the detection of PCR Samples Age (years) Week of Pregnancy products. Primer and probe sequences are available upon request. Blood NP patients (n ϭ 8, first 27.25 Ϯ 6.08 16.25 Ϯ 2.76 Immunohistochemistry and immunofluorescence and second trimester) Cytokeratin staining was used to differentiate between decidua basalis and NP patients (n ϭ 4, 29 Ϯ 6.08 30.25 Ϯ 0.50 decidua parietalis, which helped analyzing the distribution of hCG-posi- third trimester) tive cells. For cytokeratin and hCG staining, paraffin-embedded tissue sec- Tissue tions containing decidua and placental tissue were dewaxed and washed NP patients (n ϭ 19) 29.47 Ϯ 6.90 10.11 Ϯ 2.84 twice with TBS (pH 7.4). For Ag retrieval, tissue sections were cooked in SA patients (n ϭ 21) 33.69 Ϯ 6.21 8.46 Ϯ 2.45 citrate buffer for 10 min for cytokeratin and 5 min in EDTA buffer (pH 9.0) EU patients (n ϭ 15) 30.33 Ϯ 6.41 4.26 Ϯ 1.82 for hCG staining. After washing in TBS, tissues were treated with 3% hydrogen peroxide in methanol for 20 min at room temperature (RT) to block the endogenous peroxidase activity. Tissues were then blocked with 5% BSA (Sigma-Aldrich) in TBS for 20 min at RT. The primary Abs were 29, 30) and B lymphocytes (31) via induction of suppressor T cells diluted with 5% BSA in TBS as follows: monoclonal mouse anti-human (32). It has also been shown that hCG facilitates trophoblast dif- cytokeratin AE1/AE3 Ab (1/500; Dako) and polyclonal rabbit anti-human ferentiation (33) and invasion by up-regulating molecules support- CG Ab (1/600; Dako) and applied either for 60 min at RT (anti- cytokeratin) or overnight at 4°C (anti-hCG). After washing, the tissue sec- ing the implantation process such as leukemia inhibitory factor Downloaded from tions were incubated with the polyclonal goat anti-rabbit Ig (1/500; Dako) (34, 35). hCG also positively influences angiogenesis by inducing or horse anti-mouse IgG (HϩL) (1:100; Vector Laboratories) for1hatRT. the expression of vascular endothelial growth factor and matrix Then, HRP-conjugated solution (Dako) was added for 30 min at RT. Fi- metalloproteinase 9 (35). Moreover, hCG has a direct influence on nally, the samples were developed with 3-amino-9-ethylcarbazole (Dako), the decidualization process (36, 37). In the clinical practice, hCG counterstained with hematoxylin (Sigma-Aldrich), and mounted with application has been found to support a successful pregnancy out- mounting medium (Dako). Negative controls were performed by replacing the primary Ab with 5% BSA in TBS or by using diluted mouse or rabbit come in women undergoing in vitro fertilization (IVF). Several stud- serum. http://www.jimmunol.org/ ies demonstrated that hCG administration for luteal support in IVF For detection of luteinizing hormone/chorionic gonadotropin (LH/CG) cycles benefits corpus luteum function and thereby increases preg- receptor, Treg were isolated from human peripheral blood of pregnant or nancy rates compared with placebo-treated women (38, 39, 40). nonpregnant women and cocultured with JEG-3 cells for 24 h. After coculture, Treg were washed with PBS containing 1% BSA and incubated Taking into account this background, it seemed interesting to us with a polyclonal rabbit anti-LH/CG receptor Ab (dilution 1/200 in PBS to investigate whether hCG may attract Treg to the fetal-maternal plus 1% BSA) (Acris Antibodies) for1hatRT.After washing, Treg were interface. As no IL-2 is present at the human fetal-maternal inter- stained with FITC-conjugated goat anti-rabbit Ig-specific polyclonal Ab face (41), Treg are likely to migrate from the periphery immedi- (BD Pharmingen) for 30 min at 4°C in the dark. Then, cells were washed ately after being generated. Therefore, the main aim of the present and spread on a glass slide. After drying, Treg were fixed in 100% methanol for 10 min in the dark. Finally, the cells were mounted using a ready- study was to investigate whether Treg are attracted by hCG, pro- to-use mounting medium with 4Ј,6-diamidino-2-phenylindole for counter- by guest on September 24, 2021 duced by the trophoblast, during early pregnancy. staining (Vector Laboratories). Negative controls were performed by replacing the LH/CG receptor Ab with 1% BSA or diluted rabbit serum. Slides were analyzed using a fluorescence microscope (Zeiss Axio) and Materials and Methods images of LH/CG receptor-positive Treg were taken in a magnification of Sample collection ϫ400 (ϫ40 objective and ϫ10 ocular). For analyzing the levels of Treg and hCG in tissue, snap-frozen and par- Transfection of HCT116 cells with hCG plasmid DNA affin-embedded decidua and placenta tissue were obtained from the De- partment of Gynecology and Obstetrics, University of Leipzig. Tissue sam- Complete coding segments of the cDNAs for ␤-hCG3 (according to the ples were obtained from pregnant women undergoing selective termination National Resource for Molecular Biology Information database entry of pregnancy, suffering from spontaneous abortion, or having extrauterine NM_000737) and ␤-hCG7 (NM_033142) and for the hCG ␣-subunit pregnancies (Table I). The tissue sampling was approved by the University (NM_000735) were PCR amplified and cloned as KpnI/XhoI fragments of Leipzig. For migration studies, blood samples were taken from normal into the pcDNA3.1(ϩ) expression vector (Invitrogen). Transfections of pregnant women at the first or second trimester. This was approved by the HCT116 cells with the expression vectors were performed using FuGENE Ethics Board at the University of Magdeburg (study 28/08) as well as by HD transfection reagent (Roche) according to the manufacturer’s instruc- the Ethics Board at the Medical School University of Leipzig (study 254- tions. As a control, cells were transfected with lacZ- or enhanced GFP 2007). The characteristics of the patients included in the study are shown expression vectors. Sixteen hours after transfection, cells were washed in Table I. three times with sterile PBS followed by addition of Opti-MEM medium (Invitrogen). The cells were then used as the bottom layer in a migration Cell lines assay. Primary first trimester trophoblast cells were obtained as described else- ELISA for hCG determination where (42) and further cultured in Medium 199 (Invitrogen) supplemented with 10% FBS (Biochrom) and 50 mg/ml Normocin (Amaxa). Both the Supernatants from cell cultures were analyzed for their hCG content using choriocarcinoma trophoblast cell line JEG-3 and the skin cell line HaCat the ␤-hCG ELISA kit from DRG Instruments. All individual steps were were cultured in DMEM normal growth medium (Invitrogen) supple- performed according to the manufacturer’s instructions. mented with 10% FBS and 100 nM penicillin/streptomycin (Invitrogen).

Cells were cultured as monolayers at 37°C and 5% CO2. Human colon Treg isolation carcinoma 116 (HCT116) cells were cultured in a humidified atmosphere For migration assays, CD4ϩCD25ϩ Treg were isolated from peripheral with 7.5% CO2 at 37°C in McCoy’s 5A medium (Biochrom) supplemented with 10% FBS (Lonza). blood samples from normal pregnant women from the first and second trimester (n ϭ 8) as well as from the third trimester (n ϭ 4) of pregnancy. Real-time RT-PCR The Treg population was obtained using magnetic beads from Miltenyi Biotec, following the instructions of the manufacturer. After isolation, Treg Frozen tissue samples containing decidua and placenta tissue (100 mg) were counted and diluted at 4 ϫ 104 cells/ml in Opti-MEM medium and were treated with 1 ml of TRIzol (Invitrogen) and disaggregated using a used for migration assays. The purity of the isolated Treg was determined homogenizer (Ultra-Turrax T8; IKA). Isolation of RNA, cDNA synthesis, before using the cells for migration assays by staining an aliquot of the and real-time PCR were performed as described elsewhere (43). For cells with CD25-PE (provided with the kit), CD4-FITC (BD Pharmingen), 5490 HCG ATTRACTS Treg TO THE UTERUS Downloaded from http://www.jimmunol.org/ FIGURE 1. hCG mRNA and protein levels are significantly decreased in miscarriages and ectopic pregnancies compared with normal pregnancies. Tissue samples from women suffering from spontaneous abortion (SA) or extrauterine pregnancies (EU) showed significantly diminished hCG mRNA levels when compared with normal pregnant FIGURE 2. hCG protein levels are diminished in miscarriages and ec- women (NP) (A). On the protein level hCG was significantly decreased topic pregnancies compared with normal pregnancies. A–F, Representative in tissue samples from SA cases or ectopic pregnancies as compared images of hCG staining. A and B, Representative decidua basalis samples with samples from normal pregnancy in decidua basalis (B). For mRNA from normal pregnancies (NP) (A) and spontaneous abortions (SA) (B). C and protein analysis each circle represents a single patient and the lines and D, Representative decidua parietalis samples from NP (C) and SA (D) by guest on September 24, 2021 show the medians (n ϭ 4–13/group). Statistical analysis was conducted cases. E, Representative uterine sample from an extrauterine (EU) preg- p Ͻ 0.05 nancy. Nearly no hCG protein expression was detectable in spontaneous ,ء .by Kruskal-Wallis test followed by Mann-Whitney U test p Ͻ 0.01. abortion and extrauterine samples (low staining intensity) as compared ,ءء and with samples from normal pregnancy, which showed a high expression of hCG, especially in decidua basalis (high staining intensity). F, Negative control obtained after replacing the first Ab by diluted rabbit serum to and Foxp3-allophycocyanin (eBioscience) and analyzing them by flow cytometry. The purity of the isolated cells varied between 92 and 97%. About exclude unspecific staining of the Ab. All panels have an ocular magnifi- ϫ 82% of the cells expressed intracellular Foxp3. cation of 200. Migration assay using a two-chamber system and flow cytometry analysis collagen; Collagen Corp.)) at a final collagen concentration of 1.7 mg/ ml. This solution was filled into a small chamber built by a hollowed Both cell lines, JEG-3 and HaCat, were trypsinized and plated overnight at coverslip on a glass slide and allowed to polymerize for 20 min (37°C, 1 ϫ 105 cells/well in their respective growth media (Invitrogen) in 24-well 5% CO2). The remaining space in the chamber was filled with RPMI plates. For migration assays the medium of either JEG-3, HaCat, or trans- 1640 medium. The chamber was then sealed with wax. For video doc- fected HCT116 cells was changed to Opti-MEM medium (1 ml/well). Cell umentation, cells incorporated within collagen lattices were visualized inserts (8 ␮m; BD Falcon) were placed in the wells. Then, 1 ml of Opti- on a conventional upright microscope (Olympus CellR). Time-lapse 4 MEM medium containing isolated Treg (4 ϫ 10 /well for JEG-3 and Ha- video microscopy was used to record the movement of 5–10 cells vis- 4 Cat; 1–2 ϫ 10 /well for HCT116) was filled in each insert, which separates ible within one optical field over a period of up to 16 h with a rate of the Treg in the upper chamber from the adherent cells in the lower cham- three frames per minute. Subsequently, the path of one single Treg was ber. After 4, 8, 24, or 48 h, inserts were removed and supernatants from the reconstructed from the recorded film by computer-assisted cell tracking. lower chamber were taken. Cells were washed with PBS and fixed over- Therefore, time-lapse video movie was displayed on a computer screen. night with 1% paraformaldehyde solution (Carl Roth). After washing, the From the first frame of the time-lapse sequence, three cells (one Treg absolute number of migrated Treg was determined by flow cytometry and two JEG-3 cells) were selected, giving a nonbiased, representative (FACSCalibur; BD Biosciences). In addition to fluorescence microscopy, sample of the mixed cell population. Then, the movement of the Treg the expression of LH/CG receptor in Treg was determined by using flow was individually followed by a trackball over a specific time period. cytometry analysis. Data analysis and statistics Migration assay using a three-dimensional collagen matrix and computer-assisted cell tracking The patterns and intensities of the immunohistochemical staining were evaluated by two independent observers using a light microscope (Zeiss The three-dimensional collagen matrix assay was employed to visualize Axiophot) in a ϫ200 magnification (ϫ20 objective and ϫ10 ocular). The Treg migration to JEG-3 cells using video microscopy. Three-dimen- degree of staining was graduated semiquantitatively from 0 (negative) to 5 sional collagen matrices were prepared as described elsewhere (44, 45). (intense). Data from immunohistochemistry are shown as scatter plots ac- Briefly, 100,000 Treg and 100,000 JEG-3 cells were resuspended in 33 companied by representative images of decidual tissue. ␮l of RPMI 1640 medium (Invitrogen) and embedded within a total of Immunhistochemistry and RT-PCR data are presented as dots for single 100 ␮l of collagen (33 ␮l of cells and 66 ␮l of collagen (dermal bovine samples showing medians due to their non-normal distribution. Hence, The Journal of Immunology 5491

FIGURE 3. Miscarriages and ectopic pregnancies are associated with diminished levels of Foxp3, Nrp-1, IL-10, and TGF-␤ mRNA, and hCG mRNA corre- lates positively with Foxp3 mRNA levels. Foxp3 and Nrp-1 mRNA levels were signiﬁcantly lower in patients having pathological pregnancies as compared with normal pregnant women (A and B). Moreover, IL-10 (C) and TGF-␤ (D) mRNA expression was signiﬁcantly (for IL-10) diminished in women suffering from spontaneous abortion (SA) or ex-

trauterine pregnancies (EU) when com- Downloaded from pared with women with normal pregnancies (NP). Each circle represents a single patient, and the lines show the medians. Statistical analysis was conducted by Kruskal-Wallis test followed by Mann- p Ͻ ,ءء ;p Ͻ 0.05 ,ء .Whitney U test

/p Ͻ 0.001. hCG and Foxp3 http://www.jimmunol.org ,ءءء ;0.01 mRNA expression correlate positively in placenta and decidual tissue for all samples (E). The nonparametric Spearman correlation was used to analyze the cor- .p Ͻ 0.05 ,ء .relation of RT-PCR data by guest on September 24, 2021

statistical difference between all groups was performed using the Kruskal- basalis (Fig. 1B). The implantation of the fertilized egg occurs in Wallis test, while differences between two groups were calculated using the the decidua basalis, and therefore essential tolerance mechanisms nonparametric Mann-Whitney U test. The nonparametric Spearman corre- allowing the acceptance of the fetus are expected to be in this area. lation was used to analyze the correlation of RT-PCR data. Parametric data (e.g., migration assays) were analyzed using a Student’s t test to compare Interestingly, we observed a significant difference regarding differences between two groups, and two-way ANOVAs were used to com- hCG expression in decidua basalis from patients suffering from pare three or more groups. spontaneous abortion or ectopic pregnancies as compared with normal pregnancy. This difference was not so strong when an- Results alyzing decidua parietalis (Fig. 1B). Representative images of Decreased hCG mRNA and protein levels in patients suffering decidua parietalis and basalis tissue sections from all three from miscarriages or ectopic pregnancies as compared with groups are shown in Fig. 2. Taken together, our data lend ad- normal pregnant women ditional support to the already known important role of hCG for hCG has been shown to be indispensable for a successful preg- a successful pregnancy outcome. nancy outcome (46, 47). Here, the expression of hCG in tissue containing decidua and placenta at early pregnancy stages was Miscarriages and ectopic pregnancies are associated with ␤ determined at the mRNA and protein levels in samples from pa- diminished levels of Foxp3, Nrp-1, IL-10, and TGF- mRNA at tients suffering from spontaneous abortion or ectopic pregnancies the fetal-maternal interface and compared with samples from women having normal pregnan- To first analyze whether high hCG levels indicate high numbers cies that were interrupted for social reasons. We observed signif- of Treg or their associated molecules, we next quantified the icantly decreased hCG mRNA levels in tissue samples from pa- levels of Foxp3, Nrp-1, IL-10, and TGF-␤ mRNA in tissues tients with spontaneous abortions or ectopic pregnancies when whose hCG had been analyzed before. Foxp3 was described as compared with normal pregnant women (Fig. 1A). These observa- an essential transcription factor for CD4ϩCD25bright Treg re- tions were confirmed at the protein level when analyzing the sam- sponsible for the development and function of these cells (48). ples stained for hCG (Fig. 1B). Most of the patients having spon- Recently it has been discussed whether Foxp3 is as important in taneous abortions or ectopic pregnancies had nondetectable or very humans as it is in mice for Treg function (49). In addition to low hCG protein expression. In contrast, normal pregnant women Foxp3, Nrp-1 was described as a marker for Treg (50). Regarding showed a very high expression of hCG in decidua cells of decidua Nrp-1, we were able to show that abortion-prone mice express 5492 HCG ATTRACTS Treg TO THE UTERUS

FIGURE 4. LH/CG receptor expression on Treg. A, Representative dot plot showing isolated Treg from a pregnant woman stained with anti- rabbit IgG (FITC-labeled) as a control for unspeciﬁc binding of the second Ab. B, Treg from a pregnant woman stained with rabbit anti-human hCGR followed by anti-rabbit IgG staining.

At least 25% of Treg were positive for Downloaded from the LH/CG receptor (B) when compared with the control (A) for this sample and for the rest of the samples analzyed. C–H, Representative immu- noﬂuorescence images of LH/CG receptor at a single Treg level from either a pregnant woman (F–H)ora http://www.jimmunol.org/ nonpregnant control (C–E). C and F, Counterstaining of the nucleus with DAPI (4Ј,6-diamidino-2-phenylindole); D and G, staining for the LH/CG receptor; E and H show a merge out of C and D or out of F and H. Several LH/CG receptors were shown to be expressed on the surface of a single Treg. C–H, ϫ Ocular magniﬁcation 400. by guest on September 24, 2021

diminished levels of this molecule as compared with normal tissue samples, as no fresh specimens could be used for isolating pregnant mice (51). It has been found that the secretion of IL-10 Treg and analyzing their cytokine production. Our data support and TGF-␤ by Treg is associated with their protective function previous findings suggesting that the presence of Treg at the fetal- (12, 16). Here, we clearly show that patients suffering from maternal interface is associated with a normal human pregnancy spontaneous abortion or ectopic pregnancies had significantly outcome (10, 17), and we confirmed that low hCG levels are ac- lower levels of Foxp3 as compared with normal pregnant companied by low Treg occurrence. women (Fig. 3A). Moreover, Nrp-1 mRNA levels were significantly reduced in patients having spontaneous abortions when Treg are susceptible to hCG chemoattraction by expressing the compared with normal pregnant women (Fig. 3B). Patients suffer- LH/CG receptor ing from spontaneous abortions and ectopic pregnancies showed After confirming the presence of both hCG and Treg at the significantly diminished IL-10 mRNA levels and slightly dimin- fetal-maternal interface from normally progressing pregnancies ished TGF-␤ mRNA levels as compared with the normal pregnant and their correlation, we hypothesized that the presence of hCG group (Fig. 3, C and D). Although not conclusive, data on IL-10 might also be causative for the increased numbers of Treg at the and TGF-␤ are in agreement with lower Treg levels. Correlation fetal-maternal interface by directly attracting Treg. Therefore, analysis of Foxp3 mRNA and hCG mRNA levels revealed a sig- we investigated whether Treg express the LH/CG receptor on nificant positive correlation (Spearman ϭ 0.5954; p Ͻ 0.05) be- their surface, making them susceptible to hCG attraction. We tween both (Fig. 3E), emphasizing the possibility that low hCG were able to show that at least 25% of Treg isolated from pe- levels are related to low Treg number. We next analyzed the levels ripheral blood of pregnant women (week 30 of pregnancy) ex- of mRNA for cytokines known to be secreted by Treg in whole pressed the LH/CG receptor on the surface after exposure to The Journal of Immunology 5493

FIGURE 6. Three-dimensional collagen matrices show Treg contacting JEG-3 cells several times. The image depicts the migration pathway (red

line) of a single Treg (TR) contacting several times two different JEG-3 cells (J1 and J2) (movie available in supplemental material). Cell move- ments were recorded by time-lapse microscopy. TR1 indicates starting point of Treg migration; TR2, ending point of Treg migration.

normal pregnant women from the second trimester (n ϭ 4; different patients tested in independent experiments) were able to mi- Downloaded from grate efficiently to JEG-3 cells (Fig. 5A), which actively produced hCG, as we could observe by ELISA (25 IU/L per 10,000 cells). To investigate to which extent this reflected the physiological sit- uation and to exclude experimental artifacts by using a carcinoma cell line, we repeated the assay using primary trophoblast cells obtained from women undergoing selective termination of preg- http://www.jimmunol.org/ nancy (20 IU/L per 10,000 cells). Comparable results were obtained (Fig. 5B). We further observed that Treg obtained from normal pregnant women from the third trimester (n ϭ 4) were attracted in the same way by trophoblasts (data not shown). To analyze whether and how Treg contacted trophoblasts directly ex FIGURE 5. Treg are attracted by hCG-producing primary first tri- vivo, we proceeded to coincubate JEG-3 cells and isolated Treg mester trophoblast cells and JEG-3 cells. Treg were isolated from pe- together in three-dimensional collagen matrices, which express ϭ ripheral blood of pregnant women from the second trimester (n 4) of many features of true extracellular matrix (45). In fact, Treg ac- by guest on September 24, 2021 pregnancy. Using a two-chamber transwell system, the migration of tively migrated toward JEG-3 cells and contacted them several Treg to JEG-3 cells and primary first trimester trophoblast cells was determined at different time points (0, 4, 8, 24, and 48 h). Treg were efficiently attracted to both JEG-3 cells (A) and primary first trimester trophoblast cells (B).

hCG produced by JEG-3 cells (Fig. 4B). This observation is in accordance with other studies in which 2–50% of Treg were found to be positive for molecules (e.g., CCR2, CCR4, and CD62L) associated with the migration of Treg (52, 53, 54). Additionally, we found LH/CG receptors on Treg at the single- cell level (Fig. 4F–H). We also investigated the LH/CG receptor expression on Treg from age-matched nonpregnant women. We found that, as expected, Treg from nonpregnant women express the LH/CG receptor after exposure to hCG, suggesting that the presence of hCG is sufficient to stimulate the up-regulation of the receptor (Fig. 4C–E). These data clearly demonstrate that Treg have the ability to sense the attraction of hCG, supporting our hypothesis that Treg migrate from the periphery to the fetal-maternal interface along a hCG gradient generated by the trophoblast cells. hCG-producing primary first trimester trophoblast cells as well as the choriocarcinoma cell line JEG-3 attract Treg FIGURE 7. Overexpression of hCG in HCT116 cells line benefits the After confirming the presence of LH/CG receptors on the surface Treg migration. HCT116 cells transfected with both subunits (presenting of Treg, we investigated whether Treg might be attracted by hCG- whole hCG molecule) attracted Treg significantly more than HCT116 cells producing primary first trimester trophoblast cells as well as by transfected with GFP/LacZ (A). Moreover, Treg are attracted in the same choriocarcinoma cell line JEG-3. For this, we used a two-chamber way when HCT116 cells were transfected with the subunits (␣ or ␤) alone transwell system to determine the active migration of Treg to the (B). Data were analyzed using two-way ANOVA to compare two or more .p Ͻ 0.01 ,ءء p Ͻ 0.05 and ,ء .trophoblast cells. We could clearly show that Treg obtained from groups at different time points 5494 HCG ATTRACTS Treg TO THE UTERUS

transfection or by the fact that they can dimerize. Even though the concentration of hCG produced by transfected cells is much higher than those produced naturally by trophoblasts or by JEG-3 cells, the migration rates were comparable, which could be interpretated as a determined amount of hCG needed to attract Treg, which once reached is sufﬁcient for their attraction. More hCG would not mean migration of more Treg, as we did not observe differences in the migration of Treg to different hCG concentrations toward HCT116 cells upon transfection.

Lack of Treg migration to the hCG nonproducing HaCat cell line As a further validation of hCG-dependent attraction of Treg into the fetal-maternal interface, we used the hCG nonproducing keratinocyte cell line HaCat to determine whether Treg were attracted by them as they also produce several chemokines that potentially attract lymphocytes (55, 56, 57, 58). As shown in Fig. 8A,nohCG was detectable in the culture supernatant of HaCat cells as com-

pared with JEG-3 cells. By performing migration assays, we could Downloaded from clearly demonstrate that Treg were exclusively attracted by the hCG-producing JEG-3 cells but not by the hCG nonproducing Ha- Cat cells (Fig. 8B). Our data strongly suggest that hCG secreted by FIGURE 8. Treg are not attracted by a non-hCG-producing cell line trophoblast cells is necessary to attract Treg to the fetal-maternal (HaCat). The keratinocyte cell line HaCat did not produce hCG (A). In contrast, JEG-3 cells produce significant higher levels of hCG (A). The interface. This may explain the low numbers of Treg observed in two-chamber transwell system was used to determine the migration of Treg patients suffering from spontaneous abortion associated with low http://www.jimmunol.org/ (isolated from peripheral blood of 30 wk pregnant women; n ϭ 4) to both hCG levels. the JEG-3 cells and the HaCat cells (B). Nearly no migration could be observed to the non-hCG-producing HaCat cells when compared with the Discussion hCG-producing JEG-3 cells (B). Data from A were analyzed using Stu- Although it is well known that the maternal immune system tol- dent’s t test to compare differences between two groups. Data from B were erates the foreign paternal Ags expressed in the fetus during preg- analyzed using two-way ANOVA to compare two or more groups at dif- nancy, the mechanisms underlying this phenomenon are still under Ͻ ءءء Ͻ ءء Ͻ ء ferent time points. , p 0.05; , p 0.01; , p 0.001. investigation. Treg were described to play an important role in the maintenance of the tolerant state during pregnancy. This unique T cell subpopulation is known to have immunoregulatory properties by guest on September 24, 2021 times, as can be seen in Fig. 6 and in Video1 from the supple- 4 preventing autoimmune diseases (1–3) and allowing the accep- mental material. tance of allografts (4–8). It has been found that Treg increase Overexpression of hCG in HCT116 cells benefits the migration during the very early stages of pregnancy and that this expansion of Treg is essential for a successful pregnancy outcome, as women with impaired augmentation in their Treg level suffer from spontaneous After confirming that Treg migrate to choriocarcinoma cells or abortion and infertility (10, 17, 59). Moreover, it has been dem- primary trophoblasts, both known to secrete hCG, we next sought onstrated that Treg from women with recurrent spontaneous abor- to confirm that hCG is responsible for Treg migration. Therefore, tion were functionally deficient, as higher numbers were required we investigated whether transient overexpression of hCG in to exert a similar magnitude of suppression as compared with Treg HCT116 cells would attract Treg to these cells normally not pro- from fertile women (60). In mice, Treg are generated immediately ducing hCG or attracting Treg. To this end, HCT116 cells were after fertilization and expand at the periphery, being specific for ␣ cotransfected with expression vectors for the -subunit of hCG paternal Ags (61). Accordingly, paternal lymphocyte immuniza- ␤ ␤ together with expression vectors for -hCG3 or -hCG7. First, tion used in the clinical practice to improve pregnancy outcome in production of recombinant hCG by transfected HCT116 cells was women with previous recurrent abortions (62) is able to augment confirmed. Upon transfection, 500-2800 IU/L of hCG could be the number of male-specific suppressor T cells (63). detected in the supernatant depending on the experiment. In con- The pregnancy hormone hCG has been shown to be indispens- trast, control-transfected HCT116 cells were not able to produce able for the establishment of a successful pregnancy (46, 47) and hCG. By performing migration assays using the transfected has been described to have immunoregulatory properties support- HCT116 cells, we demonstrated that Treg were significantly more ing the implantation process of the fetus in the maternal endome- attracted by HCT116 cells producing recombinant hCG in trium (34–37). Treatment of PBMCs with hCG and their further comparison to control-transfected cells that did not produce hCG transfer 2 days after oocyte retrieval was shown to increase the (Fig. 7A). Our results clearly demonstrate that among all factors implantation rates of blastocysts in women suffering from repeated secreted by primary first trimester trophoblast cells or a choriocar- IVF failure (64). Thus, it seemed interesting to us to investigate cinoma cell line, hCG was highly effective in attracting Treg. whether hCG produced by trophoblasts may attract Treg to the ␣ ␤ Moreover, we found that both the - and -subunits of hCG alone fetal-maternal interface. were able to attract Treg efficiently, showing no significant differ- We first performed a descriptive study in which we searched for ␤ ence between the -hCG isoforms 3 and 7 (Fig. 7B), which might a coincidence between high hCG levels and high Treg numbers. be explained by the supraphysiological levels of expression upon We confirmed high hCG levels in normally progressing pregnancies. In sharp contrast, significantly lower mRNA and protein lev- 4 The online version of this article contains supplemental material. els of hCG were found in samples from women suffering from The Journal of Immunology 5495 spontaneous abortion and extrauterine pregnancies. To analyze share this subunit with hCG, are able to attract Treg. This was not whether low hCG levels correlate with decreased number and analyzed in the context of this study and may further open new function of Treg, we measured Foxp3, Nrp-1, IL-10, and TGF-␤ possibilities in studying Treg migration toward hormones. The ob- expression. Foxp3 and Nrp-1, described as specific markers for served high hCG levels after transfection of HCT116 cells might Treg (48, 50), were expressed significantly lower in patients hav- favor the formation of hCG homodimers (hCG␣/␣ or hCG␤/␤). In ing miscarriages as compared with normal pregnant women. Fur- this regard several publications describe the natural occurrence of thermore, hCG mRNA levels correlated significantly with Foxp3 hCG homodimers. Both the hCG␣/␣ and hCG␤/␤ homodimer are mRNA levels, emphasizing that low hCG levels are associated biological stable and activate the LH/CG receptor specifically. Ad- with low Treg number and vice versa. These data are in agreement ditionally, the homodimers can stimulate the signal transduction to with those from other research groups (10, 17). Moreover, IL-10 the same extent as hCG heterodimers (73–76). Thus, we assume levels were significantly diminished and TGF-␤ levels were that Treg migration observed to the ␣-or␤-subunit alone might be slightly diminished in pathological pregnancies compared with a result of the homodimerization of these subunits followed by an normal pregnancies, further suggesting low Treg numbers in activation of the LH/CG receptor on the Treg. As we found that the pathological pregnancies, since Treg are known to affect immune percentage of Treg migration achieved using trophoblast cells was responses by secreting antiinflammatory cytokines at the fetal-ma- higher than the one achieved using hCG-transfected HCT116 cells, ternal interface (14, 61, 67). Thus, our data on low levels of hCG it needs to be clarified further which other factors produced by the coinciding with low levels of Treg-associated molecules suggest trophoblast cells contribute to the overall chemoattractant effect, that hCG may be chiefly responsible for Treg attraction to the albeit to a much lower degree than hCG. To conclusively confirm fetal-maternal interface. that hCG and not other factors orchestrate the main Treg migra- Downloaded from To test whether migration toward hCG is possible, we first an- tion, we attempted to knock down hCG by means of siRNA. Un- alyzed whether Treg express the LH/CG receptor, necessary to fortunately, the best percentage of inhibition that could be obtained respond to hCG-mediated signals and hCG attraction. We were among all the sequences of hCG-specific siRNA was 40% (data able to show that at least 25% of Treg from human peripheral not shown). Although this was not statistically significant, we ob- blood of pregnant donors expressed the LH/CG receptor, with in- served that with this modest reduction in hCG secretion Treg mi- dividual cells having several LH/CG receptor molecules on their gration was reduced 30% as compared with the controls. Unfor- http://www.jimmunol.org/ surface. We also found Treg from nonpregnant women expressing tunately, no commercially available blocking Abs for hCG exist, the LH/CG receptor after coculture with JEG-3 cells producing and the Abs we checked were shown not to have blocking activity hCG, suggesting that the presence of hCG is sufficient to up-reg- in vitro. ulate the LH/CG receptor on the surface of Treg from nonpregnant In an in vivo study it has been shown that hCG administration women. In agreement with our observation, an older study dem- was able to prevent autoimmune diabetes resulting from destruc- onstrated the presence of the LH/CG receptor on human suppres- tion of pancreatic ␤ cells in NOD mice (77). hCG injection was sor T cells (68). However, at the time of this mentioned study it associated here with a significant increase of Treg in spleen and was not possible to define the nature of these cells by analyzing,

pancreatic lymph node, accompanied by an increase in IL-10 and by guest on September 24, 2021 for example, CD25 and Foxp3 expression. Having confirmed that TGF-␤ levels. This study suggests that Treg are attracted to the site Treg were principally susceptible to attraction by hCG by express- of inflammation by hCG to allow suppression of T effector cells ing the receptor, we went ahead studying whether in fact Treg were responsible for the onset of the disease (77). A similar hypothetical attracted by hCG using a two-chamber transwell system. We analyzed the attraction of Treg by primary first trimester trophoblast scenario can be proposed for early implantation. Our observations cells and the choriocarcinoma cell line JEG-3, which is confirmed support Treg migrating to the site in which implantation is taking to secrete hCG. Treg from the second trimester and from the third place, known to be a site of inflammatory conditions (78–80). trimester migrated efficiently to primary trophoblast cells and to In 1985, suppressor T cells were shown to be present in the human decidua (81). Recently, the presence of so-called CD4ϩ JEG-3 cells. This supports our hypothesis of an active migration of ϩ ϩ Treg into the fetal-maternal interface and suggests that hCG may CD25 Foxp3 Treg in decidual tissue was confirmed, further sup- be involved in this migration. However, at this stage we could not porting the important role of Treg at the site of fetal-maternal exclude that Treg are attracted by chemokines generally produced contact (82–85). Knowing that no IL-2 is available at the fetal- in great extent by trophoblasts (69–72). We next conducted addi- maternal interface in mice and humans (41, 65, 66) and IL-2 is tional experiments to determine to which extent hCG directed Treg necessary for Treg to survive and proliferate in vivo, we hypoth- migration toward trophoblasts. We observed no Treg migration in esized that Treg need to migrate to the decidua after generation or the direction of HaCat cells, which are unable to produce hCG as expansion. Data from mice support this hypothesis (61, 86). More- compared with the hCG-producing JEG-3 cells. To finally prove over, it was postulated that after conception macrophages and lym- that hCG is responsible for Treg migration, two isoforms of the phocytes, which produce hCG, infiltrate the endometrium, contrib- hCG ␤-chain, ␤-hCG3 and ␤-hCG7, alone or together with the uting to its function (87–89). Taking this into account, Treg might ␣-subunit, were recombinantly expressed in the hCG nonproduc- be attracted not only by hCG secreted by the trophoblast but also ing HCT116 cells. The synthesis of recombinant hCG (with or by other immune cells secreting hCG at the fetal-maternal inter- without the ␣-subunit) by these cells supported the migration of face. In this context, it was already demonstrated that stimulation Treg when compared with control-transfected HCT116 cells with of monocytes with hCG augmented the production of IL-8 (90) no difference between the different ␤-hCG isoforms used. Hence, known to attract leukocytes (91). In another in vitro study it was Treg migration does not discriminate between the ␤-hCG isoforms shown that hCG is a potent attractor of neutrophils, monocytes, 3 and 7, although in normal pregnancy mainly ␤-hCG3, ␤-hCG5, and lymphocytes at very low doses (92). Moreover, it was found and ␤-hCG8 are expressed (20). These results clearly show that the that the type of migration is dependent on positive concentration ␤-subunit, which is unique to hCG, is already sufficient to attract gradients of hCG (92). An alternative explanation for the presence the Treg. However, the observed migration of Treg to the ␣-sub- of Treg at the fetal-maternal interface may be that they are de unit alone might indicate that also luteinizing hormone, follicle- novo-generated cells with regulatory activity, as, for example, by stimulating hormone, and thyroid stimulating hormone, which TGF-␤ or IDO, both present in the decidua. 5496 HCG ATTRACTS Treg TO THE UTERUS

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