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 Why The JI? Submit online. by guest on September 24, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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 abor- tions (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 neuropi- lin-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