Which types of regulatory T cells are necessary for the maintenance of pregnancy S Saito, T Shima, K Inada, A Nakashima Department of Obstetrics and Gynecology, University of Toyama, Toyama, Japan

Regulatory T (Treg) cells play an important role in the maintenance of allogeneic pregnancy. Recent data have shown the heterogeneity of Treg cells, specific marker for Treg cells, Foxp3, transiently expressed in activated T cells in human. Therefore, we should re-evaluate which type of Treg cells play an essential role in the implantation and pregnancy maintenance of mouse and human. We established a method to detect paternal antigen-specific Treg cells in mice. Importantly, paternal antigen-specific Treg cells accumulated in uterine draining lymph nodes before implantation and also increased in pregnant uterus after implantation. Our data showed that seminal plasma was important to the induction of paternal antigen-specific Treg cells in uterine draining lymph nodes and uterus. These paternal antigen-specific Treg cells expressed CCR5 on their surface, but the population of CCR5+ Treg in decidua did not change in miscarriage with normal chromosomal embryo. Samstein et al. reported that extrathymic Treg cells in placental mammals played a central role in the maintenance of allogeneic pregnancy in mice. But our recent study showed that Hellios, a marker of extrathymic Treg cells, expressed in decidual Treg cells did not change between miscarriage with normal chromosomal embryo and miscarriage with abnormal chromosomal embryo. Further studies are needed to find a suitable marker for paternal antigen specific Treg cells in human.

Potential roles of microRNAs in preeclampsia P Xu1, Y Bai1, M Liu1, L Ji1, Y Wang2, Y Zhao2, W Gao1, H Wang1, Y-X Li1, J Qiao2, Y-l Wang1 1

State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China; 2Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China

Introduction and Objective: MicroRNAs (miRNAs) have been indicated as fine-tune factors in maintaining homoeostasis in many organs including the placenta. Identification of dysregulated miRNAs in preeclamptic placenta and clarifying the gene networks that are regulated by them are likely to be novel steps in understanding the pathogenesis of preeclampsia. Methods: Human placenta and/or plasma samples were derived from a prospective cohort including pregnant women who underwent perinatal care in Peking University Third Hospital (Beijing, China). Real time PCR, luciferase reporter assays, Western blotting, in situ hybridization, and Matrigel invasion assay were used to analyze the expression of the functions of miRNAs. Results: A total of 12 down-regulated miRNAs and 7 up-regulated miRNAs were identified in sPE placentas when compared with the normal pregnant controls. The unique changing patterns of these miRNAs in the chorionic plate and the basal plate of the placentas as well as in the maternal circulations of PE patients were well demonstrated. Several differential miRNAs (miR-195, miR-18a, miR-376c, miR-378-5p, and miR-675) were found to regulate trophoblast cell invasion and/or proliferation through targeting molecules in association with Activin/Nodal signaling. Conclusion: MicroRNAs can server as potential biomarkers for preeclampsia, and the aberrant expression of these small molecules substantially contributes to the deregulation of trophoblast cell activities during the occurrence of preeclampsia.

American Journal of Reproductive Immunology 70 (Suppl. 1) (2013) 1–10 ª 2013 John Wiley & Sons A/S



Special function of NK cells in maintaining fetomaternal tolerance B Fu, XC Li, R Sun, X Tong, B Ling, Z Tian, H Wei School of Life Sciences, University of Science & Technology of China, Hefei, Anhui, China

In contrast with that on T and B cells, the knowledge on the development and functions of NK cells is still in its infancy, especially that on decidual NK cells which have many differences in gene expression, development stages and functions when compared to those in the periphery NK cells. In order to implant and grow, the trophoblast cells from the allograft fetus need to invade the mother tissues and inevitably bring certain inflammatory reaction. How to control these inflammations and maintain tissue homeostasis is the key problem of embryo tolerance. In the current study, we characterised four novel populations defined by CD11b and CD27, which can represent the distinct stages of human NK cells from different tissues. Interestingly, we found that there were large CD11b CD27+ population of NK cells from deciduas. Furthermore, we provided evidence that TH17 cells and local inflammation could occur at the maternal-fetal interface during natural allogenic pregnancies. Additionally, we found that decidual NK cells promoted immune tolerance and successful pregnancy by dampening inflammatory TH17 cells via IFN-~ a secreted by CD56brightCD27+ NK subset. This NK cell-mediated regulatory response was lost in patients who experienced recurrent spontaneous abortions, which resulted in a prominent TH17 response and extensive local inflammation. This local inflammatory response further impacted the regulatory function of NK cells, leading to the eventual loss of maternal-fetal tolerance. Thus, our data suggest that NK cells can serve as key regulatory cells at the maternal-fetal interface by suppressing TH17-mediated local inflammation.

Chemokine CXCL12 and its receptor CXCR4 at the materno-fetal interface during human first trimester pregnancy W Zhou1, M Du2 1

Reproductive Medicine Center, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China; 2Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College, Shanghai, China

Background: To investigate the role of the chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 (CXCL12/CXCR4) axis on the crosstalk between human first-trimester trophoblast cells (TCs) and decidual stromal cells (DSCs) so as to have a better understanding of the molecular mechanism of the interaction between mother and embryo during pregnancy. Methods: Expression of CXCL12/CXCR4 in TCs and DSCs was detected by reverse transcription-polymerase chain reaction and immunochemical staining; the secretion of CXCL12 by TCs, determined by enzyme-linked immunosorbent assay; and the effect of CXCL12 on the biological functions of TCs and DSCs, analyzed using a cell viability assay, flow cytometry and matrigel invasion assay, hence a co-culture model established to investigate the modulation of CXCL12/CXCR4 in the interaction of TCs and DSCs. Results: CXCR4 was transcribed and translated by both human first-trimester TCs and DSCs. Human TCs secreted CXCL12 spontaneously in vitro, but DSCs did not. Human recombinant CXCL12 induced an increase in CXCR4 levels on DSCs by binding to CXCR4, but produced no effect on the PCNA expression of DSCs. CXCL12 induced a significant increase in the invasiveness of both TCs and DSCs via CXCR4 ligation. A co-culture model further confirmed that the enhanced invasiveness of TCs and DSCs induced by CXCL12 was inhibited by anti-CXCR4 or antiCXCL12 neutralizing antibody. Conclusions: TCs-derived CXCL12 can not only enhance their invasive ability in an autocrine manner, but promote the invasiveness of DSCs in a paracrine manner; therefore our data highlight the effect of CXCL12/CXCR4 axis on the co-operation between TCs and DSCs during human first-trimester pregnancy.

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ª 2013 John Wiley & Sons A/S


NK cytotoxicity inhibited by Tim-3Galectin-9 Pathway toward trophoblast during early pregnancy J Sun1, M Yang1, B Kong2, Y Li1, B Dong2, P Li2, W Gao1, B Song1, Q Shao1, C Ma3, X Qu1 1

Institute of Basic Medical Sciences, Qilu Hospital, Shandong University, Jinan, Shandong, China; 2 Institute of Immunology, School of Medicine, Shandong University, Jinan, Shandong, China; 3 Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, Jinan, Shandong, China

The successful immune tolerance at maternal-fetal interface is essential for normal pregnancy outcome. The underlying mechanisms for the maintenance of pregnancy have been proposed but still remain poorly clarified. In the present study, we demonstrated that Tim-3 (T cell immunoglobulin- and mucin-domain-containing molecule-3), a newly identified Th1/Th2 modulation molecule, plays an important role in immune regulation via interaction with its ligand Galectin-9 during early pregnancy. The expression of Tim-3 was significantly up-regulated in peripheral NK cells (pNK), and the abnormal expression of Tim-3 in pNK cells might be associated with unexplained recurrent spontaneous abortion (RSA). In addition, we found that decidual NK cells (dNK) expressed higher level Tim-3 than pNK, and that the increased TGF-b signal may contribute to the upregulation of Tim-3 in NK cells during early pregnancy. With the blocking antibody and siRNA we further demonstrated that Tim-3-Galectin-9 pathway modulated the cytokine production of NK cells and inhibited the cytotoxicity of NK cells toward trophoblast cell line HTR-8. In conclusion, our results demonstrate that Tim-3 may play an important role in maintaining normal pregnancy and may potentially be taken as an indicator to predict the risk of abortion during early pregnancy. Key words: Tim-3, Natural Killer Cells, Cytotoxicity, Cytokine, Early pregnancy.

Objective: To determine the expression of CCL17 and CCL22 in dendritic cells (DC) from human decidua and endometria. Methods: The decidua were collected from normal pregnant women undergoing induced abortion and recurrent spontaneous abortion(RSA) women undergoing early abortion.The endometria were collected from non-pregnant women undergoing abdominal hysterectomy. The mononuclear cells in the decidua and endometria were isolated. DCs were induced by GM -CSF and IL-4 to be cultured in vitro and identified.The expression of CCL17 and CCL22 in DC at mRNA and protein levels was analyzed through realtime PCR and ELISA. Results: The mRNA levels of CCL17 and CCL22 in decidual DC in norm al pregnancy group were 3.04 4–0.40 and 1.83  0.24, respectively, significantly higher than those in endometrial DC in non-pregnancy group (0.85  0.24 and 0.31  0.08, respectively, P < 0.01) and those in decidual DC in RSA group (1.65  0.14 and 0.96  0.09, respectively, P < 0.01). Decidual DC continually and strongly secreted CCL17 and CCL22. The levels of CCLI7 and CCL22 in the normal pregnancy group were significantly higher than those in the non-pregnancy and RSA at the same culture time point (P < 0.01). Conclusion: The expression of CCL17 and CCL22 in the decidual DC of pregnant woman can increase.This may attract more CD4 CD25 regulatory T cells to decidua, thus playing an important role in the establishment of maternal- fetal immune tolerance. Key words: Dendritic cells, Immune tolerance, CCL17, CCL22.

Leptin-enhanced extravillous trophoblast invasion is MT1-MMP dependent and requires the crosstalk between Notch1 and PI3K signaling H Wang, H Cheng, Q Shao, Z Dong, L Zhao, X Qu

Role of CCL17 and CCL22 of dendritic cells in maternal-fetal immune tolerance

Institute of Basic Medical Sciences, Qilu Hospital, Shandong University, Jinan, Shandong, China

L Meilan, L Yunkun, W Yunhui, C Hui, M Lili, Z Jian-Ping

Invasion of uterine tissue by extravillous trophoblast cells (EVT) is essential for the establishment of a successful pregnancy. Normal invasiveness of EVT cells is tightly controlled by variant factors, including cytokines, chemokines and hormones. Leptin is significantly up-regulated in the first trimester of preg-

Department of Obstetrics and Gynecology, Sun Yat-sen Memorial Hospital, Sun Yatsen University, Guangzhou, China

American Journal of Reproductive Immunology 70 (Suppl. 1) (2013) 1–10 ª 2013 John Wiley & Sons A/S



nancy and plays important roles in the regulation of reproductive function. Previous studies have suggested that leptin regulates trophoblast cells invasion, but the underlying mechanisms remain poorly understood. Membrane type 1-matrix metalloproteinase (MT1-MMP), a surface-anchored “master switch”proteinase, plays a crucial role in physiological cells migration, cancer cells invasion and metastasis. The intense expression of MT1-MMP in EVT cells of the first trimester suggests that it might also be involved in the invasion of trophoblasts. In the current study, we characterized the importance of MT1-MMP in the pro-invasion effect of leptin on EVT cells and elucidated its molecular mechanisms. Transwell assay revealed that leptin promoted invasion of the immortalized EVT cell line HTR-8/SVneo in vitro in a dose- and time-dependent manner. Further studies showed leptin promoted HTR-8/SVneo cells invasion by up-regulating MT1-MMP expression, and knockdown of MT1-MMP by small interference RNA (siRNA) blocked the pro-invasion effect of leptin. Notably, leptin promoted the expression of Notch1 receptor and activated its signaling in EVT cells, and the knockdown of Notch1 signaling by siRNA inhibited both leptin-enhanced invasiveness and MT1-MMP expression of EVT cells, which suggested an significant role of Notch1 pathway in this process. Moreover, Notch1 triggered the activation of PI3K-Akt signaling, and the blockage of PI3K pathway by specific inhibitor (LY294002) suppressed significantly leptin-promoted MT1-MMP expression. Taken together, our observations suggest that leptinenhanced MT1-MMP expression can be one of the important mechanisms in the invasion of EVT cells, and this process requires the crosstalk between Notch1 and PI3K-Akt signaling pathway. The findings contribute to a better understanding of the precise mechanism underlying EVT cells invasion, and provide new strategies for the prevention and treatment of pregnancy complications.

HCG to promote the proliferation of decidual stromal cell by up-regulating the auto-secreted IL-25 via the PI3K-AKT signaling pathway

Background: Th2 bias contributes to the establishment and maintenance of successful pregnancy. Interleukin-25 (IL-17E), the newest member of the IL-17 cytokine family, has been implicated as an important immune regulator in Th2 cell-mediated immune responses. Therefore IL-25 might play an important role in the fetal-maternal interface. Objective: To investigate the hormone regulation and role of IL-25 in decidual stromal cells (DSC) and the signaling pathway involved; to compare levels of IL-25 and its receptor in DSC from the normal pregnant women and abortion patients. Methods: We used flow cytometry (FCM) and ELISA to detect the expression and secretion of IL25 and its receptor IL-17BR in DSC, and FCM to detect whether pregnancy-related hormones could regulate the level of IL-25 and IL-17BR. In addition, we used BrdU method to detect the proliferation of DSC and Western Blot to observe the activated signaling pathway after HCG and IL-25 treatment. We compared the levels of IL-25 and IL-17BR in DSC from normal pregnant women and abortion patients via immunohistochemistry and Western Blot. Results: DSC constitutively expressed IL-17RB and secreted IL-25 in a time-dependent manner and IL25 promoted the proliferation of DSC by activating the PI3K-AKT and JNK signaling pathway. Moreover, HCG up-regulated the level of IL-25 and IL17BR and promoted the proliferation of DSC, and PI3K-specific inhibitor LY294002 completely attenuated HCG- or IL-25-induced proliferation of DSC. By Western Blot and immunohistochemistry, we confirmed that DSC from the miscarriage patients expressed reduced levels of IL-25 and IL-17BR when compared with those from the normal early pregnancy. Conclusion: Our data supported the hypothesis that DSC could promote the proliferation of itself by auto-secreting IL-25, and this secretion could be regulated by the pregnancy-related hormones in the feta-maternal interface. Our findings indicate that IL-25 in the fetal-maternal interface plays a positive role in the process of pregnancy, which might be a potential treatment target for miscarriage.

Y Wang, M Li, L Jin Hospital and Institute of Obstetrics & Gynecology, Fudan University Shanghai Medical College, Shanghai, China

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The expression and function of CXCL12/ CXCR4 at the maternal-fetal interface in human early pregnancy M Du Hospital and Institute of Obstetrics & Gynecology, Fudan University Shanghai Medical College, Shanghai, China

Chemokine CXCL12 and its receptor CXCR4 have been implicated as a pivotal player in many physiological and pathological conditions, but little is known about the expression and function of CXCL12/CXCR4 at the materno-fetal interface. Our group has demonstrated that CXCL12 was derived much more from trophoblasts and from decidual stromal cells (DSCs) at the maternal-fetal interface while CXCR4 was expressed on trophoblast, DSCs. Furthermore, around 80% of peripheral natural killer (NK) cells and 40% of decidual NK cells were CXCR4-positive. CXCL12/CXCR4 interaction triggered the activation of EGF receptor and ERK signaling pathway, involving in proliferation and invasion of human trophoblast cells. CXCL12 secreted by human trophoblasts enhanced the coordination between trophoblasts and DSCs via the regulation of MMP9 and MMP2, improving trophoblasts invasiveness, hence is a precise intercellular crosstalking at maternal-fetal interface, which contributed to placental formation. Additionally, the human first-trimester trophoblast cells recruited peripheral NK cells into decidua by expressing and secreting chemokine CXCL12. Then trophoblasts instructed the peripheral blood-origined NKs to have such characteristics as low cytotoxity, preferentially expressing immunosuppressive molecules and Th2-type cytokines via CXCL12/CXCR4 signaling. Intriguingly, decidual CXCR4+- not CXCR4 - NKs produced a high level of IL-4, and decidual CXCR4-expressing NK cells induced immune tolerance via an interleukin-4dependent Th2 differentiation and conversion of CD4+CD25+Foxp3+ T regulatory cells. Together, it can be concluded that CXCL12/CXCR4 axis plays an essential role in human early pregnancy, improving placental development and inducing maternal-fetal tolerance.

Cyclosporin A enhancing the ability of trophoblast to displace the activated human umbilical vein endothelial cell monolayers C Tang, L Chen, W Gu, M Du, M Li, Q Chen, D Li Hospital and Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College, Shanghai, China

Introduction: Transformation of the spiral arteries including the displacement of vascular endothelial cells by extravillous trophoblasts is an essential prerequisite to normal placentation. Our previous study in vitro had shown that the activated endothelial cells resisted the invasion of trophoblasts, and that CsA promoted the migration and invasion of human first-trimester trophoblast cells. In the present study, we further investigated whether CsA could promote the ability of trophoblasts to displace the activated human umbilical vein endothelial cell (HUVEC) monolayers and the possible molecular mechanism. Methods: CsA pretreated and red fluorescently labeled Jar cells were added to monolayers of green fluorescently-labeled HUVECs that had been exposed to either necrotic Jar cells or TNF-.The ability of Jar cells to displace HUVECs from the monolayers was examined using confocal microscopy. The effect of CsA on the expression of titin, E-cadherin, matrix metalloproteinases (MMPs) activity and CXCL12 secretion of Jar cells were evaluated by western blot, gelatin zymography and enzyme-linked immunosorbent assay (ELISA), respectively. Results: We found that less of the activated HUVECs were displaced from the monolayers by untreated Jar cells, but more of the activated HUVECs were displaced by CsA-pretreated Jar cells. Moreover, CsA pretreatment up-regulated titin expression, downregulated E-cadherin expression, improved MMP2 and MMP9 activity and increased the CXCL12 secretion. Conclusion: In conclusion, these results indicate that CsA may improve the trophoblast invasion to the normal and activated HUVEC monolayers through different downstream targets, thus contributing to the spiral arteries remodeling. The findings may help provide a rationale for the development of a novel therapeutic strategy in treating pregnancy disorders from insufficient trophoblast invasion.

American Journal of Reproductive Immunology 70 (Suppl. 1) (2013) 1–10 ª 2013 John Wiley & Sons A/S



TECK-drived CD4+Foxp3+ regulatory T cells enhancing proliferation and invasiveness of endometrial stromal cells via cross regulation in the endometriotic milieu

Cyclosporine A to enhance Th2 Bias at the maternal-fetal interface in early human Pregnancy with aid of the Interaction between maternal and fetal cells

M Li1, Y Wang1,2, K Chang1, Y Meng1, L Liu1, L Jin1, D Li1

H Piao


Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, Fudan University Shanghai Medical College, Shanghai, China; 2Department of Assisted Reproduction, Shanghai Ninth People’s Hospital Affiliated Shanghai JiaoTong University School of Medicine, Shanghai, China

Endometriosis is associated with an abrogated immune response, displaying some features of an autoimmune disorder. CD4+Foxp3+ regulatory T (Treg) cells play a suppressive role in the development of autoimmune diseases. Thymus-expressed chemokine (TECK)/CCL25 stimulates the invasion of endometrial stromal cells (ESCs) in an autocrine manner in the progression of endometriosis. The aim of the current study was to investigate the mechanism of ESCsderived TECK on the cross-talking between Tregs and ESCs in the endometriotic milieu. The percentage of Tregs and the concentration of TECK in peritoneal fluid were found to increase with the progression of endometriosis. The supernatant of ESCs and macrophage co-culture up-regulated CCR9 expression on Na€ıve T cells, promoted the differentiation and IL-10, TGF-b and CD73 expressions of Tregs partly by activating AKT and STAT3 signal pathways, down-regulated Fas and FasL and inhibited the apoptosis of Tregs, and further enhanced the suppressive function of Tregs to CD4+CD25 T cells. Moreover, these effects could be partly abrogated by anti-TECK neutralizing antibody. In turn, Tregs-secreted IL-10 and TGF-b increased the expression of Metalloproteinase 2/9 (MMP2/9) and Cox-2, decreased tissue inhibitor of metalloproteinase 1 (TIMP1) expression, and further stimulated the proliferation and invasiveness of ESCs in the endometriotic milieu. These results suggest that TECK derived of ESCs and macrophage interaction may up-regulate the number and function of Tregs, which can result in immunotolerance and high invasiveness of ESCs in the ectopic milieu that contributes to the onset and progression of endometriosis. Key words: regulatory T cells, endometrial stromal cells, TECK, IL-10, TGF-b, endometriosis.

Hospital and Institute of Obstetrics & Gynecology, Fudan University Shanghai Medical College, Shanghai, China

Our previous study had demonstrated that cyclosporine A (CsA) administration in vivo induced Th2 bias at the maternal-fetal interface, leading to improved murine pregnancy outcomes. In the current study, we investigated how CsA treatment in vitro induced Th2 bias at the human maternal-fetal interface in early pregnancy. The cell co-culture in vitro in different combinations of component cells there was established to investigate the regulation of CsA on cytokine production from the interaction of these cells. It was found that interferon (IFN)-c was produced only by decidual immune cells (DICs), and not by trophoblasts or decidual stromal cells (DSCs); all these cells secreted interleukin (IL)-4, IL-10, and tumor necrosis factor (TNF)-a. Treatment with CsA completely blocked IFN-c production in DICs and inhibited TNF-a production in all examined cells. CsA increased IL-10 and IL-4 production in trophoblasts co-cultured with DSCs and DICs although CsA treatment did not affect IL-10 or IL-4 production in any of the cells when cultured alone. These results suggest that CsA can promote Th2 bias at the maternal-fetal interface by increasing Th2-type cytokine production in trophoblasts with DSCs and DICs, while inhibiting Th1-type cytokine production in DICs and TNF-a production in all investigated cells. Such findings might be useful in clinical therapeutics for spontaneous pregnancy wastage and other pregnancy complications.

Angiogenesis promoted by NME1 suppression of endometrial stromal cells in the endometriotic milieu by stimulating the secretion of IL-8 and VEGF K Chang, D Li Hospital and Institute of Obstetrics & Gynecology, Fudan University Shanghai Medical College, Shanghai, China

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Nometastatic gene 23-H1 (NME1, also known as nm23-H1) is a wide-spectrum tumor metastasis suppressor gene that plays an important role in suppressing the proliferation, adhesion and invasion of endometrial stromal cells (ESCs). The present study was undertaken to explore the mechanism by which NME1 in ESCs from endometriosis modulates the angiogenesis and herein participates in the pathogenesis of endometriosis. The expression of NME1 in the primary ESCs from normal endometrium without endometriosis was found to be higher than that from eutopic endometrium and ectopic lesion with endometriosis. Silencing NME1 stimulated the secretion of angiogenic factors interleukin-8 (IL-8) and vascular-endothelial growth factor (VEGF) of the eutopic ESCs from women with endometriosis, and these effects could be abrogated by MAPK/ERK1/2 or AKT inhibitor. In addition, the supernatant of NME1-silenced ESCs increased the expression of angiogenesis-relative molecules CD62E and CD105, and promoted angiogenesis of human umbilical vein endothelial cells (HUVECs). Anti-human IL-8 or VEGF neutralizing antibody reversed the effect on angiogenesis of HUVECs induced by NME1-silenced ESCs. Our current results suggest that the abnormal lower expression of NME1 in ESCs can secrete more IL-8 and VEGF through activation of MAPK/ERK and AKT signal pathways, up-regulate the level of CD62E and CD105, thus leading to numerous angiogenesis of vascular endothelial cells in the endometriotic milieu, which is beneficial to the origin and development of endometriosis.

The modulation of collagen from human trophoblasts and decidual stromal cells in early pregnancy on decidual NK cells Q Fu, Y Tao, H Piao, Y Li, M Du, DJ Li Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics & Gynecology, Shanghai Medical School, Fudan University, Shanghai, China

Objective: To determine the effect of collagen expressed at the maternal-fetal interface on decidual NK cells phenotype and cytokines secretion. Methods: Decidual and villious samples were collected from women with normal pregnancy and miscarriage. The expression of collagen and its receptor LAIR-1 were analyzed by masson and immunohistochemical staining. Flow cytometry (FCM) was

employed to detect the expression of cell surface molecule (LAIR-1, NKp30, KIR2DL1) and intracellular molecule (perforin, IFN-c, TNF-a, interleukin-4 (IL-4) and IL-10) by NK cells, and enzyme-linked immunosorbent assay (ELISA), to measure the secretion of cytokine IFN-c and TNF-a produced by decidual NK cells. The coculture of dNK with trophoblast and DSCs were established to determine the effect of tropboblasts and DSCs on NK cells. Human recombinant LAIR-2 was used to block the interaction of collagen with LAIR-1. Konck-down of LAIR-1 was obtained by transfection of HTR-8 and DSC cells with plasmid pPGK-GFP-hP4H. The silence was confirmed by qPCR and ELISA assays. Results: Decidual stromal cells and placental trophoblasts from normal pregnancy showed higher collagen expression than those from miscarriage, which was accompanied by higher LAIR-1 expression on decidual NK cells from normal pregnancy. Collagen treatment increased LAIR-1 expression in dosedependent manner. Coculture of dNK with HTR-8 and DSCs upregulated LAIR-1 expression on dNK cells, which was attenuated by pretreatment with LAIR-2. Collagen treatment down-regulated cell surface active receptor NKp30 and intracellular perforin expression while up-regulating the expression of suppressive receptor KIR2DL1. The secretion of cytotoxic cytokine IFN-c and TNF-a by dNK decreased with collagen treatment. Furthermore, coculture of dNK with HTR-8/DSCs decreased perforin expression and Th1-type IFN-c and TNF-a production while upregulating Th2-type IL-10 and IL-4 production by dNK. All these effects were abrogated by pretreatment with LAIR-2. In addition, transfection of HTR-8/DSCs with pPGK-GFP-hP4H rather than control plasmid decreased significantly collagen expression. Inhibition of collagen expression in HTR-8/DSCs by shRNA attenuated significantly the regulation of HTR-8/ DSCs on dNK cytotoxity and cytokine production. Conclusion: From trophoblasts and DSCs collagen can induce LAIR-1 expression by dNK in the early human pregnancy. The interaction of collagen and LAIR-1 can modulate dNK cell cytotoxity and cytokine expression profile, favoring maternal-fetal tolerance and maintenaning normal pregnancy. The decreased expression of collagen and its receptor LAIR-1 at the maternal-fetal interface can be involved in human pregnancy loss.

American Journal of Reproductive Immunology 70 (Suppl. 1) (2013) 1–10 ª 2013 John Wiley & Sons A/S



CCL24 regulating the function of cd T cells at the maternal-fetal interface in human first-trimester pregnancy H Li, D-J Li Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, IBS, Fudan University Shanghai Medical College, Shanghai, China

Objective: To test the expression of co-stimulatory molecules, the proliferation or apoptosis associated molecules in cd T cells, and to evaluate the potential effect of CCL24/CCR3 axis on the biological function of cd T cells. Methods: We examined the levels of CCL24 and CCR3 at the maternal-fetal interface in human firsttrimester pregnancy by immunohistochemistry, ELISA and flow cytometry. We purified cd T cells from DICs (decidual immunal cells), detected the expression of nine co-stimulatory molecules on their surface, and then evaluated the regulatory function of CCL24 on co-stimulatory molecules. Additionally, we tested the expression of proliferation or apoptosis associated molecules in cd T cells, and evaluated the regulatory role of CCL24 on those molecules. Results: By immunohistochemistry, the cytomembrane and cytoplasm of trophoblasts were moderately stained for CCL24; by ELISA, trophoblasts were cultured in vitro, the secretion level of CCL24 showed a peak at 24 h and then decreased with duration along. As measured by flow cytometry, about half of T cells in DICs were CD3+ cd T cells, which expressed a high level of CCR3. By magnetic isolation, the purity of decidual cd T cells reached 90 %. About 8.62  4.78 % of cd T cells were CD69+. Among nine co-stimulatory molecules, ICOS, GITR, CD40L and NKG2D were highly expressed; OX-40 and PD-1 were moderately expressed, while the levels of CTLA-4, CD28, and NKG2A were low. Both rhCCL24 and the culture supernatant of trophoblasts inhibited the expression of ICOS, GITR, and PD-1. When anti-CCL24 or antiCCR3 was added to the supernatant, the inhibitory function of CCL24 was suppressed. Moderate levels of Ki67, Bcl2 and Fas were also detected, whereas FasL was low. After cd T cells treated with rhCCL24 or trophoblasts culture supernatant, the expression of Ki67 and Bcl2 in cd T cells was inhibited. When anti-CCL24 or anti-CCR3 was added to the culture supernatant, the inhibition was suppressed.

Conclusion: Trophoblasts secrete CCL24, and cd T cells highly express CCR3 at the maternal-fetal interface in human first-trimester pregnancy. cd T cells are rich in some co-stimulatory molecules as well as in proliferation or apoptosis associated molecules. As we proved, CCL24/CCR3 axis could hamper the expression of GITR, ICOS and PD-1, and then participate in the regulation of cd T cell activation. Also, CCL24 can inhibit proliferation associated molecules Ki67 and Bcl2, and play a negative role in cd T cells accumulation at decidua.

Characterization of human firsttrimester decidual CCR10+Tregs C Sun, S Wang, M Du, D Li Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics and Gynecology, Fudan University, Shanghai, China

Previous studies have showed that Tregs play an important role in the maintenance of maternal-fetal immune tolerance during pregnancy. Not as a homeostasis T cell subset, however, Tregs could express multiple chemokine receptors. In the present study, decidual immunocompetent cells from human first-trimester pregnancy were isolated and analyzed by flow cytometry, which turned out the results that more than 10 percent dedidual Tregs were CCR10+ decidual Tregs. In addition, the percentage of CCR10+Tregs in total Tregs from unexplained miscarriage was higher than that in normal pregnancy. Phenotype of CCR10+ decidual Tregs was then further determined by flow cytometry in the aspect of Treg-related molecules, chemokine receptors and intergrins. Our results indicated that compared to CCR10 decidual Tregs, CCR10+decidual Tregs expressed a higher level of CTLA4, TGF-b1, IL-10, CD45RA, CCR7, CXCR3, CXCR6, CD103, CD29 and aVb5, a lower level of CXCR4 and Foxp3. Additonally, over 80 percent of decidual Tregs were CXCR4+Tregs. Both CXCR4 ligand SDF-1 and CCR10 ligand CCL28 could recruit Tregs, which was evaluated by migration assay and flow cytometry. Interestingly, our results demonstrated that SDF-1 mainly recruited CCR10 Tregs not CCR10+Tregs. CCL28 treatment in vitro induced down-regulation of Foxp3 expression and up-regulation of CCR10 and another CCL28 receptor CCR3. In conclusion, decidual Tregs, a heterogeneous population, can express a variety of chemokine receptors. Decidual Treg contains a American Journal of Reproductive Immunology 70 (Suppl. 1) (2013) 1–10


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subset of CCR10+ Tregs with a unique phenotype which increases in the unexplained miscarriage. SDF-1 mainly recruits CCR10 Tregs, and CCL28 recruits Tregs and down-regulates their Foxp3 expression. CCR10 and CCR3 expression is also upregulated by CCL28. CCL28 can be a good complement of SDF-1 in terms of recruitment of CCR10 + Treg which involves in miscarriage.

CD56bright and CXCR4 reflecting different populations of natural killer cells and its function in promoting immune tolerance at human maternal-fetal interface

Hyaluronan-CD44 interaction promotes growth of decidual stromal cells in human first-trimester pregnancy

Natural killer (NK) cells accumulate at the maternal– fetal interface in large numbers, but their exact role in maintaining successful pregnancy remains poorly understood. Chemokine CXCL12 and its receptor CXCR4 have been known as important factors both in physiological and pathological circumstances. Our previous studies have demonstrated that human first-trimester trophoblast cells produced CXCL12, which in turn chemoattracted CXCR4 positive peripheral NK cells, and that more than 90% of peripheral NK cells and 40% of decidual NK cells were CXCR4 positive. Interestingly, CXCR4 positive and negative decidual NK cells were totally different subsets of decidual NK cells. CXCR4 negative decidual NK cells expressed relatively high phenotypical markers including activating and inhibitory receptors, with high secretion of TNF-a and IFN-c, while CXCR4 positive decidual NK cells expressed extremely low phenotypical markers, with high secretion of IL-4 and IL-10. When co-cultured with trophoblast cells, peripheral blood-origined CXCR4 positive NK cells were instructed into lower cytotoxity, immuno-suppressive molecules expression and Th2type cytokines secretion CXCR4 positive decidual NK phenotype via CXCL12/CXCR4 signaling. Given the findings that CD56brightCXCR4+ decidual NK cells could readily secrete high levels of IL-4 and that IL4 can inhibit the polarization of TH1 cells, we designed an in vitro co-culture system and confirmed that activated human decidual CXCR4 positive NK cells could induce TH2 differentiation through the production of IL-4. Our results indicate that decidual NK cells can antagonize directly TH1 cells through an IL-4-dependent mechanism. Furthermore, we also found that decidual CXCR4 positive NK cells could induce immune tolerance via conversion of CD4+CD25 T cells into CD4+CD25+Foxp3+ T regulatory cells. Together, our findings support a model in which NK cells especially the CD56brightCXCR4+ subset antagonize TH1cells through IL-4 secretion and regulatory T cells conversion at maternal-fetal

R Zhu, S-C Wang, C Sun, Y Tao, H-L Piao, X-Q Wang, M-R Du, D-J Li Hospital and Institute of Obstetrics & Gynecology, Fudan University Shanghai Medical College, Shanghai, China

Hyaluronan (HA) and its receptor CD44 are expressed at the maternal-fetal interface, but its role in early pregnancy remains unclear. Here, we found that primary decidual stromal cells (DSCs) continuously secreted HA and expressed its receptor CD44. Pregnancy-associated hormones up-regulated HA synthetase (HAS)2 transcription and HA release from DSCs. High molecular weight-HA (HMW-HA), but not medium molecular weight (MMW-HA) or low molecular weight (LMW-HA), promoted proliferation and inhibited apoptosis of DSCs in a CD44dependent manner. The in-cell Western analysis revealed HMW-HA activated PI3K/AKT and mitogen-activated protein kinase (MAPK)/ERK1/2 signaling pathways time-dependently. Blocking these pathways by specific inhibitor LY294002 or U0126 abrogated HMW-HA-regulated DSC proliferation and apoptosis. Finally, we have found that HA content, HA molecular weight, HAS2 mRNA level, and CD44 expression were significantly decreased in DSCs from unexplained miscarriage compared with the normal pregnancy. Collectively, our results indicate that higher level and greater molecular mass of HA at maternal-fetal interface contributes to DSC growth and maintenance of human early pregnancy. Key words: Hyaluronan, CD44, decidual stromal cell, proliferation, apoptosis.

Y Tao, D Li Hospital and Institute of Obstetrics & Gynecology, Fudan University Shanghai Medical College, Shanghai, China

American Journal of Reproductive Immunology 70 (Suppl. 1) (2013) 1–10 ª 2013 John Wiley & Sons A/S



interface and is critical for maintaining tolerance at early human pregnancy .

Functional regulation of Tim-3 on the maternal-fetal crosstalk in human early pregnancy S Wang, M Du, D Li Hospital and Institute of Obstetrics & Gynecology, Fudan University Shanghai Medical College, Shanghai, China

moted the local tolerance and help maintain the function of DSC, suggesting that the abnormality of Tim-3 in pregnant woman may be connected to pregnancy loss.

Regulatory T cells and gestational diabetes: the link between exhausted phenotype and disease risk S Sharma Women & Infants Hospital-Brown University, Providence, RI, USA

It is important for the result of pregnancy to have the local tolerance and well-functional decidual stromal cells at the maternal-fetal interface. In the current study, we found that during normal pregnancy, Th1, Th17, Treg, DC and CD8+T Tim-3 were higher than those during miscarriage; that Th1, Th2, Th17 and CD8+T Tim-3 were higher in decidua than in PBMC during normal pregnancy; both CD4 and CD8+T cells Tim-3/PD-1 expression were higher than that of miscarriage and PBMC; and that the dual or single expression of Tim-3 and PD-1 on Th2 cells was higher than that of Th1. Moreover, we found that the Tim-3+PD-1+population contained the largest fraction of effector/memory (CD44hiCD62Llow) cells but the lowest fraction of central memory (CD44hiCD62Lhi) cells. And the majority of the expanded population of T cells at the maternal-fetal interface was Tim-3+PD-1+, while the proliferation of Tim-3 PD-1 T cells was restricted. More importantly, Tim-3/PD-1 both positive T cells, the production of IL-4, IL-10 and TGF-b1 was much higher. Tim-3 preserved DSCs from apoptosis through MEK signaling. Ligands of different TLRs induced apoptosis of DSCs while raising the expression of Tim-3 on DSCs; consequnetly the up-regulated Tim-3 repressed TLRLs-mediated DSC apoptosis, and this protection of Tim-3 also accomplished via MEK signaling. Tim-3 down-regulated INF-r and IL-17A while up-regulating IL-4, IL-5, IL-10 and TGF-b1of DSC, respectively. Additionally, Tim-3 was higher in DSC and deciduas to miscarriage when compared to normal pregnancy. In conclusion, our data showed that an immune regulatory molecule Tim-3, through its up-regulation at maternal-fetal interface, pro-

It is hypothesized that inflammation-associated metabolic syndrome resulting in excessive increase in insulin resistance and high free glucose levels at the maternal-fetal interface and in circulation is associated with gestational diabetes mellitus (GDM). However, the definitive cause of this excessive inflammation and autoimmune type phenomenon associated with GDM is still unknown. As regulatory T cells (Tregs) are vital players in maintaining immune tolerance during pregnancy, we evaluated the phenotype and function of Tregs in peripheral blood from GDM patients and age-matched normal pregnancy individuals. We demonstrate that although Tregs are represented in similar numbers with signature phenotypic (CD4+CD25+FoxP3+) features in both GDM and normal pregnancy, GDM Tregs harbor “exhausted” phenotype, characterized by high expression of negative regulatory receptors such as PD-1 as compared to normal pregnancy Tregs. Uncharacteristically, GDM Tregs lose their signature phenotype ex vivo. Notably, PD-1 blockade upregulated Foxp3 expression and rescued immunosuppressive activity of GDM Tregs. PD-1 expression on GDM Tregs could be detected as early as 14 weeks of pregnancy. Expression of PD-1 on GDM Tregs is an epigenetic event whereby the PD-1 locus undegrgoes demethylation. These studies demonstrate for the first time that PD-1 expression negatively regulates Treg function in GDM and could serve as a predictive marker for this disease.

American Journal of Reproductive Immunology 70 (Suppl. 1) (2013) 1–10


ª 2013 John Wiley & Sons A/S

Abstracts of the Third International Conference on Reproductive Immunology. September 28-30, 2013. Shanghai, China.

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