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An efficient immune defense against pathogens requires sufficient basal sensing mechanisms that can deliver prompt responses. Type I IFNs are protective against acute viral infections and respond to viral and bacterial infections, but their efficacy depends on constitutive basal activity that promotes the expression of downstream genes known as IFN-stimulated genes (ISGs). Type I IFNs and ISGs are constitutively produced at low quantities and yet exert profound effects essential for numerous physiological processes beyond antiviral and antimicrobial defense, including immunomodulation, cell cycle regulation, cell survival, and cell differentiation. Although the canonical response pathway for type I IFNs has been extensively characterized, less is known regarding the transcriptional regulation of constitutive ISG expression. Zika virus (ZIKV) infection is a major risk for human pregnancy complications and fetal development and depends on an appropriate IFN-β response. However, it is poorly understood how ZIKV, despite an IFN-β response, causes miscarriages. We have uncovered a mechanism for this function specifically in the context of the early antiviral response. Our results demonstrate that IFN regulatory factor (IRF9) is critical in the early response to ZIKV infection in human trophoblast. This function is contingent on IRF9 binding to Twist1. In this signaling cascade, Twist1 was not only a required partner that promotes IRF9 binding to the IFN-stimulated response element but also an upstream regulator that controls basal levels of IRF9. The absence of Twist1 renders human trophoblast cells susceptible to ZIKV infection.

TRX-E-002 -1 is active against chemosensitive ovarian cancer cells and spheroid cultures obtaine from ovarian cancer stem cells. (A) TRX-E-002-1 efficacy was tested against two clones of chemosensitive CD44-/MyD88- OCCs. The IC50 was calculated to be at the nM range; (B) phase contrast images showing spheroid integrity/collapse after treatment with TRX-E-002-1.

The effects of Î"b-Twist1 overexpression on spheroids formation and migration. (A) Representative images of spheroid forming assay. (B) Representative images of the migrated cells in migration assay. (C) Quantification of the migrated cells.*P< 0.05 compared to control group.

Establishing a cancer screening biomarker's intended performance requires “phase III” specimens obtained in asymptomatic individuals before clinical diagnosis rather than “phase II” specimens obtained from symptomatic individuals at diagnosis. We used specimens from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial to evaluate ovarian cancer biomarkers previously assessed in phase II sets. Phase II specimens from 180 ovarian cancer cases and 660 benign disease or general population controls were assembled from four Early Detection Research Network or Ovarian Cancer Specialized Program of Research Excellence sites and used to rank 49 biomarkers. Thirty-five markers, including 6 additional markers from a fifth site, were then evaluated in PLCO proximate specimens from 118 women with ovarian cancer and 474 matched controls. Top markers in phase II specimens included CA125, HE4, transthyretin, CA15.3, and CA72.4 with sensitivity at 95% specificity ranging from 0.73 to 0.40. Except for transthyretin, these markers had similar or better sensitivity when moving to phase III specimens that had been drawn within 6 months of the clinical diagnosis. Performance of all markers declined in phase III specimens more remote than 6 months from diagnosis. Despite many promising new markers for ovarian cancer, CA125 remains the single-best biomarker in the phase II and phase III specimens tested in this study. Cancer Prev Res; 4(3); 365–74. ©2011 AACR.

Perspectives on This Article from Ovarian Cancer Biomarker Performance in Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial Specimens

Supplementary Figure 1 from Targeting the Mitochondria Activates Two Independent Cell Death Pathways in Ovarian Cancer Stem Cells

TRX-E-002-1 decreases tumor burden and activates c-jun in vivo. Mice were treated i.p. daily with 50 mg/kg or 100 mg/kg TRX-E-002-1 . (A) quantification of total i.p. tumor burden at end of study; (B) western blot analysis for p-c-Jun and total c-Jun in residual tumors from C, control mice or T, mice treated with TRX-E-002-1 100 mg/kg. Representative western blot from two controls and two TRX-E-002-1- treated mice; (C) quantification total i.p. tumor burden at end of maintenance treatment

TRX-E-002 is more potent than current standard of careMorphological analysis of OCSCs exposed to Cisplatin, Paclitaxel, or TRX-E-002 for 24h.

Supplementary Tables

TRX-E-002-1 given daily at 150 mg/kg is toxic. Once tumors were established and detected by live imaging, mice were treated i.p. with 150 mg/kg TRX-E-002-1 three times weekly. (A) tumor growth curves as measured using mCherry fluorescence area. * p < 0.0001 ; (B) quantification of total i.p. tumor burden at end of study, * p = 0.1245; (C) change in animal weight by the end of study, * p = 0.0013; (D) WBC count, * p < 0.001; (E) Neutrophil count; (F) Lymphocyte count.

The protein levels of Twist1 (A) and p53 (B) in tumor samples. Protein levels were determined by the quantification of western blot band intensity comparing to GAPDH bands as loading control.

Supplementary Figure Legends 1-3 from Targeting the Mitochondria Activates Two Independent Cell Death Pathways in Ovarian Cancer Stem Cells

Cancer stem cells are responsible for tumor initiation and chemoresistance. In ovarian cancer, the CD44+/MyD88+ ovarian cancer stem cells are also able to repair the tumor and serve as tumor vascular progenitors. Targeting these cells is therefore necessary to improve treatment outcome and patient survival. The previous demonstration that the ovarian cancer stem cells are resistant to apoptotic cell death induced by conventional chemotherapy agents suggests that other forms of targeted therapy should be explored. We show in this study that targeting mitochondrial bioenergetics is a potent stimulus to induce caspase-independent cell death in a panel of ovarian cancer stem cells. Treatment of these cells with the novel isoflavone derivative, NV-128, significantly depressed mitochondrial function exhibited by decrease in ATP, Cox-I, and Cox-IV levels, and by increase in mitochondrial superoxide and hydrogen peroxide. This promotes a state of cellular starvation that activates two independent pathways: (i) AMPKα1 pathway leading to mTOR inhibition; and (ii) mitochondrial MAP/ERK kinase/extracellular signal-regulated kinase pathway leading to loss of mitochondrial membrane potential. The demonstration that a compound can specifically target the mitochondria to induce cell death in this otherwise chemoresistant cell population opens a new venue for treating ovarian cancer patients. Mol Cancer Ther; 10(8); 1385–93. ©2011 AACR.

Expression of p53-associated E3 ligase in cancer cell lines. (A) RNA expression of MDM2, Pirh2, and E6-AP1, and TOPORS assessed by QPCR in R182 (p53high/Twist-) and Tara R182 (p53low/Twist+) cells. GADPH was used as reference gene. (B) Protein expression of Pirh2 assessed by Western blot. β-actin was used as loading control.

TP53 mutation status and protein levels of p53 and Twist1 ovarian cancer cell lines. * u = unknown.

A panel of biomarkers may improve predictive performance over individual markers. Although many biomarker panels have been described for ovarian cancer, few studies used prediagnostic samples to assess the potential of the panels for early detection. We conducted a multisite systematic evaluation of biomarker panels using prediagnostic serum samples from the Prostate, Lung, Colorectal, and Ovarian Cancer (PLCO) screening trial.Using a nested case–control design, levels of 28 biomarkers were measured laboratory-blinded in 118 serum samples obtained before cancer diagnosis and 951 serum samples from matched controls. Five predictive models, each containing 6 to 8 biomarkers, were evaluated according to a predetermined analysis plan. Three sequential analyses were conducted: blinded validation of previously established models (step 1); simultaneous split-sample discovery and validation of models (step 2); and exploratory discovery of new models (step 3). Sensitivity, specificity, sensitivity at 98% specificity, and AUC were computed for the models and CA125 alone among 67 cases diagnosed within one year of blood draw and 476 matched controls. In step 1, one model showed comparable performance to CA125, with sensitivity, specificity, and AUC at 69.2%, 96.6%, and 0.892, respectively. Remaining models had poorer performance than CA125 alone. In step 2, we observed a similar pattern. In step 3, a model derived from all 28 markers failed to show improvement over CA125.Thus, biomarker panels discovered in diagnostic samples may not validate in prediagnostic samples; utilizing prediagnostic samples for discovery may be helpful in developing validated early detection panels. Cancer Prev Res; 4(3); 375–83. ©2011 AACR.

Perspectives on This Article from A Framework for Evaluating Biomarkers for Early Detection: Validation of Biomarker Panels for Ovarian Cancer

Supplementary Figure 3 from Targeting the Mitochondria Activates Two Independent Cell Death Pathways in Ovarian Cancer Stem Cells

Cells treated with vehicle control

3-D spheroids treated with vehicle control

Supplementary Figure 2 from Targeting the Mitochondria Activates Two Independent Cell Death Pathways in Ovarian Cancer Stem Cells

TRX-E-002-1 given daily at 100 mg/kg is tolerated. Mice were treated i.p. daily with 50 mg/kg or 100 mg/kg TRX-E-002-1 . (A) change in animal weight by the end of study, * p = 0.0147; (B) WBC count, p > 0.05.

Supplementary Materials from A Framework for Evaluating Biomarkers for Early Detection: Validation of Biomarker Panels for Ovarian Cancer

Pregnancy is a unique condition where the maternal immune system is continuously adapting in response to the stages of fetal development and signals from the environment. The placenta is a key mediator of the fetal/maternal interaction by providing signals that regulate the function of the maternal immune system as well as provides protective mechanisms to prevent the exposure of the fetus to dangerous signals. Bacterial and/or viral infection during pregnancy induce a unique immunological response by the placenta, and type I interferon is one of the crucial signaling pathways in the trophoblast cells. Basal expression of type I interferon-β and downstream ISGs harbors physiological functions to maintain the homeostasis of pregnancy, more importantly, provides the placenta with the adequate awareness to respond to infections. The disruption of type I interferon signaling in the placenta will lead to pregnancy complications and can compromise fetal development. In this review, we focus the important role of placenta-derived type I interferon and its downstream ISGs in the regulation of maternal immune homeostasis and protection against viral infection. These studies are helping us to better understand placental immunological functions and provide a new perspective for developing better approaches to protect mother and fetus during infections.

Lactic acid (LA) metabolism in the tumor microenvironment contributes to the establishment and maintenance of immune tolerance. This pathway is characterized in tumor associated macrophages. However, the role and pathway of LA metabolism at maternal-fetal interface during early pregnancy, especially in decidual macrophage differentiation, are still unclear. Herein, for the first time, we discovered that LA can trigger either M2 or M1 macrophage polarization via oxidative phosphorylation and glycolysis regulation under normoxia or hypoxia, respectively. Also, LA metabolism played a vital role in decidual macrophages-mediated recurrent pregnancy loss (RPL), through HIF-1α/SRC/LDHA pathway. Moreover, blockade of LA intake with AZD3965 (MCT-1 inhibitor) could rescue pregnancy in an abortion-prone mouse model, suggesting a potential therapeutic target in RPL. Collectively, the present study identifies the previously unknown functions of LA metabolism in the differentiation of decidual macrophages in early normal pregnancy and RPL, and provides a potential therapeutic strategy in RPL by manipulating decidual macrophages' functions through LA metabolic pathway.

A successful pregnancy requires the maternal immune system to tolerate an allogeneic fetus. The incidence of preeclampsia and other complications related to impaired fetal tolerance is lower during the second pregnancy than during the first pregnancy. At the same time, compared with normal pregnant women in the previous pregnancy, patients with pregnancy complications in the previous pregnancy also have an increased risk of the disease when they become pregnant again. This difference may be related to the immunological memory of pregnancy. Regulatory T cells (Tregs) are immunosuppressive CD4

Trophoblasts differentiate and form the placenta during pregnancy in a complex and finely orchestrated process, which is dependent on the establishment of maternal-fetal immune tolerance and the proper function of trophoblasts. Trophoblasts express HLA-C and non-classical HLA-Ib molecules (HLA-E, HLA-F, and HLA-G). Numerous studies have shown that the unique expression pattern of the HLA molecules is closely linked to the successful acceptance of allogeneic fetus by the mother during pregnancy. However, some controversies still exist concerning the exact expression and recognition patterns of HLA molecules in different trophoblast subpopulations and cell lines. Thus, we summarize three types of trophoblast subpopulations as well as the common trophoblast lineages. Then, the classification and structural characteristics of HLA molecules were elucidated. Finally, the presence of HLA-C and non-classical HLA-Ib molecules (HLA-E, HLA-F, and HLA-G) in various trophoblasts and cell lines, as well as their potential role in establishing and maintaining normal pregnancy were also discussed. Together, this review will help people comprehensively understand the complex immune interactions between maternal and fetal crosstalk during pregnancy and ultimately better understand the physiological and pathological etiologies of pregnancy.

{Supplementary Data} from Diagnostic Markers for Early Detection of Ovarian Cancer

Purpose: Early detection would significantly decrease the mortality rate of ovarian cancer. In this study, we characterize and validate the combination of six serum biomarkers that discriminate between disease-free and ovarian cancer patients with high efficiency.Experimental Design: We analyzed 362 healthy controls and 156 newly diagnosed ovarian cancer patients. Concentrations of leptin, prolactin, osteopontin, insulin-like growth factor II, macrophage inhibitory factor, and CA-125 were determined using a multiplex, bead-based, immunoassay system. All six markers were evaluated in a training set (181 samples from the control group and 113 samples from OC patients) and a test set (181 sample control group and 43 ovarian cancer).Results: Multiplex and ELISA exhibited the same pattern of expression for all the biomarkers. None of the biomarkers by themselves were good enough to differentiate healthy versus cancer cells. However, the combination of the six markers provided a better differentiation than CA-125. Four models with Conclusions: We describe the first blood biomarker test with a sensitivity of 95.3% and a specificity of 99.4% for the detection of ovarian cancer. Six markers provided a significant improvement over CA-125 alone for ovarian cancer detection. Validation was performed with a blinded cohort. This novel multiplex platform has the potential for efficient screening in patients who are at high risk for ovarian cancer.

Correction: Clin Cancer Res 2008 14: 1065-1072 from Diagnostic Markers for Early Detection of Ovarian Cancer

Supplementary Table 1 from Biological Significance of Prolactin in Gynecologic Cancers

There is increasing evidence that prolactin (PRL), a hormone/cytokine, plays a role in breast, prostate, and colorectal cancers via local production or accumulation. Elevated levels of serum PRL in ovarian and endometrial cancers have been reported, indicating a potential role for PRL in endometrial and ovarian carcinogenesis. In this study, we show that serum PRL levels are significantly elevated in women with a strong family history of ovarian cancer. We show dramatically increased expression of PRL receptor in ovarian and endometrial tumors as well as in endometrial hyperplasia, signifying the importance of PRL signaling in malignant and premalignant conditions. PRL mRNA was expressed in ovarian and endometrial tumors, indicating the presence of an autocrine loop. PRL potently induced proliferation in several ovarian and endometrial cancer cell lines. Binding of PRL to its receptor was followed by rapid phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, mitogen-activated protein kinase/ERK kinase 1, signal transducer and activator of transcription 3, CREB, ATF-2, and p53 and activation of 37 transcription factors in ovarian and endometrial carcinoma cells. PRL also activated Ras oncogene in these cells. When human immortalized normal ovarian epithelial cells were chronically exposed to PRL, a malignant transformation occurred manifested by the acquired ability of transformed cells to form clones, grow in soft agar, and form tumors in severe combined immunodeficient-beige mice. Transformation efficiency was diminished by a Ras inhibitor, providing proof that PRL-induced transformation uses the Ras pathway. In summary, we present findings that indicate an important role for PRL in ovarian and endometrial tumorigenesis. PRL may represent a risk factor for ovarian and endometrial cancers. [Cancer Res 2009;69(12):5226–33]

Background: The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the cause of the Coronavirus Diseases of 2019 (COVID-19). This virus has been responsible for the global pandemic of 2020 and 2021 with over 107 million confirmed cases and over 2.3 million deaths. An important population that deserves meticulous consideration during the COVID-19 pandemic is pregnant woman; however, in many places in the world there is inadequate screening for SARS-COV-2 during pregnancy. We aimed to determine the impact of maternal and neonatal cord blood SARS-COV-2 antibodies and placental transfer ratios in a region with a low screening plan. Methods: We performed a blind study in Babahoyo, Ecuador; one of the SARS-CoV-2epicenters in South America. Blood samples were collected from 100 serum samples collected from pregnant women at the time of delivery along with corresponding neonatal cord blood. SARS-CoV-2 IgG specific antibodies were determined using a enzyme linked immunosorbent assay (ELISA) for IgG and IgM SARS-CoV-2-specific nucleocapsid and spike antigens (DiaPro, Milano, Italy Cat number COV19G.CE). Findings: We observed 32% of pregnant women to be serological positive in areas lacking an appropriate screening program. We also show data that suggests an efficient passive immunization of the fetus to SARS-CoV-2 during pregnancy. Interpretation: There is a high incidence of SARS-CoV-2 infection during pregnancy. The best prevention for protecting the mother from infection is vaccination, which, will provide the fetus and neonate with a strong protection against SARS-CoV-2 infection through passive placental transfer of SARS-CoV-2 specific antibodies. This is especially important because newborns are highly susceptible to many viral infections. Funding: NIAID 1R01AI145829-01 Declaration of Interest: None to declare. Ethical Approval: This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines. This was retrospective study done with samples sent for tests evaluation that were originally not related to this study. A consent waiver was approved since the samples were not collected for the study and the samples used were defined as dischargeable material.

Due to their crucial roles in embryo implantation, maternal-fetal tolerance induction, and pregnancy progression, immune checkpoint molecules (ICMs), such as programmed cell death-1, cytotoxic T-lymphocyte antigen 4, and T cell immunoglobulin mucin 3, are considered potential targets for clinical intervention in pregnancy complications. Despite the considerable progress on these molecules, our understanding of ICMs at the maternal-fetal interface is still limited. Identification of alternative and novel ICMs and the combination of multiple ICMs is urgently needed for deeply understanding the mechanism of maternal-fetal tolerance and to discover the causes of pregnancy complications. Leukocyte immunoglobulin-like receptor subfamily B (LILRB) is a novel class of ICMs with strong negative regulatory effects on the immune response. Recent studies have revealed that LILRB is enriched in decidual immune cells and stromal cells at the maternal-fetal interface, which can modulate the biological behavior of immune cells and promote immune tolerance. In this review, we introduce the structural features, expression profiles, ligands, and orthologs of LILRB. In addition, the potential mechanisms and functions mediated by LILRB for sustaining the maternal-fetal tolerance microenvironment, remodeling the uterine spiral artery, and induction of pregnancy immune memory are summarized. We have also provided new suggestions for further understanding the roles of LILRB and potential therapeutic strategies for pregnancy-related diseases.

Environmental contaminants in ambient air pollution pose a serious risk to long-term metabolic health. Strong evidence shows that prenatal exposure to pollutants can significantly increase the risk of Type II Diabetes (T2DM) in children and all ethnicities, even without the prevalence of obesity. The central nervous system (CNS) is critical in regulating whole-body metabolism. Within the CNS, the hypothalamus lies at the intersection of the neuroendocrine and autonomic systems and is primarily responsible for the regulation of energy homeostasis and satiety signals. The hypothalamus is particularly sensitive to insults during early neurodevelopmental periods and may be susceptible to alterations in the formation of neural metabolic circuitry. Although the precise molecular mechanism is not yet defined, alterations in hypothalamic developmental circuits may represent a leading cause of impaired metabolic programming. In this review, we present the current knowledge on the links between prenatal pollutant exposure and the hypothalamic programming of metabolism.

Genetic and environmental factors impact on the interindividual variability of susceptibility to communicable and non-communicable diseases. A class of ubiquitous chemicals, Per- and polyfluoroalkyl substances (PFAS) have been linked in epidemiological studies to immunosuppression and increased susceptibility to viral infections, but possible mechanisms are not well elucidated. To begin to gain insight into the role of PFAS in susceptibility to one such viral infection, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), male and female C57BL/6 J mice were exposed to control water or a mixture of 5 PFAS (PFOS, PFOA, PFNA, PFHxS, Genx) for 12 weeks and lungs were isolated for examination of expression of SARS-CoV-2-related receptors Angiotensin-Converting Enzyme 2 (ACE2) and others. Secondary analyses included circulating hormones and cytokines which have been shown to directly or indirectly impact on ACE2 expression and severity of viral infections. Changes in mRNA and protein expression were analyzed by RT-qPCR and western blotting and circulating hormones and cytokines were determined by ELISA and MESO QuickPlex. The PFAS mixture decreased Ace2 mRNA 2.5-fold in male mice (p 0.0001), with no significant change observed in females. In addition, TMPRSS2, ANPEP, ENPEP and DPP4 (other genes implicated in COVID-19 infection) were modulated due to PFAS. Plasma testosterone, but not estrogen were strikingly decreased due to PFAS which corresponded to PFAS-mediated repression of 4 representative pulmonary AR target genes; hemoglobin, beta adult major chain (Hbb-b1), Ferrochelatase (Fech), Collagen Type XIV Alpha 1 Chain (Col14a1), 5'-Aminolevulinate Synthase 2 (Alas2). Finally, PFAS modulated circulating pro and anti-inflammatory mediators including IFN-γ (downregulated 3.0-fold in females; p = 0.0301, 2.1-fold in males; p = 0.0418) and IL-6 (upregulated 5.6-fold in males; p = 0.030, no change in females). In conclusion, our data indicate long term exposure to a PFAS mixture impacts mechanisms related to expression of ACE2 in the lung. This work provides a mechanistic rationale for important future studies of PFAS exposure and subsequent viral infection.

In healthy couples over half of the conceptions result in failed pregnancy and around 30% of them occur during implantation defining it as a rate-limiting step for the success of native and in vitro fertilization. The understanding of the factors regulating each step of implantation and immune recognition is critical for the pregnancy outcome. Creation of 3D-cell culture models, such as spheroids and organoids, is in the focus of placental tissue engineering in attempt to resemble the in vivo complexity of the maternal-fetal interface and to overcome the need of laboratory animals and human embryos. We constructed stable, reliable, and reproducible trophoblast Sw71 spheroids which are functional independently of the serum level in the culture media. These models resemble the hatched human blastocyst in size, shape and function and are useful for in vitro studies of the in vivo concealed human implantation. Since Sw71 spheroids produce HLA-C, the only classical MHC molecule indispensable for establishment of the immune tolerance and proper human implantation, they are applicable for the evaluation not only of implantation itself but also of maternal-trophoblasts immune interactions. In addition, Sw71-blastocyst-like spheroids are manipulable in low-volume platform, easy to monitor and analyze automatically under treatment with favorable/detrimental factors.

The tumor microenvironment of ovarian cancer is the peritoneal cavity wherein adipose tissue is a major component. The role of the adipose tissue in support of ovarian cancer progression has been elucidated in several studies from the past decades. The adipocytes, in particular, are a major source of factors, which regulate all facets of ovarian cancer progression such as acquisition of chemoresistance, enhanced metastatic potential, and metabolic reprogramming. In this review, we summarize the relevant studies, which highlight the role of adipocytes in ovarian cancer progression and offer insights into unanswered questions and possible future directions of research.

STUDY QUESTION What is the mechanism of Tim-3+ regulatory T (Treg)-cell accumulation in the decidua during early pregnancy and is its disruption associated with recurrent pregnancy loss (RPL)? SUMMARY ANSWER IL-27 and Gal-9 secreted by trophoblasts activate the Tim-3 signaling pathway in CD4+ T cells and Treg cells and so promote accumulation of Tim-3+ Treg cells, the abnormal expression of IL-27 and Gal-9 is associated with impaired immunologic tolerance in RPL patients. WHAT IS KNOWN ALREADY Tim-3+ Treg cells are better suppressors of Teff cell proliferation, and display higher proliferative activity than Tim-3− Treg cells. Tim-3+ Treg cells are tissue-specific promoters of T-cell dysfunction in many tumors. These cells express a unique factor that influences and shapes the tumor microenvironment. STUDY DESIGN, SIZE, DURATION The animal study included 80 normal pregnant mice. In human study, decidua tissues in the first trimester for flow cytometry analysis were collected from 32 normal pregnant women and 23 RPL patients. Placenta tissues for immunohistochemistry analysis were collected from 15 normal pregnant women. Placenta tissues for western blot analysis were collected from 5 normal pregnant women, 5 RPL patients and 5 women who have experienced one miscarriage. Blood samples for in vitro experiments were collected from 30 normal pregnant women. This study was performed between January 2017 and March 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS In this study, we investigated the kinetics of Tim-3+ CD4+ T-cell accumulation, and the proportions of Tim-3+ Treg cells throughout murine pregnancies using flow cytometry. We compared Tim-3 expression on decidual CD4+ T cells and Treg cells during normal pregnancies with expression on the same cell populations in women suffering from RPL. IL-27 and Gal-9 transcription and protein expression in the placenta were determined by RT-PCR and western blot, respectively. An in vitro co-culture model consisting of peripheral CD4+ T cells and primary trophoblasts from early pregnancy was used to mimic the maternal–fetal environment. MAIN RESULTS AND THE ROLE OF CHANCE The percentage of Tim-3+ Treg cells present in mouse uteri fluctuates as gestation proceeds but does not change in the spleen. Levels of Tim3+ Treg cells in uteri peaked at pregnancy Day 6.5 (E 6.5), then progressively diminished, and fell to non-pregnant levels by E18.5. In pregnant mice, Tim-3+ Treg cells constituted 40–70% of Treg cells in uteri but were present at much lower abundance in spleens. About 60% of decidual Treg cells were Tim-3 positive at E6.5. Of these decidual Tim3+ Treg cells, nearly 90% were PD-1 positive. However, only about 16% of Tim3− Treg cells expressed PD-1. Blocking the Tim-3 signaling pathway decreased the proportion of Treg cells and led to embryo resorption. Moreover, much lower Tim-3 expression was observed on CD4+ T cells and Treg cells in women who had suffered from RPL at 6–9 gestational weeks compared with those who had normal pregnancies at matched gestations. In a normal pregnancy, Tim-3 expression on decidual CD4+ T cells is induced initially by IL-27. Then Gal-9-Tim-3 interaction promotes differentiation of decidual Tim-3+ CD4+ T cells into Treg cells. IL-27 and Gal-9 cooperatively induced Tim-3+ Treg cells in vitro. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION We did not investigate the kinetics of human decidual Tim-3+ CD4+ T and Tim-3+ Treg cell populations throughout pregnancy due to limited availability of second and third trimester decidua. In addition, functional suppressive data on the decidual Tim-3+ Treg cells are lacking due to limited and low quantities of these cells in decidua. WIDER IMPLICATIONS OF THE FINDINGS These findings might have therapeutic clinical implications in RPL. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by research grants from the National Natural Science Foundation of China (No. 81871186) and National Key Research & Developmental Program of China (2018YFC1003900, 2018YFC1003904). The authors declare no conflict of interest.

Introduction The limited cell number of primary trophoblasts and contamination of trophoblast cell lines promote us to develop a novel stable trophoblast cell line. Method of study Primary trophoblast cells were isolated from first-trimester placenta and telomerase-induced immortalization was used to immortalize these cells. Subsets of cells were then evaluated by flow cytometry using CK7, HLA-G, CD45 and CD14, specific markers for trophoblast cells, extra-villous trophoblast, pan leucocyte and monocyte/macrophage, respectively. Immunofluorescence staining and immunocytochemistry were used to detect CK7 expression in trophoblast cells. The level of secreted human Chorionic Gonadotropin (hCG) was measured by electrochemiluminescence (ECL). The Bio-Plex MAGPIX System was used to analyze the cytokines and chemokines produced by AL07 cell line. Results We were able to isolate primary trophoblast cells from several first-trimester placentas. One clone, AL07 trophoblast cells, isolated from a week 7 placenta, was morphologically stable and positive for the expression of CK7 by immunofluorescence and immunocytochemistry staining. Characterization of AL07 cells reveled that they are CD45 or CD14 negative and had constitutive secretion of hCG and low HLA-G expression. Furthermore, clone AL07 secret high levels of several cytokines and chemokines, including IL-6, IL-8 and VEGF, and moderately secreted MCP-1 IP-10 and RANTES. Discussion We report the successful isolation, immortalization and characterization of AL07 cells, a novel cell clone isolated from first trimester human placenta. The clone is free of contamination of immune cells, and exhibits similar cytokine profile as other trophoblast cell lines. This new cytotrophoblast-like AL07 cell, can be a valuable tool for in-vitro trophoblast studies in the future.

Decidual macrophages are in close contact with trophoblast cells during placenta development, and an appropriate crosstalk between these cellular compartments is crucial for the establishment and maintenance of a healthy pregnancy. During different phases of gestation, macrophages undergo dynamic changes to adjust to the different stages of fetal development. Trophoblast-secreted factors are considered the main modulators responsible for macrophage differentiation and function. However, the phenotype of these macrophages induced by trophoblast-secreted factors and the factors responsible for their polarization has not been elucidated. In this study, we characterized the phenotype and function of human trophoblast-induced macrophages. Using in vitro models, we found that human trophoblast educated macrophages (TEMs) were CD14(+)CD206(+)CD86(−) and presented an unusual transcriptional profile in response to TLR4/LPS activation characterized by the expression of type 1 Interferon beta (IFN-β) expression. IFN-β further enhance the constitutive production of soluble programmed cell death ligand 1 (PD-L1) from trophoblast cells. PD-1 blockage inhibited trophoblast induced macrophage differentiation. Soluble PD-L1 was detected in the blood of pregnant women and increased throughout the gestation. Collectively, our data suggest the existence of a regulatory circuit at the maternal fetal interface wherein IFN-β promotes sPD-L1 expression/secretion by trophoblast cells, which can then initiate a PD-L1/PD-1 mediated macrophage polarization towards an M2 phenotype, consequently decreasing inflammation. Macrophages then maintain the expression of sPD-L1 by the trophoblasts through IFN-β production induced through TLR4 ligation. Circulating PD-L1 might also function as a marker for normal trophoblast immune modulation during early pregnancy.

Deaths from ovarian cancer usually occur when patients succumb to overwhelmingly numerous and widespread micrometastasis. Whereas epithelial-mesenchymal transition is required for epithelial ovarian cancer cells to acquire metastatic potential, the cellular phenotype at secondary sites and the mechanisms required for the establishment of metastatic tumors are not fully determined. Using in vitro and in vivo models we show that secondary epithelial ovarian cancer cells (sEOC) do not fully re-acquire the molecular signature of the primary epithelial ovarian cancer cells from which they are derived. Despite displaying an epithelial morphology, sEOC maintains a high expression of the mesenchymal effector, TWIST-1. TWIST-1 is however transcriptionally non-functional in these cells as it is precluded from binding its E-box by the PcG protein, CBX7. Deletion of CBX7 in sEOC was sufficient to reactivate TWIST-1-induced transcription, prompt mesenchymal transformation, and enhanced tumorigenicity in vivo. This regulation allows secondary tumors to achieve an epithelial morphology while conferring the advantage of prompt reversal to a mesenchymal phenotype upon perturbation of CBX7. We also describe a sub-classification of ovarian tumors based on CBX7 and TWIST-1 expression, which predicts clinical outcomes and patient prognosis.

Introduction Placental viral infections are associated with fetal inflammation and adverse pregnancy outcomes. However, there have been limited studies on how placental macrophages in the villous and adjacent fetal umbilical endothelial cells respond to a viral insult. This study aimed to evaluate the communication between Hofbauer cells (HBCs) and human umbilical vein endothelial cells (HUVECs) during a viral infection. Methods HBCs were either uninfected or infected with the γ-herpesvirus, MHV-68, and the conditioned medium (CM) collected. HUVECs were exposed to HBC CM and the levels of the pro-neutrophilic response markers: IL-8; E-selectin; intercellular adhesion molecule 1 (ICAM-1); and vascular adhesion molecule 1 (VCAM-1) measured by ELISA and qPCR. The role of HBC-derived IL-1β was investigated using an IL-1β blocking antibody (Ab) or IL-1 receptor antagonist (IL-1Ra). Results MHV-68 infection of HBCs induced a significant increase in IL-1β secretion. CM from infected HBCs induced HUVEC expression of IL-8, E-selectin, VCAM-1, ICAM-1 mRNA, and secretion of IL-8. The HUVEC response to the CM of MHV-infected HBCs was inhibited by a neutralizing IL-1β Ab and by IL-1Ra. Discussion Virally-induced HBC IL-1β activates HUVECs to generate a pro-neutrophilic response. This novel cell-cell communication pathway may play an important role in the genesis of fetal inflammation associated with placental viral infection.

Successful pregnancy is a unique situation requires the maternal immune system to recognize and tolerate a semi-identical fetus and allow normal invasion of trophoblast cells. Although efforts have been made, the deep mechanisms of the maternal-fetal crosstalk have not yet been fully deciphered. Immune checkpoint molecules (ICMs) are a group of negative modulators of the immune response that avoid immune damage. They have been extensively studied in the fields of oncology and transplantation, while the latest evidence suggests that they are closely associated with pregnancy outcomes via multiple inhibitory mechanisms. Although studies have mostly demonstrated the regulatory role of the well-known PD-1, CTLA-4 at the maternal-fetal interface, what is unique about the newly discovered multiple ICMs remains a mystery. Here, we review the latest knowledge on ICMs, focusing on the first generation of checkpoints (PD-1, CTLA-4) and the next generation (Tim-3, Tigit, Lag-3, VISTA) highlighting their immunoregulatory roles in maternal-fetal tolerance and decidual vascular remodeling, and their involvement in pathological pregnancies. The content covers three aspects: the characteristics they possess, the dynamic expression profile of their expression at the maternal-fetal interface, and their involvement in pathological pregnancy. In immunotherapy strategies for pregnancy complications, upregulation of immune checkpoints may play a role. Meanwhile, the impact on pregnancy outcomes when using ICMs in clinical cancer treatment during pregnancy is a topic worth exploring. These may serve as a guide for future basic research and clinical applications of maternal-fetal immunity.