HLA-G Expression/Secretion and T-Cell Cytotoxicity in Missed Abortion in Comparison to Normal Pregnancy.

The main role of HLA-G is to protect the semi-allogeneic embryo from immune rejection by proper interaction with its cognate receptors on the maternal immune cells. Spontaneous abortion is the most common adverse pregnancy outcome, with an incidence rate between 10% and 15%, with immunologic dysregulation being thought to play a role in some of the cases. In this study, we aimed to detect the membrane and soluble HLA-G molecule at the maternal-fetal interface (MFI) and in the serum of women experiencing missed abortion (asymptomatic early pregnancy loss) in comparison to the women experiencing normal early pregnancy. In addition, the proportion of T cells and their cytotoxic profile was evaluated. We observed no difference in the spatial expression of HLA-G at the MFI and in its serum levels between the women with missed abortions and those with normal early pregnancy. In addition, comparable numbers of peripheral blood and decidual total T and γδT cells were found. In addition, as novel data we showed that missed abortion is not associated with altered extravilous invasion into uterine blood vessels and increased cytotoxicity of γδT cells. A strong signal for HLA-G on non-migrating extravilous trophoblast in the full-term normal placental bed was detected. In conclusion, HLA-G production at the MFI or in the blood of the women could not be used as a marker for normal pregnancy or missed abortions.

Validation of the Sw71-spheroid model with primary trophoblast cells.

PROBLEM: Human implantation is a limiting factor for the success of natural and IVF reproduction since about 60% of pregnancy losses occur in the peri-implantation period. The in vitro modeling of human implantation challenges the researchers in accurate recreation of the complex in vivo differentiation and function of human blastocyst in the peri-implantation period. In previous studies, we constructed Sw71-spheroid models, which like human blastocyst undergo compactization, attaches to the endometrial epithelium, invade, and migrate. The aim of this study was to validate the trophoblast Sw71-spheroid model with primary trophoblast cells, derived from healthy women in early pregnancy. METHOD OF STUDY: We performed a direct comparison of Sw71-spheroid model with placenta-derived primary trophoblasts regarding their hybrid phenotype and HLA status, as well as the ability to generate spheroids able to migrate and invade. From the primary trophoblast cells, isolated by mild enzymatic treatment and Percoll gradient separation, were generated long-lived clones, which phenotype was assessed by FACS and immunocytochemistry. RESULTS: Our results showed that cultured primary trophoblasts have the EVT phenotype (Vim+/CK7+/HLA-C+/HLA-G+), like Sw71 cells. In both 3D culture settings, we obtained stable, round-shaped, multilayered spheroids. Although constructed from the same number of cells, the primary trophoblast spheroids were smaller. The primary trophoblast spheroids migrate successfully, and in term of invasion are equally potent but less stable as compared to Sw71 spheroids. CONCLUSIONS: The Sw71 cell line and cultured native trophoblast cells are interchangeable regarding their EVT phenotype (HLA-C+/HLA-G+/Vim+/CK7+). The blastocyst-like spheroids sourced by both types of cells differentiate in the same time frame and function similarly. We strongly advise the use of Sw71 spheroids as blastocyst surrogate for observation on trophectoderm differentiation and function during early human implantation.

Twist1-IRF9 Interaction Is Necessary for IFN-Stimulated Gene Anti-Zika Viral Infection.

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-ß response. However, it is poorly understood how ZIKV, despite an IFN-ß 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.

Functional HLA-C expressing trophoblast spheroids as a model to study placental-maternal immune interactions during human implantation.

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.

Immunity at maternal-fetal interface: KIR/HLA (Allo)recognition.

Both KIR and HLA are the most variable gene families in the human genome. The recognition of the semi-allogeneic embryo-derived trophoblasts by maternal decidual NK (dNK) cells is essential for the establishment of the functional placenta. This recognition is based on the KIR-HLA interactions and trophoblast expresses a specific HLA profile that constitutes classical polymorphic HLA-C and non-classical oligomorphic HLA-E, HLA-F, and HLA-G molecules. This review highlights some features of the KIR/HLA-C (allo)recognition by decidual NK (dNK) cells as a main immune cell population specifically enriched at maternal-fetal interface during human early pregnancy. How KIR/HLA-C axis operates in pregnancy disorders and in the context of transplacental infections is discussed as well. We summarized old and new data on dNK-cell functional plasticity, their selective expression of KIR and fetal maternal/paternal HLA-C haplotypes present. Results showed that KIR-HLA-C combinations and the corresponding axis operate differently in each pregnancy, determined by the variability of both maternal KIR haplotypes and fetus' maternal/paternal HLA-C allotype combinations. Moreover, the maturation of NK cells strongly depends on if or not HLA allotypes for certain KIR are present. We suggest that the unique KIR/HLA combinations reached in each pregnancy (normal and pathological) should be studied according to well-defined guidelines and unified methodologies to have comparable results ease to interpret and use in clinics.