Development of properly-polarized trophoblast stem cell-derived organoids to model early human pregnancy.

The development of human trophoblast stem cells (hTSC) and stem cell-derived trophoblast organoids has enabled investigation of placental physiology and disease and early maternal-fetal interactions during a stage of human pregnancy that previously had been severely restricted. A key shortcoming in existing trophoblast organoid methodologies is the non-physiologic position of the syncytiotrophoblast (STB) within the inner portion of the organoid, which neither recapitulates placental villous morphology in vivo nor allows for facile modeling of STB exposure to the endometrium or the contents of the intervillous space. Here we have successfully established properly-polarized human trophoblast stem cell (hTSC)-sourced organoids with STB forming on the surface of the organoid. These organoids can also be induced to give rise to the extravillous trophoblast (EVT) lineage with HLA-G + migratory cells that invade into an extracellular matrix-based hydrogel. Compared to previous hTSC organoid methods, organoids created by this method more closely mimic the architecture of the developing human placenta and provide a novel platform to study normal and abnormal human placental development and to model exposures to pharmaceuticals, pathogens and environmental insults. Motivation: Human placental organoids have been generated to mimic physiological cell-cell interactions. However, those published models derived from human trophoblast stem cells (hTSCs) or placental villi display a non-physiologic "inside-out" morphology. In vivo , the placental villi have an outer layer of syncytialized cells that are in direct contact with maternal blood, acting as a conduit for gas and nutrient exchange, and an inner layer of progenitor, single cytotrophoblast cells that fuse to create the syncytiotrophoblast layer. Existing "inside-out" models put the cytotrophoblast cells in contact with culture media and substrate, making physiologic interactions between syncytiotrophoblast and other cells/tissues and normal and pathogenic exposures coming from maternal blood difficult to model. The goal of this study was to develop an hTSC-derived 3-D human trophoblast organoid model that positions the syncytiotrophoblast layer on the outside of the multicellular organoid.

Modeling human peri-implantation placental development and function†.

It is very difficult to gain a better understanding of the events in human pregnancy that occur during and just after implantation because such pregnancies are not yet clinically detectable. Animal models of human placentation are inadequate. In vitro models that utilize immortalized cell lines and cells derived from trophoblast cancers have multiple limitations. Primary cell and tissue cultures often have limited lifespans and cannot be obtained from the peri-implantation period. We present here two contemporary models of human peri-implantation placental development: extended blastocyst culture and stem-cell derived trophoblast culture. We discuss current research efforts that employ these models and how such models might be used in the future to study the "black box" stage of human pregnancy.

Regulatory T cells decrease invariant natural killer T cell-mediated pregnancy loss in mice.

Pregnancy loss is the commonest complication of pregnancy. The causes of pregnancy loss are poorly understood. It has been reported that stimulation of invariant natural killer T (iNKT) cells using α-galactosylceramide (αGC) induces pregnancy loss in mice. Here we investigated the mechanisms, especially the role of regulatory T (Treg) cells, in iNKT cell-mediated pregnancy loss. We found that injection of αGC rapidly induced fetal resorption, activated decidual iNKT cells, decreased the percentage of decidual Treg cells and their interleukin (IL)-10 and transforming growth factor (TGF)-ß production, and upregulated the levels of interferon (IFN)-γ, tumor necrosis factor-α, IL-4, and IL-10 in serum. Adoptive transfer of iNKT cells from wild-type (WT) and IL-4-/- mice but not IFN-γ-/- mice into αGC-treated iNKT cell-deficient Jα18-/- mice restored αGC-induced pregnancy loss. Adoptive transfer of Treg cells downregulated α-GC-induced pregnancy loss in WT mice. Finally, co-culture with αGC-stimulated decidual iNKT cells decreased the production of IL-10 and TGF-ß in decidual Treg cells and inhibited their suppressive activity. These findings suggest that activation of iNKT cells induces pregnancy loss in mice in an IFN-γ-dependent manner. In addition, inhibition of the function of decidual Treg cells has an important role in iNKT cell-mediated pregnancy loss.

Comparison of extravillous trophoblast cells derived from human embryonic stem cells and from first trimester human placentas.

INTRODUCTION: Preeclampsia and other placental pathologies are characterized by a lack of spiral artery remodeling associated with insufficient invasion by extravillous trophoblast cells (EVT). Because trophoblast invasion occurs in early pregnancy when access to human placental tissue is limited, there is a need for model systems for the study of trophoblast differentiation and invasion. Human embryonic stem cells (hESC) treated with BMP4- differentiate to trophoblast, and express HLA-G, a marker of EVT. The goals of the present study were to further characterize the HLA-G(+) cells derived from BMP4-treated hESC, and determine their suitability as a model. METHODS: HESC were treated with BMP4 under 4% or 20% oxygen and tested in Matrigel invasion chambers. Both BMP4-treated hESC and primary human placental cells were separated into HLA-G(+) and HLA-G(-)/TACSTD2(+) populations with immunomagnetic beads and expression profiles analyzed by microarray. RESULTS: There was a 10-fold increase in invasion when hESC were BMP4-treated. There was also an independent, stimulatory effect of oxygen on this process. Invasive cells expressed trophoblast marker KRT7, and the majority were also HLA-G(+). Gene expression profiles revealed that HLA-G(+), BMP4-treated hESC were similar to, but distinct from, HLA-G(+) cells isolated from first trimester placentas. Whereas HLA-G(+) and HLA-G(-) cells from first trimester placentas had highly divergent gene expression profiles, HLA-G(+) and HLA-G(-) cells from BMP4-treated hESC had somewhat similar profiles, and both expressed genes characteristic of early trophoblast development. CONCLUSIONS: We conclude that hESC treated with BMP4 provide a model for studying transition to the EVT lineage.

Murine female reproductive tract intraepithelial lymphocytes display selection characteristics distinct from both peripheral and other mucosal T cells.

Despite immense effort, the development of vaccines effective at mucosal sites has proceeded at a faltering pace. Efforts concentrating on humoral immunity but neglecting cellular immunity may be misdirected by ignoring many viral mucosal pathogens. Improved understanding of the development and maintenance of lymphocytes populating the reproductive tract (rtIELs) may inform advances in vaccination strategies for sexually transmitted diseases. Recent studies highlight tissue-specific differences in the development of mucosal immunity and suggest that the local milieu may play a role in selection, maintenance and function of resident lymphocytes. Here, we describe MHC class I and thymus dependence of subpopulations of rtIELs. TCRalphabeta+ CD8alphabeta+ T cells in the periphery, intestine, and female reproductive tract are all developmentally dependent on classical class I MHC and the thymus. TCRalphabeta+ CD8alphaalpha+ are absent from the periphery and the rtIELs, but are present and classical MHC class I-independent, in the intestine. In contrast to intestinal TCRgammadelta+ cells, TCRgammadelta+ rtIELs are CD8 negative and thymus dependent. In contrast to peripheral TCRgammadelta+ cells, murine TCRgammadelta+ rtIELs express not a diverse array of Vdelta genes, but rather, a canonical Vdelta1. In summary, lymphocytes isolated from the murine female reproductive tract have characteristics distinct from both peripheral T cells and those found at other mucosal sites. Therefore, for the purpose of vaccination strategies, the female reproductive tract should be regarded neither as peripheral nor mucosal, but rather as a tissue with distinctive immunological characteristics.

Human cytomegalovirus US2 endoplasmic reticulum-lumenal domain dictates association with major histocompatibility complex class I in a locus-specific manner.

The human cytomegalovirus-encoded US2 glycoprotein targets endoplasmic reticulum-resident major histocompatibility complex (MHC) class I heavy chains for rapid degradation by the proteasome. We demonstrate that the endoplasmic reticulum-lumenal domain of US2 allows tight interaction with class I molecules encoded by the HLA-A locus. Recombinant soluble US2 binds properly folded, peptide-containing recombinant HLA-A2 molecules in a peptide sequence-independent manner, consistent with US2's ability to broadly downregulate class I molecules. The physicochemical properties of the US2/MHC class I complex suggest a 1:1 stoichiometry. These results demonstrate that US2 does not require additional cellular proteins to specifically interact with soluble class I molecules. Binding of US2 does not significantly alter the conformation of class I molecules, as a soluble T-cell receptor can simultaneously recognize class I molecules associated with US2. The lumenal domain of US2 can differentiate between the products of distinct class I loci, as US2 binds several HLA-A locus products while being unable to bind recombinant HLA-B7, HLA-B27, HLA-Cw4, or HLA-E. We did not observe interaction between soluble US2 and either recombinant HLA-DR1 or recombinant HLA-DM. The substrate specificity of US2 may help explain the presence in human cytomegalovirus of multiple strategies for downregulation of MHC class I molecules.

Qa-2-dependent selection of CD8alpha/alpha T cell receptor alpha/beta(+) cells in murine intestinal intraepithelial lymphocytes.

Murine intestinal intraepithelial lymphocytes (iIELs) are made up of a heterogeneous mix of T cells with unique phenotypes. Whereas CD8(+) T cells in peripheral lymphoid organs use CD8alpha/beta and are selected on MHC class Ia molecules, a majority of iIELs use CD8alpha/alpha. Here, we report that the presence of CD8alpha/alpha TCR-alpha/beta cells in iIELs is independent of classical MHC class I molecules K(b) and D(b), as illustrated by their presence in K(b)/D(b) double-knockout mice and in mice lacking a nonclassical MHC class I molecule, CD1d. Most strikingly, their presence is decreased by approximately 70% in mice lacking transporter associated with antigen processing (TAP). The TAP-dependent nonclassical MHC class I molecule Qa-2 is strongly implicated in the presence of these cells, as inferred from the low numbers of CD8alpha/alpha TCR-alpha/beta T cells in mice deficient in Qa-2 genes. Second, a Qa-2-transgenic mouse made in a Qa-2(-) strain showed an increase in the numbers of CD8alpha/alpha cells among its iIELs. Thus, the presence of CD8alpha/alpha TCR-alpha/beta cells in iIELs is mainly dependent on the nonclassical MHC class I molecule Qa-2.

Viral subversion of the immune system.

This review describes the diverse array of pathways and molecular targets that are used by viruses to elude immune detection and destruction. These include targeting of pathways for major histocompatibility complex-restricted antigen presentation, apoptosis, cytokine-mediated signaling, and humoral immune responses. The continuous interactions between host and pathogens during their coevolution have shaped the immune system, but also the counter measures used by pathogens. Further study of their interactions should improve our ability to manipulate and exploit the various pathogens.

Can viruses help us to understand and classify the MHC class I molecules at the maternal-fetal interface?

Placental expression of HLA-C, HLA-E, and HLA-G locus products is now well described. However, to date, the functional relevance of these MHC class I products at the maternal-fetal interface is incompletely described. We propose here that HLA-C, -E, and -G comprise a distinct and cohesive group of MHC class I products. This hypothesis is supported by a growing body of data, including that obtained through the study of viral immune evasion. Continued investigation of viral interactions with MHC class I products promises to help us to define those specific attributes of HLA-C, -E, and -G that define their common characteristics.

HLA-G, -E, -F preworkshop: tools and protocols for analysis of non-classical class I genes transcription and protein expression.

Non-classical MHC class I HLA-E, -F, and -G molecules differ from classical class I histocompatibility antigens by specific patterns of transcription, protein expression, and immunological functions. Restriction of the expression pattern of these non-classical antigens may play a key role in modulation of immune responses during pregnancy and diseases but remains to be additionally defined. A specific component of the second International Conference on HLA-G and the 13th HLA-G Histocompatibility Workshop will be dedicated to the analysis of transcription and expression of non-classical class I genes in normal and pathological tissues. In a first step, referred to as the preworkshop, we here report the analysis and conclusions of a working group which was constituted to gather and validate optimal reagents and protocols allowing RT-PCR analysis of HLA-E, -F, -G transcript levels and flow cytometry and immunochemistry analysis of HLA-G expression in cells and tissues. As a result of this work, use of specific primers and probes detecting alternative transcripts of HLA-E, -F, and G have been validated in transfected cells expressing differential pattern of HLA class I antigens. Analysis of the specificity and affinity of collected antibodies has allowed definition of reagents to be proposed for immunochemistry and flow cytometry analysis of HLA-G expression in normal and pathological tissues during the workshop. This work has allowed constitution of an extended workshop group which is now initiating analysis of non-classical class I transcription and expression in various cells and tissues, a collective contribution that will additionally refine our view of the expression of these antigens in normal and pathological situations.

HLA-G and HLA-C at the feto-maternal interface: lessons learned from pathogenic viruses.

Immunoevasive viruses which effect antigen presentation by class I major histocompatibility complex (MHC) molecules have helped to broaden our understanding of the intracellular transport and processing of HLA-G and HLA-C in the placenta. Cellular infection with herpes simplex virus (HSV) and human cytomegalovirus (HCMV) are each associated with the downregulation of surface expression of HLA-A and -B, albeit by remarkably distinct mechanisms. Investigations on the effects of HSV and HCMV infection on HLA-G and HLA-C in the trophoblast have revealed both hypothesized similarities and surprising differences between trophoblast and classical class I products.

Trophoblast class I major histocompatibility complex (MHC) products are resistant to rapid degradation imposed by the human cytomegalovirus (HCMV) gene products US2 and US11.

US11 and US2 encode gene products expressed early in the replicative cycle of human cytomegalovirus (HCMV), which cause dislocation of human and murine major histocompatibility complex (MHC) class I molecules from the lumen of the endoplasmic reticulum to the cytosol, where the class I heavy chains are rapidly degraded. Human histocompatibility leukocyte antigens (HLA)-C and HLA-G are uniquely resistant to the effects of both US11 and US2 in a human trophoblast cell line as well as in porcine endothelial cells stably transfected with human class I genes. Dislocation and degradation of MHC class I heavy chains do not appear to involve cell type-specific factors, as US11 and US2 are fully active in this xenogeneic model. Importantly, trophoblasts HLA-G and HLA-C possess unique characteristics that allow their escape from HCMV-associated MHC class I degradation. Trophoblast class I molecules could serve not only to block recognition by natural killer cells, but also to guide virus-specific HLA-C- and possibly HLA-G-restricted cytotoxic T-lymphocytes to their targets.

Why certain antibodies cross-react with HLA-A and HLA-G: epitope mapping of two common MHC class I reagents.

Antigen presentation at the maternal-fetal interface has been characterized by a reported lack of classical MHC class I products and the presence of a tissue-restricted, non-classical class I product with limited polymorphism, HLA-G. The lack of HLA-A, -B, and -C products at this interface would allow escape from T-cell mediated attack, while the presence of HLA-G may enable evasion of NK cell-mediated destruction. We provide evidence that in addition to HLA-G, the classical class I product HLA-C is also present in trophoblast. Specifically, cDNA from the trophoblast-derived JEG 3 cell line encodes the HLA-C-locus product, HLA-Cw*0401. This protein, obtained by in vitro transcription/translation, has biochemical characteristics identical to MHC class I products immunoprecipitated directly from the same cells. These findings are in agreement with RNA analysis and immunohistochemistry on both cell lines and primary trophoblast tissues. We report here the preferential reactivity in JEG 3 cells of two widely used monoclonal anti-MHC class I heavy chain antibodies, HC10 and HCA2, with HLA-C and HLA-G, respectively. We have mapped the epitopes recognized by these reagents to distinct areas of the alpha1 domain of the MHC class I heavy chain. HCA2 recognizes the motif xLxTLRGx spanning amino acids 77 84 present in both HLA-A and HLA-G. In contrast, HC10 may recognize a discontinuous epitope, with essential elements of the recognized motif surrounding residue 60 in the alpha1 domain of the class I heavy chain, as shown by truncation analysis. These results adequately explain the immunochemical cross-reactivity of HLA-A and HLA-G.

Herpes simplex virus blocks intracellular transport of HLA-G in placentally derived human cells.

Spontaneous fetal loss is associated with herpes simplex virus (HSV) infection as deduced from epidemiologic data. To date, the underlying mechanisms remain to be elucidated, but an immune component is suspected. HLA-G is a class I MHC molecule selectively expressed on extravillous cytotrophoblast; this cell type does not express conventional HLA-A or -B, whereas expression of novel HLA-C-like products has been reported. While its function remains unclear, a role for HLA-G in silencing NK cells that would otherwise attack cells devoid of classical class I molecules has been invoked. We here show that expression of HLA class I molecules is abrogated in HSV-infected choriocarcinoma cells, a phenomenon mediated by the virally encoded inhibitor of the transporter associated with Ag presentation, ICP47. These observations may provide a link between HSV infection and spontaneous fetal loss.

Correlation of serum cytokine and adhesion molecule determinations with pregnancy outcome.

OBJECTIVE: To test the hypothesis that serum levels of T helper [Th]-1-type (interleukin [IL]-2, interferon [IFN]-gamma, tumor necrosis factor [TNF]-alpha) and Th2-type (IL-10) cytokines and the soluble intracellular adhesion molecule-1 (sICAM-1) are associated with pregnancy outcome in women with a history of recurrent spontaneous abortion. METHODS: Peripheral serum was obtained from 104 women between 29 and 42 years of age with a history of two or more (range two to 12, mode four) prior pregnancy losses. Levels of IL-2, IFN-gamma, TNF-alpha, IL-10, and sICAM-1 were measured by enzyme-linked immunosorbent assay in sera from 29 of the 104 individuals when not pregnant and 92 of these women when pregnant between 6 and 7 weeks of gestation. Seventeen women provided samples before and during pregnancy. RESULTS: Interleukin-2, IFN-gamma, and IL-10 were not detectable in any serum sample. Low levels of TNF-alpha were detected in 46 sera from pregnant women but did not correlate with pregnancy outcome. In contrast, sICAM-1 was demonstrable in all sera. However, no differences were observed between pregnant and nonpregnant women. Similarly, there was no difference in serum levels of sICAM-1 between women who subsequently aborted (n = 13) and those who successfully carried to term (n = 15). Neither patient age nor number of prior losses significantly correlated with serum cytokine and sICAM-1 levels. CONCLUSION: Peripheral serum levels of selected Th1-type and Th2-type cytokines and sICAM-1 were not associated with pregnancy outcome in women with a history of recurrent spontaneous abortion.

Progesterone-induced immunosuppression is not mediated through the progesterone receptor.

Progesterone is a known immunosupressant in humans and may be important in treatment regimens for women with immunological and endocrinological reproductive failure. The molecular mechanism of progesterone-mediated immunosuppression remains controversial. We used the reverse transcriptase polymerase chain reaction (RT-PCR) technique to detect progesterone receptor RNA in human peripheral blood mononuclear cells (PBMCs). No expression could be documented in PBMCs from men or women representing various reproductive states. We also used the glucocorticoid receptor antagonist RU 43044 to address the hypothesis that progesterone exerts immunomodulatory effects via interactions with the glucocorticoid receptor. Both hydrocortisone (10(-6) and 10(-7) M) and progesterone (10(-5), 10(-6) and 10(-7) M) inhibited phytohaemagglutinin-induced lymphocyte proliferation in a dose-dependent fashion. RU 43044 (10(-5) M) significantly reversed the immunosuppressive effect od hydrocortisone but not that of progesterone. These studies indicate that human PBMCs do not express the classical progesterone receptor. Our results further suggest that progesterone does not mediate its immunomodulatory effects via interaction with the glucocorticoid receptor. Interaction with other members of the steroid and thyroid hormone receptor superfamily, local conversion to other steroid substances or non-classical receptor-mediated mechanisms may be involved.