Tamara Tilburgs

Evidence for a Selective Migration of Fetus-Specific CD4+CD25bright Regulatory T Cells from the Peripheral Blood to the Decidua in Human Pregnancy

During pregnancy, the maternal immune system has to tolerate the persistence of fetal alloantigens. Many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. Murine studies suggest that CD4+CD25+ T cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. Previous studies by our group demonstrate that a significantly higher percentage of activated T cells and CD4+CD25bright T cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy. In this study, we examined the phenotypic and functional properties of CD4+CD25bright T cells derived from maternal peripheral blood and decidual tissue. Depletion of CD4+CD25bright T cells from maternal peripheral blood demonstrates regulation to third party umbilical cord blood cells comparable to nonpregnant controls, whereas the suppressive capacity to umbilical cord blood cells of her own child is absent. Furthermore, maternal peripheral blood shows a reduced percentage of CD4+CD25brightFOXP3+ and CD4+CD25brightHLA-DR+ cells compared with peripheral blood of nonpregnant controls. In contrast, decidual lymphocyte isolates contain high percentages of CD4+CD25bright T cells with a regulatory phenotype that is able to down-regulate fetus-specific and fetus-nonspecific immune responses. These data suggest a preferential recruitment of fetus-specific regulatory T cells from maternal peripheral blood to the fetal-maternal interface, where they may contribute to the local regulation of fetus-specific responses.

Two Unique Human Decidual Macrophage Populations

Several important events occur at the maternal–fetal interface, including generation of maternal–fetal tolerance, remodeling of the uterine smooth muscle and its spiral arteries and glands, and placental construction. Fetal-derived extravillous trophoblasts come in direct contact with maternal decidual leukocytes. Macrophages represent ∼20% of the leukocytes at this interface. In this study, two distinct subsets of CD14+ decidual macrophages (dMɸs) are found to be present in first-trimester decidual tissue, CD11cHI and CD11cLO. Gene expression analysis by RNA microarray revealed that 379 probes were differentially expressed between these two populations. Analysis of the two subsets revealed several clusters of coregulated genes that suggest distinct functions for these subsets in tissue remodeling, growth, and development. CD11cHI dMɸs express genes associated with lipid metabolism and inflammation, whereas CD11cLO dMɸs express genes associated with extracellular matrix formation, muscle regulation, and tissue growth. The CD11cHI dMɸs also differ from CD11cLO dMɸs in their ability to process protein Ag and are likely to be the major APCs in the decidua. Moreover, these populations each secrete both proinflammatory and anti-inflammatory cytokines that may contribute to the balance that establishes fetal–maternal tolerance. Thus, they do not fit the conventional M1/M2 categorization.

Fetal–maternal HLA-C mismatch is associated with decidual T cell activation and induction of functional T regulatory cells

Human leukocyte antigen-C (HLA-C) is the only polymorphic classical histocompatibility antigen expressed by fetal trophoblasts at the fetal-maternal interface. Interactions between HLA-C and decidual natural killer (NK) cells may facilitate trophoblast invasion into maternal tissue. Thus far no evidence has been provided that decidual T cells specifically recognize and respond to fetal alloantigens at the fetal-maternal interface. In this study, we show that pregnancies containing a HLA-C mismatched child induce an increased percentage of CD4(+)CD25(dim) activated T cells in decidual tissue. In addition, HLA-C mismatched pregnancies exhibit a decidual lymphocyte response to fetal cells and contain functional CD4(+)CD25(bright) regulatory T cells in decidual tissue, whereas HLA-C matched pregnancies do not. This suggests that decidual T cells specifically recognize fetal HLA-C at the fetal-maternal interface but are prevented from inducing a destructive immune response in uncomplicated pregnancies.

Human Decidual Tissue Contains Differentiated CD8+ Effector-Memory T Cells with Unique Properties

During pregnancy, maternal lymphocytes at the fetal–maternal interface play a key role in the immune acceptance of the allogeneic fetus. Recently, CD4+CD25bright regulatory T cells have been shown to be concentrated in decidual tissue, where they are able to suppress fetus-specific and nonspecific immune responses. Decidual CD8+ T cells are the main candidates to recognize and respond to fetal HLA-C at the fetal–maternal interface, but data on the characteristics of these cells are limited. In this study we examined the decidual and peripheral CD8+ T cell pool for CD45RA, CCR7, CD28, and CD27 expression, using nine-color flow cytometry. Our data demonstrate that decidual CD8+ T cells mainly consist of differentiated CD45RA−CCR7− effector-memory (EM) cells, whereas unprimed CD45RA+CCR7+ naive cells are almost absent. Compared with peripheral blood EM CD8+ T cells, the decidual EM CD8+ T cells display a significantly reduced expression of perforin and granzyme B, which was confirmed by immunohistochemistry of decidual tissue sections. Interestingly, quantitative PCR analysis demonstrates an increased perforin and granzyme B mRNA content in decidual EM CD8+ T cells in comparison with peripheral blood EM CD8+ T cells. The presence of high levels of perforin and granzyme B mRNA in decidual EM T cells suggests that decidual CD8+ T cells pursue alternative means of EM cell differentiation that may include a blockade of perforin and granzyme B mRNA translation into functional perforin and granzyme B proteins. Regulation of decidual CD8+ T cell differentiation may play a crucial role in maternal immune tolerance to the allogeneic fetus.

Comparison of Macrophage Phenotype Between Decidua Basalis and Decidua Parietalis by Flow Cytometry

The two regions of the maternal decidua, decidua basalis and decidua parietalis, differ in the extent of trophoblast invasion and consequently in cytokines and other biological mediators, extracellular matrix and cellular components. Our aim was to compare the phenotypic features of macrophages from the two decidual regions across a broad gestational age range. We isolated macrophages by enzymatic digestion from healthy decidua samples obtained after elective abortions, at 9-18-week and at 19-23-weeks, or after term deliveries (caesarean sections at term and spontaneous term vaginal deliveries). Macrophages were analysed by flow cytometry applying the same instrument settings to all the samples to allow semi-quantitative comparison of the expression of a particular marker between different samples. We found higher expressions of CD80, CD86 and HLA-DR, suggestive of a more activated phenotype of decidual macrophages, at early/mid pregnancy than at term. Marginal differences were found between term decidual macrophages obtained after spontaneous vaginal deliveries or caesarean sections which imply that the parturient process is not associated with decidual macrophage activation. The expressions of CD105, DC-SIGN and MMR were the strongest in decidua basalis of mid pregnancy and indicate the importance of decidual macrophages in tissue homeostasis at the uteroplacental interface.

Human HLA-G+ extravillous trophoblasts: Immune-activating cells that interact with decidual leukocytes

Significance Fetal extravillous trophoblasts (EVT) invade uterine tissue and interact with maternal immune cells during pregnancy. EVT express human leukocyte antigen-C (HLA-C) and -G (HLA-G). Although polymorphic HLA-C can elicit a maternal immune response, HLA-G has been associated with induction of immune tolerance. We have succeeded in isolating all maternal immune cell types as well as EVT from human placental tissue. These methods were used to elucidate the unique charateristics of EVT as well as their interaction with maternal immune cells. We demonstrate that EVT are specialized cells whose properties are not imitated by HLA‐G–expressing surrogate cell lines. Studies using primary EVT are crucial for understanding maternal–fetal tolerance and development of pregnancy complications such as preeclampsia and miscarriages. Invading human leukocyte antigen-G+ (HLA‐G+) extravillous trophoblasts (EVT) are rare cells that are believed to play a key role in the prevention of a maternal immune attack on foreign fetal tissues. Here highly purified HLA‐G+ EVT and HLA‐G− villous trophoblasts (VT) were isolated. Culture on fibronectin that EVT encounter on invading the uterus increased HLA‐G, EGF-Receptor-2, and LIF-Receptor expression on EVT, presumably representing a further differentiation state. Microarray and functional gene set enrichment analysis revealed a striking immune-activating potential for EVT that was absent in VT. Cocultures of HLA‐G+ EVT with sample matched decidual natural killer cells (dNK), macrophages, and CD4+ and CD8+ T cells were established. Interaction of EVT with CD4+ T cells resulted in increased numbers of CD4+CD25HIFOXP3+CD45RA+ resting regulatory T cells (Treg) and increased the expression level of the Treg-specific transcription factor FOXP3 in these cells. However, EVT did not enhance cytokine secretion in dNK, whereas stimulation of dNK with mitogens or classical natural killer targets confirmed the distinct cytokine secretion profiles of dNK and peripheral blood NK cells (pNK). EVT are specialized cells involved in maternal–fetal tolerance, the properties of which are not imitated by HLA‐G–expressing surrogate cell lines.

Elsevier Trophoblast Research Award Lecture: Unique properties of decidual T cells and their role in immune regulation during human pregnancy.

Maternal lymphocytes at the fetal-maternal interface play a key role in the immune acceptance of the allogeneic fetus. Most studies focus on decidual NK cells and their interaction with fetal trophoblasts, whereas limited data are available on the mechanisms of fetus specific immune recognition and immune regulation by decidual T cells at the fetal-maternal interface. The aim of this review is to describe the phenotypic characteristics of decidual T cell subsets present at the fetal-maternal interface, their interaction with HLA-C expressed by fetal trophoblasts and their role in immune recognition and regulation at the fetal-maternal interface during human pregnancy.

Expression of NK cell receptors on decidual T cells in human pregnancy

Specific receptors enable NK cells to discriminate between cells with normal expression of MHC class I and cells that have low or absent expression of MHC class I molecules. In addition to NK cells, these receptors can be expressed on T cell subsets, mainly on CD8+ T cells but also on gammadeltaTCR+ T cells and CD4+ T cells. Although the function of NK cell receptor expression on T cells is not completely understood, various studies have shown that they are involved in down regulation of T cell receptor (TCR)-mediated activation and influence effector functions, like cytotoxicity and cytokine production. The aim of this study was to analyze expression of NK cell receptors on peripheral blood and decidual T cells during human pregnancy using flow cytometry. We demonstrate that a proportion of decidual T cells express HLA-C specific killer immunoglobulin-like receptors (KIRs). Furthermore, a small proportion of decidual T cells express the HLA-E specific CD94-NKG2A inhibitory and CD94-NKG2C activating receptors. Decidual KIR+ and CD94-NKG2+ T cells mainly display a CD3+CD4-CD8- phenotype. However, decidual tissue also contains higher percentages of KIR and CD94-NKG2 expressing CD4+ and CD8+ T cells compared to peripheral blood. So far, the functional capacities of decidual T cells expressing the NK cell receptors are unknown but NK cell receptor expression on decidual T cells may provide an alternative means by which decidual T cells distinguish self (maternal) cells from allogeneic fetal cells, and act to modulate the decidual immune response.

CD8+ Effector T Cells at the Fetal–Maternal Interface, Balancing Fetal Tolerance and Antiviral Immunity

During pregnancy CD8+ effector T cells need optimal immune regulation to prevent a detrimental response to allogeneic fetal cells while providing immune protection to infections. A significant proportion of (prospective) mothers carry naïve or memory CD8+ T cells with a TCR that can directly bind to paternal MHC molecules. In addition, a high percentage of pregnant women develop specific T cell responses to fetal minor histocompatibility antigens (mHags). Under normal conditions, fetal–maternal MHC and mHag mismatches lead to elevated lymphocyte activation but do not induce pregnancy failure. Furthermore, viral infections alter the maternal CD8+ T cell response by changing the CD8+ T cell repertoire and increasing the influx of CD8+ T cells to decidual tissue. The normally high T cell activation threshold at the fetal–maternal interface may prevent efficient clearance of viral infections. Conversely, the increased inflammatory response due to viral infections may break fetal–maternal tolerance and lead to pregnancy complications. The aim of this review is to discuss the recent studies of CD8+ T cells in pregnancy, identify potential mechanisms for antigen‐specific immune recognition of fetal extravillous trophoblast (EVT) cells by CD8+ T cells, and discuss the impact of viral infections and virus‐specific CD8+ T cells during pregnancy.

Human Cytomegalovirus Inhibits Cytokine-Induced Macrophage Differentiation

ABSTRACT Human cytomegalovirus (HCMV) infection in immunocompromised patients is associated with impaired immunological function. Blood monocytes, which differentiate into macrophage effector cells, are of central importance for immune reactivity. Here, we demonstrate that HCMV transiently blocks cytokine-induced differentiation of monocytes into functionally active phagocytic macrophages. In HCMV-treated cultures, the cells had classical macrophage markers but lacked the classical morphological appearance of macrophages and had impairments in migration and phagocytosis. Even at very low multiplicities of infection, macrophage differentiation was almost completely inhibited. The inhibition appeared to be mediated by a soluble factor released upon viral treatment of monocytes. Human immunodeficiency virus or measles virus had no such effects. These findings suggest that HCMV impairs immune function by blocking certain aspects of cytokine-induced differentiation of monocytes and demonstrate an efficient pathway for this virus to evade immune recognition that may have clinical implications for the generalized immunosuppression often observed in HCMV-infected patients.