Characterization of murine decidual natural killer (NK) cells and their relevance to the success of pregnancy

The identification of lytic cells in 6.5-day to 9.5-day murine decidua as NK cells has been extended. The cells with natural killer (NK) activity in early decidua were nonphagocytic and heterogeneous in size as assessed by velocity sedimentation at unit gravity. The numbers of lytic cells were reduced by treatment with anti-asialo GM1 in vivo and they were absent from the decidua of bg/bg mice. Thus, decidual NK cells were not distinct from NK cells in other tissues. The decline in the levels of decidual NK activity as pregnancy progressed was attributed to their regulation by other cells present in decidua by midgestation. The development of NK activity in decidua was dependent upon the presence of an embryo, however, decidual NK cells were not essential for successful pregnancy because viable offspring were obtained from mice lacking decidual NK activity. It was shown that NK cells from either spleen or decidua were unlikely to cause damage to embryos during the first half of pregnancy as freshly dissociated 9.5- and 11.5-day embryonic cells resisted NK lysis. Furthermore, blastocysts were not damaged by coincubation with splenic or decidual NK cells and were viable upon subsequent embryo transfer. These studies indicate that decidual NK cells are not essential for successful pregnancy and are not necessarily detrimental to early embryos. It is suggested that decidual NK cells may play other nonimmunological roles during embryonic development.     

Characterization of immune effector cells present in early murine decidua

It has been suggested that murine decidual cells act as an important immunoregulatory population localized to the pregnant uterus. We have examined early murine decidua to determine if immune effector cells occur in the decidual environment in proximity to the conceptus. High levels of natural killer (NK) cell activity were found consistently in decidual cell suspensions with peak activity occurring on Day 6.5 of gestation. NK activity declined as pregnancy proceeded and was not significant by Day 12.5 of gestation. Decidual cell suspensions did not appear to contain significant numbers of functional B or T effector cells. No antipaternal T-cell response could be demonstrated even in the decidua of immune mice. Lack of T-cell responses was attributed to the absence of T cells from decidua rather than to their inactivation because precursors of cytotoxic T lymphocytes (pCTL) could not be detected in decidual cell suspensions. Furthermore, the levels of pCTL detectable in spleen cell suspensions could not be reduced by mixing spleen cells with 7.5-day decidual cells. These results suggest that B cells and T cells may not occur in early decidua while NK cells are present and regulated independently.     

Normal murine and porcine embryos recruit NK cells to the uterus

Decidual NK cells, indistinguishable from those found in lymphoid tissues, are present in cell suspensions prepared from maternal decidua of random-bred mice between Days 6.5 and 10.5 of first gestation. The stringency of the correlation between NK cells and normal embryos during successful pregnancy is unknown. Our previous finding that active NK cells were unable to mediate lysis of fresh embryonic tissues at any stage during gestation suggests that if NK cells play a functional role in normal pregnancy it would be a noncytolytic role. Before studies on the function of uterine NK cells were undertaken, evidence that the association of NK cells with normal embryos is widespread was sought by assessing NK cell activity in cell suspensions from decidua of syngeneically mated mice, from decidua of multiparous, random-bred mice, and from the endometrium of pigs during first pregnancy. Neither parity nor maternal-fetal compatibility changed the pattern of high levels of decidual NK cell activity early in pregnancy followed by decline. Porcine NK cell activity was not detected in uterine cells isolated from cycling pigs but in pregnant animals it gradually increased during the preattachment period and reached levels greater than those in blood, postattachment (Day 28). Some of this activity was hormone dependent but sustained increases in NK cell activity required the presence of an embryo. These studies demonstrate that the association of functional NK cells with normal embryos is widespread during early pregnancy.     

Prostaglandin-mediated inactivation of natural killer cells in the murine decidua

Previous studies from this laboratory have demonstrated a large influx of null lymphocytes into the murine decidua during pregnancy. We had also shown that trophoblast cells of the murine placenta bear target structures recognized by NK cells. Since NK lineage cells belong to the null category of lymphocytes, we examined whether cells of this lineage appear in the murine decidua, and if so, whether their activity is locally regulated by NK suppressor cells. We further investigated the identity of the suppressor cells as well as their suppressor products. NK lineage cells, irrespective of their activation status, were identified morphologically in radioautographic preparations as the non-T, non-B (null) lymphocytes capable of binding YAC-1 lymphoma targets. NK activity of nucleated cells was measured with a 4-hr 51Cr-release assay against labeled YAC-1 targets. Studies with outbred CD1 mice, and to a smaller extent, inbred CBA mice revealed that the incidence of NK lineage cells remained fairly constant within the decidua throughout pregnancy, but their activity decreased steadily to negligible levels by Day 12-14 of gestation. This was found to result from an inactivation caused by NK-suppressor cells in the decidua. A mixing of Ficoll-Paque-separated nucleated cells of the decidua with normal splenic effector cells (at 1:1 ratio) led to a suppression of their NK activity tested immediately or after a 20-hr coculture. This suppression was MHC unrestricted. Suppressor cells were identified both in plastic nonadherent fraction highly enriched for typical decidual cells as well as in the plastic adherent fraction containing decidual cells and macrophages. Addition of indomethacin (10(-5) M), an inhibitor of prostaglandin synthesis, or anti PGE2 antibody, revived the NK activity in the mixed population, as well as in the decidua, suggesting a PGE2-mediated suppression. High levels of PGE2 were detectable in decidual cell supernatants with a sensitive radioimmunoassay. Addition of pure PGE2 (10(-7)-10(-6) M) but not PGF2 alpha (10(-6) M) during the NK assay or to the effector cells for a 20-hr period prior to the assay led to an inhibition of NK activity. These results reveal that NK cells appearing in the murine decidua are progressively inactivated by PGE2 produced by decidual cells and decidual macrophages.     

Immunophenotype and cytokine profiles of rhesus monkey CD56bright and CD56dim decidual natural killer cells

The primate endometrium is characterized in pregnancy by a tissue-specific population of CD56(bright) natural killer (NK) cells. These cells are observed in human, rhesus, and other nonhuman primate decidua. However, other subsets of NK cells are present in the decidua and may play distinct roles in pregnancy. The purpose of this study was to define the surface marker phenotype of rhesus monkey decidual NK (dNK) cell subsets, and to address functional differences by profiling cytokine and chemokine secretion in contrast with decidual T cells and macrophages. Rhesus monkey decidual leukocytes were obtained from early pregnancy tissues, and were characterized by flow cytometry and multiplex assay of secreted factors. We concluded that the major NK cell population in rhesus early pregnancy decidua are CD56(bright) CD16(+)NKp30(-) decidual NK cells, with minor CD56(dim) and CD56(neg) dNK cells. Intracellular cytokine staining demonstrated that CD56(dim) and not CD56(bright) dNK cells are the primary interferon-gamma (IFNG) producers. In addition, the profile of other cytokines, chemokines, and growth factors secreted by these two dNK cell populations was generally similar, but distinct from that of peripheral blood NK cells. Finally, analysis of multiple pregnancies from eight dams revealed that the decidual immune cell profile is characteristic of an individual animal and is consistently maintained across successive pregnancies, suggesting that the uterine immune environment in pregnancy is carefully regulated in the rhesus monkey decidua.     

Human decidual natural killer cells express the receptor for and respond to the cytokine interleukin 15

The natural killer (NK) cells that are present in the uterine mucosa (decidua) during early pregnancy have a distinctive phenotype, CD56(bright) CD16(-). These cells have previously been shown to proliferate and be activated by interleukin (IL)-2. However, IL-2 is absent from the decidua and placenta, and we have therefore investigated whether IL-15 is present in the uterus and can act on decidual NK cells. Both IL-15 mRNA and protein were found in a variety of cells but particularly in decidual macrophages. IL-15 induced a proliferative response in decidual NK cells that was blocked by anti-IL-15 and was augmented by stem cell factor. The cytolytic activity of decidual NK cells against K562 was augmented. Interestingly, in contrast to IL-2, although activation with IL-15 resulted in some killing of JEG-3 choriocarcinoma cells, normal trophoblast cells remained resistant to lysis. These findings suggest that IL-15 is a candidate cytokine responsible for NK cell proliferation in vivo in the progesterone-dominated secretory endometrium and early decidua.     

Mesenchymal stem cells alter the frequency and cytokine profile of natural killer cells in abortion-prone mice

Natural killer cells, which play a pivotal role in the establishment and maintenance of normal pregnancy, are the most abundant leukocytes at the fetomaternal interface that their subsets frequencies and cytokine profile are influential factors in the preservation of the decidual tolerogenic microenvironment. Any imbalance in NK cells' frequency and functions could be associated with pregnancy failure. Mesenchymal stem cells (MSCs) are shown to have immunomodulatory effects on NK cells and their cytokine profile. The purpose of this study is to evaluate the impact of MSCs therapy on the cytokine profiles and subpopulations of NK cells in a murine model of recurrent pregnancy loss. Adipose-derived MSCs were injected intraperitoneally to the abortion-prone mice on Day 4.5 of gestation. The abortion rate was determined after MSCs administration and the frequency and cytokine profiles of the different subsets of NK cells were determined using the flow cytometry. Our results showed that, in abortion-prone mice, the frequency of CD49b⁺ NK cells was significantly higher than normal pregnant mice that decreased after therapy. We also demonstrated that MSCs downregulated the production of IFN-γ and upregulated IL-4 and IL-10 production by uNK cells. These findings indicate that MSCs can decrease the infiltration of CD49b⁺ NK cells to the fetomaternal interface and modulate the cytokine profile of NK cells from inflammatory to tolerogenic profile and thereby improve the tolerogenic microenvironment at the fetomaternal interface in benefit of pregnancy maintenance.     

The uterine NK cell population requires IL-15 but these cells are not required for pregnancy nor the resolution of a Listeria monocytogenes infection

During pregnancy in mice, uterine natural killer (uNK) cells abundantly accumulate on the mesometrial side of the placenta. In this study, we show that the presence of both mature and immature uNK cells requires IL-15. Bone marrow transplantation of NK cell-negative mice due to null mutations in the recombination-activating gene (Rag) 2/common cytokine receptor gamma-chain (Rag2(-/-)gamma(c)(-/-)) genes indicated that uNK cells originate from the bone marrow and require IL-15 to develop. NK cells are thought to be central players in the immune response to intracellular pathogens such as Listeria monocytogenes, a bacterium that also has a predilection for replication in the placenta. However, IL-15(-/-), NK cell-deficient mice were relatively protected from this infection compared with wild-type mice, and during pregnancy the absence of NK cells did not compromise the immune response at this site. The loss of uNK cells results in decidual abnormalities, including thickening of the arterial walls with luminal narrowing and a hypocellular decidua basalis. These defects were rescued by bone marrow transplantation of the Rag2(-/-)gamma(c)(-/-) mice that restored the uNK cell population. The decidual abnormalities in the IL-15(-/-) mice however did not result in infertility as gestation times and litter sizes were comparable to those of wild-type mice. Fetal weights were mildly compromised, consistent with the arterial pathologies. These results show that uNK cells are not required for successful pregnancy and that NK cells are not essential for an adequate immune response to L. monocytogenes in either pregnant or nonpregnant mice.     

Dendritic cell editing by activated natural killer cells results in a more protective cancer-specific immune response

Over the last decade, several studies have extensively reported that activated natural killer (NK) cells can kill autologous immature dendritic cells (DCs) in vitro, whereas they spare fully activated DCs. This led to the proposal that activated NK cells might select a more immunogenic subset of DCs during a protective immune response. However, there is no demonstration that autologous DC killing by NK cells is an event occurring in vivo and, consequently, the functional relevance of this killing remains elusive. Here we report that a significant decrease of CD11c(+) DCs was observed in draining lymph nodes of mice inoculated with MHC-devoid cells as NK cell targets able to induce NK cell activation. This in vivo DC editing by NK cells was perforin-dependent and it was functionally relevant, since residual lymph node DCs displayed an improved capability to induce T cell proliferation. In addition, in a model of anti-cancer vaccination, the administration of MHC-devoid cells together with tumor cells increased the number of tumor-specific CTLs and resulted in a significant increase in survival of mice upon challenge with a lethal dose of tumor cells. Depletion of NK cells or the use of perforin knockout mice strongly decreased the tumor-specific CTL expansion and its protective role against tumor cell challenge. As a whole, our data support the hypothesis that NK cell-mediated DC killing takes place in vivo and is able to promote expansion of cancer-specific CTLs. Our results also indicate that cancer vaccines could be improved by strategies aimed at activating NK cells.     

Immunology of pregnancy: renewed interest

The long-standing question of pregnancy immunological paradox has been generating renewed interest. Recent insights have emerged from studies in pregnant mice and humans demonstrating a number of mechanisms that prevent potentially harmful effects of maternal anti-paternal allo-antibodies (complement inhibition, partial deletion of maternal B cells specific of paternal antigens), cytotoxic CD8+ T cells (lack of HLA-A and HLA-B expression on trophoblast, local immunosuppressive molecules, transient tolerance of paternal allo-antigens specific T cells) and uterine NK cells directed against fetal-derived trophoblast cells (limited NK cytotoxic potential, trophoblast resistance to NK killing). Interestingly, it appears that not only decidual NK cell/trophoblast interactions are not harmful for the fetus but are beneficial for the placental vascularization and its subsequent development. A recent report has indeed demonstrated that during pregnancy most of the combinations of uterine KIR (killer cell immunoglobulin-like receptor) NK cell receptors and fetal HLA-C molecules expressed by trophoblast led to normal pregnancies, whereas mothers lacking activating KIR of the AA genotype when the fetus possessed HLA-C of the C2 group were at a greatly increased risk of severe preeclampsia pathology.     

Trypanosoma cruzi: the expansion of NK, T, and NKT cells in the experimental infection

T and NK cells play a key role in resistance to Trypanosoma cruzi infections, mainly through IFN-gamma production. The expression of T and NK cells surface markers was studied in NWNA spleen cells of resistant C3H and susceptible BALB/c mice that release IFN-gamma in the early and late acute infection, respectively. In the progressively enlarged spleens, we found: (a) an increased percentage and number of NK blast cells as early as at 2 days post-infection (pi), (b) an enrichment of T and NK cells, in both the total and blast populations, during the late acute phase. At 17 days pi, there was also an accumulation of TCR- alphabeta+DX5+, NKT cells, mainly in resistant mice. At 21 days pi, the enrichment of NK cells ceased, while spleen cells and the T cell compartment continued their expansion. In the chronic stage, TCR-alphabeta+ blasts were expanded in both mouse strains, but NK blasts increased only in BALB/c that, unlike C3H mice, release IFN-gamma. As T and NK cell proliferation is not always associated to IFN-gamma release the experimental downregulation of their expansion to avoid tissue damage could be explored.     

Single-cell Immune Landscape of Human Recurrent Miscarriage

Successful pregnancy in placental mammals substantially depends on the establishment of maternal immune tolerance to the semi-allogenic fetus. Disorders in this process are tightly associated with adverse pregnancy outcomes including recurrent miscarriage(RM). However, an indepth understanding of the systematic and decidual immune environment in RM remains largely lacking. In this study, we utilized single-cell RNA-sequencing(sc RNA-seq) to comparably analyze the cellular and molecular signatures of decidual and peripheral leukocytes in normal and unexplained RM pregnancies at the early stage of gestation. Integrative analysis identifies 22 distinct cell clusters in total, and a dramatic difference in leukocyte subsets and molecular properties in RM cases is revealed. Specifically, the cytotoxic properties of CD8+effector T cells, nature killer(NK), and mucosal-associated invariant T(MAIT) cells in peripheral blood indicates apparently enhanced pro-inflammatory status, and the population proportions and ligand–receptor interactions of the decidual leukocyte subsets demonstrate preferential immune activation in RM patients.The molecular features, spatial distribution, and the developmental trajectories of five decidual NK(d NK) subsets have been elaborately illustrated. In RM patients, a d NK subset that supports embryonic growth is diminished in proportion, while the ratio of another d NK subset with cyto-toxic and immune-active signature is significantly increased. Notably, a unique pro-inflammatory CD56+CD16+d NK subset substantially accumulates in RM decidua. These findings reveal a comprehensive cellular and molecular atlas of decidual and peripheral leukocytes in human early pregnancy and provide an in-depth insight into the immune pathogenesis for early pregnancy loss.     

NK and CD4 cells collaborate to protect against melanoma tumor formation in the brain

NK cells represent a potent immune effector cell type that have the ability to recognize and lyse tumors. However, the existence and function of NK cells in the traditionally "immune-privileged" CNS is controversial. Furthermore, the cellular interactions involved in NK cell anti-CNS tumor immunity are even less well understood. We administered non-Ag-loaded, immature dendritic cells (DC) to CD8alpha knockout (KO) mice and studied their anti-CNS tumor immune responses. DC administration induced dramatic antitumor immune protection in CD8alpha KO mice that were challenged with B16 melanoma both s.c. and in the brain. The CNS antitumor immunity was dependent on both CD4+ T cells and NK cells. Administration of non-Ag-loaded, immature DC resulted in significant CD4+ T cell and NK cell expansion in the draining lymph nodes at 6 days postvaccination, which persisted for 2 wk. Finally, DC administration in CD8alpha KO mice was associated with robust infiltration of CD4+ T cells and NK cells into the brain tumor parenchyma. These results represent the first demonstration of a potent innate antitumor immune response against CNS tumors in the absence of toxicity. Thus, non-Ag-loaded, immature DC administration, in the setting of CD8 genetically deficient mice, can induce dramatic antitumor immune responses within the CNS that surpass the effects observed in wild-type mice. Our results suggest that a better understanding of the cross-talk between DC and innate immune cells may provide improved methods to vaccinate patients with tumors located both systemically and within the CNS.     

Persistence of decidual NK cells and KIR genotypes in healthy pregnant and preeclamptic women: a case-control study in the third trimester of gestation

Background  

Natural Killer (NK) cells are the most abundant lymphocytes in the decidua during early gestation. The interactions of NK cells with the extravillous cytotrophoblast have been associated with a normal spiral artery remodeling process, an essential event for a successful pregnancy. Recent data indicate that alterations in the amount of decidual NK (dNK) cells contribute to the development of preeclampsia (PE). Moreover, genetic studies suggest that Killer Immunoglobulin-like Receptors (KIR) expressed in dNK cells influence the susceptibility to PE. Although dNK cells have been well characterized during early pregnancy, they have been scarcely studied in the third trimester of gestation. The aim of this work was to characterize dNK cells at the last trimester of gestation and to analyze the KIR genotype of healthy and PE women.     

Qa-1(b)-dependent modulation of dendritic cell and NK cell cross-talk in vivo

Dendritic cells (DC) trigger activation and IFN-gamma release by NK cells in lymphoid tissues, a process important for the polarization of Th1 responses. Little is known about the molecular signals that regulate DC-induced NK cell IFN-gamma synthesis. In this study, we analyzed whether the interaction between Qa-1(b) expressed on DC and its CD94/NKG2A receptor on NK cells affects this process. Activation of DC using CpG-oligodeoxynucleotides in Qa-1(b)-deficient mice, or transfer of CpG-oligodeoxynucleotide-activated Qa-1(b)-deficient DC into wild-type mice, resulted in dramatically increased IFN-gamma production by NK cells, as compared with that induced by Qa-1(b)-expressing DC. Masking the CD94/NKG2A inhibitory receptor on NK cells in wild-type mice similarly enhanced the IFN-gamma response of these cells to Qa-1(b)-expressing DC. Furthermore, NK cells from CD94/NKG2A-deficient mice displayed higher IFN-gamma production upon DC stimulation. These results demonstrate that Qa-1(b) is critically involved in regulating IFN-gamma synthesis by NK cells in vivo through its interaction with CD94/NKG2A inhibitory receptors. This receptor-ligand interaction may be essential to prevent unabated cytokine production by NK cells during an inflammatory response.     

Natural killer (NK) cell activity of first trimester human decidua

The NK cell functional capacity of first trimester human decidua against K562 targets was assessed in a 3-hr CRA. Collagenase dispersal combined with plastic adherence, nylon wool passage, and density gradient centrifugation yielded NKH-1 (Leu 19) positive enriched decidual large granular lymphocyte fraction (mean 75% positive). Decidual effectors displayed reduced lytic activity compared with autologous PB effectors at every effector:target ratio but this difference in cytotoxicity was abolished by short-term culture before the CRA. Decidual effectors treated with 50 units rIL-2 showed increased lytic activity compared to untreated decidual cells. By FACS analysis the majority of NKH-1 positive decidual effectors were CD3 and CD16 negative which corresponds with a minority PB NK population. The implications of a population of functional NK cells in early pregnancy decidua for the materno-fetal relationship is discussed.     

Influence of relative NK–DC abundance on placentation and its relation to epigenetic programming in the offspring

Normal placentation relies on an efficient maternal adaptation to pregnancy. Within the decidua, natural killer (NK) cells and dendritic cells (DC) have a critical role in modulating angiogenesis and decidualization associated with pregnancy. However, the contribution of these immune cells to the placentation process and subsequently fetal development remains largely elusive. Using two different mouse models, we here show that optimal placentation and fetal development is sensitive to disturbances in NK cell relative abundance at the fetal–maternal interface. Depletion of NK cells during early gestation compromises the placentation process by causing alteration in placental function and structure. Embryos derived from NK-depleted dams suffer from intrauterine growth restriction (IUGR), a phenomenon that continued to be evident in the offspring on post-natal day 4. Further, we demonstrate that IUGR was accompanied by an overall reduction of global DNA methylation levels and epigenetic changes in the methylation of specific hepatic gene promoters. Thus, temporary changes within the NK cell pool during early gestation influence placental development and function, subsequently affecting hepatic gene methylation and fetal metabolism.Maternal adaptations to pregnancy are critical during mammalian development. Cumulative evidence suggests that immune-competent cells in the developing decidua are important for placental development and embryo survival. For instance, the cross-talk between natural killer (NK) cells and dendritic cells (DC) during early pregnancy has emerged as a key regulator of angiogenesis and decidualization,^{1, 2} both of which are critical events for normal placentation. In this context, we have shown that NK cells prevent the development of alloimmunity during normal pregnancy by modulating the immune-regulatory functions of DC during early gestation.^{2, 3} Furthermore, unbalanced relative NK cell abundance (i.e., upon in vivo NK cell depletion following fms-like tyrosine kinase 3 ligand (FL)-mediated DC expansion) upregulated the expression of anti-angiogenic growth factors, suggesting that NK cells are required for early vascular responses associated with pregnancy. Indeed, NK cells regulate the remodeling of maternal spiral arteries, transforming them from low capacity high resistance into high capacity low resistance vessels.⁴ As for DC, these cells not only influence maternal immune tolerance to fetal alloantigens,³ but also coordinate pregnancy-associated vascular adaptations including vessel permeability and blood flow to the implantation site via the CXCL12/CXCR4 pathway.^{1, 5}The placenta is the key organ supporting the survival and growth of the fetus during gestation. It constitutes an interface between the maternal and fetal circulations, allowing nutrient uptake, waste elimination and metabolic/gas exchange.⁶ In mice, placental development begins early in the blastocyst (embryonic day E3.5) and continues to show dramatic changes throughout gestation. The mouse placenta consists of three layers: the labyrinth (comprising highly branched villi that maximize nutrient transport), the junction zone (containing trophoblast giant cells and spongiotrophoblast cells that invade the decidua and maternal vessels) and the maternal decidua (including immune and decidual cells and the maternal vasculature).⁷ Inadequate differentiation of these layers leads to detrimental changes in placental architecture, which affect the supply of nutrients or oxygen to the fetus. Faulty placental development has been implicated in the pathophysiology of several human pregnancy disorders, including preeclampsia, intrauterine growth restriction (IUGR) and preterm labor.⁷ In this context, it is likely that the fetus adapts to the pathological situation by modifying its energy metabolism in order to sustain development and continue pregnancy.Changes in fetal metabolism are thought to occur via epigenetic modification of mitochondrial gene expression patterns, causing changes in mitochondrial number or function.^{8, 9} Indeed, animal models of IUGR have shown a reduction in mitochondrial DNA (mtDNA) content, accompanied by reduced expression of mtDNA encoded genes and epigenetic changes in fetal growth factors (e.g., insulin-like growth factor I (IgfI) and IgfII). In addition, many studies have suggested a role for nuclear receptors in the observed fetal metabolic adaptations toward adverse conditions in utero. These include receptors and associated coactivators, such as the peroxisome proliferator-activated receptor alpha (Pparα), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Pgc1α), hepatocyte nuclear factor 4 alpha (Hnf4α) and the glucocorticoid receptor (Nr3c1).⁸ During adaptation to restricted fetal development, modifications to nuclear receptors act to optimize growth and differentiation. In a process called fetal programming, these modifications can persist through adulthood possibly contributing to the observed increase in late-onset disorders at an adult age.^{10, 11}As the cross-talk between NK cells and DC influences both decidualization and angiogenesis, in this work we analyzed how in vivo manipulation of relative DC–NK cell abundance within the decidua impacts the placentation process and subsequently fetal development. We show that although manipulation of the relative abundance of DC and NK cells in early gestation does not seem to compromise fetal survival, both placental function and structure are specifically altered compared with control mice. Using a candidate gene approach, we demonstrate that these structural changes in the placenta are associated with epigenetic changes in the methylation of specific hepatic gene promoters in pups principally after NK cell depletion. The gene-specific changes were further accompanied by an overall reduction of global DNA methylation levels. Our data confirm that temporary changes within the NK and/or the DC pool during early gestation influence placental development and function, subsequently affecting hepatic gene methylation and fetal metabolism.     

Leukocyte subpopulations in the uteri of leukemia inhibitory factor knockout mice during early pregnancy

Leukemia inhibitory factor (LIF) is transiently expressed on Day (D) 1 of pregnancy by the uterine epithelium and on D4 specifically by the glandular epithelium. The Lif knockout female mice are infertile because of uterine defects that affect embryo implantation, but pregnancy can be rescued in these mice by injections of LIF on D4 of pregnancy. Many of the specific actions of LIF in the uterus are unknown, especially with regard to uterine cell biology. Leukocytes, such as macrophages, natural killer (NK) cells, and eosinophils, are present in the pregnant uterus and are thought to be beneficial, because alterations in their proportions can adversely affect pregnancy. Immunocytochemistry and cell counting were used to compare the distributions and dynamics of leukocyte subpopulations in wild-type and Lif knockout mice. The percentage of macrophages was reduced by more than half in the Lif knockout mice on D3 of pregnancy, and their distribution was disrupted, suggesting that LIF is a chemokine for these cells. The NK cells were detected as early as D3 of pregnancy, but the Lif knockout mice had double the percentage of NK cells compared to wild-type mice at this time, indicating that LIF restricts the migration of NK cells to the uterus. The Lif knockout mice also had significantly higher percentages of eosinophils in the outer stroma on D3, and in the midstroma on D4, of pregnancy, suggesting that LIF also may restrict eosinophil migration to the uterus. These alterations of the uterine leukocyte subpopulations in Lif knockout mice may disrupt pregnancy and contribute to failure of implantation.     

Decidual cell-specific surface antigen(s) recognized by monoclonal antibodies: tissue and species distribution

Decidual cells are direct descendants of endometrial stromal cells and the ultimate progeny of bone marrow-derived precursors. In view of their bone marrow genealogy and demonstrated immunoregulatory role during pregnancy, this study attempted to identify a lineage-specific differentiation marker(s) on murine decidual cells with the hope of tracing their developmental pathway and exploring their familial relationship to other lymphomyeloid cells. Two protein A-binding, IgG2b isotype monoclonal antibodies (secreted by clones 16F12 and 2G4F8) were raised by immunizing virgin CBA mice with syngeneic decidual cells. The presence and the density of the antigenic marker(s) recognized by these antibodies were examined by radioautography on various cell types in single cell suspensions of the decidua, placenta, and lymphomyeloid organs after a sandwich labeling with hybridoma supernatants followed by 125I-protein A. Both antibodies appeared to recognize antigen(s) unique for the decidual cell lineage in mice, humans, and rats. The incidence of antigen-bearing decidual cells increased with gestational age in CBA, C3H, and CD1 mice between days 8 and 14, and in humans between 6 and 10.5 wk; in rats, however, some decline was noted between days 8 and 14. The binding was always higher with 16F12 than with 2G4F8 supernatants. No significant binding of either antibody to trophoblast cells of the placenta or leukocytes within the decidua was noted in any of the above mouse strains or species. Little or no labeling of any cell type was seen on lymphomyeloid cells of the virgin or pregnant CBA mice, but a consistent labeling of a rare blast-type cell in the blood was observed with both antibodies, raising the possibility that this cell may represent the circulating precursor of the decidual cell lineage. It remains to be investigated whether these antibodies are recognizing the same or different differentiation antigen(s) on the decidual cells, and whether a conservation of this antigen(s) during speciation signifies its functional importance.