The uterus in early pregnancy is a non-lymphoid organ that is enriched in natural killer (NK) cells. Studies to address the role of these abundant human NK cells at the maternal/fetal interface have focused on their response to the major histocompatibility complex (MHC) molecules on fetal trophoblast cells that they contact. The interaction of maternal NK cell receptors belonging to the killer cell immunoglobulin-like receptor (KIR) family with trophoblast MHC class I molecules in pregnancy can regulate NK cell activation for secretion of pro-angiogenic factors that promote placental development. This review will cover the role of KIR at the maternal/fetal interface and focus on KIR2DL4, a KIR family member that is uniquely poised to play a role in pregnancy due to the restricted expression of its ligand, human leukocyte antigen (HLA)-G, by fetal trophoblast cells early in pregnancy. The pathways by which KIR2DL4–HLA-G interactions induce the cellular senescence of NK cells and the role of the resulting senescence-associated secretory phenotype (SASP) in vascular remodeling will be discussed in the context of reproduction.
Natural killer (NK) cells are white blood cells that are important for immune defense and reproduction.1,2 Their role in immune defense has been well studied. They participate in innate immunity as the first line of defense through their ability to kill target cells and secrete cytokines. More recently, the identification of several features of NK cells, such as their capacity for memory responses and ability to control adaptive immune cells, underscores their importance as players in adaptive immunity.3,4
In contrast to what is known about their role in immune defense, much less is known about their role in reproduction. Unlike in the peripheral blood, where NK cells make up 2%–5% of lymphocytes, they are the most abundant immune cells (50%–70%) at the maternal/fetal interface during implantation and early pregnancy. Altered cell numbers and genetic association studies involving these NK cells in women with reproductive disorders have shown that they may play a relevant role in reproduction.2
Although systematic manipulation of NK cells in the uterus of pregnant women at different times following implantation is not feasible, much has been learnt from alternative approaches, such as samples from first trimester abortions, term human placenta and timed endometrial biopsies from non-pregnant women.5 This is complemented by studies in pregnant rodents and humanized mice, where findings can be extrapolated to human NK cell functions.6 In this regard, studies in mice identified spiral artery remodeling as a major uterine NK function that results in the conversion of maternal vessels to low-resistance, high-volume conduits to increase blood supply to the fetus.7
NK cells in pregnancy
NK cells are the dominant lymphocyte population that accumulates in the decidua basalis of a healthy pregnancy. At this site, NK cells are CD56bright and CD16neg and phenotypically distinct from peripheral blood NK cells that are predominantly CD56dim and CD16+.2 These uterine NK cells exist in lower numbers in the non-pregnant endometrium and their numbers increase in early pregnancy. The peak human decidual NK cell numbers were identified between 8 and 13 weeks of gestation.8 Recent work has shown a role for the soluble factors secreted by these cells during placental development, both in spiral artery remodeling and in the regulation of trophoblast invasion.8,9
Decidual NK cells are poorly cytotoxic in a normal pregnancy, even though these cells contain lytic granules and are fully capable of cytotoxic activity against classical NK targets, such as K562 cells.10 Recent studies have revealed that decidual NK cells have the potential for efficient pathogen control and clearance as a result of pathogen mediated signals.11 Coculture of human cytomegalovirus-infected decidual fibroblasts with decidual NK cells from early gestation resulted in significant induction of an array of cytokines and chemokines, such as vascular endothelial growth factor-A, IL-6, chemokine (C–X–C motif) ligand 1 and macrophage chemoattractant protein (MCP)-1.12 A combination of the modulation of expression of NK receptors and pathogen-induced ligands allows NK cells to overcome the inhibition of decidual NK killing seen in normal pregnancy. There are regulatory mechanisms in place to prevent killing at the maternal/fetal interface during a healthy pregnancy. These include inhibitory receptors on NK cells belonging to the killer cell immunoglobulin-like receptor (KIR), CD94/NKG2 and ILT families of receptors that recognize major histocompatibility complex (MHC) class I molecules on extravillous trophoblast cells and block cytotoxic responses.2
Of the two types of fetal trophoblast cells at the maternal/fetal interface, villous trophoblast cells, in contact with maternal blood flowing through the intervillous space, do not express any MHC class I molecules and are considered to be immunologically inert. Extravillous trophoblast cells that migrate through the decidua towards the maternal spiral arteries in early pregnancy express HLA-C, HLA-E and HLA-G. In contrast, the classical, polymorphic MHC molecules (HLA-A and HLA-B) are not present on trophoblasts at the maternal/fetal interface. While the inhibitory 2 domain (2D) KIR (KIR2DL1, KIR2DL2 and KIR2DL3) recognize HLA-C alleles, HLA-E is recognized by the inhibitory CD94/NKG2A and the activating CD94/NKG2C heterodimers. Receptors on NK cells for HLA-G include the inhibitory ILT2 (LILRB1) and the activating KIR2DL4.13
Since all extravillous trophoblast cells express HLA-E (which relies on the signal peptides of HLA-C and HLA-G for surface expression) and all decidual NK cells express CD94/NKG2A, this interaction is thought to be responsible for the lack of killing of trophoblasts by NK cells. In addition, inhibition is also provided by interactions of inhibitory KIR with HLA-C and by ILT2 with HLA-G.13
What is the role of HLA-G, a non-classical MHC molecule that is unusual in that it is uniquely restricted to fetal extravillous trophoblast cells under normal physiological conditions? The dogma that HLA-G has a key role in tolerance at the maternal/fetal interface by protecting the fetus from attack by NK cells of the mother 14 is losing ground, as recent findings do not support such a role.15,16,17,18 Earlier reports on the inhibitory effects of HLA-G used total peripheral blood mononuclear cells to study NK cell cytotoxic activity.19,20,21 The inhibitory effect of HLA-G was found to be due to T cells and not NK cells, with HLA-G inhibiting T-cell proliferation and killing while activating decidual NK cells to proliferate and secrete soluble factors.16,17 Thus, the view of a tolerogenic HLA-G is being challenged by more recent evidence that points to a more constructive role in promoting changes in placentation. The presence of soluble HLA-G in human embryo culture after in vitro fertilization has been associated with better pregnancy rates and reduced HLA-G levels in maternal circulation has been reported in disorders of pregnancy such as recurrent spontaneous abortion and pre-eclampsia.22 There are important vascular changes in response to MHC-dependent signals that appear to contribute to the spiral artery remodeling and control of trophoblast invasion that is critical to a successful pregnancy. This is reflected in pathologies related to defects in this vascularization, such as intrauterine growth restriction and pre-eclampsia. Thus, decidual NK responses to MHC class I molecules including HLA-G on trophoblast cells have important consequences for reproductive fitness down the line.
The interplay of KIR and their MHC ligands in pregnancy
Several studies have focused on KIR responses to their MHC ligands on trophoblasts and assessed the impact of these responses on successful placentation.23 KIR family members can be either inhibitory or activating, and NK cell activation is the result of a complex interplay between these different receptors that are stochastically expressed on NK cells.24
Genetic association studies indicate that the interaction between maternal KIR and fetal HLA-C during pregnancy may influence the delivery of sufficient blood supply for the fetus.25 Combinations of fetal HLA-C and maternal KIR that resulted in a potential for increased inhibitory interactions showed association with pre-eclampsia, a condition of pregnancy characterized by inadequate trophoblast invasion and impaired vascular remodeling. The strongest association was seen with maternal KIR of the AA haplotype (lacking activating KIR) in combination with HLA-C2 in the fetus.26 Similar epidemiological evidence was also reported in women with recurrent miscarriage.27 These association studies underscore the importance of MHC–KIR interactions to the regulation of placentation and show that too much inhibition is detrimental to successful placentation. Experimental evidence for a role for excessive NK cell inhibition in compromising reproductive success has been elegantly provided in the mouse system. By using mice that differ in a single additional MHC (expressed by either maternal or fetal tissues) that confers additional inhibitory potential upon recognition of Ly49 family members, it was shown that excess inhibition compromises decidual vascular remodeling and results in fetal growth restriction.28
In a normal pregnancy, it is well documented that decidual NK cells produce soluble factors that influence placental development. These include pro-angiogenic factors, such as vascular endothelial growth factor, angiopoietin-1, angiopoietin-2 and placenta growth factor.9,29,30 The release of these soluble mediators is the result of engagement of activating receptors (in addition to KIR) that are expressed by decidual NK cells, such as NKp30 and NKp46, by ligands on the extravillous trophoblasts and decidual stromal cells.9,31 These receptors also induce the secretion of cytokines and chemokines, such as interferon (IFN)-γ, tumor-necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein-1α and macrophage inflammatory protein-1β. They also secrete chemokines such as IL-8 and IP10 that interact with chemokine receptors on trophoblast cells.9 These chemokines favor the migration of extravillous trophoblast cells into the decidua basalis where they invade the spiral arteries to promote uterine vascular remodeling. The effect of soluble factors, and IFN-γ in particular, on mesometrial spiral artery remodeling, has also been shown in seminal experiments in the mouse.7,32
The KIR family of receptors contains activating isoforms that recognize specific HLA ligands. Engagement of KIR2DS1 on decidual NK cells resulted in the secretion of GM-CSF, a factor that enhances trophoblast migration.33 Likewise, HLA-Cw4-mediated activation of KIR2DS4 on decidual NK cells resulted in the secretion of IL-8, IP10, vascular endothelial growth factor and placenta growth factor.9 Unlike the rest of the KIR2D receptors that are stochastically expressed on all NK cells, KIR2DL4 is an activating receptor that is expressed by all NK cells.34,35 Its relevance at the maternal/fetal interface is due to the trophoblast-specific expression of its ligand, HLA-G, in healthy individuals.
KIR2DL4–HLA-G interactions and endosomal signaling
KIR2DL4 is an unusual KIR family member in many respects, including its structure, expression, localization and signaling function. Unlike the other 2D KIR with a D1–D2 domain structure, it has a hybrid D0–D2 domain structure, utilizing the D0 domain of the 3D KIR (which have D0, D1 and D2 domains). As the anchor gene in the middle of the KIR complex, KIR2DL4 is present in all haplotypes and is constitutively expressed by all NK cells. Although it carries a long cytoplasmic tail characteristic of inhibitory KIR, it carries only a single ITIM and shows weak inhibitory potential.36 It has a charged arginine residue in its transmembrane domain that allows it to pair with FcεRIγ to signal for weak cytotoxicity and cytokine secretion at the cell surface.37
The non-classical MHC-I molecule HLA-G is the ligand for KIR2DL4. The soluble form of HLA-G is likely to be the natural ligand as it accumulates into endosomes containing KIR2DL4.38 Alternate splicing of the transmembrane isoform HLA-G1 results in the soluble HLA-G5. Soluble HLA-G can also be generated by shedding of the extracellular portion of HLA-G1 from the cell surface due to the action of metalloproteases.39 Recombinant KIR2DL4-immunoglobulin fusion proteins bound transfectants expressing high levels of HLA-G on their surface,34,40 and this binding was blocked with antibodies to HLA-G (G233) or KIR2DL4 (#33).38 However, direct binding of soluble forms of HLA-G and KIR2DL4 has not been detected by surface plasmon resonance,41 possibly due to low intrinsic affinity, as seen with the activating KIR and their MHC ligands.42 Tetrameric HLA-G bound ILT4 on monocytic cells, but did not bind either ILT2 or KIR2DL4,43 owing to the very low to negligible surface expression of these two receptors on NK cells. On the cell surface, HLA-G can be expressed as either monomers or as a disulphide-linked homodimer.41,44 While dimeric HLA-G is the preferred ligand of ILT2, whether KIR2DL4 favors monomers or dimers is unknown. From the crystal structure of dimeric HLA-G and modeling of KIR2DL4–HLA-G interactions, it is unlikely that KIR2DL4 can bind HLA-G in its dimeric state.45
In contrast to all the other KIR family members that are expressed well at the cell surface, KIR2DL4 is undetectable at the cell surface of primary, resting NK cells from the peripheral blood. Surface levels of this receptor can be transiently increased upon IL-2 activation.38,46 In decidual NK cells isolated from first trimester abortions, there is evidence of low cell surface expression of KIR2DL4.31 Despite negligible surface expression, in resting NK cells, activation of KIR2DL4 with soluble agonists resulted in strong cytokine responses in the absence of killing.15 This is explained by its intracellular localization in Rab5+ early endosomes.38 This endosomal targeting is unique to KIR2DL4 as other KIR family members are expressed at high levels on the cell surface. Endosomal localization of KIR2DL4 is controlled by its immunoglobulin domains and not by the cytoplasmic tail, where endocytic signals normally reside.47 It was recently reported that syndecan 4, a member of the syndecan family of cell surface proteoglycans, colocalizes with KIR2DL4 in endosomes and that this interaction was reduced in the presence of heparin sulfate.48 However, its potential role as a partner chain responsible for its trafficking to endosomes and signaling there awaits further study.
Analysis of wild-type and chimeric mutants of KIR2DL4 in 293T cells indicated that this receptor needed to be in endosomes to signal, as chimeric receptors targeted to the cell surface could not signal upon crosslinking at the cell surface.38 Signaling by KIR2DL4 is distinct from that of other KIR as it is independent of both ITIM and ITAM-mediated signaling and resistant to inhibitors of Src family kinases and phosphatidylinositide 3-kinase.49 Unexpectedly, Akt phosphorylation (at serine 473) was identified by kinase phosphorylation-profiling upon activation through KIR2DL4. Importantly, this Akt activation upon KIR2DL4 engagement required its endocytosis. Mass spectrometry analysis of proteins associated with endosomal KIR2DL4 was used to identify regulators of Akt activation. This approach identified DNA-PKcs, a DNA damage signaling kinase, as the kinase responsible for phosphorylation of Akt at serine 473.49
KIR2DL4 has a conserved binding motif in its cytoplasmic tail for the ubiquitin ligase TNF-receptor-associated factor 6. Its association with TNF-receptor-associated factor 6 results in phosphorylation of the kinase TAK1 and links it to nuclear factor kappa B activation pathways.50 The ubiquitin ligase TNF-receptor-associated factor 6 is known to couple proximal signals from endosomal receptors, such as the Toll-like receptors, through TAK1 for nuclear factor kappa B-dependent pro-inflammatory responses. In this way, KIR2DL4 activates nuclear factor kappa B responses via the canonical pathway to generate pro-inflammatory/pro-angiogenic signals. Negative regulation of KIR2DL4 signaling occurs through a different ubiquitin ligase, Triad3A that binds the cytoplasmic tail of KIR2DL4 and promotes its degradation.51
Thus, endosomal signaling by KIR2DL4 offers the advantage of sustained signals for the secretion response that involves the secretion of cytokines and chemokines, such as IFN-γ, TNF-α, IL-1α, IL-1β, IL-6 and IL-8.47 Signaling from intracellular endosomal compartments offers the advantage of escaping the regulation by inhibitory NK receptors at the NK cell surface. In addition, endosomes provide a platform that allows optimization of signals that may otherwise be weak due to factors such as low affinity and limiting ligand concentrations.52
Induction of cellular senescence through KIR2DL4–HLA-G interactions
It was surprising that signaling from endosomes for a pro-inflammatory and pro-angiogenic response would involve DNA-PKcs, a kinase involved in DNA damage response signaling pathways. Since DNA damage response signaling occurs during the induction of cellular senescence, the induction of the cyclin kinase inhibitor, p21, a key determinant of senescence was evaluated. Primary NK cells was stimulated with the ligand of KIR2DL4, sHLA-G upregulated p21 and, this was sensitive to inhibitors of DNA-PKcs.53 These cells underwent changes characteristic of senescence, such as changes in cell shape and size, and staining for senescence-associated β-galactosidase. They also remained arrested at the G0/G1 stage of the cell cycle and were refractory to apoptosis.53
The induction of cellular senescence and the cell cycle arrest associated with it has been implicated in aging, cancer and tissue repair.54 Although senescent cells do not divide or undergo apoptosis, they are metabolically very active and secrete an array of soluble mediators collectively termed the senescent-associated secretory phenotype (SASP).55,56 The SASP includes pro-inflammatory cytokines, growth factors and proteases. Once a cell becomes senescent, it has altered interactions with its surrounding milieu via the SASP. This SASP can be both detrimental by promoting tumor growth and age-related pathologies and useful as in the case of tissue repair during wound healing.
Micro-array studies of NK cells stimulated via KIR2DL4 revealed evidence of both senescence markers and SASP. Moreover, the senescence secretome of such cells was capable of inducing both vascular permeability and angiogenesis, using in vitro readouts for permeability induction and tube formation in human umbilical vein endothelial cells.53 Thus, the SASP induced by NK cells in response to sHLA-G could play a favorable role in promoting neovascularization and tissue remodeling at sites of HLA-G expression. These studies used primary, resting NK cells from human peripheral blood to study responses as a result of KIR2DL4–sHLA-G interactions. It is not possible to study the early events of activation when decidual NK cells first encounter fetal trophoblast cells. The typical source of decidual NK cells are cells isolated from first trimester abortions, and in these cells, the early stimulation by trophoblasts secreting soluble HLA-G has already occurred.
To test the hypothesis that decidual NK cells would display a senescent phenotype as a result of early responses to soluble HLA-G from trophoblasts during pregnancy, a retrospective analysis of micro-array data of decidual NK cells from first trimester abortions (gestational age: 6–12 weeks) compared to resting peripheral blood NK cells57 was performed. There was upregulation of genes involved in senescence and SASP in the decidual samples, including genes critical for the induction and maintenance of senescence, such as IL-6, IL-8, IL-1β and CDKN1A (p21).53 It is noteworthy that the cell cycle inhibitor p21, a major determinant of senescence, was upregulated throughout the gestational period studied (6–12 weeks).57 This evidence of a senescence signature in decidual NK cells was validated by gene set enrichment analysis against the molecular signature of oncogene-induced senescence.53 Thus, as seen in NK cells from peripheral blood, the increased expression of genes involved in senescence and SASP is also a feature of NK cells isolated directly from the pregnant uterus in the first trimester.
A role for senescence in reproduction
The decidua in early pregnancy is a setting where the senescence and SASP produced by KIR2DL4–HLA-G interactions may contribute to the normal vascular adaptations that occur to support fetal development (Figure 1).58 NK cells at the implantation site would sense trophoblast invasion by responding to the soluble HLA-G produced by these invading fetal cells. Endosomal signaling by KIR2DL4 in response to HLA-G would result in the reprogramming of NK cells to a senescent state. The resulting SASP would include the production of pro-inflammatory factors, such as TNF-α, IL-1β and IFN-γ, and pro-angiogenic factors, such as IL-6 and IL-8. They also include proteins of the urokinase plasminogen activator system (e.g., uPAR) that contribute to the extracellular matrix breakdown required for both trophoblast invasion and spiral artery remodeling. The resulting SASP would shape and remodel the local environment to increase vascularization required for a growing fetus. In such a situation, senescence would promote reproductive success.
As neither KIR2DL4 nor HLA-G molecules are present in mice, responses generated by their interactions cannot be studied in mice. However, Croy and colleagues5 have noted, using morphological criteria, the presence of large, post-mitotic, heavily granulated senescent cells with nuclear changes in the mid-gestation decidual basalis of pregnant mice. In the mouse, normal vascular remodeling occurs after gestation day 8.5 and is completed by gestation day 10.5. At this time, these senescent cells were seen within lumens of the decidual vessels, embedded within arterial walls and intermingled with decidual cells. Moreover, studies of mouse uterine NK cells showed an increase in the inhibitory receptor KLRG1 with gestational age.59 KLRG1 is a marker of senescence in mouse and human NK and T cells and is thought to block proliferation by interfering with Akt activation.60 Thus, there is indirect evidence of a senescent phenotype of the mouse uterine NK cells during the key period of remodeling to permit placental blood flow. Systematic analysis with other markers of senescence on uterine NK cells at different gestational times is needed.
During normal placental development, the process of cytotrophoblast fusion to form the syncytiotrophoblast continues throughout pregnancy. Markers of senescence, such as SAβG, cyclin-dependent kinase inhibitors p21 and p16, and tumor suppressor p53, were detected on these cells upon cell fusion.61 This cell fusion induced senescence would allow the multinucleate, postmitotic syncytium to sustain pregnancy, while allowing the maintenance of cell cycle arrest.62 Whether senescence occurs in other cells at different stages of placental development and what role, if any it has in placental pathologies merits investigation.
Historically, cellular senescence has been well studied as a cell fate triggered by different stress stimuli, such as DNA damage or oncogene activation. The outcomes of senescence induction and the resulting SASP have been contradictory and confusing.63 The cell cycle arrest associated with senescence is a tumor suppressive mechanism, but the SASP can paradoxically promote cancer and also contributes to age-related pathologies. The SASP can have apparently opposing effects by promoting proliferation and tumor growth on one hand,56 while having the potential to induce paracrine senescence of neighboring cells.64 It is in the context of pregnancy that all the seemingly contradictory features of senescence would be integrated to favor fetal development. Senescence of decidual NK cells upon sensing soluble HLA-G secreted by trophoblast cells would arrest cells, while promoting the secretion of factors that would favor remodeling at this site.58 In this regard, two studies have revealed an unexpected role for senescence during normal development.65,66 Markers of senescence such as SAβG were used to identify senescent cells at specific stages and in distinct patterns in the developing embryo. The presence of these senescent cells in mouse, chick and human embryos revealed senescence to be a conserved feature of vertebrate embryonic development. Developmental senescence involves p21 upregulation, and expression of a subset of SASP proteins, but is independent of DNA damage and other characteristic mediators of classical senescence, such as p53 and p16.65,66
The emerging consensus from the latest developments in senescence research is that cellular senescence arose during evolution as a critical feature of normal development in the removal of unwanted structures and in patterning of the embryo, and in remodeling the maternal vasculature to support fetal development.67 This program is being reused later in life as stress or damage-induced senescence to protect against tumorigenesis and tissue damage by eliminating potentially harmful cells.
NK cells, abundant at the maternal fetal/interface, contribute to reproductive success through a host of receptor–ligand interactions that are subject to spatial and temporal regulation at this site. Vascular remodeling is carried out by cooperative interactions between NK cells and fetal trophoblast cells. NK responses to the soluble HLA-G secreted by extravillous trophoblasts in early pregnancy result in the reprogramming of NK cells towards a senescent state. Cellular senescence is a molecular switch that leads to a regulated and sustained secretory response upon engagement of KIR2DL4 by HLA-G that can promote vascular remodeling. There is a growing appreciation that senescence arose during evolution to serve the function of remodeling and patterning of the embryo in normal development. In the same manner, NK senescence during early gestation would favor reproduction through sustained NK cell activation to remodel maternal vasculature early in pregnancy to support the growing fetus.
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I thank Dr E O Long for his support. This work was supported by the Intramural Research Program of the NIAID, NIH.
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Rajagopalan, S. HLA-G-mediated NK cell senescence promotes vascular remodeling: implications for reproduction. Cell Mol Immunol 11, 460–466 (2014). https://doi.org/10.1038/cmi.2014.53
- cellular senescence
- natural killer cells
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