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The Immunology of Implantation
6
The Immunology of Implantation ASHLEY MOFFETT, Y.W. LOKE AND ANDREW SHARKEY
KEY POINTS • The extravillous pathway of trophoblast differentiation is essential for the development of the fetoplacental blood supply. • As they invade into the maternal decidua, extravillous trophoblast cells express a unique array of human leukocyte antigen (HLA) class I molecules, HLA-G, HLA-E and HLA-C. • The main population of maternal immune cells in the decidua during placentation are uterine natural killer (uNK) cells. • Interaction between polymorphic killer immunoglobulinlike receptors (KIRs) on maternal uNK cells and their HLA-C ligands on fetal trophoblast cells may regulate the depth and extent of vascular modification by trophoblast. • KIR–HLA-C interactions resulting in uNK inhibition are associated with reduced trophoblast invasion and increased risk for the great obstetric syndromes (GOS): pre-eclampsia, stillbirth and fetal growth restriction. • Conversely, KIR–HLA-C interactions that activate uNK are associated with increased birth weight and higher risk for obstructed labour. Hence, the maternal immune system plays a role in regulating human birth weight.
Introduction The traditional way to study pregnancy immunology follows the classical transplantation model, which views the fetus as an allograft. A more recent approach focuses on the unique, local uterine immune response to the implanting placenta. This requires a detailed knowledge of implantation and placental structure because this impacts greatly on the type of immune response produced by the mother. At the implantation site, cells from the mother and the fetus intermingle during pregnancy. Unravelling what happens here is crucial to our understanding of why some human pregnancies are successful but others are not.
Nidation The invasive implantation undertaken by the human embryo brings fetally derived trophoblast cells into direct contact with maternal cells in the uterine mucosa. Initial contact is followed by adhesion between the embryonic trophectoderm of the blastocyst and the uterine surface epithelium.1 As the blastocyst penetrates through the surface epithelium into the uterine mucosa, this trophectoderm layer differentiates into an outer multinucleated syncytiotrophoblast (primitive syncytium) and an inner layer 48
of primitive mononuclear cytotrophoblast. Lacunae soon appear in the syncytium, and these rapidly enlarge by fusing with each other. The uteroplacental circulation is potentially established when this lacuna system erodes through the uterine capillaries. The intervillous space of the definitive placenta is a derivation of these lacunae. The subsequent differentiation of trophoblast occurs along two main pathways, villous and extravillous (Fig. 6.1). Villous trophoblast is in contact with maternal blood in the intervillous space, and its main functions are transport of nutrients and oxygen to the fetus and secretion of hormones. In contrast, extravillous trophoblast is involved in the establishment of the placental blood supply and intermingles with maternal uterine tissues.2 At the tips of some chorionic villi, cytotrophoblast cells proliferate into cytotrophoblast columns that anchor these villi to the underlying decidua. From these columns, individual trophoblast cells break off to invade the decidua. These interstitial extravillous trophoblast cells appear to move towards the decidual spiral arteries, encircling these vessels, which then show endothelial swelling and a characteristic ‘fibrinoid’ destruction of the smooth muscle of the media. How trophoblast cells induce these changes in the vessel wall is unknown. When migrating trophoblast cells reach the decidual–myometrial junction, many become multinucleated placental bed giant cells. These can be regarded as the endpoint of the extravillous pathway of trophoblast differentiation. Cytotrophoblast columns that lie over the openings of the decidual spiral arteries form a plug of cells that are known as endovascular trophoblast. Early in gestation, these plugs occlude the lumen of the vessels (see Fig. 6.1A). This limits the influx of blood in the first trimester so that there is only seepage of serum into the intervillous space. This means that early in pregnancy, the embryo in the first trimester exists in a low-oxygen environment.3 From these plugs, some endovascular trophoblasts move down the inside of the artery, replacing the endothelium, and become incorporated into the vessel wall. At around 10 weeks of gestation, the endovascular plugs disperse, and maternal blood flow to the intervillous space is established. Transformation of the spiral arteries by trophoblast is crucial to successful implantation because these changes convert the arteries from muscular vessels into flaccid sacs capable of transmitting the increased blood flow required for the developing fetoplacental unit. Failure of this arterial transformation will result in reduced conductance and poor perfusion of the placenta, which will affect the development of the villous tree. This in turn will lead to clinical conditions such as miscarriage, stillbirth, fetal growth restriction and pre-eclampsia (Fig. 6.2).4
CHAPTER 6 The Immunology of Implantation
Villous
49
Villous stem cell
ST
Villous cytotrophoblast (CT)
CT COL TS
Extravillous cytotrophoblast column (COL)
ET Epithelial gland F
A
Trophoblast shell (TS)
IT uNK
IT A
Villous syncytiotrophoblast (ST)
F
Interstitial trophoblast (IT)
Endovascular trophoblast (ET)
S GC
T
M
A
B
Placental giant cell (GC)
• Fig. 6.1 Schematic representation of the implantation site. A, Placental villi (top) are shown with anchoring cytotrophoblast cell columns and trophoblast invasion into the maternal decidua (bottom). Maternal blood from decidual spiral arteries (A) fills the intervillous space in direct contact with syncytiotrophoblast (ST). Distinct trophoblast populations are shown. Villous trophoblast comprises: cytotrophoblast (CT) syncytiotrophoblast form the two layers covering placental villi and do not stain for human leukocyte antigen (HLA)-G. Extravillous trophoblast includes cytotrophoblast cell columns (COL), interstitial trophoblast (IT), endovascular trophoblast (ET) and placental bed giant cells (GCs); all stain strongly for HLA-G. Anchoring cell columns coalesce to form a continuous trophoblast shell (TS). From this shell, interstitial trophoblasts (ITs) invade through the decidual stroma to encircle and destroy the arterial media, which is replaced by fibrinoid material (F). ETs move in retrograde fashion down spiral arteries, displacing endothelial cells. On reaching the inner layer of the myometrium, trophoblast cells differentiate to multinuclear giant cells (GCs). The inset shows a representation of cellular interactions within the decidua. ITs are seen between large decidual stromal cells (S). Maternal leukocytes present are mainly uterine natural killer NK (uNK) cells with a few macrophages (Ms) and occasional T cells (Ts). B, Pathways of trophoblast differentiation and trophoblast subtypes at the implantation site. (Panel A adapted from Moffett-King A. Natural killer cells and pregnancy. Nat Rev Immunol 2(9):656–663, 2002.)
Decidualisation The uterine endometrial mucosa into which trophoblast invades is transformed into decidua during pregnancy.5 Morphologically, the most obvious changes occur in the stromal cells, which become rounded and glycogen rich. There is also infiltration by large numbers of bone marrow–derived cells. These changes begin during the luteal phase of the menstrual cycle (predecidual change), but if pregnancy occurs, the decidualisation process continues. This is unlike the situation in most other species in which decidualisation only begins at implantation. Decidualisation is under the control of sex hormones oestrogen and progesterone. Both glandular and stromal cells of the endometrium increasingly express oestrogen and progesterone receptors until the time of ovulation, and expression then declines soon after in the glands. Expression of progesterone receptors continues in the stroma throughout the secretory phase and in early decidua. Prolonged exposure to progesterone results in large, rounded cells that secrete high levels of prolactin and insulin growth factor binding protein-1. Other changes include secretion of interleukin-15 (IL-15), metalloproteinases and chemokines and the laying down of a pericellular rim of matrix proteins, particularly fibronectin.
Current opinion favours the view that decidualisation facilitates implantation by providing an appropriate substrate for trophoblast migration and a fertile soil for nourishment of the developing fetus throughout gestation. However, it is also possible that decidua provides a restraining influence against overinvasion by trophoblast.4,5 This accords with observations that, in situations in which decidualisation is inadequate, such as in ectopic pregnancies or implantation over a previous caesarean section scar, trophoblast invasion is unrestrained, leading to conditions such as placenta accreta. It is likely that decidua provides a balance, allowing migration of trophoblast but only to a certain depth. Thus mammalian reproduction may be considered as a parental tugof-war between the requirements of the fetus to derive as much nourishment as possible from the mother and the defence of the mother to reduce this nutritional burden for the sake of her own health and for future pregnancies.
Trophoblast Interaction with Extracellular Matrix Cell migration depends on the expression of adhesion molecules which bind to extracellular matrix (ECM) proteins. For example,
50
SE C T I O N 2 The Placenta
Myometrium
Chorion
Decidua parietalis
Amnion Amniotic cavity Cervical canal
Allantoic vessels in umbilical cord Placenta vascularised by allantoic vessels Decidua basalis Remnants of yolk sac Radial artery
A
Arcuate artery
Uterine artery Fetus Placenta
Villous trophoblast cell
Placenta Maternal blood in intervillous space
Spiral arterial wall replaced by trophoblast cells (endovascular)
Extravillous trophoblast cells (interstitial)
Decidua basalis
Placental bed giant cells
Decidua basalis Spiral artery remains narrowed in this segment
Basal artery
Media
Media
Myometrium
Endothelium Radial artery
Placental villous tree has fewer branches because of altered blood flow characteristics
Endothelium Radial artery
B C Arcuate artery Arcuate artery • Fig. 6.2 Disorders of human pregnancy resulting from abnormal placentation. A, The blood supply to a human pregnant uterus. B, Normal pregnancy. Maternal blood flow to the intervillous space begins at around 10 weeks’ gestation. The spiral arteries of the placental bed are converted to uteroplacental arteries by the action of migratory extravillous trophoblast cells. Both the arterial media and the endothelium are disrupted by trophoblast cells, converting the artery into a wide-calibre vessel that can deliver blood to the intervillous space at low pressure. The small basal arteries are not involved and remain as nutritive vessels to the inner myometrium and decidua basalis. C, Pre-eclampsia and fetal growth restriction. When trophoblast cell invasion is inadequate, there is deficient transformation of the spiral arteries. The disturbed pattern of blood flow leads to reduced growth of the branches of the placental villous tree, which results in poor fetal growth.
the physiological migration of epithelial cells in wound healing and the pathological invasion of cancer cells require cell–matrix interactions. Trophoblast migration into decidua appears to use similar mechanisms. There are four families of adhesion molecules, of which the most important for adhesion to the ECM are the integrins. These are transmembrane glycoproteins consisting of noncovalently associated α and β subunits. Different α and β subunits exist and the way they combine determines the ligand specificity of the integrin. For example, the heterodimers α1β1 and α6β4 are receptors for the ECM protein, laminin, while α5β1, α4β1 and α4β7 bind fibronectin. Using monoclonal antibodies specific for various subunits, the pattern of expression of integrins by different trophoblast populations at the implantation site is now well documented. The α6β4 integrin is expressed on the villous cytotrophoblast layer and the cytotrophoblast cells of the cell columns nearest the villous core. This integrin disappears further out in the cell columns to be replaced by the heterodimers α5β1 that continue to be expressed by the interstitial trophoblast invading into decidua. Thus the α6β4 laminin receptor is downregulated with a concomitant upregulation of the α5β1 fibronectin receptor as trophoblast
invades the decidua. This observation is similar to that seen during the healing of a skin wound where the sessile keratinocytes that form the normal epidermis express α6β4, but keratinocytes which migrate to close over the wound express α5β1. Binding of trophoblast to fibronectin results in signalling through integrins to the trophoblast cell with changes in gene expression that will affect trophoblast function.6 In pre-eclampsia, trophoblast fails to downregulate β4 as seen in normal pregnancy, indicating that dysregulation of these integrins could contribute to the inadequate trophoblast invasion of decidua associated with this pathological condition.
Matrix Degradation by Trophoblast Besides adhesion to ECM proteins, cellular migration also requires degradation of the matrix. This involves the production of proteolytic enzymes by the migrating cell.7 The two main groups of enzymes are members of the plasminogen activator (PA) system of serine proteases and the family of matrix metalloproteinases (MMPs). The MMP family comprises three main classes based on their substrate specificities: the collagenases, the gelatinases and
CHAPTER 6 The Immunology of Implantation
the stromelysins. There is an intricate interaction between the PA and MMP systems, and together they can break down the major components of ECM. The activity of these proteases is subjected to close control by specific inhibitors. There are two inhibitors for PA (PAI), designated as PAI-1 and PAI-2, and two tissue inhibitors for MMP (TIMP), designated as TIMP-1 and TIMP-2. Trophoblast cells possess proteolytic activity which can be demonstrated in vitro by their digestion of the surrounding matrix on which the cells are seeded. Zymogram studies have shown that trophoblast cells produce a wide array of proteases, this production being greater in first-trimester trophoblast compared with trophoblast later in gestation, which therefore mirrors the invasive capacity of early trophoblast. These observations have led to the conclusion that PA, MMP, PAI and TIMP together provide an intricate network that controls matrix degradation during trophoblast invasion. Although it is clear that changes in integrin and protease expression play an important role in regulating trophoblast differentiation and invasion, the factors that control these changes in normal and pathological pregnancies are poorly understood.
Trophoblast Expression of Major Histocompatibility Complex Antigens Another group of molecules that alter expression as trophoblast invasion occurs are the major histocompatibility complex (MHC) class I and class II antigens. These serve as important recognition molecules for immune cells. There is now good evidence that interactions between MHC antigens and maternal immune cells may regulate the extent of trophoblast invasion. In humans, these MHC molecules are known as human leukocyte antigens (HLAs). HLA class I antigens are expressed on nearly all nucleated cells and class II antigens on specialised cells involved in antigen presentation such as dendritic cells and activated macrophages. Both HLA class I and class II antigens are highly polymorphic and incompatibility for these antigens between donor and recipient is the basis of graft rejection. None of the trophoblast cell populations express HLA class II antigens, and villous trophoblast is also negative for HLA class
I. Maternal T cells cannot therefore directly recognise paternal alloantigens presented by HLA class I on villous trophoblast.8 However, extravillous trophoblast cells that invade decidua and interact with uterine tissues express an unusual array of HLA class I antigens. Presently, there are six HLA class I loci that code for an expressed protein: three classical loci (HLA-A, -B and -C) and three nonclassical loci (HLA-E, -F and -G). Normal somatic cells express HLA-A, -B -C, and the antigens expressed by extravillous trophoblast are HLA-C, -E and -G.4 Of these only HLA-C is highly polymorphic and varies significantly among pregnancies (Table 6.1). By contrast, HLA-E and -G are essentially invariant. In healthy individuals, the expression of HLA-G appears to be restricted to extravillous trophoblast, and this suggests that it might have a role to play in implantation.
Leukocyte Populations in Decidua Analysis of the leukocyte populations in decidua has shown that the predominant cell type is natural killer (NK) cells, with relatively few classical lymphocytes, T or B cells.9 The uterine NK (uNK) cells have prominent cytoplasmic granules and the unusual phenotype of CD56bright CD16-ve. This differentiates them from classical NK cells in peripheral blood, which are CD56dim CD16+. Unlike blood NK cells, uNK cells are weakly cytotoxic against normal NK targets and do not kill trophoblast cells.10 The number of these NK cells in the uterine mucosa varies throughout the menstrual cycle. They are sparse during the proliferative phase, increase significantly by the secretory phase and remain in high numbers in decidua during early gestation. Current evidence suggests their recruitment is hormonally controlled most probably by IL-15 secretion by stromal cells in response to progesterone. The numbers then decline as pregnancy progresses, and very few cells remain by term. During the first trimester, these NK cells are particularly abundant in the decidua basalis in close contact with invading trophoblast cells. This temporal and spatial association with the implanting placenta has led to the proposal that these uNK cells might play an important role in the control of trophoblast migration and differentiation.
TABLE HLA Class I Polymorphism: Expression on Somatic and Extravillous Trophoblast Cells and Corresponding 6.1 Major Receptors
HLA
Protein Sequences
Somatic Cells
Trophoblast
Receptors on
HLA-A
2396
+++
-
T cells (TCR)
HLA-B
3131
+++
-
T cells (TCR)
HLA-C
2089
+
+++
NK cells C1 epitope, KIR2DL2/3 C2 epitope, KIR2DL1 or KIR2DS1 T cells (TCR; rare)
HLA-E
7
+
+
NK cells CD94/NKG2A/D
HLA-F
4
(+)
-
(Poorly defined) KIR3DS1, LILRB1
HLA-G
16
-
+++
NK and myeloid cells LILRB1, LILRB2, KIR2DL4?
+, Low expression; +++, high expression; HLA, human leukocyte antigen; NK, natural killer; TCR, T cell receptor. From Robinson J, Halliwell JA, Hayhurst JH, Flicek P, Parham P, Marsh SGE: The IPD and IPD-IMGT/HLA Database: allele variant databases. Nucleic Acids Research (2015) 43:D423–431.
51
52
SE C T I O N 2 The Placenta
3DL3
C1
C2
2DL3
2DL1
Some C1/C2 2DL4 3DL1
2DS4
3DL2
KIR haplotype A 3DL3 2DS2
2DL2
2DL1
C1
C2
2DL4 3DS1
2DL5 2DS5 2DS1
3DL2
KIR haplotype B
Framework genes
Activating receptors
C2 Inhibitory receptors
Inactivated in >60% of individuals
• Fig. 6.3 Representative killer immunoglobulin-like receptor (KIR) A and B haplotypes of the KIR gene family with known binding of human leukocyte antigen (HLA)-C epitopes (C1 or C2) depicted above their cognate receptors. KIR2DS4 binds a few C1 and C2 allotypes, but more than 60% of individuals have a truncated form of KIR2DS4. KIR2DS4 only recognises some HLA allotypes carrying that epitope. Framework KIR genes that are present in all haplotypes are shown as black boxes. Activating KIRs are shown as blue boxes and inhibitory KIR as red boxes.
Uterine Natural Killer Cell Recognition of Trophoblast
Maternal KIR–Fetal HLA-C Combinations Influence Reproductive Success
Uterine NK cells express an array of receptors, some of which are known to bind to the HLA class I molecules expressed by extravillous trophoblast.4 Unlike blood NK cells, all uNK cells express high levels of the C-type lectin family member CD94/NKG2A, which binds to HLA-E, resulting in inhibition of NK-cell cytotoxicity. Neither ligand or receptor shows significant polymorphism, and this is likely to be the signal that stops uNK cells from killing trophoblast and the maternal cells in the decidua. Uterine NK cells also express members of the killer immunoglobulin-like receptors (KIRs) family of receptors. These are carried together as a haplotype on chromosome 19 and have different binding specificities.11 The KIR also differ in the length of their cytoplasmic tail that either results in an inhibitory or an activating signal to the NK cell. Those that have a short tail (S) are activating (KIR2DS), and those that are long (L) are inhibitory (KIR2DL) receptors. In all populations, there are two main KIR haplotypes, A and B; these differ in the presence of additional activating receptors in the B haplotype. HLA-C, which is the only polymorphic MHC class I molecule expressed by extravillous trophoblast, is the dominant ligand for several KIR receptors. These HLA-C allotypes all fall into two groups, C1 and C2, based on a dimorphism at amino acid 80 of the α1 domain. KIR that bind HLA-C distinguish between C1 and C2 as mutually exclusive epitopes as shown in Fig. 6.3. The maternal–fetal immunologic interaction that occurs at the site of implantation between uNK and trophoblast therefore involves two gene systems, maternal KIR and fetal HLA-C molecules. Because these are both polymorphic and both maternal and paternal HLA-C allotypes are expressed on trophoblasts, the exact KIR–HLA-C interaction differs in each pregnancy. Some KIR–HLA-C combinations appear to be more favourable to trophoblast invasion than others, thus affecting reproductive outcome.
Genetic studies of large pregnancy cohorts have now shown that mothers with two KIR A haplotypes (KIR AA genotype) are at increased risk for disorders of pregnancy, including preeclampsia and other GOS if the fetus carries an HLA-C allele with a C2 epitope inherited from the father.12 Conversely, mothers with a KIR B haplotype (containing activating KIR2DS1 that can also bind C2 epitopes) are at low risk, but instead these mothers have an increased risk for delivering a large baby.13 When the fetus is C1/C1 homozygous, the mother’s KIR genotype has no effect, so a C2 epitope is the crucial fetal ligand (Fig. 6.4). Three major conditions of pregnancy–recurrent miscarriage, FGR and pre-eclampsia–all show the same association. Overall our results suggest that receptor–ligand interactions leading to strong uNK inhibition result in decreased trophoblast invasion and compromised fetal development. Findings in mice support this. Binding of the inhibitory receptor Ly49A on murine uNK cells to a single extra MHC molecule on trophoblast results in decreased uterine vascular remodelling and reduced fetal growth.14 A key question remains: how does the presence of the KIR B haplotype reduce this risk? In Europeans, the protective genes on the KIR B haplotype map to the region where the activating KIR for C2 (KIR2DS1) is located. Protection from pre-eclampsia is likely to be due to counterbalancing uNK activation when KIR2DS1 binds C2. Indeed, when KIR2DS1 on uNK cells binds to C2, this increases secretion of cytokines that enhance trophoblast invasion in vitro.15 If trophoblast invasion in vivo is correspondingly enhanced, this could lead to improved placental perfusion and better fetal growth. In support of this model, we find that highbirth-weight pregnancies are associated with mothers who have inherited the activating receptor KIR2DS1 on the KIR B haplotype and a fetus with an HLA-C allele bearing a C2 epitope. The
CHAPTER 6 The Immunology of Implantation
Baby’s HLA-C Mother’s KIR haplotype C1 C1
KIR2DL1 ↓ A A ↑ A B
B B
C1 C2
C2 C2
↓ ↑
↑ KIR2DS1
↓
↓
↑
↑ KIR2DS1
•
Fig. 6.4 Certain combinations of maternal killer immunoglobulin-like receptor (KIR) and fetal human leukocyte antigen (HLA)-C genotypes increase susceptibility to pre-eclampsia, recurrent miscarriage or fetal growth restriction. A cross (x) indicates the increased risk for a poor clinical outcome. Maternal KIR A haplotype carries the inhibitory KIR2DL1 that can bind the C2 epitope carried by some fetal HLA-C alleles. KIR B haplotypes can also include the activating KIR2DS1 that binds C2.
effect is significant resulting in an estimated average increase in birth weight of some 200 g.13 Conversely, KIR AA mothers who have two copies of KIR2DL1 the inhibitory receptor for C2 and have a fetus with a C2 epitope show reduced birth weight compared with fetuses that lack C2. These effects on both large and small babies are most significant when the fetal C2-bearing allele is paternally derived (Fig. 6.5). Pregnancies at both extremes of birth weight more likely to experience serious obstetric complications. Large babies are at risk for fetal obstruction, which can result in prolonged labour, fetal death from asphyxia and postpartum haemorrhage.16 On the other hand, when spiral artery modification is inadequate, poor placental perfusion can manifest as increased risk for preeclampsia, recurrent miscarriage or FGR, with increased maternal and fetal morbidity. Because humans have a large brain and narrow pelvis relative to other primates, the obstetric dilemma is particularly acute, and there is strong selective pressure to maintain human birth weight between these two extremes. Although many genes and environmental influences have an impact on fetal growth, the genetic studies suggest that KIR–HLA-C interactions play a role in maintaining an optimal birth weight in human populations.
0.4
Birth weight frequency
Population frequency
Special care transfer frequency 0.3
0.2
0.1
0.0
1000
2000
3000
53
4000
5000
6000
Birth weight (g)
Low Increased frequency of KIR AA+ paternal C2
Normal
High Increased frequency of KIR2DS1+ paternal C2
Poor spiral artery transformation
Normal transformation
Exceptional spiral artery transformation?
• Fig. 6.5 The presence of maternal KIR2DS1 is associated with increased birth weight if fetus has inherited a human leukocyte antigen (HLA)-C allele with a C2 epitope. Distribution of birth weights in Norwegian MoBa cohort is shown together with percentage of babies transferred to special care baby unit. The cohort was divided into high- (>90th percentile), normal- (6th–89th percentile) and low- (<5th percentile) birthweight babies. Whereas small babies show increased frequency of maternal killer immunoglobulin-like receptor (KIR) AA and fetal C2, frequency of KIR2DS1 and fetal C2 is higher in large babies compared with normal pregnancies. The schematic below indicates the extent of maternal spiral artery vascular conversion corresponding to each condition. The enhanced vascular conversion depicted in mothers with KIR2DS1 and a fetus with C2 is hypothesised. Evidence directly demonstrating improved blood supply to the placenta in such pregnancies is not yet available. (Original data from Hiby SE, Apps R, Chazara O, et al. Maternal KIR in combination with paternal HLA-C2 regulate human birth weight. J Immunol 192:5069–5073, 2014.)
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SE C T I O N 2 The Placenta
The third HLA class I molecule expressed by trophoblast is HLA-G, which binds with high affinity to leukocyte immunoglobulin-like receptors (LILRs) expressed by myelomonocytic cells. This interaction results in the induction of a ‘tolerogenic’ population of dendritic cells which, in a transplantation setting, leads to tolerance. The idea that the placenta itself (via HLA-G) is modifying the maternal immune reactivity locally in the uterus to downregulate damaging alloreactive T-cell responses during pregnancy is attractive. Thus HLA-G could act as a ‘placental’ signal to the decidual innate immune system through LILRB1 on myelomonocytic cells to induce pregnancy-specific immune functions in the uterus.4,17 Adults homozygous for HLA-G null alleles have been identified, showing that expression of membrane-bound HLA-G by trophoblast is not essential for successful pregnancy.18 It is likely that HLA-G provides only one of several redundant mechanisms, including absence of HLA-A or HLA-B on trophoblast, that favour T-cell tolerance in the decidua. Migration of antigen presenting cells from decidua to local lymph nodes is poor, and chemokines that recruit T cells are epigenetically silenced. These effects work together with antigen-independent mechanisms such as expression of indoleamine 2,3-dioxygenase (IDO), galectins and secretion of immunosuppressive cytokines such as transforming growth factor β, to create a local tolerogenic immune environment.11 This favours the generation of regulatory T cells (Tregs) that could act to limit the development of effector T cells at the maternal–fetal interface. Although Treg depletion in mice leads to fetal loss, indicating an important role, the mechanism and specificity of these cells are unclear. Tregs are also generated in HLA-C–mismatched human pregnancies to a greater degree than in HLA-C– matched pregnancies, but there still is no convincing evidence to date that demonstrates that T cell–mediated mechanisms are responsible for human pregnancy failure.19 Further work is required to understand the role played by T cells at the maternofetal interface.
Conclusion Adequate trophoblast invasion and vascular remodelling is required for proper placental and fetal growth. In addition, epidemiological data have shown that growth retardation in utero is associated with increased incidence of certain diseases in adulthood. Thus any dysregulation of placentation has farreaching consequences. Recent genetic and functional studies provide strong evidence that interactions of KIR on uNK cells with HLA-C on trophoblast play an important role in regulating the depth of trophoblast invasion. KIR–HLA-C combinations that cause excessive uNK inhibition are associated with reduced vascular remodelling and increased risk for GOS, including pre-eclampsia, recurrent miscarriage, stillbirth and FGR. Conversely, combinations that enhance uNK activation may contribute to increased birth weight. A direct effect of uNK cells on spiral artery structure and function is also likely. The relative importance of interactions between the three components– uNK cells, trophoblast and arteries–probably varies in different species. It is also clear that maternal recognition of pregnancy results in changes in decidual T cells, including Tregs, but there is no compelling evidence that maternal T cells specific for fetal HLA molecules are directly involved in pregnancy disorders.9,19 Whatever mechanisms are involved, the maternal immune system must provide a balance between fetal intrusion into the mother’s resources and the need to protect the mother from excessive fetal demand. In studying this, the view of the uterus as a ‘privileged site’ is no longer valid because all anatomical sites have unique immune features, and this applies particularly to mucosal surfaces. The unusual features of the decidua result in a peaceful physiological environment in which specific combinations of maternal KIR and HLA-C contribute to successful reproduction and act to maintain birth weight between two extremes. Access the complete reference list online at ExpertConsult.com. Self-assessment questions available at ExpertConsult.com.
References 1. Cha J, Sun X, Dey SK. Mechanisms of implantation: strategies for successful pregnancy. Nat Med. 2012;18:1754–1767. 2. Pijnenborg R, Vercruysse L, Hanssens M. The uterine spiral arteries in human pregnancy: facts and controversies. Placenta. 2006;27: 939–958. 3. Burton GJ, Jauniaux E, Watson AL. Maternal arterial connections to the placental intervillous space during the first trimester of human pregnancy: the Boyd collection revisited. Am J Obstet Gynecol. 1999;181:718–724. 4. Moffett-King A. Natural killer cells and pregnancy. Nat Rev Immunol. 2002;2:656–663. 5. Gellersen B, Brosens IA, Brosens JJ. Decidualization of the human endometrium: mechanisms, functions, and clinical perspectives. Semin Reprod Med. 2007;25:445–453. 6. Pollheimer J, Fock V, Knöfler M. Review: the ADAM metalloproteinases—novel regulators of trophoblast invasion? Placenta. 2014;35(suppl):S57–S63. 7. Aghababaei M, Beristain AG. The Elsevier Trophoblast Research Award Lecture: importance
of metzincin proteases in trophoblast biology and placental development: a focus on ADAM12. Placenta. 2015;36(suppl 1):S11– S19. 8. Nancy P, Erlebacher A. T cell behavior at the maternal-fetal interface. Int J Dev Biol. 2014;58:189–198. 9. Moffett A, Colucci F. Uterine NK cells: active regulators at the maternal-fetal interface. J Clin Invest. 2014;124:1872–1879. 10. King A, Birkby C, Loke YW. Early human decidual cells exhibit NK activity against the K562 cell line but not against first trimester trophoblast. Cell Immunol. 1989;118:337–344. 11. Moffett A, Hiby SE. How does the maternal immune system contribute to the development of pre-eclampsia? Placenta. 2007;28(suppl A):S51–S56. 12. Hiby SE, Walker JJ, O’shaughnessy KM, et al. Combinations of maternal KIR and fetal HLA-C genes influence the risk of preeclampsia and reproductive success. J Exp Med. 2004;200:957–9565. 13. Hiby SE, Apps R, Chazara O, et al. Maternal KIR in combination with paternal HLAC2 regulate human birth weight. J Immunol. 2014;192:5069–5073.
14. Kieckbusch J, Gaynor LM, Moffett A, et al. MHC-dependent inhibition of uterine NK cells impedes fetal growth and decidual vascular remodelling. Nat Commun. 2014;5:3359. 15. Xiong S, Sharkey AM, Kennedy PR, et al. Maternal uterine NK cell-activating receptor KIR2DS1 enhances placentation. J Clin Invest. 2013;123:4264–4272. 16. Wittman AB, Wall LL. The evolutionary origins of obstructed labor: bipedalism, encephalization, and the human obstetric dilemma. Obstet Gynecol Surv. 2007;62:739–748. 17. Li C, Houser BL, Nicotra ML, et al. HLA-G homodimer-induced cytokine secretion through HLA-G receptors on human decidual macrophages and natural killer cells. Proc Natl Acad Sci U S A. 2009;106:5767–5772. 18. Ober C, Aldrich C, Rosinsky B, et al. HLAG1 protein expression is not essential for fetal survival. Placenta. 1998;19:127–132. 19. Tilburgs T, Scherjon SA, van der Mast BJ, et al. Fetal-maternal HLA-C mismatch is associated with decidual T cell activation and induction of functional T regulatory cells. J Reprod Immunol. 2009;82:148–157.
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The Immunology of Implantation ASHLEY MOFFETT, Y.W. LOKE AND ANDREW SHARKEY
KEY POINTS • The extravillous pathway of trophoblast differentiation is essential for the development of the fetoplacental blood supply. • As they invade into the maternal decidua, extravillous trophoblast cells express a unique array of human leukocyte antigen (HLA) class I molecules, HLA-G, HLA-E and HLA-C. • The main population of maternal immune cells in the decidua during placentation are uterine natural killer (uNK) cells. • Interaction between polymorphic killer immunoglobulinlike receptors (KIRs) on maternal uNK cells and their HLA-C ligands on fetal trophoblast cells may regulate the depth and extent of vascular modification by trophoblast. • KIR–HLA-C interactions resulting in uNK inhibition are associated with reduced trophoblast invasion and increased risk for the great obstetric syndromes (GOS): pre-eclampsia, stillbirth and fetal growth restriction. • Conversely, KIR–HLA-C interactions that activate uNK are associated with increased birth weight and higher risk for obstructed labour. Hence, the maternal immune system plays a role in regulating human birth weight.
Introduction The traditional way to study pregnancy immunology follows the classical transplantation model, which views the fetus as an allograft. A more recent approach focuses on the unique, local uterine immune response to the implanting placenta. This requires a detailed knowledge of implantation and placental structure because this impacts greatly on the type of immune response produced by the mother. At the implantation site, cells from the mother and the fetus intermingle during pregnancy. Unravelling what happens here is crucial to our understanding of why some human pregnancies are successful but others are not.
Nidation The invasive implantation undertaken by the human embryo brings fetally derived trophoblast cells into direct contact with maternal cells in the uterine mucosa. Initial contact is followed by adhesion between the embryonic trophectoderm of the blastocyst and the uterine surface epithelium.1 As the blastocyst penetrates through the surface epithelium into the uterine mucosa, this trophectoderm layer differentiates into an outer multinucleated syncytiotrophoblast (primitive syncytium) and an inner layer 48
of primitive mononuclear cytotrophoblast. Lacunae soon appear in the syncytium, and these rapidly enlarge by fusing with each other. The uteroplacental circulation is potentially established when this lacuna system erodes through the uterine capillaries. The intervillous space of the definitive placenta is a derivation of these lacunae. The subsequent differentiation of trophoblast occurs along two main pathways, villous and extravillous (Fig. 6.1). Villous trophoblast is in contact with maternal blood in the intervillous space, and its main functions are transport of nutrients and oxygen to the fetus and secretion of hormones. In contrast, extravillous trophoblast is involved in the establishment of the placental blood supply and intermingles with maternal uterine tissues.2 At the tips of some chorionic villi, cytotrophoblast cells proliferate into cytotrophoblast columns that anchor these villi to the underlying decidua. From these columns, individual trophoblast cells break off to invade the decidua. These interstitial extravillous trophoblast cells appear to move towards the decidual spiral arteries, encircling these vessels, which then show endothelial swelling and a characteristic ‘fibrinoid’ destruction of the smooth muscle of the media. How trophoblast cells induce these changes in the vessel wall is unknown. When migrating trophoblast cells reach the decidual–myometrial junction, many become multinucleated placental bed giant cells. These can be regarded as the endpoint of the extravillous pathway of trophoblast differentiation. Cytotrophoblast columns that lie over the openings of the decidual spiral arteries form a plug of cells that are known as endovascular trophoblast. Early in gestation, these plugs occlude the lumen of the vessels (see Fig. 6.1A). This limits the influx of blood in the first trimester so that there is only seepage of serum into the intervillous space. This means that early in pregnancy, the embryo in the first trimester exists in a low-oxygen environment.3 From these plugs, some endovascular trophoblasts move down the inside of the artery, replacing the endothelium, and become incorporated into the vessel wall. At around 10 weeks of gestation, the endovascular plugs disperse, and maternal blood flow to the intervillous space is established. Transformation of the spiral arteries by trophoblast is crucial to successful implantation because these changes convert the arteries from muscular vessels into flaccid sacs capable of transmitting the increased blood flow required for the developing fetoplacental unit. Failure of this arterial transformation will result in reduced conductance and poor perfusion of the placenta, which will affect the development of the villous tree. This in turn will lead to clinical conditions such as miscarriage, stillbirth, fetal growth restriction and pre-eclampsia (Fig. 6.2).4
CHAPTER 6 The Immunology of Implantation
Villous
49
Villous stem cell
ST
Villous cytotrophoblast (CT)
CT COL TS
Extravillous cytotrophoblast column (COL)
ET Epithelial gland F
A
Trophoblast shell (TS)
IT uNK
IT A
Villous syncytiotrophoblast (ST)
F
Interstitial trophoblast (IT)
Endovascular trophoblast (ET)
S GC
T
M
A
B
Placental giant cell (GC)
• Fig. 6.1 Schematic representation of the implantation site. A, Placental villi (top) are shown with anchoring cytotrophoblast cell columns and trophoblast invasion into the maternal decidua (bottom). Maternal blood from decidual spiral arteries (A) fills the intervillous space in direct contact with syncytiotrophoblast (ST). Distinct trophoblast populations are shown. Villous trophoblast comprises: cytotrophoblast (CT) syncytiotrophoblast form the two layers covering placental villi and do not stain for human leukocyte antigen (HLA)-G. Extravillous trophoblast includes cytotrophoblast cell columns (COL), interstitial trophoblast (IT), endovascular trophoblast (ET) and placental bed giant cells (GCs); all stain strongly for HLA-G. Anchoring cell columns coalesce to form a continuous trophoblast shell (TS). From this shell, interstitial trophoblasts (ITs) invade through the decidual stroma to encircle and destroy the arterial media, which is replaced by fibrinoid material (F). ETs move in retrograde fashion down spiral arteries, displacing endothelial cells. On reaching the inner layer of the myometrium, trophoblast cells differentiate to multinuclear giant cells (GCs). The inset shows a representation of cellular interactions within the decidua. ITs are seen between large decidual stromal cells (S). Maternal leukocytes present are mainly uterine natural killer NK (uNK) cells with a few macrophages (Ms) and occasional T cells (Ts). B, Pathways of trophoblast differentiation and trophoblast subtypes at the implantation site. (Panel A adapted from Moffett-King A. Natural killer cells and pregnancy. Nat Rev Immunol 2(9):656–663, 2002.)
Decidualisation The uterine endometrial mucosa into which trophoblast invades is transformed into decidua during pregnancy.5 Morphologically, the most obvious changes occur in the stromal cells, which become rounded and glycogen rich. There is also infiltration by large numbers of bone marrow–derived cells. These changes begin during the luteal phase of the menstrual cycle (predecidual change), but if pregnancy occurs, the decidualisation process continues. This is unlike the situation in most other species in which decidualisation only begins at implantation. Decidualisation is under the control of sex hormones oestrogen and progesterone. Both glandular and stromal cells of the endometrium increasingly express oestrogen and progesterone receptors until the time of ovulation, and expression then declines soon after in the glands. Expression of progesterone receptors continues in the stroma throughout the secretory phase and in early decidua. Prolonged exposure to progesterone results in large, rounded cells that secrete high levels of prolactin and insulin growth factor binding protein-1. Other changes include secretion of interleukin-15 (IL-15), metalloproteinases and chemokines and the laying down of a pericellular rim of matrix proteins, particularly fibronectin.
Current opinion favours the view that decidualisation facilitates implantation by providing an appropriate substrate for trophoblast migration and a fertile soil for nourishment of the developing fetus throughout gestation. However, it is also possible that decidua provides a restraining influence against overinvasion by trophoblast.4,5 This accords with observations that, in situations in which decidualisation is inadequate, such as in ectopic pregnancies or implantation over a previous caesarean section scar, trophoblast invasion is unrestrained, leading to conditions such as placenta accreta. It is likely that decidua provides a balance, allowing migration of trophoblast but only to a certain depth. Thus mammalian reproduction may be considered as a parental tugof-war between the requirements of the fetus to derive as much nourishment as possible from the mother and the defence of the mother to reduce this nutritional burden for the sake of her own health and for future pregnancies.
Trophoblast Interaction with Extracellular Matrix Cell migration depends on the expression of adhesion molecules which bind to extracellular matrix (ECM) proteins. For example,
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SE C T I O N 2 The Placenta
Myometrium
Chorion
Decidua parietalis
Amnion Amniotic cavity Cervical canal
Allantoic vessels in umbilical cord Placenta vascularised by allantoic vessels Decidua basalis Remnants of yolk sac Radial artery
A
Arcuate artery
Uterine artery Fetus Placenta
Villous trophoblast cell
Placenta Maternal blood in intervillous space
Spiral arterial wall replaced by trophoblast cells (endovascular)
Extravillous trophoblast cells (interstitial)
Decidua basalis
Placental bed giant cells
Decidua basalis Spiral artery remains narrowed in this segment
Basal artery
Media
Media
Myometrium
Endothelium Radial artery
Placental villous tree has fewer branches because of altered blood flow characteristics
Endothelium Radial artery
B C Arcuate artery Arcuate artery • Fig. 6.2 Disorders of human pregnancy resulting from abnormal placentation. A, The blood supply to a human pregnant uterus. B, Normal pregnancy. Maternal blood flow to the intervillous space begins at around 10 weeks’ gestation. The spiral arteries of the placental bed are converted to uteroplacental arteries by the action of migratory extravillous trophoblast cells. Both the arterial media and the endothelium are disrupted by trophoblast cells, converting the artery into a wide-calibre vessel that can deliver blood to the intervillous space at low pressure. The small basal arteries are not involved and remain as nutritive vessels to the inner myometrium and decidua basalis. C, Pre-eclampsia and fetal growth restriction. When trophoblast cell invasion is inadequate, there is deficient transformation of the spiral arteries. The disturbed pattern of blood flow leads to reduced growth of the branches of the placental villous tree, which results in poor fetal growth.
the physiological migration of epithelial cells in wound healing and the pathological invasion of cancer cells require cell–matrix interactions. Trophoblast migration into decidua appears to use similar mechanisms. There are four families of adhesion molecules, of which the most important for adhesion to the ECM are the integrins. These are transmembrane glycoproteins consisting of noncovalently associated α and β subunits. Different α and β subunits exist and the way they combine determines the ligand specificity of the integrin. For example, the heterodimers α1β1 and α6β4 are receptors for the ECM protein, laminin, while α5β1, α4β1 and α4β7 bind fibronectin. Using monoclonal antibodies specific for various subunits, the pattern of expression of integrins by different trophoblast populations at the implantation site is now well documented. The α6β4 integrin is expressed on the villous cytotrophoblast layer and the cytotrophoblast cells of the cell columns nearest the villous core. This integrin disappears further out in the cell columns to be replaced by the heterodimers α5β1 that continue to be expressed by the interstitial trophoblast invading into decidua. Thus the α6β4 laminin receptor is downregulated with a concomitant upregulation of the α5β1 fibronectin receptor as trophoblast
invades the decidua. This observation is similar to that seen during the healing of a skin wound where the sessile keratinocytes that form the normal epidermis express α6β4, but keratinocytes which migrate to close over the wound express α5β1. Binding of trophoblast to fibronectin results in signalling through integrins to the trophoblast cell with changes in gene expression that will affect trophoblast function.6 In pre-eclampsia, trophoblast fails to downregulate β4 as seen in normal pregnancy, indicating that dysregulation of these integrins could contribute to the inadequate trophoblast invasion of decidua associated with this pathological condition.
Matrix Degradation by Trophoblast Besides adhesion to ECM proteins, cellular migration also requires degradation of the matrix. This involves the production of proteolytic enzymes by the migrating cell.7 The two main groups of enzymes are members of the plasminogen activator (PA) system of serine proteases and the family of matrix metalloproteinases (MMPs). The MMP family comprises three main classes based on their substrate specificities: the collagenases, the gelatinases and
CHAPTER 6 The Immunology of Implantation
the stromelysins. There is an intricate interaction between the PA and MMP systems, and together they can break down the major components of ECM. The activity of these proteases is subjected to close control by specific inhibitors. There are two inhibitors for PA (PAI), designated as PAI-1 and PAI-2, and two tissue inhibitors for MMP (TIMP), designated as TIMP-1 and TIMP-2. Trophoblast cells possess proteolytic activity which can be demonstrated in vitro by their digestion of the surrounding matrix on which the cells are seeded. Zymogram studies have shown that trophoblast cells produce a wide array of proteases, this production being greater in first-trimester trophoblast compared with trophoblast later in gestation, which therefore mirrors the invasive capacity of early trophoblast. These observations have led to the conclusion that PA, MMP, PAI and TIMP together provide an intricate network that controls matrix degradation during trophoblast invasion. Although it is clear that changes in integrin and protease expression play an important role in regulating trophoblast differentiation and invasion, the factors that control these changes in normal and pathological pregnancies are poorly understood.
Trophoblast Expression of Major Histocompatibility Complex Antigens Another group of molecules that alter expression as trophoblast invasion occurs are the major histocompatibility complex (MHC) class I and class II antigens. These serve as important recognition molecules for immune cells. There is now good evidence that interactions between MHC antigens and maternal immune cells may regulate the extent of trophoblast invasion. In humans, these MHC molecules are known as human leukocyte antigens (HLAs). HLA class I antigens are expressed on nearly all nucleated cells and class II antigens on specialised cells involved in antigen presentation such as dendritic cells and activated macrophages. Both HLA class I and class II antigens are highly polymorphic and incompatibility for these antigens between donor and recipient is the basis of graft rejection. None of the trophoblast cell populations express HLA class II antigens, and villous trophoblast is also negative for HLA class
I. Maternal T cells cannot therefore directly recognise paternal alloantigens presented by HLA class I on villous trophoblast.8 However, extravillous trophoblast cells that invade decidua and interact with uterine tissues express an unusual array of HLA class I antigens. Presently, there are six HLA class I loci that code for an expressed protein: three classical loci (HLA-A, -B and -C) and three nonclassical loci (HLA-E, -F and -G). Normal somatic cells express HLA-A, -B -C, and the antigens expressed by extravillous trophoblast are HLA-C, -E and -G.4 Of these only HLA-C is highly polymorphic and varies significantly among pregnancies (Table 6.1). By contrast, HLA-E and -G are essentially invariant. In healthy individuals, the expression of HLA-G appears to be restricted to extravillous trophoblast, and this suggests that it might have a role to play in implantation.
Leukocyte Populations in Decidua Analysis of the leukocyte populations in decidua has shown that the predominant cell type is natural killer (NK) cells, with relatively few classical lymphocytes, T or B cells.9 The uterine NK (uNK) cells have prominent cytoplasmic granules and the unusual phenotype of CD56bright CD16-ve. This differentiates them from classical NK cells in peripheral blood, which are CD56dim CD16+. Unlike blood NK cells, uNK cells are weakly cytotoxic against normal NK targets and do not kill trophoblast cells.10 The number of these NK cells in the uterine mucosa varies throughout the menstrual cycle. They are sparse during the proliferative phase, increase significantly by the secretory phase and remain in high numbers in decidua during early gestation. Current evidence suggests their recruitment is hormonally controlled most probably by IL-15 secretion by stromal cells in response to progesterone. The numbers then decline as pregnancy progresses, and very few cells remain by term. During the first trimester, these NK cells are particularly abundant in the decidua basalis in close contact with invading trophoblast cells. This temporal and spatial association with the implanting placenta has led to the proposal that these uNK cells might play an important role in the control of trophoblast migration and differentiation.
TABLE HLA Class I Polymorphism: Expression on Somatic and Extravillous Trophoblast Cells and Corresponding 6.1 Major Receptors
HLA
Protein Sequences
Somatic Cells
Trophoblast
Receptors on
HLA-A
2396
+++
-
T cells (TCR)
HLA-B
3131
+++
-
T cells (TCR)
HLA-C
2089
+
+++
NK cells C1 epitope, KIR2DL2/3 C2 epitope, KIR2DL1 or KIR2DS1 T cells (TCR; rare)
HLA-E
7
+
+
NK cells CD94/NKG2A/D
HLA-F
4
(+)
-
(Poorly defined) KIR3DS1, LILRB1
HLA-G
16
-
+++
NK and myeloid cells LILRB1, LILRB2, KIR2DL4?
+, Low expression; +++, high expression; HLA, human leukocyte antigen; NK, natural killer; TCR, T cell receptor. From Robinson J, Halliwell JA, Hayhurst JH, Flicek P, Parham P, Marsh SGE: The IPD and IPD-IMGT/HLA Database: allele variant databases. Nucleic Acids Research (2015) 43:D423–431.
51
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SE C T I O N 2 The Placenta
3DL3
C1
C2
2DL3
2DL1
Some C1/C2 2DL4 3DL1
2DS4
3DL2
KIR haplotype A 3DL3 2DS2
2DL2
2DL1
C1
C2
2DL4 3DS1
2DL5 2DS5 2DS1
3DL2
KIR haplotype B
Framework genes
Activating receptors
C2 Inhibitory receptors
Inactivated in >60% of individuals
• Fig. 6.3 Representative killer immunoglobulin-like receptor (KIR) A and B haplotypes of the KIR gene family with known binding of human leukocyte antigen (HLA)-C epitopes (C1 or C2) depicted above their cognate receptors. KIR2DS4 binds a few C1 and C2 allotypes, but more than 60% of individuals have a truncated form of KIR2DS4. KIR2DS4 only recognises some HLA allotypes carrying that epitope. Framework KIR genes that are present in all haplotypes are shown as black boxes. Activating KIRs are shown as blue boxes and inhibitory KIR as red boxes.
Uterine Natural Killer Cell Recognition of Trophoblast
Maternal KIR–Fetal HLA-C Combinations Influence Reproductive Success
Uterine NK cells express an array of receptors, some of which are known to bind to the HLA class I molecules expressed by extravillous trophoblast.4 Unlike blood NK cells, all uNK cells express high levels of the C-type lectin family member CD94/NKG2A, which binds to HLA-E, resulting in inhibition of NK-cell cytotoxicity. Neither ligand or receptor shows significant polymorphism, and this is likely to be the signal that stops uNK cells from killing trophoblast and the maternal cells in the decidua. Uterine NK cells also express members of the killer immunoglobulin-like receptors (KIRs) family of receptors. These are carried together as a haplotype on chromosome 19 and have different binding specificities.11 The KIR also differ in the length of their cytoplasmic tail that either results in an inhibitory or an activating signal to the NK cell. Those that have a short tail (S) are activating (KIR2DS), and those that are long (L) are inhibitory (KIR2DL) receptors. In all populations, there are two main KIR haplotypes, A and B; these differ in the presence of additional activating receptors in the B haplotype. HLA-C, which is the only polymorphic MHC class I molecule expressed by extravillous trophoblast, is the dominant ligand for several KIR receptors. These HLA-C allotypes all fall into two groups, C1 and C2, based on a dimorphism at amino acid 80 of the α1 domain. KIR that bind HLA-C distinguish between C1 and C2 as mutually exclusive epitopes as shown in Fig. 6.3. The maternal–fetal immunologic interaction that occurs at the site of implantation between uNK and trophoblast therefore involves two gene systems, maternal KIR and fetal HLA-C molecules. Because these are both polymorphic and both maternal and paternal HLA-C allotypes are expressed on trophoblasts, the exact KIR–HLA-C interaction differs in each pregnancy. Some KIR–HLA-C combinations appear to be more favourable to trophoblast invasion than others, thus affecting reproductive outcome.
Genetic studies of large pregnancy cohorts have now shown that mothers with two KIR A haplotypes (KIR AA genotype) are at increased risk for disorders of pregnancy, including preeclampsia and other GOS if the fetus carries an HLA-C allele with a C2 epitope inherited from the father.12 Conversely, mothers with a KIR B haplotype (containing activating KIR2DS1 that can also bind C2 epitopes) are at low risk, but instead these mothers have an increased risk for delivering a large baby.13 When the fetus is C1/C1 homozygous, the mother’s KIR genotype has no effect, so a C2 epitope is the crucial fetal ligand (Fig. 6.4). Three major conditions of pregnancy–recurrent miscarriage, FGR and pre-eclampsia–all show the same association. Overall our results suggest that receptor–ligand interactions leading to strong uNK inhibition result in decreased trophoblast invasion and compromised fetal development. Findings in mice support this. Binding of the inhibitory receptor Ly49A on murine uNK cells to a single extra MHC molecule on trophoblast results in decreased uterine vascular remodelling and reduced fetal growth.14 A key question remains: how does the presence of the KIR B haplotype reduce this risk? In Europeans, the protective genes on the KIR B haplotype map to the region where the activating KIR for C2 (KIR2DS1) is located. Protection from pre-eclampsia is likely to be due to counterbalancing uNK activation when KIR2DS1 binds C2. Indeed, when KIR2DS1 on uNK cells binds to C2, this increases secretion of cytokines that enhance trophoblast invasion in vitro.15 If trophoblast invasion in vivo is correspondingly enhanced, this could lead to improved placental perfusion and better fetal growth. In support of this model, we find that highbirth-weight pregnancies are associated with mothers who have inherited the activating receptor KIR2DS1 on the KIR B haplotype and a fetus with an HLA-C allele bearing a C2 epitope. The
CHAPTER 6 The Immunology of Implantation
Baby’s HLA-C Mother’s KIR haplotype C1 C1
KIR2DL1 ↓ A A ↑ A B
B B
C1 C2
C2 C2
↓ ↑
↑ KIR2DS1
↓
↓
↑
↑ KIR2DS1
•
Fig. 6.4 Certain combinations of maternal killer immunoglobulin-like receptor (KIR) and fetal human leukocyte antigen (HLA)-C genotypes increase susceptibility to pre-eclampsia, recurrent miscarriage or fetal growth restriction. A cross (x) indicates the increased risk for a poor clinical outcome. Maternal KIR A haplotype carries the inhibitory KIR2DL1 that can bind the C2 epitope carried by some fetal HLA-C alleles. KIR B haplotypes can also include the activating KIR2DS1 that binds C2.
effect is significant resulting in an estimated average increase in birth weight of some 200 g.13 Conversely, KIR AA mothers who have two copies of KIR2DL1 the inhibitory receptor for C2 and have a fetus with a C2 epitope show reduced birth weight compared with fetuses that lack C2. These effects on both large and small babies are most significant when the fetal C2-bearing allele is paternally derived (Fig. 6.5). Pregnancies at both extremes of birth weight more likely to experience serious obstetric complications. Large babies are at risk for fetal obstruction, which can result in prolonged labour, fetal death from asphyxia and postpartum haemorrhage.16 On the other hand, when spiral artery modification is inadequate, poor placental perfusion can manifest as increased risk for preeclampsia, recurrent miscarriage or FGR, with increased maternal and fetal morbidity. Because humans have a large brain and narrow pelvis relative to other primates, the obstetric dilemma is particularly acute, and there is strong selective pressure to maintain human birth weight between these two extremes. Although many genes and environmental influences have an impact on fetal growth, the genetic studies suggest that KIR–HLA-C interactions play a role in maintaining an optimal birth weight in human populations.
0.4
Birth weight frequency
Population frequency
Special care transfer frequency 0.3
0.2
0.1
0.0
1000
2000
3000
53
4000
5000
6000
Birth weight (g)
Low Increased frequency of KIR AA+ paternal C2
Normal
High Increased frequency of KIR2DS1+ paternal C2
Poor spiral artery transformation
Normal transformation
Exceptional spiral artery transformation?
• Fig. 6.5 The presence of maternal KIR2DS1 is associated with increased birth weight if fetus has inherited a human leukocyte antigen (HLA)-C allele with a C2 epitope. Distribution of birth weights in Norwegian MoBa cohort is shown together with percentage of babies transferred to special care baby unit. The cohort was divided into high- (>90th percentile), normal- (6th–89th percentile) and low- (<5th percentile) birthweight babies. Whereas small babies show increased frequency of maternal killer immunoglobulin-like receptor (KIR) AA and fetal C2, frequency of KIR2DS1 and fetal C2 is higher in large babies compared with normal pregnancies. The schematic below indicates the extent of maternal spiral artery vascular conversion corresponding to each condition. The enhanced vascular conversion depicted in mothers with KIR2DS1 and a fetus with C2 is hypothesised. Evidence directly demonstrating improved blood supply to the placenta in such pregnancies is not yet available. (Original data from Hiby SE, Apps R, Chazara O, et al. Maternal KIR in combination with paternal HLA-C2 regulate human birth weight. J Immunol 192:5069–5073, 2014.)
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SE C T I O N 2 The Placenta
The third HLA class I molecule expressed by trophoblast is HLA-G, which binds with high affinity to leukocyte immunoglobulin-like receptors (LILRs) expressed by myelomonocytic cells. This interaction results in the induction of a ‘tolerogenic’ population of dendritic cells which, in a transplantation setting, leads to tolerance. The idea that the placenta itself (via HLA-G) is modifying the maternal immune reactivity locally in the uterus to downregulate damaging alloreactive T-cell responses during pregnancy is attractive. Thus HLA-G could act as a ‘placental’ signal to the decidual innate immune system through LILRB1 on myelomonocytic cells to induce pregnancy-specific immune functions in the uterus.4,17 Adults homozygous for HLA-G null alleles have been identified, showing that expression of membrane-bound HLA-G by trophoblast is not essential for successful pregnancy.18 It is likely that HLA-G provides only one of several redundant mechanisms, including absence of HLA-A or HLA-B on trophoblast, that favour T-cell tolerance in the decidua. Migration of antigen presenting cells from decidua to local lymph nodes is poor, and chemokines that recruit T cells are epigenetically silenced. These effects work together with antigen-independent mechanisms such as expression of indoleamine 2,3-dioxygenase (IDO), galectins and secretion of immunosuppressive cytokines such as transforming growth factor β, to create a local tolerogenic immune environment.11 This favours the generation of regulatory T cells (Tregs) that could act to limit the development of effector T cells at the maternal–fetal interface. Although Treg depletion in mice leads to fetal loss, indicating an important role, the mechanism and specificity of these cells are unclear. Tregs are also generated in HLA-C–mismatched human pregnancies to a greater degree than in HLA-C– matched pregnancies, but there still is no convincing evidence to date that demonstrates that T cell–mediated mechanisms are responsible for human pregnancy failure.19 Further work is required to understand the role played by T cells at the maternofetal interface.
Conclusion Adequate trophoblast invasion and vascular remodelling is required for proper placental and fetal growth. In addition, epidemiological data have shown that growth retardation in utero is associated with increased incidence of certain diseases in adulthood. Thus any dysregulation of placentation has farreaching consequences. Recent genetic and functional studies provide strong evidence that interactions of KIR on uNK cells with HLA-C on trophoblast play an important role in regulating the depth of trophoblast invasion. KIR–HLA-C combinations that cause excessive uNK inhibition are associated with reduced vascular remodelling and increased risk for GOS, including pre-eclampsia, recurrent miscarriage, stillbirth and FGR. Conversely, combinations that enhance uNK activation may contribute to increased birth weight. A direct effect of uNK cells on spiral artery structure and function is also likely. The relative importance of interactions between the three components– uNK cells, trophoblast and arteries–probably varies in different species. It is also clear that maternal recognition of pregnancy results in changes in decidual T cells, including Tregs, but there is no compelling evidence that maternal T cells specific for fetal HLA molecules are directly involved in pregnancy disorders.9,19 Whatever mechanisms are involved, the maternal immune system must provide a balance between fetal intrusion into the mother’s resources and the need to protect the mother from excessive fetal demand. In studying this, the view of the uterus as a ‘privileged site’ is no longer valid because all anatomical sites have unique immune features, and this applies particularly to mucosal surfaces. The unusual features of the decidua result in a peaceful physiological environment in which specific combinations of maternal KIR and HLA-C contribute to successful reproduction and act to maintain birth weight between two extremes. Access the complete reference list online at ExpertConsult.com. Self-assessment questions available at ExpertConsult.com.
References 1. Cha J, Sun X, Dey SK. Mechanisms of implantation: strategies for successful pregnancy. Nat Med. 2012;18:1754–1767. 2. Pijnenborg R, Vercruysse L, Hanssens M. The uterine spiral arteries in human pregnancy: facts and controversies. Placenta. 2006;27: 939–958. 3. Burton GJ, Jauniaux E, Watson AL. Maternal arterial connections to the placental intervillous space during the first trimester of human pregnancy: the Boyd collection revisited. Am J Obstet Gynecol. 1999;181:718–724. 4. Moffett-King A. Natural killer cells and pregnancy. Nat Rev Immunol. 2002;2:656–663. 5. Gellersen B, Brosens IA, Brosens JJ. Decidualization of the human endometrium: mechanisms, functions, and clinical perspectives. Semin Reprod Med. 2007;25:445–453. 6. Pollheimer J, Fock V, Knöfler M. Review: the ADAM metalloproteinases—novel regulators of trophoblast invasion? Placenta. 2014;35(suppl):S57–S63. 7. Aghababaei M, Beristain AG. The Elsevier Trophoblast Research Award Lecture: importance
of metzincin proteases in trophoblast biology and placental development: a focus on ADAM12. Placenta. 2015;36(suppl 1):S11– S19. 8. Nancy P, Erlebacher A. T cell behavior at the maternal-fetal interface. Int J Dev Biol. 2014;58:189–198. 9. Moffett A, Colucci F. Uterine NK cells: active regulators at the maternal-fetal interface. J Clin Invest. 2014;124:1872–1879. 10. King A, Birkby C, Loke YW. Early human decidual cells exhibit NK activity against the K562 cell line but not against first trimester trophoblast. Cell Immunol. 1989;118:337–344. 11. Moffett A, Hiby SE. How does the maternal immune system contribute to the development of pre-eclampsia? Placenta. 2007;28(suppl A):S51–S56. 12. Hiby SE, Walker JJ, O’shaughnessy KM, et al. Combinations of maternal KIR and fetal HLA-C genes influence the risk of preeclampsia and reproductive success. J Exp Med. 2004;200:957–9565. 13. Hiby SE, Apps R, Chazara O, et al. Maternal KIR in combination with paternal HLAC2 regulate human birth weight. J Immunol. 2014;192:5069–5073.
14. Kieckbusch J, Gaynor LM, Moffett A, et al. MHC-dependent inhibition of uterine NK cells impedes fetal growth and decidual vascular remodelling. Nat Commun. 2014;5:3359. 15. Xiong S, Sharkey AM, Kennedy PR, et al. Maternal uterine NK cell-activating receptor KIR2DS1 enhances placentation. J Clin Invest. 2013;123:4264–4272. 16. Wittman AB, Wall LL. The evolutionary origins of obstructed labor: bipedalism, encephalization, and the human obstetric dilemma. Obstet Gynecol Surv. 2007;62:739–748. 17. Li C, Houser BL, Nicotra ML, et al. HLA-G homodimer-induced cytokine secretion through HLA-G receptors on human decidual macrophages and natural killer cells. Proc Natl Acad Sci U S A. 2009;106:5767–5772. 18. Ober C, Aldrich C, Rosinsky B, et al. HLAG1 protein expression is not essential for fetal survival. Placenta. 1998;19:127–132. 19. Tilburgs T, Scherjon SA, van der Mast BJ, et al. Fetal-maternal HLA-C mismatch is associated with decidual T cell activation and induction of functional T regulatory cells. J Reprod Immunol. 2009;82:148–157.
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