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Trophoblast cells

Trophoblast cells are the earliest extra-embryonic cells to differentiate from the cells of the mammalian embryo. They surround the conceptus throughout gestation and are in direct contact with maternal tissues.

Blastocyst

After fertilization, the potential embryo undergoes mitotic division and, at the 128-cell stage in humans, two distinct cell lineages are present. Trophoblast cells are derived from the trophectoderm that surrounds the blastocyst and the inner cell mass gives rise to the embryo.

King’s College, Cambridge CB2 1ST, UK. Correspondence to A.M. e-mail: am485@cam.ac.uk

doi:10.1038/nri1897

Immunology of placentation in eutherian mammals

Ashley Moffett and Charlie Loke

Abstract | The traditional way to study the immunology of pregnancy follows the classical transplantation model, which views the fetus as an allograft. A more recent approach, which is the subject of this Review, focuses on the unique, local uterine immune response to the implanting placenta. This approach requires knowledge of placental structure and its variations in different species, as this greatly affects the type of immune response that is generated 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 whereas others are not.

For immunologists, ruminations about the immune sys- tem during pregnancy are mostly centred on the acqui- sition of maternal tolerance to the allogeneic fetus 1,2 . This view is probably too simplistic because it does not take into consideration the anatomical fact that it is the maternal relationship with the placenta rather than with the fetus that holds the key to our understanding of the ‘immunological paradox’ of pregnancy. In particular, the focus should be on the intermingling of placental and maternal cells in the uterine wall, as this is where direct tissue contact occurs during placentation. Failure to dis- tinguish between the local uterine immune response to the placenta and the systemic immune response to fetal cells (which usually cross to the mother at delivery) has led to a great deal of confusion. To understand maternal uterine immune responses to the placenta requires knowledge of the sequen- tial anatomical and physiological events that occur during placentation. Herein lies a difficulty, in that each species has developed its own strategy and this results in a great divergence of types of placentation in mammals 35 . One of the most obvious differences is the extent of invasion into the uterus by placental trophoblast cells. This can range from no invasion at all (as in epitheliochorial placentation) to very extensive invasion (as in haemochorial placentation), whereby trophoblast cells penetrate through uterine blood ves- sels to come into direct contact with maternal blood. Humans have haemochorial placentae, as do many laboratory animals, such as mice, rats, guinea pigs and rabbits, but even among this group, the human placenta is particularly invasive.

Comparison of divergent placental strategies must also encompass the maternal reaction that each placental type evokes. Here there is also much diversity. In haemo- chorial placentation, the uterine mucosa is transformed into a highly specialized tissue known as the decidua (a process referred to as decidualization). This does not occur in species with non-invasive epitheliochorial placentae. In primates, decidualization correlates closely with the degree of invasion, so the most marked decidual change is seen in those species with the most invasive placentae. A conspicuous feature of the decidua is the influx of a distinctive lymphocyte population of maternal uterine natural killer (NK) cells 6 . NK cells are emerging as important players in the uterine immune response to invasive forms of placentation, although the precise role they have is still unclear. The cells that define the boundary between the mother and fetus are trophoblast cells 7 . These cells are derived from the outer layer of the blastocyst and have many unusual characteristics that tend to be ignored by immunologists 8 (BOX 1). Because trophoblast cells are freed from the developmental constraints that affect the rest of the embryo, they have a unique pattern of pater- nal and maternal gene expression. Of most relevance to immunologists is the expression of MHC and MHC-like genes by trophoblast cells, which would be the poten- tial ligands for immune receptors on uterine NK cells, lymphocytes and myelomonocytic cells. Human tropho- blast cells have been studied extensively and express a unique and intriguing array of HLA class I molecules, the functions of which might hold the key to the successful temporary coexistence of two individuals.

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Eutherian placenta

Eutherian mammals include all mammalian species except marsupials and egg-laying monotremes. The eutherian placenta is well developed compared with the marsupial placenta and has a great diversity of forms.

Amniote egg

Eggs of amniote vertebrates provide an interface between the embryo and its immediate environment, therefore allowing increased respiratory and excretory capacity as well as nutrient provision.

Yolk sac

The first of the four extra- embryonic membranes of amniote eggs to form during embryogenesis. It surrounds the mass of yolk in reptile and bird eggs and is connected to the midgut by the yolk stalk. The yolk sac is also formed in mammals, despite the absence of yolk.

Amnion

The innermost membranous sac of amniote eggs. It is filled with a serous fluid and encloses the embryo of an amniote (reptile, bird or mammal).

It is therefore clear that a detailed knowledge of the anatomical and molecular interactions between the pla- centa and the uterus at the implantation site is necessary if we are to understand nature’s allograft. The starting point of this Review will be the eutherian placenta. The evolution from the extra-embryonic membranes of amniote eggs to the formation of the definitive placenta is traced and the diverse characteristics of placentae that are seen in extant mammalian species is emphasized. The unique characteristics of trophoblast cells and the adaptation of the uterine mucosa by transformation into decidua are described. Also, the immunological implica- tions of these divergent placental forms are considered. It is hoped that this approach will provide a more solid framework on which to discuss the immunology of mam- malian reproduction, especially from the standpoint of the success or failure of human pregnancy.

Evolution of viviparity Viviparity (the bearing of live young) has evolved inde- pendently many times in many groups of vertebrates, including fish, reptiles and mammals 911 . The selective pressures for viviparity include protection of offspring from cold, from inhospitable environments and from predators. The spectrum of viviparity seen today ranges from a mother simply holding yolky eggs in her body until they hatch (ovoviviparity) to the development of a complex placenta that extracts nutrients from the mother. The placenta is formed when fetal membranes become closely attached to the uterine wall to facilitate physiologi- cal exchange of gases, nutrients and waste products. The first step in the emergence of placentation was the evolu- tion of the amniote egg, which was an important verte- brate innovation 10,11 . This paved the way for the transition from oviparity to viviparity and a shift from yolk-sac nutrition to nourishment delivered by the mother 4,12 . The

Box 1 | Characteristics and functions of trophoblast cells

Characteristics

Paternal X chromosome inactivation

Unmethylated DNA

Expression of endogenous retroviral products (such as syncytin)

Expression of oncofetal proteins (such as carcinoembryonic antigen, α-fetoprotein and human placental lactogen)

Formation of multinucleated cells by fusion or endoreduplication

Lack of expression of MHC class II antigens and variable expression of MHC class I antigens

Functions

Anchoring the placenta to the uterine wall

Transport of nutrients and oxygen to the fetus

Removal of waste products

Secretion of hormones and other placental proteins

Physical barrier between maternal and fetal circulations

Site of contact between the maternal immune system and the conceptus

Transfer of maternal immunoglobulins to the fetus*

Phagocytosis of red blood cells for acquisition of iron*

*Species-specific functions. For more details, see REFS 8,85.

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crucial modification from the anamniote to amniote egg is the development of four extra-embryonic membranes,

consisting of the yolk sac, amnion, chorion and allantois. The

egg shell was subsequently lost during the evolution of viviparous animals, but all amniote embryos retain these extra-embryonic membranes. Only minor modifications were then required for the evolution of these into the definitive placenta. Mammals can be divided into three subclasses that became separated from the reptile-like mammals 120 million years ago, and these are known as the monotremes (for example, duckbill platypus), marsupi- als (for example, kangaroos) and eutherians (for exam- ple, humans). Monotremes are oviparous and the egg is retained in the oviduct until shortly before the young hatches. The eggs of marsupials hatch in the oviduct at the 10-somite stage of development, when the embryo implants briefly and superficially with a simple placenta. The eutherians have the most complex placental develop- ment. The main evolutionary change in mammalian placental development was the emergence of trophoblast cells as a distinctive cell type from the outer epithelium (chorion) of the amniote egg.

Anatomy of placentation in eutherian mammals

Bringing order to the seeming chaos of placental diversity is difficult, but for immunologists the most important consideration is the invasive potential of trophoblast cells in each species and how this is regu- lated. Traditionally, the various complex types of placen- tation seen in eutherian mammals have been viewed as three simplified groups, based on the number of inter- vening cellular layers between the maternal and fetal circulations 13 (FIG. 1). Trophoblast cells are always the outermost layer of fetal cells that overlie an inner core of mesenchyme and fetal capillaries. In epitheliochorial placentation, the trophoblast cells can attach (and even fuse with) the surface epithelium of the uterus but there is no invasion by the trophoblast cells. Trophoblast-cell infiltration through the surface epithelium of the uterus is characteristic of other placental forms. For example, trophoblast cells can migrate to abut maternal blood vessels (in endotheliochorial placentation). The most invasive form is seen when trophoblast cells infiltrate through the maternal vessels to come into direct contact with maternal blood (in haemochorial placentation). In this haemochorial form, trophoblast cells disrupt the endothelial cells and, in some cases, the muscle coat (media) of the uterine arteries as well. Molecular phylogenetics has allowed placental struc- ture to be viewed in a new context, although some ques- tions still remain 14 . All extant eutherian mammals can be grouped into four superorders (or clades): Afrotheria, Xenarthra, Laurasiatheria and Euarchontoglires (or Supraprimates) 15 . Although the relationship between the clades is still disputed, by studying retroposed elements, the basic eutherian divergence was found to be between Xenarthra and the other clades 16 . All placentae examined so far from eutherian mammals of the Xenarthra clade (such as armadillos and sloths) are either haemochorial or endotheliochorial, whereas non-invasive epitheliochorial

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Chorion

In birds and reptiles, the chorion adheres to the shell and is highly vascularized to function in gas exchange. In mammals, it forms the fetal contribution to the placenta, made by an outer layer of trophoblast cells and inner layer of extra-embryonic mesoderm, which contains blood vessels that allow exchange of materials with the maternal circulation.

Allantois

The extra-embryonic membrane that emerges as a sac from the posterior part of hindgut of the embryo. It fuses with the chorion to form the chorio-allantoic placenta. The connection it makes between the embryo and the placenta becomes the umbilical cord.

Retroposed elements

Retroposons randomly insert into the genomes with little likelihood of the same element integrating into the orthologous position in different species. Analysis of the patterns of presence or absence of retroposons is a reliable method for studying the evolutionary history of organisms.

Convergent evolution

The process whereby organisms that are not closely related independently acquire similar characteristics while evolving in separate and sometimes varying ecosystems.

Haemolytic disease of the newborn

If there is rhesus-blood-group incompatibility between the mother and her fetus, the mother makes an antibody response against fetal red blood cells that access the mother’s circulation at delivery. These IgG antibodies cross the placenta during a subsequent pregnancy, which results in the destruction of fetal red blood cells, leading to haemolytic disease of the new born.

Maternal and fetal microchimerism

The presence of fetal cells in the mother or maternal cells in the fetus. Fetal or maternal cells generally cross the placenta at delivery and might persist for many years.

a Epitheliochorial Fetal Fetal vessel mesenchyme Trophoblast cell Uterine Maternal epithelium uterine vessel
a
Epitheliochorial
Fetal
Fetal vessel
mesenchyme
Trophoblast cell
Uterine
Maternal
epithelium
uterine vessel
b Endotheliochorial Fetal vessel Trophoblast cell Maternal uterine vessel Endometrium
b
Endotheliochorial
Fetal vessel
Trophoblast cell
Maternal
uterine vessel
Endometrium
c Haemochorial
c
Haemochorial

Fetal vessel

Cytotrophoblast cell
Cytotrophoblast cell
Maternal blood Syncytiotrophoblast in intervillous layer space Maternal uterine vessel
Maternal blood
Syncytiotrophoblast
in intervillous
layer
space
Maternal
uterine vessel

Figure 1 | Types of placentation. Schematic representation of the three main types of placentation, showing the relationship between the fetal trophoblast cells and maternal blood. a | Epitheliochorial. Trophoblast cells of the placenta are in direct apposition with the surface epithelial cells of the uterus but there is no trophoblast-cell invasion beyond this layer. b | Endotheliochorial. The uterine epithelium is breached and trophoblast cells are in direct contact with endothelial cells of maternal uterine blood vessels. c | Haemochorial. Maternal uterine blood vessels are infiltrated by trophoblast cells causing rupture and release of blood into the intervillous space. The outer layer of the chorionic villi (syncytiotrophoblast) is now bathed in blood ‘like a mop in a bucket of blood’.

placentation (as is found in marsupials) is not seen 17,18 . It is still uncertain what the primordial form of eutherian placentation is and there are compelling arguments that this was the endotheliochorial form 14,17 . However, a recent phylogenetic analysis combined with morphological and molecular data indicates that the ancestral placenta was haemochorial and invasive 19 . The observation that haemochorial placentae are found in diverse species belonging to all four eutherian superorders is consistent with convergent evolution and the presence of strong selective pressures that favour this condition, presumably to provide the fetus with easy access to nutrients directly from the maternal blood. However, the disadvantage of this form of placentation is that the mother and fetus are no longer separated by an intact layer of epithelial cells and this allows exposure of the trophoblast cells to potential allogeneic immune responses by the mother. Uterine immune responses must therefore allow the placenta access to maternal supplies but at the same time prevent excessive invasion. In addition, the transfer of cells between the mother and the fetus becomes more likely in haemochorial placen- tation. In humans, fetal cells invariably cross into the maternal circulation at birth and a maternal antibody response to incompatible red blood cell antigens such as

Rhesus can result in haemolytic disease of the newborn in

subsequent pregnancies 20 . A long-term consequence of this cellular deportation is microchimerism, in which fetal cells persist in the mother for several decades. The pres- ence of increased numbers of fetal cells is associated with diseases such as systemic sclerosis, giving rise to the idea that such diseases might have an alloimmune rather than autoimmune pathogenesis 21 . Haemochorial placentation can be viewed as a trade-off between the risk of these adverse immunological reactions and the need for an efficient way of obtaining nutrients from the mother.

Maternal uterine response to placentation

Concomitant with the marked degree of placental diversity in different species, there is also variation in the uterine response to placentation that correlates closely with the extent of trophoblast-cell invasion. In epitheliochorial placentation, there are minimal changes in the stroma of the uterine mucosa during pregnancy, apart from local angiogenesis, which is needed to increase the blood flow and deliver nutri- ents to the uterine surface. By contrast, haemochorial placentation is characterized by two changes in the uterus, the differentiation of the endometrium into decidua and the transformation of the uterine spiral arteries. In the two extremes of placental types, the non-invasive epitheliochorial form and the invasive haemochorial form, it is obvious that the mechanisms for increasing the blood flow to the feto–placental unit are completely different. In epitheliochorial placentation, this is achieved by expansion of the size of the vascular bed in the uterus by angiogenesis. By contrast, in human haemochorial placentation, there is lowering of resistance in the vessels of the placental bed caused by modification of the walls of pre-existing arteries, resulting in increased low-pressure blood

flow 22,23 (FIG. 2).

The changes of decidualization involve all the cel- lular elements of the uterine mucosa and are most pronounced in humans. The most obvious features are enlargement of the uterine stromal cells and the presence of a distinct lymphocyte population of uter- ine NK cells 6 (BOX 2). In all species, the hallmark of the decidua might indeed be the presence of uterine NK cells. Similar cells are not found in other tissues, and whenever decidual tissue is formed uterine NK cells are present (even in ectopic locations such as in

endometriotic foci) 24 .

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Systemic sclerosis

A chronic autoimmune disease

that causes a hardening of the

skin. The skin thickens because

of increased deposits of

collagen. Compared with the localized form of the disease (scleroderma), systemic sclerosis causes more widespread skin changes and

can be associated with damage

to the lungs, heart and kidneys.

Endometriotic foci

Foci of endometrial tissue outside the endometrium or myometrium (muscle wall) of the uterus. They are usually found in the peritoneum.

Reproductive failure in humans

In humans, it is clear that disruption of the normal balance between the itinerant trophoblast cells and the uterine tis-

sues they colonize during placentation can result in vari- ous clinical problems. These conditions give insight into how the delineation of the territorial boundary between

a

two individuals is achieved. Because of the close corre- lation between the invasion of trophoblast cells and the extent of decidualization, it was argued that the decidual tissue has a permissive influence that favours trophoblast- cell invasion into the uterus 25 . The alternative view was that the decidua provides a defensive riposte to the highly

Myometrium Chorion Amnion Amniotic cavity Decidua parietalis Cervical canal Allantoic vessels in umbilical cord
Myometrium
Chorion
Amnion
Amniotic cavity
Decidua parietalis
Cervical
canal
Allantoic vessels
in umbilical cord
Placenta vascularized
by allantoic vessels
Remnants
of yolk sac
Decidua basalis
Arcuate artery
Radial artery
Uterine artery
Fetus
c
b
Normal pregnancy
Pre-eclampsia and fetal growth restriction
Placenta
Placenta
Villous
trophoblast
cell
Placental villous
tree has fewer
branches
because
of altered
blood flow
characteristics
Maternal
blood in
intervillous
space
Decidua
Extravillous
basalis
Spiral arterial
trophoblast
wall replaced
by trophoblast
cells
cells
(interstitial)
(endovascular)
Placental
bed giant
Decidua
cells
basalis
Spiral artery
remains
narrowed
in this segment
Basal
artery
Media
Media
Myometrium
Endothelium
Endothelium
Radial artery
Radial artery
Arcuate artery
Arcuate artery

Figure 2 | Disorders of human pregnancy resulting from abnormal placentation. a | The blood supply to the human

pregnant uterus is shown. b | Normal pregnancy. 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.

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Box 2 | Characteristics and functions of human uterine NK cells

CD56 hi CD16 uterine natural killer (NK) cells are similar to the minor CD56 hi CD16 NK-cell population in the blood but with phenotypic differences from both CD56 low and CD56 hi blood NK-cell subsets.

They represent 70% of leukocytes at the implantation site.

They might arise ab initio from a separate lymphoid lineage or differentiate in the endometrial microenvironment from blood CD56 hi NK cells.

They produce a range of soluble products, including angiogenic cytokines (such as angiopoietin-2 and vascular endothelial growth factor C 88 ) and lytic enzymes (such as granzymes and perforin).

The diagnostic tests used to evaluate NK-cell phenotype and activity in the peripheral blood of women with reproductive failure give no information regarding uterine NK-cell function 89 .

Therapeutic regimes to downregulate NK-cell ‘activities’ or numbers (including steroids or intravenous immunoglobulins) to treat pregnancy failure have little scientific basis 89 .

Similar cells are found in species with haemochorial placentation. These have been called granulated metrial gland cells in rodents or Kurloff cells in guinea-pigs 9092 . In all species, they are always associated with the spiral arteries that supply the placenta but their spatial association with trophoblast cells is more variable.

Their functions are unknown, but possible roles include: first, to maintain the mucosa and stability of blood vessels (in the non-pregnant endometrium). NK cells are only found in the non-pregnant endometrium in menstruating primates when there is an associated pronounced decidual reaction 93 . Second, to modify the walls of spiral arteries (in the decidua). Third, to control trophoblast-cell invasion of the decidua, myometrium and arteries (at the implantation site).

For more details, see REFS 6,86,87.

Tubal pregnancy

An ectopic pregnancy occurs

when the blastocyst implants

at a site outside the uterus.

Most ectopic pregnancies occur in the fallopian tube so the terms ectopic pregnancy and tubal pregnancy are nearly synonymous.

Placenta creta

A condition when placental

trophoblast cells invade deeply into the muscle coat (myometrium) of the uterus, usually because of the absence

of decidua. This can lead to

uterine rupture, torrential haemorrhage and failure of the placenta to separate after delivery.

Procrustean bed

In Greek mythology, Procrustes

(whose name means he who stretches) was a host who adjusted his guests to their bed. If they were longer than the bed, he cut off the redundant part; if shorter, he

stretched them till they fitted it. Any attempt to reduce men

to one standard, one way of

thinking or one way of acting,

is called placing them on a

Procrustean bed.

invasive trophoblast cells 26 . Mothers compromise, and it now seems probable that both ideas are correct and that the decidua allows orderly access of trophoblast cells to the maternal nutrient supply by achieving the right

balance between under- and over-invasion. Trophoblast-cell penetration of the uterine epithe- lium and invasion into the uterine wall and arteries is potentially highly dangerous, particularly in humans. Uncontrolled trophoblast-cell invasion is seen when the decidua is deficient or absent, as in tubal pregnancy

or when the placenta implants on scar tissue from a previous Caesarian section, a condition known as placenta creta 27 . Without medical intervention, these conditions result in maternal death from haemorrhage.

Early studies in which trophoblast cells were trans- planted to ectopic sites in mice and pigs showed the inherent invasive proclivities of trophoblast cells 26 . The decidua can be considered to behave as a Procrustean bed, violently forcing conformity on its guests what- ever their shape or size — a harsher view of maternal

compromise 28 . At the opposite extreme, excessive restraint of tro- phoblast cells by the decidua can result in pregnancies in which trophoblast-cell invasion into the arteries and uterine wall is inadequate. In this case, the territo- rial boundary has moved in favour of the mother and the blood supply to the fetus becomes poor. The main

problems that result from such reduced blood supply

are fetal prematurity, fetal growth restriction, still-birth

and pre-eclampsia, and in many of these pregnancies the main defect is reduced trophoblast-cell infiltration

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into the uterus 22 (FIG. 2c). The extent to which all of these conditions occur in apes and monkeys is difficult to ascertain, but pre-eclampsia seems to be restricted to humans. Pre-eclampsia has a high maternal and fetal mortality rate and mainly affects first-time mothers. Why should such a devastating disease be maintained despite the strong selective pressures for reproductive success? The answer probably lies in the delicate nego- tiation between trophoblast-cell invasion and decidua that is required during every human pregnancy. The few mothers dying from pre-eclampsia can be viewed as an evolutionary consequence, or indeed sacrifice, because of the need to control the aggressive behaviour of human trophoblast cells.

Placentation in primates

Interestingly, not all primates have haemochorial pla- centae but they are seen in all higher primates such as monkeys, apes and humans, although the pattern of trophoblast-cell invasion differs 29 (FIG. 3a). In particular, interstitial invasion into the decidual stroma and myo- metrium is a prominent feature in humans, whereas only vascular migration has been seen in Rhesus monkeys 30 . The pattern of trophoblast-cell infiltration in the great apes is not known. Humans clearly have evolved a uniquely invasive form of placentation that is potentially dangerous to the mother. So, what selective pressures might be driving these changes? The most obvious dif- ferences between humans and apes are bipedalism and enlarged brain size. Both these characteristics could influence reproductive strategies. The physiological responses that are necessary to redistribute blood flow to the uterus are likely to alter with bipedalism 31 . The cardiac output is affected because of compression of the inferior vena cava by the larger uterus and also by the increased sympathetic tone (that is, increased peripheral vascular resistance and heart rate) required to ensure perfusion of blood to the brain against the pull of gravity. These conse- quences of bipedalism might place selective pressures to increase structural transformation of the uterine arter- ies by trophoblast cells to ensure that the uteroplacental blood flow that is required for fetal development can be achieved throughout pregnancy. In the third trimester of human pregnancies, to sup- port development of a large brain, 60% of total nutri- tional needs are directed to the fetal brain compared with only 20% in other mammalian pregnancies 32 . However, large brains are not limited to species with haemochorial placentation, as dolphins (which have epitheliochorial placentae) have the second largest brain after humans. Evolution of the large human brain, at a time when the form of haemochorial placenta in higher primates was already in place, required modifications of the primate haemochorial placenta to allow increased delivery of oxygen and nutrients through the uterine arteries. To achieve this, trophoblast cells might have to invade deeper into the uterine wall to modify the struc- ture of the uterine arteries so that they are converted to the high-conductance vessels that are required for the development of our large brains.

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a Implantation site of rhesus monkey Cord and fetus Fetal vessel Placenta Maternal blood Radial
a Implantation site of rhesus monkey
Cord and fetus
Fetal vessel
Placenta
Maternal
blood
Radial artery
Radial artery
Radial artery

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b Implantation site of mouse at day 13 of pregnancy Cord and fetus

Intervillous space

Placental villous

tree covered

by villous

syncytiotrophoblast

Trophoblast-cell

shell

Decidua

Trophoblast cells infiltrate the endothelium and media of spiral arteries to replace the wall

Myometrium

Media

Endothelium

Central arterial canal lined by trophoblast cells Maternal blood Fetal vessel space Placenta Myometrium Junctional
Central arterial
canal lined by
trophoblast cells
Maternal blood
Fetal vessel
space
Placenta
Myometrium
Junctional zone
with glycogen cells
Spongiotrophoblast
cell
Mesometrium
Decidua basalis
Mesometrial triangle
containing NK cells

Uterine artery

Labyrinth

area

Figure 3 | Placentation in rhesus monkeys and mice. a | In contrast to humans, the structural and destructive changes to the uterine vessels of Rhesus monkeys are not so marked (FIG. 2). The arteries are only invaded in an endovascular manner, with trophoblast cells migrating down the lumen of the blood vessels and replacing the endothelium and eventually the media 94 . Interstitial trophoblast-cell invasion and the decidual reaction is very limited and no invasion of the myometrium occurs. Instead, there is a well-developed trophoblast-cell shell that forms a clear demarcation line between the placenta and uterine tissues. A similar pattern is found in baboons 95 . b | In mice, the area of placental exchange of nutrients is the labyrinth, which is provided by extensive branching of the chorionic villi and is analogous to the villous placenta in humans. Natural killer (NK) cells are abundant in the decidua basalis on days 8–10 of pregnancy. By day 13 of pregnancy, there are very few NK cells remaining in the decidua basalis and they are found in the mesometrial triangle, an area formed by the two layers of the myometrium.

Pre-eclampsia

Eclampsia (in Greek meaning bolt from the blue) describes grand mal seizures (epileptic fits) occurring towards the end of pregnancy. Pre-eclampsia describes the symptoms that precede eclampsia, which include oedema, proteinuria and hypertension.

Inferior vena cava

The large vein that carries de-oxygenated blood from the lower half of the body to the heart.

Placenta–uterine immune interaction

So, what does placental development have to do with the immunology of reproduction? The standard approach of immunology text books is to view the fetus as an allograft, a situation that can be associated with strong antibody and T-cell-mediated responses to the allogeneic MHC molecules expressed by the vascularized graft. This approach does not distinguish between fetal cells in the circulation or trophoblast cells in the uterus nor does it encompass the different placental forms. For example, in haemochorial placentation, although the decidua avoids classic allogeneic rejection of trophoblast cells, the depth of trophoblast-cell invasion is regulated. To understand the mechanisms involved, both the anatomy of placenta- tion and the maternal leukocytes present in the lining of the uterus are clearly important considerations. In addi- tion, it is crucial that the MHC status of trophoblast cells is considered, as these molecules can function as ligands for uterine immune cells, including T cells, NK cells and myelomonocytic cells. Trophoblast-cell populations are

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always negative for MHC class II expression, indicating that they cannot present antigen directly to maternal CD4 + T cells. The most definitive data on MHC expres- sion are for the human placenta, whereas the picture in other species is limited and confusing. Obviously, these species differences in placentation and MHC expression mean that humans and other primates need to be consid- ered separately from species using other placental strat- egies (particularly those without decidua), as the local uterine immune responses are likely to be dissimilar.

Immune responses in epitheliochorial placentation. In most cases of epitheliochorial placentation, the allan- tochorion trophoblast cells that contact the uterine epithelium lack expression of both MHC class I and II molecules. There are however species-specific excep- tions, with MHC-class-I-expressing trophoblast cells described at certain sites and stages of gestation in several species. For example, in horses, MHC-class-I- positive trophoblast cells do invade into the uterus to form

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Table 1 | Differences between mouse and human placentation

Characteristics

Mouse

Human

Intravascular trophoblast -cell invasion

Minimal*

Extensive

Arterial transformation

Largely independent of trophoblast cells

Caused by perivascular and endovascular trophoblast cells

Decidua

Formed after implantation only at site of placentation

Formed in late-secretory non-pregnant endometrium, involving entire uterine mucosa

NK cells

Infiltrate the media of arteries and disrupt vascular architecture

Encircle adventitia of arteries. Probably have some direct effect on arterial function but mainly act through indirect effects on trophoblast-cell invasion

NK-cell receptors

KIR genes not functional. The Ly49 family fulfils the same function as human KIRs, but Ly49 expression by uterine NK cells and MHC expression by trophoblast cells are unknown

Highly diverse KIR genes. The KIR ligands HLA-G and HLA-C are present on migratory trophoblast cells

*In guinea-pigs 91 , trophoblast cells migrate through the media out of the uterus into the mesometrial artery. This species has a long gestation period. Hamsters 90 have prominent granulated lymphocytes that form a sheath around the arteries. In rats, in late gestation 39 , trophoblast cells extend as far as the mesometrial triangle. KIR, killer-cell immunoglobulin-like receptor; NK, natural killer.

Endometrial cups

A focal collection of

trophoblast cells that penetrate the uterus of horses. These cells are responsible for secretion of equine chorionic gonadotrophin.

Ectoplacental cone

A core of rapidly dividing

trophoblast cells with an outer layer of giant cells that is present in the developing mouse conceptus at 7.5 days post-coitum.

transient endometrial cups that are surrounded and even- tually destroyed by maternal lymphocytes. In addition, maternal antibody responses to paternal MHC class I antigens are often generated in horses, but cytotoxic T-cell responses against paternal alloantigens are reduced compared with such responses before pregnancy, indi- cating an asymmetric immune response to the fetus 33 . Subpopulations of bovine trophoblast cells seem to express mRNAs that are encoded by both classical and non-classical MHC class I genes 34 . In sheep, binucleate trophoblast cells fuse with the epithelial cells that line the uterus, creating a condition known as synepitheliochorial placentation. Although the binucleate cells express MHC class I molecules 35 , the potential immunological effects of this fusion between two allogeneic cells are not known. In species that use epitheliochorial placentae, the simple apposition between the placenta and uterine epithelium might not provoke any damaging immune responses by the mother. The conceptus in these species could be regarded as similar to commensal bacteria in the gut, generating minimal immune recognition by the host unless they breach the epithelial-cell barrier. In other words, the conceptus is non-self, settled innocuously in an epithelial-cell-lined lumen. Although intraepithelial granulated lymphocytes, which are characteristic of mucosal surfaces, have been described, the endometrial stroma in epitheliochorial placentation lacks NK cells 36 .

This indicates that a different immune response to the placenta occurs in species that use epitheliochorial pla- centae than in those species that have trophoblast-cell invasion and decidualization.

Immunology of rodent placentation. Haemochorial pla- centation is a feature of most rodents (FIG. 3b) but it differs

from that in humans with regard to the depth of inva- sion by trophoblast cells and the pattern of distribution of uterine NK cells around the spiral arteries that supply the placenta 3740 (TABLE 1). Even among rodents, there are significant variations. In mice, the presence of uterine NK

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cells in the media of the arteries indicates that they might have a direct physiological role in regulating the blood pressure and flow to the placenta. In support of this, preg- nant mice with no uterine NK cells retain the vascular architecture that is typical of the non-pregnant state 41 . Mouse uterine NK cells might also indirectly modify the blood flow through an effect on trophoblast-cell behav- iour (as seems to be the case in humans), because direct contact occurs between uterine NK cells and trophoblast cells when the ectoplacental cone moves into the decidual tissue on day 8 of gestation. The receptors expressed by uterine NK cells might give insights into how these cells function, but information on the expression by uterine NK cells of members of the Ly49 family (which carry out the same function as killer-cell immunoglobulin-like receptors (KIRs) in primates) and about their cognate ligands on trophoblast cells is sparse. It is therefore not clear whether uterine NK cells have the same role or use the same molecular mechanisms in mice and humans. T-cell recognition of paternal alloantigens expressed by the fetus was shown to occur in mice in which all T cells expressed a transgenic T-cell receptor specific for pater- nal alloantigens, and this resulted in transient tolerance of the transgenic T cells 42 . But where in the feto-placental unit are these alloantigens expressed? The most glaring omission that has so far prevented a clear understanding of mouse reproductive immunology is the lack of defini- tive information on the MHC expression status of mouse trophoblast cells. It seems that the labyrinthine trophoblast cells are MHC class I and class II negative. By contrast, the spongiotrophoblast cells that separate the labyrinth layer from the decidua have been shown to express polymor- phic paternally derived MHC class I molecules 43,44 . It is not known which MHC class I loci encode these products nor whether any non-classical MHC molecules are present 45 . Disruption of many immunological pathways can lead to reproductive failure in mouse models, but cau- tion is needed in interpreting the results because it is often unclear whether the failure is caused by a classical

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Syncytiotrophoblast

The outermost trophoblast-cell layer covering the chorionic villi that is formed by fusion of the underlying layer of mononuclear trophoblast cells to become a multinucleated syncytium, which forms a barrier between the fetus and the mother.

allogeneic response or due to some other immunological mechanism such as inflammation. Paradoxically, the ‘nor- mal’ controls used in many of these models are syngeneic pregnancies, clearly something of an oxymoron when thinking of normal human pregnancy 4648 . Furthermore, in another classic mouse model of abortion — CBA/J mice (H2 k ) crossed with DBA/2 mice (H2 d ) — the nor- mal control mating is with a BALB/c male (which is also H2 d ), so it is not certain whether the fetal losses in CBA/J × DBA/2 matings have an alloimmune basis owing to MHC differences 4951 . In another model, T-cell reactivity to the conceptus could be implicated because ligation of the T-cell-expressed co-stimulatory molecule CD40 ligand led to pregnancy loss 47 . However, the mechanism of pregnancy failure proved to be caused by dysfunc- tional ovaries (ovarian insufficiency) resulting from excessive inflammation in the ovary. Despite these caveats, lessons can be learnt. When syngeneic matings are used, analysis of gene- knockout animals has indicated that pregnancy failure (resorption) results from the lack of genes that seem to have functions that prevent excessive inflammation at the implantation site 52,53 . These genes encode proteins such as the complement regulator Crry (complement- receptor-related protein) and CD95 ligand (CD95L, also known as FASL) 52,53 . Other models using allogeneic mat- ings have helped to explain how adverse T-cell responses might be avoided; for example, by mechanisms involving indoleamine 2,3-dioxygenase (IDO), T-cell co-stimulatory molecules (such as CD80, CD86 and programmed death ligand 1 (PDL1)) and immune deviation to T helper 2 (T H 2)-type responses 46,50,51,54,55 . There is presumably redundancy in the system, because mice deficient in IDO or T H 2-type cytokines reproduce normally 56,57 . Notably, many of these pathways might affect the generation of regulatory T cells, which are known to be increased in mouse as well as human pregnancies both systemically and in the uterus 48,58,59 . However, regulatory T cells seem to be driven by hormonal rather than antigen-dependent mechanisms, as they are also increased in number in syngeneic pregnancies. Depletion of regulatory T cells leads to failure of pregnancies following allogeneic but not syngeneic matings, indicating that regulatory T cells might regulate damaging allospecific effector T-cell responses 48 . Crucially, it is still not established whether the failed pregnancies occur because of T-cell reactivity to either trophoblast-cell antigens or to fetal antigens. Furthermore, the effector mechanisms are unclear, as it has not been shown that uterine T cells can kill murine trophoblast cells.

Adaptive immune responses in humans and other pri- mates. In primates, placental trophoblast cells encounter the maternal immune system in two main areas — the villous trophoblast cells interact with the maternal blood and the extravillous trophoblast cells interact with the uterine tissue. The first area of interaction is between the layer of syncytiotrophoblast that overlies the chorionic villi and is bathed by maternal blood that is delivered by the spiral arteries into the intervillous space (FIG. 3a). In humans, the syncytiotrophoblast is therefore in contact

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with the systemic but not the uterine immune com- ponents of the mother. The syncytiotrophoblast expresses no MHC antigens on its surface, which is consistent with the concept that the placenta is immunologically neutral 6 . Indeed, it has been difficult to demonstrate any systemic maternal T- or B-cell responses to trophoblast

cells (as opposed to fetal cells that cross into the maternal circulation) during human pregnancy 60 . Hints that there are qualitative differences in all systemic T- and B-cell responses in pregnancy come from the altered clinical course of autoimmune diseases and viral infections dur- ing pregnancy. For example, the symptoms of rheumatoid arthritis (which is T H 1-cell mediated) improve during pregnancy, whereas those of systemic lupus erythema- tosus (which is T H 2-cell mediated) worsen and this is presumably caused by the bias away from T H 1- towards

T H 2-cell responses 61,62 . Notably, these responses are to all

antigens, not just to those expressed by components of

the feto-placental unit. This shift to T H 2-cell responses in pregnancy might be an epiphenomenon that is secondary to the flux of hormones and cytokines that are secreted into the circulation, because there is no evidence that it is essential for pregnancy success in humans. Overall,

it is improbable that classical allogeneic rejection of the

villous placenta is responsible for reproductive failure. The second area of contact is between invasive extra- villous trophoblast cells and immune cells in the decidua. In contrast to the syncytiotrophoblast, extravillous tropho- blast cells express an unusual combination of HLA-C, HLA-G and HLA-E molecules 6 . High level expression of HLA-G is restricted to the trophoblast cells that infiltrate the uterus. The polymorphic HLA-A and HLA-B molecules, which initiate allograft rejection, are not expressed, and HLA-C is the only HLA molecule expressed by trophoblast cells that shows any appreciable polymorphism. In those species that have been studied in detail, such as humans and mice, there is no large influx of T or B cells to the implantation site in normal pregnancies. Any T cells present in failed pregnancies might be recruited following the demise of the fetus and the resulting inflammatory changes. As in mice, an important role for T-cell damage to trophoblast cells infiltrating the decidua that results in pregnancy loss in humans has not been established. So, how are adverse maternal T-cell responses to paternally expressed HLA-C molecules or other unidenti- fied trophoblast-cell antigens avoided? MHC-class-II- expressing macrophages and dendritic cells (DCs) are present in the placental bed and could present trophoblast- cell-derived antigens indirectly to the maternal immune system 63 . These decidual antigen-presenting cells might be pivotal in the expansion of both CD4 + CD25 + and CD8 + regulatory T-cell populations that are present in utero during human pregnancy 59,64 . Interestingly, the CD8 + regulatory T cells in the uterus are not MHC restricted but are specific for a member of the carcinoembryonic antigen family, an oncofetal trophoblast molecule, and selectively use the T-cell receptor Vβ9 64 . Another possible mechanism to explain the lack of uterine T-cell activa- tion in normal pregnancies depends on the high-avidity binding of HLA-G to leukocyte immunoglobulin-like

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receptors (LILRs) expressed by myelomonocytic cells 65 . Increased expression of LILRB1 is associated with the induction of a ‘tolerogenic’ population of DCs, which, in

a transplantation setting, results in tolerance 66,67 . Recent data have indicated that this HLA-G-induced tolerance

was due to decreased MHC class II peptide presentation by the tolerogenic DCs 68 . The idea that the placenta itself

is modifying the maternal immune reactivity, locally in

the uterus, through a trophoblast-cell-specific monomor- phic HLA molecule or an oncofetal protein to downregu-

late T-cell responses during pregnancy is attractive. In the non-pregnant endometrium, T-cell responses are normal as evidenced by rapid production of granulomas follow- ing infection of the endometrium with Mycobacterium tuberculosis 69 . Of the non-human primates studied, Rhesus monkeys (Macaca mulatta) express a MHC molecule (Mamu-AG) that has many of the characteristics of HLA-G, includ- ing that of having a soluble variant 70 . A similar MHC molecule is also present in baboons 71 . However, the pat- tern of expression is different, as the baboon MHC-like molecules are expressed by the syncytiotrophoblast. This might reflect the limited interstitial invasion by extravillous trophoblast cells in these species. The role of these HLA-G-like molecules in immunomodulation

is unexplored.

Uterine NK-cell recognition of trophoblast cells. Predecidual changes in the endometrium and the influx of uterine NK cells, which occur before implantation, are unique to primates. Given the lack of evidence for T-cell responses to trophoblast cells, it is compelling to think that uterine NK cells provide the main mechanism by which the maternal immune system recognizes tropho- blast cells. In humans, 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 cells 6 . Unlike blood NK cells, all uterine NK 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 72 . All NK cells also express the KIR-family member KIR2DL4, which can bind HLA-G. HLA-G is endocytosed into KIR2DL4-containing endo- somal compartments. The subsequent interaction results in upregulation of expression of pro-inflammatory and pro-angiogenic cytokines, indicating a mechanism by which the placenta can increase its own blood supply 73 . In addition, any soluble HLA-G molecules in the maternal circulation could bind KIR2DL4 expressed by blood NK cells and as a result could contribute to the inflammatory and vascular changes that are characteristic of all preg- nancies 74 . Therefore, a trophoblast-cell MHC molecule can signal to the decidual innate immune system through both KIR2DL4 on NK cells and LILRB1 (or LILRB2) on myelomonocytic cells. By alerting two different cell types, HLA-G might be acting as a ‘placental’ signal that induces pregnancy-specific functions in the uterus. HLA-C is the only known polymorphic MHC or MHC-like molecule that is expressed by trophoblast cells and is the dominant ligand for the members of the KIR family of receptors that have two immunoglobulin-like

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domains (KIR2D). These might be activating (KIR2DS) or inhibitory (KIR2DL) receptors. KIR haplotypes com-

prise two groups, A and B, the main difference being that there are more activating receptors in the B hap- lotype 75 . In any pregnancy, the maternal KIR genotype could be AA (no activating KIR) or AB/BB (presence of between one and five activating KIRs). The HLA-C ligands for KIRs on trophoblast cells can belong to two groups, HLA-C1 and HLA-C2, which are defined by

a dimorphism at position 80 of the α1 domain. This

maternal–fetal immunological interaction that occurs

at the site of placentation, therefore involves two poly-

morphic gene systems, maternal KIRs and fetal HLA-C

molecules. NK-cell function is therefore likely to vary in each pregnancy. Some KIR/HLA-C combinations might be more favourable to trophoblast-cell invasion, result- ing in a greater increase in in utero placental blood flow than other combinations as a result of the overall signals that the NK cell receives. This hypothesis is supported by a recent study show- ing that the occurrence of pre-eclampsia is associated with an increased frequency of maternal KIRs of the AA genotype but only when this is combined with the presence of an HLA-C2 allotype in the fetus 76 . How do these genetic results translate to functional events

at the implantation site? The KIR phenotype of uterine

NK cells is skewed towards increased expression of the

KIR2D receptors that bind to the two HLA-C groups compared with blood NK cells 77 . Stronger inhibitory sig- nals are delivered to NK cells by the HLA-C2–KIR2DL1 interaction compared with the HLA-C1–KIR2DL2 or HLA-C1–KIR2DL3 interactions 75 . We propose that in pregnancies with a fetus that expresses HLA-C2, the strong inhibitory signal needs to be overcome for suf- ficient trophoblast-cell invasion to occur and this will happen if the mother has activating KIRs, otherwise the feto-placental blood supply will be inadequate. When trophoblast cells are homozygous for HLA-C1, there is only weak inhibition that does not require the presence of compensatory activating KIRs. To summarize, uterine NK cells do express KIRs that are specific for HLA-C ligands expressed by trophoblast cells, and genetic polymorphisms of this system can affect reproduc- tive outcome. This predicts that there is strong selec- tion against those HLA-C–KIR combinations that are detrimental to reproduction. Population analysis has demonstrated reciprocal frequencies of HLA-C2 and KIR AA genotypes in different human populations 76 . Because they segregate independently, a situation might have evolved so that pregnancies with HLA-C2–KIR AA combinations (which are associated with pre-eclampsia) do not occur too frequently in any population. However, individuals must not only reproduce but their offspring need to survive, and balancing selection (which would maintain the gene frequencies) for KIRs and HLA-C might come from immune recognition of pathogens 78 . Comparison of the human KIR-gene family with that

of chimpanzees, gorillas, bonobos, orangutans and rhesus

macaques indicates that this system is rapidly evolving 7983 . With regard to placentation, the lineage of KIR genes that recognizes MHC-C molecules is only present in apes.

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In rhesus macaques, in which there is a well-defined tro- phoblast-cell shell, minimal infiltration of the decidual stroma and modification of the arteries only by endovas- cular trophoblast cells, MHC-C–KIR interactions do not occur 80 . MHC-C is only present in half of orangutans and all the alleles belong to the C1 group. In this species, the KIR genes that are predicted to bind to MHC-C would all bind the C1 epitope and there are none specific for MHC-C2 (REF. 81). Chimpanzees, gorillas and bonobos have KIRs that can bind MHC-C of both C1 and C2 groups. This shows species-specific co-evolution of both KIR and MHC-C genes. The MHC-C C1 group, when in combination with the KIR AA genotype, seems to be neutral as far as the risk of pre-eclampsia is concerned. This is the only combination that occurs in orangutans and so the strong KIR inhibition mediated by MHC-C C2 is a later addition in the great apes. This has possibly arisen as a result of selective pressures imposed by the increasingly dangerous placental invasion.

Concluding remarks

Although structural variations in eutherian placentae provide endless fascination for comparative anatomists, they can present difficulties when extrapolating results from animal studies to human pregnancy. Structural characteristics are important in the study of pregnancy immunology because the more invasive the placenta, the greater the interaction it is likely to have with the maternal immune system. Placental anatomical varia- tion is reflected in the considerable difference in the gene repertoire for both immunity and reproduction in the mouse and human genome 84 . The two gene systems that have diverged most are the MHC genes and the NK-cell- receptor genes and these now seem to have important roles in both reproduction and immunity.

Although the immunological characteristics of human placentation are fairly well documented, the situation in other species, including mice, is still sparse and often con- flicting. It is clear that the placenta is not immunologically neutral because MHC antigens are expressed by tropho- blast-cell subpopulations in most of the species studied. In humans, these are ligands for receptors on innate immune cells, and whether MHC-restricted T-cell recognition of trophoblast cells occurs in normal or abnormal pregnan- cies is unclear. It will be a challenge to determine how regulatory T cells, HLA-G, oncofetal antigens and other potential mechanisms to avoid adverse T-cell responses to trophoblast cells are generated and whether failure of T-cell control ever does have a role in pregnancy failure. The role of NK cells in pregnancy is also uncertain, although in humans, there is an indication that HLA-C– KIR interactions between trophoblast cells and NK cells do regulate the depth of trophoblast-cell invasion. It is also probable that there is a direct effect of uterine NK cells on spiral artery structure and function (possibly modified by soluble trophoblast-cell-derived factors). The relative importance of interactions between the three components — uterine NK cells, trophoblast cells and arteries — prob- ably vary in different species. Whatever mechanisms are involved, the maternal immune system must provide a balance between the need for fetal intrusion into the mother’s resources and the need to protect the mother from excessive fetal greed. In studying this, the view of the uterus as a ‘privileged site’ is no longer valid, as all anatomical sites have unique immune features and this applies particularly to mucosal surfaces. The comparison of the uterine mucosa to the gut or the nose (in which CD56 hi NK-like cells are also frequently found) would seem far more informative than to the traditional sites of immune privilege, such as the eye, brain or testis.

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Acknowledgements

The authors thank D. Antczak, G. Burton, S. Ellis, S. Murphy,

P. Parham, R. Pijneneborg, A. Sharkey and P. Wooding for helpful comments.

Competing interests statement

The authors declare no competing financial interests.

DATABASES

The following terms in this article are linked online to:

Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene CD40 ligand | CD80 | CD86 | CD95L | HLA-A | HLA-B | HLA-C | HLA-E | HLA-G | KIR2DL4 | LILRB1 | NKG2A | PDL1

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