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REVIEW ARTICLE

Adaptive Immune Responses During Pregnancy


Ana Claudia Zenclussen
Experimental Obstetrics and Gynecology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany

Keywords
Pregnancy, placenta, cells, chemokines, Treg,
adaptive immunity
Correspondence
Ana Claudia Zenclussen, Experimental
Obstetrics and Gynecology, Medical Faculty,
Otto-von-Guericke University Magdeburg,
Gerhart-Hauptmann-Str 35, 39108,
Magdeburg, Germany.
E-mail: ana.zenclussen@med.ovgu.de
Submission January 23, 2013;
accepted January 23, 2013.
Citation
Zenclussen AC. Adaptive immune responses
during pregnancy. Am J Reprod Immunol
2013; 69: 291303

It has long been believed that there is no immune interaction between


mother and conceptus during pregnancy. This concept changed after
evidence was provided that the maternal immune system is aware of
the semiallogeneic conceptus and develops strategies to tolerate it. Since
then, finely regulated mechanisms of active tolerance toward the fetus
have been described. This Special Issue of the American Journal of
Reproductive Immunology deals with these mechanisms. It begins with
the description of minor histocompatibility antigens in the placenta; it
further goes through adaptive immune responses toward paternal fetal
antigens, mostly concentrating on regulatory T cells and molecules modulating the Th1/Th2 balance. The participation of antibody-producing B
cells in normal and pathological pregnancies is also discussed. This introductory chapter resumes the concepts presented throughout the Issue
and discusses the clinical applications raised from these concepts.

doi:10.1111/aji.12097

Introduction: the beginnings


In 1953, the Brazilian Sir Peter Medawar initiated
the modern immunology of pregnancy by asking:
how does the pregnant mother contrive to nourish
within itself, for many weeks or months, a fetus that
is an antigenically foreign body?.1 In his article,
three theories were proposed to explain the lack of
an immunological reaction from the mother against
the fetus:
1. The anatomical separation of the fetus from the mother:
according to this theory, a barrier impermeable to
cells would separate fetalmaternal and blood circulations. This theory was proved to be wrong,
because it is now known that the fetalmaternal
interface is a bi-directional exchange surface. Not
only maternal immune cells are present at the feto
maternal interface, but also fetal cells leak and
are found in maternal circulation. Maternal cells
can be found in the babies as well. This phenomenon is called fetomaternal microchimerism, being

microchimerism defined as a small non-host cell


population (or DNA quantity) from one individual
harbored by another individual.2 Many reports
indicate that microchimerism persists in mother
and child even decades later,35 and new studies
reveal that fetomaternal chimerism occurs even
very early in pregnancy.6,7 This clearly changes
the once accepted concept of anatomical separation between mother and fetus that wrongly persists in many text books and dismisses therefore
the idea that no adverse immune reactions
between two different individuals can take place as
they clearly see each other.
2. The antigenic immaturity of the fetus: this theory
pointed out that the fetus is antigenically immature, not expressing histocompatibility antigens
and being therefore not able to provoke classical
adaptive immune responses. This theory collapsed
very quickly, because fetal skin dendritic cells
that are positive for MHC class I but negative for
MHC class II are very potent accessory cells in

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ZENCLUSSEN

polyclonal T-cell responses.8 Furthermore, in the


last years, a new concept emerged by which
fetuses can protect themselves from maternal
immune reactions as their CD4+ cells strongly differentiate into tolerogenic Tregs that actively tolerate maternal cells that reside in fetal tissues.9
This concept is highlighted in the review by Dr.
Trevor Burt and will be also discussed later during this introductory chapter.
3. The immunological inertness of the mother: A pregnant mother does not die if she gets a cold. It is
even showed that pregnancy is associated with
inflammation rather than with inertness.10 This
alone dismisses the concept of immunological
inertness. However, this initial theory gave rise to
the concept of active tolerance mechanisms
against the fetus. Nowadays, it is considered that
the mother achieves a state of tolerance against
the fetus, still being able to elicit normal immune
responses against infections. This was first shown
by Dr. Ana Tafuri et al. in a paper where she
demonstrated that paternal T cells are aware of
the presence of paternal antigens during pregnancy, where they acquire a transient state of
tolerance specific for paternal antigens.11 This
groundbreaking piece of information was the
basis for many studies concentrating on the
mechanisms as to how the maternal immune system tolerates the fetus rather than ignoring it. In
the 1990s, many studies concentrated on the
cytokines secreted by T cells. Later, regulatory T
cells (Treg), whose main function is to prevent
autoimmunity,12 emerged as important players in
regulating tolerance toward paternal and fetal
antigens, and this is discussed in three reviews
within this issue from different optics (Dr. Robertson, Dr. Saito, Teles). It is clear that not a single cell but rather a network of communicating
cells and molecules is responsible for the successful outcome of pregnancy, and this is not only
discussed in many but not all reviews but also
highlighted in this introductory chapter.

Paternal and fetal antigens are seen by the


maternal immune system, and minor
histocompatibility antigens are expressed at the
fetalmaternal interface
Tafuri11 elegantly showed that maternal T cells are
aware of paternal components in fetal cells and

actively protect them during pregnancy. This transient state is only specific to paternal antigens.11
Since this publication, much effort has been done in
understanding how paternal antigens are recognized.
The review by Peggy Petroff and collaborators extensively revises this issue, as also do Drs. Tilburgs and
Strominger. In the last years, special attention has
been paid to minor histocompatibility antigens
(mHAgs). Its role in eliciting an immune response
has been clearly highlighted in transplantation studies. It is known that they modulate graft rejection
and graft versus host disease in HLA-matched transplant recipients.13 mHAgs can be both protective and
dangerous for the transplant acceptance.14 The role
of mHAgs for pregnancy has been first suspected
after observing that parous female donors are more
likely to elicit graft-versus-host disease in transplant
recipients when compared to either non-parous or
male donors (reviewed in ref.15). In mice, presentation of fetal antigens to maternal T cells can begin,
as already discussed, as early as at copulation
(reviewed by Sarah Robertson). In women, T cells
specific for mHAgs were described (reviewed in
Lindscheids review). These cells can be still present
up to 20 years after birth. The current hypothesis is
that these cells are of tolerogenic or suppressive nature, which at pregnancy allows the survival of the
fetus. Whether at later stages their persistence is
beneficial or rather detrimental because of the possibility of eliciting autoimmune responses is a matter
of debate.
Where it all begins: antigens present in the
seminal fluid activate the adaptive immune
response that tolerates the fetus
After the emergence of the new concept that postulates the existence and the need of a protective
adaptive immune response necessary to protect the
fetus,11 investigators concentrated on the cells
responsible for this state of active tolerance. It is of
general consensus that Treg mediate in large part
the state of active immune tolerance that prevent
maternal lymphocytes to cause cytotoxic damage to
the fetus.1621 It was first wrongly believed that the
expansion of this unique cell subpopulation was driven by pregnancy itself, for example, hormones, and
not by alloantigens.16 This could, however, not
explain why Treg are necessary before implantation17,22 and the fact that Treg from non-pregnant
mice or from pregnant females carrying third party
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fetuses cannot confer fetal protection in a model of


disturbed tolerance.17,23 It was Sarah Robertsons
pioneer work that introduced the concept of seminal
fluid as the first antigen source that activates the
maternal immune system and prepares for pregnancy establishment.24 In their review, Robertson
and collaborators summarize current evidences as
how the seminal fluid elicits a female immune
response and particularly concentrate on the events
leading to generation and expansion of Treg in the
peri-conception and peri-implantation period. There
is plenty of evidence that associates the early expansion of the Treg pool with the exposure to seminal
fluid (Robertsons review). Treg must first encounter
antigens presented by antigen-presenting cells, as for
example, dendritic cells (DCs) in an appropriate
cytokine environment,to proliferate and be functional. The encounter of seminal fluid with maternal
DCs present either in vaginal lumen or in endometrial tissue at the time of mating25 represents therefore the first event leading to a protective adaptive
immune response. It has been proved that uterine
DCs are rather tolerogenic DCs than mature DCs26
and Heme Oxygenase-1 (HO-1) seems to be pivotal
in maintaining maternal DCs in an immature state,
which contributes to the expansion of the peripheral
Treg population.27 Not only does the seminal fluid
provide the antigens to be presented to APCs but
also recruits Treg into the uterus or drives their
expansion and the draining lymph nodes as demonstrated by the lack of expansion of Treg after in
females mated with males without seminal vesicles
but not with vasectomized males19 (A. Teles, A.
Schumacher and A. Zenclussen, unpublished observations). Furthermore, seminal fluid contains potent
immune suppressive molecules that contribute to
Treg induction or conversion of conventional T cells
into Treg, such as TGFbeta and PGE-2-related prostaglandins in the plasma fraction (reviewed in Robertson). It was recently showed that seminal plasma
promotes the differentiation of human DCs to tolerogenic ones.29
CD4 cells: from Th1/Th2 to Treg/Th17
Much attention was focused in the production of
cytokines by CD4 T helper cells particularly after
Piccinni and colleagues showed that decidual T cells
from women with unexplained recurrent abortions
produced abnormally low Leukemia inhibtory factor
(LIF), IL-4, and IL-10 levels.29 This was further sup-

ported by data from patients who presented a Th1


phenotype in cases of abortion versus a Th2 phenotype in normal pregnancies.30 The very popular socalled Th1/Th2 paradigm collapsed after the report
of normal pregnancies in mice knockout for IL-10
and even in quadruple knockouts for Th2 cytokines.31,32 Thus, it was clear that the Th1/Th2 ratio
was a marker for successful or failing pregnancy but
not the cause of it and that much more complex
mechanisms are involved in pregnancy establishment and maintenance.
The existence of cells with suppressive capacity
was already suspected in the early 70s, but the first
confirmation and characterization of these cells was
performed by Shimon Sakaguchi in 1995, who called
these cells regulatory T cells (Treg).12 Treg are a subtype of CD4+ T cells that also express CD25 and are
able to actively suppress self-reactive lymphocytes
and thus to maintain immunological self-tolerance.12
Since then, further characteristics of these cells are
known as, for example, that they express the transcription factor Forkhead box p3 (Foxp3)33 and that
they can derive from the thymus (the so-called naturally occurring Treg) or be induced in the periphery
(iTreg) upon different conditions (Teles Review). It
was first proposed by Somerset that Treg may be
important for human pregnancy as they were elevated in normal pregnancy.34 At the same time, Aluvihare and colleagues show that the reconstitution
of Rag / mice with T cells lacking the CD25+ fraction was related to a higher rate of abortions as compared to mice that received the whole T-cell
fraction.16 The therapeutic potential of Treg for pregnancy was described by us shortly thereafter in a
model of disturbed tolerance during pregnancy.17 It
is nowadays known that Treg fluctuate in number in
blood and uterus during the receptive phase of the
menstrual or estrus cycle,35 (A. Teles et al., unpublished data), which is interpreted as a requisite for
pregnancy establishment further underlined by the
fact that impaired increase or diminished suppressive
capacity is associated with infertility or pregnancy
complications.3638 As it was discussed already, seminal fluid is pivotal in expanding Treg39 and local
application of TGF-b in the mice had the same
effect.40 The antigen specificity of Treg was demonstrated both in a mouse model by Schumacher and
Zhao in 200723,41 and in human by Tilburgs.42 As
for the mechanisms of action of Treg during pregnancy, it has been proposed that they act by creating
a local tolerant microenvironment43 that IL-10 and

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ZENCLUSSEN

PD-1 but not TGF-b or CTLA-4 are relevant for pregnancy.23,44 In humans, however, CTLA-4 expressed
in Treg cells up-regulates IDO expression on decidual
and peripheral blood DC and monocytes by the
induction of IFN-c production.45 Decidual Treg seem
to work by cellcell contact46 and suppression of Tcell responses.47 Dr. Saito extensively revises the
type of Treg present during pregnancy and discusses
the importance of correctly identifying their phenotypes for future clinical applications.
The identification of Th17 cells has improved our
understanding of the cellular regulation during
pregnancy.48 IL-17 acts mainly against extracellular
bacteria or fungal pathogens. It seems that an imbalance of Th17/Treg proportion is associated with
recurrent pregnancy loss and pre-eclampsia.48,49
Tolerance from the fetus toward the mother
In the last years, we have learned more and more
how Treg protect the fetus from immunological
attack by the maternal immune system, but it was
not until recently that it became clear that the fetal
immune system is also active and could potentially
danger the mother.9,50 This does not happen because
the fetus actively generates tolerance to maternal
antigens, mostly mediated by fetal Treg.9 As discussed in Dr. Burts review, Treg are abundant in
the developing fetus. In fact, the frequency of Treg
in fetal tissues is much higher than the frequency of
Treg in any tissue compared with any other time in
development (Dr. Burt). Maternal cells were found
to be present in fetal lymph nodes and in cord
blood.9 In vitro, fetal immune cells were rather suppresive against maternal antigens as compared to
responses against unrelated alloantigens.9 This suggested that fetal immune cells are already primed
against maternal antigens. The existence of a normal
immune response toward maternal antigens upon
depletion of CD25+ fraction in the fetal T cells
revealed the existence of fetal Treg that are educated
to react toward maternal antigens.9 Although the
nature of fetal Treg is still a matter of debate, it is
tempting to speculate that fetal Treg are derived
from conventional T cells that become functional
suppressor cells, thus Treg, upon antigen stimulation
(Dr. Burt). It is a challenge for the near future to
understand how and at which time point of pregnancy this system comprised mostly of cells that are
programmed to suppress convert into a system with
a majority of cells with fully potential to elicit a nor-

mal aggressive immune response. This is crucial to


understand how immunity to pathogens can be
reached at different neonatal ages. In this regard, Dr.
Burt revises the hypothesis of the layered immune
system.
Modulators of the immune responses during
pregnancy
Several molecules modulate and influence the cells
that are directly involved in the generation and
maintenance of an active immunotolerance toward
the fetus.
Pregnancy hormones are of enormous importance
for pregnancy maintenance. It is now known that
they also influence the immune system. Estradiol
was claimed to stimulate the expansion of Treg in
mice,51 although this alone does not count for the
expansion observed upon pregnancy establishment as
discussed before. Estradiol application further decreases
the production of IL-17 by T cells.52 Elevated
progesterone during pregnancy inhibits the development of Th1 immune responses during pregnancy.53
Progesterone in synergy with Galectin-1 (Gal-1) is
reportedly involved in pregnancy maintenance,54
and the application of one progesterone derivate,
dydrogesterone, can abrogate abortion triggered by
stress in a mouse model because of a deviation of
the immune response to a Th2 one.55,56 Estrogen
and progesterone in combination were necessary for
the recruitment of mast cells (MCs) to the uterus.57
MCs were recently reported to be pivotal for
implantation and placentation.58 The most important
pregnancy hormone, the human chorionic gonadotropin (hCG) secreted by the trophoblasts, was
shown to attract regulatory T cells to the fetal
maternal interface59 but also to foster their suppressive function.60 Thus, hormones are important components of the adaptive immune answer necessary
to guarantee the survival of the fetus within the
maternal uterus without eliciting classical maternal
immune responses that would target the fetus and
reject it.
Besides hormones, chemokines produced by
trophoblasts are thought to recruit Treg into the fetal
maternal interface.61 Chemokines were also
reported as important factors as their epigenetic
silencing blocks the access of classical T cells to the
fetalmaternal interface.62 The review by Drs. Perez
Leiros and Ramhorst concentrates on the recruitment of immune cells that contribute to tolerance
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by immune polypeptides that also contribute to tolerance maintenance. Regulated on activation, normal T
cell expressed and secreted (RANTES) can suppress
maternal allogenic immune responses to paternal
antigens in mixed lymphocyte cultures.63 RANTES is
produced by the peri-implantation endometrium, by
human endometrial T cells and by trophoblasts
(reviewed by Perez Leiros and Ramhorst). This molecule can additionally induce apoptosis of potentially
harmful maternal CD3+ cells and increases the frequency of Treg.64 Another molecule in the focus of
the review by Perez Leiros is vasoactive intestinal
peptide (VIP), whose anti-inflammatory and tolerogenic effects were already known.65 It is now known
that VIP levels rise at the fetalmaternal interface at
early gestation peaking at placentation begin.66 Its
role in embryogenesis was revealed after observing
that its blockade during midgestation ends in
induced growth retardation and microcephaly.66 This
is further confirmed in VIP / fetuses that highlights
the role of maternal VIP for early neural development.67 Current data position VIP as an important
immunomodulatory molecule as it can increase the
frequency of Treg and LIF at implantation sites in
mice68 and supports a tolerogenic macrophage phenotype.64 It seems that both hormones and polypeptides are involved in the recruitment of immune
cells into the fetalmaternal interface and in the
generation of a tolerogenic immune response toward
the fetus.
Accumulating evidence points galectins, a family of
endogenous glycan-binding proteins as an important
regulator of pregnancy, and this is discussed in detail
in the Review by Drs. Blidner and Rabinovich within
this Special Issue. It has been shown that the interaction between endogenous glycan-binding proteins
and glycosylated receptors is of crucial importance for
immunological homeostasis (reviewed in ref.69).
Within these processes, galectins emerge as important
regulators in several physiological and pathological
processes (reviewed in Blidner and Rabinovich). In
particular, galectins were lately reported as regulators
of feto-maternal tolerance. Galectin-1, expressed in T
and B cells, inflammatory macrophages tolerogenic
DCs, uterine NK cells, uterine MCs, and Treg, gained
much attention over the last 5 years in the reproduction research field. Gal-1 is known to define autoimmunity,70 inflammatory neurodegeneration,71
cardiac inflammation,72 and tumor escape.73 Lgals1 / mice, lacking Gal-1 expression, show increased
Th1 and Th17 responses, more immunogenic DCs,

aberrant microglia and display more autoimmune


pathology than their wild-type counterparts
(reviewed in Bildner and Rabinovich). The presence
of Gal-1 at the fetalplacental interface is long
known,74 and recent data confirm that it is not only
expressed by the placenta itself75 but also in uterine
NK cells,76 tolerogenic DCs,55 and recent data show
its importance when expressed in uterine MCs.58
Pregnancies of Gal-1-deficient mice were first
described as normal as the number of born embryos
was apparently unaffected by this mutation.77 However, they present a higher abortion rate than wildtype controls if paired allogenically,55,58 a defect that
can be completely reverted when transferring bone
marrow-derived MCs from wild-type animals.58 It
seems therefore that it is the Gal-1 secreted by uterine
MCs the decisive factor in preventing pregnancy
abnormalities. Gal-1 secreted by MCs positively influences spiral artery formation and thus placentation,
which finally allows the normal growth and development of the fetus within the uterus.58
Another molecule influencing pregnancy at various checkpoints is the heme-degrading enzyme
heme oxygenase-1 (HO-1). HO-1-deficient females
were initially reported as sterile after observing that
no progeny could be obtained after mating Hmox1 /
females with Hmox1 / males.78 We have observed
that this is not the cause. Hmox1 / females are not
sterile or even infertile. They do get pregnant, but
their fetuses do not survive to term if they are
homozygote while heterozygote fetuses usually do
survive.79 Fetuses that die intrauterine do so because
of the accumulation of toxic-free heme that leads to
defective implantation and placentation, and this
defect can be completely corrected after inhalation
of low doses of carbon monoxide, the most prominent HO-1 metabolite.79 We also found HO-1
expression to define oocyte ovulation and its fertilization.80 Besides this prominent role in ovulation,
implantation, and placentation, HO-1 was recently
found to be important for the acquisition of immunotolerance toward the fetus. HO-1 supports a tolerogenic phenotype for DCs that in turns expands the
Treg population. Consequently, the inhibition of
HO-1 activity resulted in miscarriages even after
transfer of pregnancy-protective Treg.27
The review by Martinez and collaborators concentrated on the role of pregnancy-specific glycoprotein
1a (PSG1a) in regulating the adaptive immune
responses during pregnancy. PSG1a belongs to the
family of PSG that are mainly synthesized by the

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placenta and represent early biochemical markers of


syncitiotrophoblast formation.81 It has been reported
that PSG1a can turn macrophages tolerogenic and
increase their ability to produce IL-10 and TGFbeta.82,83 PSGs and particularly PSG-1a can inhibit
T-cell proliferation but not directly, they rather do
this via macrophages (reviewed in Martinez). Similarly to what is reported for Gal-154 and HO-127
among others, PSG1a seems to modulate DC phenotype and maturation that finally promotes the secretion of IL-17.84 Other PSGs are also of relevance for
establishment of pregnancy as elegantly demonstrated by the group of Gabriela Dveksler.85,86
CD8 cells: their transformation during pregnancy
In addition to allo-specific responses to paternal antigens by CD4+ cells, recent data reveal the presence of
highly differentiated CD8+ memory cells that are
present at the fetalmaternal interface.87 This special
topic is intensively discussed in the review by Tilburgs and Strominger. The existence of memory
CD8+ cells at the fetalmaternal interface implies the
possibility of antigen presentation. The target specificity for decidual CD8+ cells that constitute the most
abundant T-cell subset in this tissue is unclear, and
the source of the antigens to which CD8+ cells would
be specific is discussed in the mentioned review. It is
known that many mothers carry nave or memory
CD8+ T cells with a T-cell receptor (TCR) that can
directly bind and respond to paternal MHC molecules
that are expressed by fetal trophoblast cells. It is further known that pregnant women develop T-cells
responses that are specific to fetal mHAgs through
the indirect allorecognition pathway (Review by Dr.
Petroff and collaborators). The nature of this immune
response is controversially discussed in the literature.
Whether some of the decidual CD8+ cells are protective is also unknown. Some evidence indicate this
may be the case, for example, Clark proposed that
TGF-b intravaginal application recruits CD8+ Foxp3+
cells,40 but this concept was no longer followed.
Interestingly, CD8+ decidual cells were reported to be
responsible for the protective effects of progesterone
application.88 An important aspect is the fact that
viral infections can skew the CD8+ cell repertoire and
can alter the dynamic of CD8+ cells during pregnancy. A pro-inflammatory profile may augment the
influx of T cells into the fetalmaternal interface, and
Treg may not be longer able to protect from effector
mechanisms and/or activate antigen-specific CD8+

cells to attack fetal structures. Whether this is the


case after viral infections remains to be elucidated.
The final players: B cells, directing the immune
response to a protective or a pathologic one
The final arm of the immune system is depicted by B
cells. These cells are in charge of producing antigenspecific antibodies that are in charge of bind and
destroy foreign antigens so that they can be easily
phagocytized and also activate the complement system. Different B-cell types circulate in blood and can
become fully activated after binding the foreign antigen through the B-cell receptor (BCR) and the signal
from T helper cells. This will finally lead to the transformation into plasma cell and the antibody secretion.89 B cells can, however, also present antigen and
produce cytokines. Little is known about the participation of B cells in pregnancy, and their study has
been mostly concentrated on the production of autoantibodies during pathologic pregnancies. The
review by Damian Muzzio and collaborators gives an
overview about the current knowledge of B-cell participation in pregnancy. In the 1980s and 1990s, several groups concentrated on the production of
pregnancy-protective antibodies by B cells. It was first
observed that the cytotoxic effect of maternal lymphocytes to trophoblast was hampered in the presence
of maternal serum,90 which introduced the concept of
antibodies against paternal components that are protective and not deleterious and thus support pregnancy. Mowbray proposed that the modulation of
HLA expression in trophoblasts is crucial for pregnancy outcome and that the absence of maternal antibodies against these antigens is a cause of recurrent
spontaneous abortion.91,92 Regarding the nature of
these antibodies, Margni and collaborators have demonstrated the existence of so-called asymmetric antibodies that are characterized by the presence of a
high-mannose residue in one F(ab) regions. This
makes these antibodies able to bind to the antigen but
unable to trigger the classical immunological mechanisms aiming to destroy it.93 It has been postulated
that these antibodies are the ones that protect from a
destructive maternal immune response to paternal
antigens. Not only have these antibodies been identified in the placenta94 but also found to be specific
against paternal antigens.94 Patients suffering from
RSA have reportedly a diminished proportion of these
particular antibodies.95 The nature of B cells producing these antibodies has not been so far studied.
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B cells are also able to produce antibodies that are


harmful for pregnancy. The most studied antibodies
in relation to infertility, spontaneous abortion, and
pre-eclampsia are antibodies in the context of the
antiphospholipid syndrome (APS). Women affected
by APS produce one or more antiphospholipid antibodies (aPL) that are directed against phospholipids
(e.g., the ones that are present in the trophoblast
membrane and get exposed to the external surface
upon tissue remodeling) but also against molecules
like lupus anticoagulant protein, cardiolipin, b2 glycoprotein 1, prothrombin, annexin, phosphatidyl
ethanolamine, and phosphatidyl inositol (reviewed
in ref.96). APS manifests often for the first time
when patients get pregnant and suffer a miscarriage,
being the predominant obstetric complication the
recurrent occurrence of spontaneous abortions.97 As
for the mechanisms, aPL themselves may not be the
cause of fetal loss. It is proposed that aPL induce a
procoagulant phenotype that results in fetal growth
restriction.98 Inflammation seems to be needed to
cause placental injury in patients with APS as not all
pregnancies in patients with APS result in a complication.99 In mice, aPLs can bind to the invading trophoblast and hinder implantation or diminish
placenta perfusion causing infarction.100 Girardi and
colleagues have shown that inflammation-driven
complement activation is an important pathway
resulting in thrombosis and endothelial cell activation.101 They recently proposed tissue factor (TF) to
be an important effector in aPL-related inflammation.102
Several other autoantibodies have been lately
related to pregnancy complications, being the most
prominent one the autoantibody against angiotensin
II type I receptor (AT1-AA). The review by Herse and
LaMarca highlights its participation in pre-eclampsia
(PE) (Herse & LaMarca). AT1-AA were first described
by Wallukat and colleagues in sera from pregnant
women developing pregnancy-induced hypertension.103 During PE, the reninangiotensin system is
dysregulated and leads to the presence of activation
AT1-AA in the circulation of these patients. Several
methods confirmed the binding of AT1-AA to the
AT1-receptor.103 In the last years, it became clear,
however, that AT1-AA are not specific for PE as they
can be found in normotensive pregnant whose
fetuses suffered from uterine growth restriction104, in
kidney-transplanted patients who presented a refractory vascular rejection,105 in patients with systemic
sclerosis,106 and patients with malignant secondary

hypertension.107 As for the mechanisms of AT1-AAinduced pathology, it could be demonstrated that IgG
isolated from pre-eclamptic women (a fraction containing AT1-AA) activates the complement system in
kidney and placenta when administered to mice.108
Besides, animals exposed to AT1-AA presented preeclampatic symptoms109 as well as elevated levels of
soluble fms-related tyrosine kinase (sFlt-1) and soluble endoglin (sEng),110 which directly links AT1-AA
to two well-characterized PE markers. Federico Jensen and colleagues recently described the subtype of
B cells, which is able to secrete AT1-AA in PE
patients, the B1aB cells. These cells produce without
antigenic stimulus antibodies that are polyreactive
and can turn autoreactive depending on the conditions. hCG, reported to be elevated in PE patients,
was identified as one factor modulating the AT1-AA
production by B1aB cells.111 The application of an
antibody that reduces the B-cell population could
restore blood pressure and endothelin production in
a rat model for PE.112
Methodological advances in studying immune
cells at the fetal-maternal interface
The study of immune cells at the fetalmaternal
interface fascinated immunologists many years ago
already. The first methods employed to study cells
and their distribution were based on their immunohistochemical staining in fixed samples. This allowed
understanding which immune cells were present at
the fetalmaternal interface and whether there were
differences between normal and pathological specimens. Later, with the description of techniques to
isolate and keep in culture different immune cells,
we could learn more about their function. The use
of flow cytometry allowed and allows a better characterization of their phenotype. With growing
knowledge of the role of immune cells at the fetal
maternal interface, it became clear that the interaction of cells and not one single cell was responsible
for physiological or pathological processes. This led
to the establishment of co-culture systems and system employing chambers to understand both the
interaction between cells and the migration of cells
to gradients or other cell types. Seminal work on
this field has been performed by the group of Dr.
Mor who could show how trophoblast cells secrete
molecules and educate the immune system to best
tolerate the conceptus.113 The more we learned from
the cells we studied, the more became clear that

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these cells may act differently in vivo as isolation protocols and mostly their maintenance in culture
change their phenotype and therefore most probably
their functionality as well. The review by Drs. Olivieri and Tadokoro deals with this interesting topic.
One of first experiments to visualize processes in vivo
at isolated organs was described by Ruttner et al. in
a superfusion chamber at which the microvascular
flow could be observed.114 Later, a skin transplanted

uterus was in vivo analyzed, and the effects of several agents were observed mostly for endometriosis
research.115 Dr. Tadokoro recently described for the
first time the in vivo imagining of immune cells at
the uterus and the placenta by using a 2-photon
microscopy.116,117 This amazing method will help us
understand how cells behave in their natural environment. With this method, Dr. Tadokoro described
that DCs accumulate at the estrus phase of the estrus

APC
Paternal antigens
(seminal vesicle)
Initial contact and antigen
presentation

CD8

B cells

Teff

uMCs

Antibodies

Macrophages

uNKs

DCs

Fetal Treg

Th17

Maternal Treg

TGF-beta

Tolerogenic DCs
B cells
uNKs
uMCs

Treg

Gal-1
Maternal Treg

Vaginal lumen

Secondary antigen
presentation

hCG

Maternal
Treg

Fetal Treg

DCs
CD8 cells
hCG
HO-1
RANTES
PSG-1

Teff
Treg
Tolerogenic DCs

Paraaortic lymph nodes

Th17

Fetal Treg

Peripheral blood

Feto-maternal interface

Fig. 1 This hypothetical scenario depicts the current knowledge about the pathways involved in recognition and tolerance of the foreign fetus as
discussed throughout this Special Issue. Paternal antigens are presented to the maternal immune system in the vaginal lumen after the encounter
of maternal/paternal immune cells with antigens present in the seminal fluid. The seminal fluid contains also substances that promote the
conversion of dendritic cells (DCs) in tolerogenic ones. This promotes the conversion and expansion of regulatory T cells (Treg). The continuous
release of paternal antigens to the circulation allows that Treg continue emerging and expanding throughout pregnancy in, for example, the paraaortic lymph nodes. In peripheral blood, Treg are likely involved in the suppression of maternal effector T cells as, for example, Th17 cells that
could be harmful to fetal antigens present here as well as in several maternal tissues. In a normal pregnancy, B cells secrete antibodies that, once
at the feto-maternal interface, protect paternal antigens present in the trophoblast. Treg migrate to the fetalmaternal interface via human
chorionic gonadotropin(hCG). At the fetomaternal interface, a broad spectrum of molecules produced or secreted by the trophoblast itself like
hCG, HO-1, RANTES, and PSG modulate the phenotype of function of immune cells, that is, DCs, that turn or stay immature and thus tolerogenic.
Additionally, molecules secreted by cells of the innate immune system like Gal-1 secreted by uNKs and uMCs can positively influence the
physiology of the trophoblast while helping maternal T cells to become or stay tolerant toward the fetus. The presence of CD8+ cells at the feto
maternal interface is well documented, but their function is still under investigation. This overview does not pretend to cover all information
available but the research topics covered within this Issue.
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cycle in clusters at the probably future implantation


sites and that their number and density is much
higher as we suspected because of immunohistochemistry and flow cytometry studies. The use of
different genetically modified mice whose cell fluoresce in different colors will allow us to understand
how and where immune cells interact with each
other, how they migrate, and how they respond to
several stimuli.
Clinical applications: are we there yet?
Much knowledge has been obtained in the last few
years about processes that enable the tolerance of
the growing fetus in the maternal uterus and how
disturbances of these fine regulated processes can
lead to pathologies with devastating consequences
for both mother and fetus. It is now our challenge
to transform all this information, mostly obtained
from experimental models either in animals or with
in vitro studies to develop strategies tending to help
reproductive challenged couples who cannot procreate because of aberrant immune responses to the
fetus.
In vitro, human seminal fluid can keep human
DCs in a tolerogenic status28 and promote the induction of Treg phenotype in T cells.19 Understanding
the molecules involved in this will help improving
IVF protocols. The more we know about mHAgs that
are related to spontaneous abortions,118 the more
we will be able to faster diagnosis the causes for
miscarriages and designing strategies for their
treatments.
It has been described that hCG efficiently attracts
Treg to the fetalmaternal interface,59 and now it is
clear that it also foster a protective immune
response.60 This lead to a clinical trial addressing the
question whether hCG application can improve
implantation (http://www.clinicaltrials.gov/ct2/show/
NCT01064219).
In the last years, the idea of paternal antigens
leading to a protective Treg-mediated immune
response was confirmed in studies where Treg
increase after paternal lymphocyte immunization
therapy119 or in vitro fertilization treatment.120 This
information is very useful and may lead to re-think
about the so controversial therapy with paternal leukocytes121 and to dissect which patients should be
treated. The large list of molecules that positively
regulate the adaptive immune response during pregnancy will surely lead to the design of strategies to

modulate this also for patients suffering from pregnancy complications.


The understanding how the fetal immune system
rapidly switches from suppression to active immunity is very important for combating neonate infections naturally. The concepts introduced in this
Introductory Chapter are resumed in (Fig. 1).
Summary
The maternal immune system undergoes profound
transformations already at the very beginning of
pregnancy. These prominent changes are directed to
protect the fetus from a detrimental immune
response. Growing evidence body document that
factors secreted by the fetal trophoblast itself contribute to these changes. Accordingly, the fetal immune
system is also programmed to tolerate the mother
and therefore survive the pregnancy until term. The
investigation of cells, molecules, and pathways
involved in these processes is vital to help patients
with infertility or pregnancy complications as well as
neonates.
Acknowledgments
This work was supported by grants from the Deutsche Forschungsgemeinschaft (ZE 526/4-1, ZE 526/
4-2, ZE 526/5-1, ZE 526/6-1, ZE 526/7-1. I am most
grateful to Maria Laura Zenclussen for the helpful
advice and great discussions.
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