adaptive immune responses during pregnancy

Adaptive Immune Responses During PregnancyAna 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: [email protected]

Submission January 23, 2013;

accepted January 23, 2013.

Citation

Zenclussen AC. Adaptive immune responses

during pregnancy. Am J Reprod Immunol

2013; 69: 291–303

doi:10.1111/aji.12097

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 mod-

ulating the Th1/Th2 balance. The participation of antibody-producing B

cells in normal and pathological pregnancies is also discussed. This intro-

ductory chapter resumes the concepts presented throughout the Issue

and discusses the clinical applications raised from these concepts.

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 fetal–maternal and blood cir-

culations. This theory was proved to be wrong,

because it is now known that the fetal–maternal

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 phenome-

non is called feto–maternal 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,3–5 and new studies

reveal that feto–maternal chimerism occurs even

very early in pregnancy.6,7 This clearly changes

the once accepted concept of anatomical separa-

tion between mother and fetus – that wrongly per-

sists 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 imma-

ture’, 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

American Journal of Reproductive Immunology 69 (2013) 291–303

ª 2013 John Wiley & Sons A/S 291

REVIEW ARTICLE

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 dif-

ferentiate into tolerogenic Tregs that actively tol-

erate 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 dur-

ing this introductory chapter.

3. The immunological inertness of the mother: A preg-

nant 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 preg-

nancy, 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 sys-

tem 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. Rob-

ertson, Dr. Saito, Teles). It is clear that not a sin-

gle cell but rather a network of communicating

cells and molecules is responsible for the success-

ful 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

fetal–maternal interface

Tafuri11 elegantly showed that maternal T cells are

aware of paternal components in fetal cells and

actively protect them during pregnancy. This tran-

sient 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 exten-

sively 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 stud-

ies. It is known that they modulate graft rejection

and graft versus host disease in HLA-matched trans-

plant 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, presenta-

tion 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

Lindscheid’s 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 nat-

ure, which at pregnancy allows the survival of the

fetus. Whether at later stages their persistence is

beneficial or rather detrimental because of the possi-

bility 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 postu-

lates 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.16–21 It was first wrongly believed that the

expansion of this unique cell subpopulation was dri-

ven by pregnancy itself, for example, hormones, and

not by alloantigens.16 This could, however, not

explain why Treg are necessary before implanta-

tion17,22 and the fact that Treg from non-pregnant

mice or from pregnant females carrying third party


292 ª 2013 John Wiley & Sons A/S

ZENCLUSSEN

fetuses cannot confer fetal protection in a model of

disturbed tolerance.17,23 It was Sarah Robertson’s

pioneer work that introduced the concept of seminal

fluid as the first antigen source that activates the

maternal immune system and prepares for preg-

nancy 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 expan-

sion of the Treg pool with the exposure to seminal

fluid (Robertson’s 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 func-

tional. The encounter of seminal fluid with maternal

DCs present either in vaginal lumen or in endome-

trial tissue at the time of mating25 represents there-

fore 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 demon-

strated 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 obser-

vations). 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 prosta-

glandins in the plasma fraction (reviewed in Robert-

son). It was recently showed that seminal plasma

promotes the differentiation of human DCs to tolero-

genic 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 pheno-

type in normal pregnancies.30 The very popular so-

called Th1/Th2 paradigm collapsed after the report

of normal pregnancies in mice knockout for IL-10

and even in quadruple knockouts for Th2 cyto-

kines.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 establish-

ment 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 sub-

type 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 tran-

scription factor Forkhead box p3 (Foxp3)33 and that

they can derive from the thymus (the so-called nat-

urally 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 ele-

vated in normal pregnancy.34 At the same time, Alu-

vihare and colleagues show that the reconstitution

of Rag�/� mice with T cells lacking the CD25+ frac-

tion was related to a higher rate of abortions as com-

pared to mice that received the whole T-cell

fraction.16 The therapeutic potential of Treg for preg-

nancy 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., unpub-

lished 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.36–38 As it was discussed already, semi-

nal 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 demon-

strated 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 preg-

nancy, it has been proposed that they act by creating

a local tolerant microenvironment43 that IL-10 and



IMMUNE RESPONSES DURING PREGNANCY

PD-1 but not TGF-b or CTLA-4 are relevant for preg-

nancy.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 cell–cell contact46 and suppression of T-

cell responses.47 Dr. Saito extensively revises the

type of Treg present during pregnancy and discusses

the importance of correctly identifying their pheno-

types 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 imbal-

ance 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 dis-

cussed in Dr. Burt’s 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 sup-

presive against maternal antigens as compared to

responses against unrelated alloantigens.9 This sug-

gested 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 preg-

nancy 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 develop-

ment 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 gonado-

tropin (hCG) secreted by the trophoblasts, was

shown to attract regulatory T cells to the fetal–maternal interface59 but also to foster their suppres-

sive function.60 Thus, hormones are important com-

ponents 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

fetal–maternal interface.62 The review by Drs. Perez

Leiros and Ramhorst concentrates on the recruit-

ment of immune cells that contribute to tolerance



ZENCLUSSEN

by immune polypeptides that also contribute to tol-

erance 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 mole-

cule can additionally induce apoptosis of potentially

harmful maternal CD3+ cells and increases the fre-

quency of Treg.64 Another molecule in the focus of

the review by Perez Leiros is vasoactive intestinal

peptide (VIP), whose anti-inflammatory and tolero-

genic effects were already known.65 It is now known

that VIP levels rise at the fetal–maternal 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 develop-

ment.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 phe-

notype.64 It seems that both hormones and polypep-

tides are involved in the recruitment of immune

cells into the fetal–maternal 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 interac-

tion 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 reproduc-

tion research field. Gal-1 is known to define au-

toimmunity,70 inflammatory neurodegeneration,71

cardiac inflammation,72 and tumor escape.73 Lgals-

1�/� 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 fetal–placental 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 How-

ever, they present a higher abortion rate than wild-

type 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 influ-

ences spiral artery formation and thus placentation,

which finally allows the normal growth and develop-

ment of the fetus within the uterus.58

Another molecule influencing pregnancy at vari-

ous 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 promi-

nent HO-1 metabolite.79 We also found HO-1

expression to define oocyte ovulation and its fertil-

ization.80 Besides this prominent role in ovulation,

implantation, and placentation, HO-1 was recently

found to be important for the acquisition of immu-

notolerance toward the fetus. HO-1 supports a toler-

ogenic 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 concen-

trated 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




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 TGF-

beta.82,83 PSGs and particularly PSG-1a can inhibit

T-cell proliferation but not directly, they rather do

this via macrophages (reviewed in Martinez). Simi-

larly to what is reported for Gal-154 and HO-127

among others, PSG1a seems to modulate DC pheno-

type and maturation that finally promotes the secre-

tion of IL-17.84 Other PSGs are also of relevance for

establishment of pregnancy as elegantly demon-

strated by the group of Gabriela Dveksler.85,86

CD8 cells: their transformation during pregnancy

In addition to allo-specific responses to paternal anti-

gens by CD4+ cells, recent data reveal the presence of

highly differentiated CD8+ memory cells that are

present at the fetal–maternal interface.87 This special

topic is intensively discussed in the review by Til-

burgs and Strominger. The existence of memory

CD8+ cells at the fetal–maternal interface implies the

possibility of antigen presentation. The target speci-

ficity 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 na€ıve 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 fur-

ther 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 protec-

tive 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 preg-

nancy. A pro-inflammatory profile may augment the

influx of T cells into the fetal–maternal 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 antigen-

specific antibodies that are in charge of bind and

destroy foreign antigens so that they can be easily

phagocytized and also activate the complement sys-

tem. Different B-cell types circulate in blood and can

become fully activated after binding the foreign anti-

gen through the B-cell receptor (BCR) and the signal

from T helper cells. This will finally lead to the trans-

formation into plasma cell and the antibody secre-

tion.89 B cells can, however, also present antigen and

produce cytokines. Little is known about the partici-

pation of B cells in pregnancy, and their study has

been mostly concentrated on the production of au-

toantibodies during pathologic pregnancies. The

review by Damian Muzzio and collaborators gives an

overview about the current knowledge of B-cell par-

ticipation in pregnancy. In the 1980s and 1990s, sev-

eral groups concentrated on the production of

pregnancy-protective antibodies by B cells. It was first

observed that the cytotoxic effect of maternal lym-

phocytes to trophoblast was hampered in the presence

of maternal serum,90 which introduced the concept of

antibodies against paternal components that are pro-

tective and not deleterious and thus support preg-

nancy. Mowbray proposed that the modulation of

HLA expression in trophoblasts is crucial for preg-

nancy outcome and that the absence of maternal anti-

bodies against these antigens is a cause of recurrent

spontaneous abortion.91,92 Regarding the nature of

these antibodies, Margni and collaborators have dem-

onstrated the existence of so-called asymmetric anti-

bodies 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 mecha-

nisms 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 identi-

fied 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 produc-

ing these antibodies has not been so far studied.



ZENCLUSSEN

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 anti-

bodies (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 gly-

coprotein 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 compli-

cation.99 In mice, aPLs can bind to the invading tro-

phoblast 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 activa-

tion.101 They recently proposed tissue factor (TF) to

be an important effector in aPL-related inflamma-

tion.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 hyperten-

sion.103 During PE, the renin–angiotensin 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 refrac-

tory vascular rejection,105 in patients with systemic

sclerosis,106 and patients with malignant secondary

hypertension.107 As for the mechanisms of AT1-AA-

induced pathology, it could be demonstrated that IgG

isolated from pre-eclamptic women (a fraction con-

taining AT1-AA) activates the complement system in

kidney and placenta when administered to mice.108

Besides, animals exposed to AT1-AA presented pre-

eclampatic symptoms109 as well as elevated levels of

soluble fms-related tyrosine kinase (sFlt-1) and solu-

ble endoglin (sEng),110 which directly links AT1-AA

to two well-characterized PE markers. Federico Jen-

sen 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 condi-

tions. 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 fetal–maternal

interface fascinated immunologists many years ago

already. The first methods employed to study cells

and their distribution were based on their immuno-

histochemical staining in fixed samples. This allowed

understanding which immune cells were present at

the fetal–maternal interface and whether there were

differences between normal and pathological speci-

mens. 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 char-

acterization of their phenotype. With growing

knowledge of the role of immune cells at the fetal–maternal interface, it became clear that the interac-

tion of cells and not one single cell was responsible

for physiological or pathological processes. This led

to the establishment of co-culture systems and sys-

tem 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




these cells may act differently in vivo as isolation pro-

tocols and mostly their maintenance in culture

change their phenotype and therefore most probably

their functionality as well. The review by Drs. Olivi-

eri 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 sev-

eral 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 envi-

ronment. With this method, Dr. Tadokoro described

that DCs accumulate at the estrus phase of the estrus

Vaginal lumen

Initi

al c

onta

ct a

nd a

ntig

enpr

esen

tatio

nSe

cond

ary

antig

enpr

esen

tatio

n

Paraaortic lymph nodes Feto-maternal interface

Treg

APC

Paternal antigens(seminal vesicle)

hCG

Treg

Tolerogenic DCs

Peripheral blood

Maternal Treg

B cells

Teff

Fetal Treg

Fetal Treg Th17

hCGHO-1 RANTES PSG-1

CD8 cells

Gal-1

Maternal Treg

uMCs

DCs

CD8

Antibodies

Fetal Treg

B cells

Macrophages

Th17

Teff

uNKs

uMCs

uNKs

MaternalTreg DCs

Tolerogenic DCs

TGF-beta

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 para-

aortic 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 fetal–maternal interface via human

chorionic gonadotropin(hCG). At the feto–maternal 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.



ZENCLUSSEN

cycle in clusters at the probably future implantation

sites and that their number and density is much

higher as we suspected because of immunohisto-

chemistry and flow cytometry studies. The use of

different genetically modified mice whose cell fluo-

resce 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 procre-

ate because of aberrant immune responses to the

fetus.

In vitro, human seminal fluid can keep human

DCs in a tolerogenic status28 and promote the induc-

tion 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 fetal–maternal 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 leu-

kocytes121 and to dissect which patients should be

treated. The large list of molecules that positively

regulate the adaptive immune response during preg-

nancy will surely lead to the design of strategies to

modulate this also for patients suffering from preg-

nancy complications.

The understanding how the fetal immune system

rapidly switches from suppression to active immu-

nity is very important for combating neonate infec-

tions 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 contrib-

ute 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 Deut-

sche 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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