REVIEWS

Mechanisms of Tcell tolerance

towards the allogeneic fetus
Adrian Erlebacher

Abstract | Work on the mechanisms of fetomaternal tolerance has undergone a renaissance

in recent years, and the general outlines of a solution to this long-standing paradox of
transplantation immunology have come into view. Here, we discuss several mechanisms,
recently described in mice, that either minimize the activation of maternal Tcells with
fetal or placental specificity, or minimize the possibility that such Tcells, if activated, are
able to harm the fetus. The Tcell response to antigens expressed by the conceptus serves
as a paradigm for the study of tissue-specific immune tolerance and is relevant to the
pathogenesis of immune-mediated pregnancy complications.

Pre-eclampsia
A disease specific to pregnancy
that is triggered by placental
dysfunction. It is characterized
by diverse systemic symptoms,
including hypertension and
proteinuria. The incidence varies
from 3% to 10% of pregnancies,
and this disease is the leading
cause of maternal mortality and
fetal morbidity and mortality
throughout the world.

Immune privilege
Immune-privileged sites are
areas in the body with a
decreased immune response
to foreign antigens, including
tissue grafts. Classic sites of
immune privilege include the
brain, eyes and testes.

Department of Pathology
and NYU Cancer Institute,
NYU School of Medicine,
Langone Medical Center,
550 First Avenue, New York,
New York 10016, USA.
e-mail: adrian.erlebacher@
nyumc.org
doi:10.1038/nri3361
Published online
14 December 2012

The question of how the fetus and placenta avoid rejection by the maternal immune system has intrigued
immunologists and reproductive biologists alike for
nearly 60years1. Until the 1990s, a great deal of work
on this immunological paradox had been conducted
from the perspective that the fetal allograft was akin
to a surgically transplanted organ. This perspective fell
somewhat into disfavour following the realization that
the leukocyte populations that reside within the pregnant uterus are not typical of surgical organ transplants.
Instead, they are comprised predominantly of a unique
type of natural killer (NK) cell that is not present outside
the context of pregnancy. These cells were subsequently
shown to perform a crucial trophic function by helping
to remodel the spiral arterioles of the uterus that supply the placenta with blood (for reviews, see REFS2,3).
Failure to sufficiently remodel these vessels leads to
inadequate placental perfusion, which in turn leads
to intrauterine growth restriction and pre-eclampsia, two
important obstetrical complications. Together, these
findings led to a shift in emphasis from considering the
potential threat to fetal survival posed by uterine leukocytes to considering the potential value of these cells for
reproductive success3.
This view, although compelling in its own right, does
not give a satisfying answer for why the rules of classical
transplantation immunology do not apply to the case of
pregnancy. At the very least, the placenta expresses its
own set of tissue-specific antigens that should be able
to both prime maternal Tcells and render the conceptus susceptible to Tcell-mediated attack. Furthermore,
although pregnancy appears to be associated with
some level of antigen-nonspecific immunosuppression,

pregnant mice and humans are clearly able to mount

robust systemic immune responses to a multitude of
pathogens (for examples, see REFS4,5). Indeed, pathogens with impaired clearance during pregnancy are typically those that colonize the maternalfetal interface,
which appears to provide some level of immune privilege6.
Thus, my discussion of fetomaternal tolerance here takes
the more traditionalist perspective of viewing the fetus
and placenta as an allograft. Moreover, my emphasis is
on Tcell-mediated immune responses that are evoked
in an antigen-specific manner, as these are the primary
responses that mediate graft rejection. As such, I do not
discuss NK cell responses to MHC molecules expressed
by the placenta, or immune-mediated mechanisms of
fetal demise that do not directly involve Tcells (for
example, complement activation). These topics have
been discussed elsewhere3,7.

The anatomy of the pregnant uterus

The term fetomaternal tolerance is to some extent a misnomer, as the fetus does not directly contact maternal
tissue. Rather, it is the placenta that is expected to be the
predominant source of antigens for Tcell priming, as
well as the direct target of any evoked maternal immune
response. For the haemochorial mode of placentation seen
in mice and humans, contact between maternal and placental tissue occurs through two distinct surfaces3,8 (for
other modes of placentation, see REF.3). First, the outer
surface of the placenta is embedded within the maternal decidua, a specialized stromal tissue layer derived
from the endometrium that provides the physical substrate for placental development. The juxtaposition of
the placenta and decidua creates what I refer to as the

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Lymphatic vessel
Decidua

NK cell

Decidual
stromal cell

Myometrium

Dendritic
cell

Spiral arteriole
Maternal
blood

Trophoblast
giant cell

Decidua

Labyrinth

Spongiotrophoblast
layer

Placental antigen

Endovascular
trophoblast

Fetal
endothelium

Fetal blood

Haemochorial mode of
placentation
A form of placentation in which
trophoblast cells erode the
maternal vasculature, which
results in the direct contact of
maternal blood with
trophoblasts.

Figure 1 | Anatomy of the pregnant mouse uterus. The figure shows a schematized cross-section of an implantation
site at embryonic day 11.5 (E11.5). The mouse placenta is comprised of three major tissue layers: the labyrinth, where
nutrient and gas exchange occurs between fetal capillaries and maternal blood; the spongiotrophoblast
layer;
and an
Nature Reviews
| Immunology
outer layer comprised in part of trophoblast giant cells. The layer formed by these trophoblast giant cells extends
around the entire implanted conceptus (that is, all of the structures derived from the fertilized egg, including all
trophoblast subtypes). Embryo implantation in mice, which occurs at around E4.5, induces the formation of the decidua,
which is a specialized stromal structure derived from the uterine endometrium. This structure encases the entire
conceptus and contains the various maternal leukocyte subsets that populate the maternalfetal interface. Formation
of the decidua involves the developmental transformation of endometrial stromal cells into decidual stromal cells.
The decidua also contains the spiral arterioles that supply the placenta with maternal blood. Near to the surface of
the placenta, the endothelial lining of these vessels is replaced by a specialized trophoblast subtype known as an
endovascular trophoblast. This topological arrangement means that antigens expressed by the placenta can be released
directly into the maternal circulation. In mice, lymphatic vessels are confined to the myometrium, which is the smooth
muscle layer of the uterus. NK, natural killer.

Decidua
The specialized endometrial
stromal tissue that encases the
implanted conceptus. The
decidua is predominantly
comprised of decidual stromal
cells, which differentiate from
endometrial stromal cells
following embryo implantation
in the mouse. The decidua also
contains various types of
maternal leukocytes, and it
makes direct contact with the
trophoblasts on the outer
surface of the conceptus to
form the maternalfetal
interface.

Trophoblasts
The earliest extra-embryonic
cells to differentiate from the
cells of the mammalian
embryo. They constitute the
predominant cellular
component of the placenta,
surround the conceptus
throughout gestation and make
direct contact with maternal
tissues.

maternalfetal interface, as this is where placental trophoblasts and uterine leukocytes most obviously come into
contact and where any frontal immunological assault on
the placenta would be staged. Second, maternal blood
bathes the trophoblasts that reside within the body of
the placenta (the placental villous tree in humans and
the labyrinth in mice). This second interface allows for
placental antigens and other material to be shed directly
into maternal blood to systemically modulate maternal immune responses (FIG.1). It is unknown whether
this second interface can also serve as a point of placental attack by effector Tcells, as the ability of Tcells
to extravasate directly into the placental villous tree or
labyrinth has not been evaluated.
Importantly, human fetal leukocytes can traffic at
low levels through the placenta, enter maternal blood
and establish long-lived states of microchimerism in the
mother. This phenomenon is thought to have a role in
the pathogenesis of autoimmune disease (reviewed in
REF.9); however, its contribution to the total load of conceptus-derived antigens sensed by maternal Tcells during pregnancy is currently unknown. It is also possible

that the placenta transports cell-free alloantigens from

fetal to maternal blood. Thus, even though the anatomy
of the pregnant uterus would suggest that the placenta
is by far the greatest source of antigens relevant to Tcell
responses during pregnancy, it remains possible that
conceptus-derived antigens are both fetal and placental
inorigin.

Mechanisms relevant to Tcell priming

Absence of direct allorecognition of paternal MHC
molecules. As with all T cell-mediated immune
responses, the rejection of surgical organ transplants
can be divided into two phases. In the priming phase,
activated dendritic cells (DCs) migrate from the graft
site via the regional lymphatics to the draining lymph
nodes and activate host Tcells. In the effector phase,
activated Tcells exit the lymph nodes and home via the
blood to the graft, where they perform the effector functions that lead to graft demise. However, the immune
response to an organ transplant has a unique feature in
that it can be mediated by Tcell receptor (TCR) interactions with either donor or self MHC molecules (for

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Box 1 | Visualizing antigen-specific Tcell responses to the conceptus
Antigen-specific Tcell responses to the fetus and placenta can be visualized using
transgenic systems in which a well-characterized model antigen is expressed exclusively
by cells of the conceptus. In the Act-mOVA system13,14, male mice hemizygous for the
Act-mOVA transgene are mated with non-transgenic females. This generates
pregnancies in which ~50% of the conceptuses express a transmembrane form of
ovalbumin (mOVA) from the ubiquitously active actin promoter. Although mOVA
is expressed throughout all cells of the fetus and placenta, it is particularly highly
expressed by trophoblasts that invade into the decidua, as well as by trophoblasts that
have replaced the endothelial cells of the spiral arterioles of the decidua13,14. The latter
site of expression means that membranes containing mOVA are in direct contact with
maternal blood, thereby allowing mOVA-containing material to be shed directly into
the maternal circulation to reach the spleen and all lymph nodes.
OVA-specific Tcell responses can be visualized by adoptively transferring Tcell
receptor-transgenic Tcells with known specificity for OVA at a gestation day of the
researchers choice. To determine simply whether the cells have been exposed to
OVA, the cells are labelled with the fluorescent dye 5,6carboxyfluorescein diacetate
succinimidyl ester (CFSE) before transfer and their proliferative response (as determined
by the dilution of the CFSE dye) is measured several days later. Proliferation does
not occur when the cells are transferred into pregnant mice that were mated to
control males of the same background as those carrying the Act-mOVA transgene
(C57BL/6 in this case).

Minor histocompatibility
antigens
Polymorphic peptides derived
from normal cellular proteins
that can be recognized by
Tcells when presented on
MHC molecules. Immune
responses against these
polymorphic antigens can
result in graft-versus-host
reactions, graft rejection or
beneficial antitumour
responses.

a review, see REF.10). Thus, the activation of Tcells in

the lymph nodes can be induced by interactions with
donor-derived DCs that present peptides on donor
MHC molecules (a process termed direct allorecognition) or by interactions with recipient DCs that present
donor-derived peptides from both MHC molecules
and minor histocompatibility antigens on self MHC
molecules (a process termed indirect allorecognition).
Similarly, in the graft site itself, effector Tcells can
directly interact either with donor MHC molecules that
are variously expressed by the different donor cell types
that comprise the graft or with self MHC molecules that
are expressed predominantly by host antigen-presenting
cells (APCs) that have infiltrated the graft. The distinction between direct and indirect allorecognition is a
crucial one, as direct allorecognition involves the activation of about tenfold more Tcells and thus provides
a much greater threat to graft survival11. Moreover,
direct allorecognition of donor MHC classI molecules
by CD8+ cytotoxic T lymphocytes (CTLs) allows for the
direct killing of graft parenchymalcells.
Until recently, the extent to which maternal Tcells
directly engage paternal MHC molecules during pregnancy had remained unclear. An early study that used
TCR-transgenic female mice bearing a largely monoclonal population of Tcells suggested that maternal CD8+
Tcells directly interact with paternal MHC classI molecules expressed by cells of the conceptus12. The Tcells
in this study were specific for H-2Kb, and evidence for
TCRMHC interactions was inferred from the changes
in Tcell surface-marker phenotypes that occurred in
pregnant transgenic females following mating with H-2b
allogeneic males but not after mating with syngeneic
(H-2k) or third-party allogeneic (H-2s) males.
However, these findings were called into question
by work that visualized the proliferation of adoptively
transferred TCR-transgenic Tcells in otherwise wildtype recipient females13,14, which is a more physiological

way of detecting antigen-specific Tcell responses (BOX1).

Antigen expression in these studies relied on the use of
the Act-mOVA transgene, which directs expression of a
transmembrane form of ovalbumin (mOVA) from the
ubiquitously active actin promoter 15. Thus, the mating
of wild-type females with either Act-mOVA-transgenic
or non-transgenic (control) males allowed for the generation of experimental and control pregnancies that
differed only with respect to a single conceptus-derived
antigen. The Act-mOVA transgene directs mOVA
expression throughout the conceptus, with particularly
high levels in placental trophoblasts that have invaded
into the decidua and in endovascular trophoblasts that
have invaded into maternal spiral arterioles13.
The use of the Act-mOVA system directly demonstrated the presentation of conceptus-derived antigens
to maternal Tcells during pregnancy, as CD4+ and CD8+
OVA-specific Tcells were found to proliferate in pregnant female mice that had been mated with Act-mOVAtransgenic males but not in those mated with control
C57BL/6 males13,14. However, these studies also clearly
showed that antigen presentation is exclusively mediated
by maternal APCs and that direct recognition of paternal
MHC molecules by maternal Tcells does not occur to
an appreciable extent during mouse pregnancy. Thus,
unlike the response to a surgical organ transplant (in
which both directly and indirectly alloreactive Tcells
contribute to graft rejection), the fetal allograft only
allows for the participation of indirectly alloreactive
Tcells, which are tenfold less numerous.
One explanation for this result is that DCs do not
appear during mouse ontogeny until late in gestation16,
thus severely limiting the potential contribution of the
cells whose counterparts (that is, donor DCs) initiate direct allorecognition in the case of surgical organ
transplants. Moreover, the absence of detectable direct
recognition of paternal MHC molecules in pregnant
mice argues against there being a significant degree of
direct recognition of any kind of fetal cell that has trafficked across the placenta into the maternal circulation. In addition, it is notable that the mouse placenta
does not express MHC classII molecules and expresses
only minimal levels of MHC classI molecules during
most of gestation13,1719. This too would be expected to
minimize the extent of direct allorecognition of the
conceptus.
Interestingly, recent work has revealed elevated
proportions of activated CD4+ Tcells in the human
decidua in pregnancies characterized by a mismatch
in the allotype of maternal and paternal HLA-C molecules20, which are the only classical MHC molecules
to be expressed by placental trophoblasts at the human
maternalfetal interface. However, similar elevations
were not evident in the same study for CD4+ or CD8+
Tcells in pregnancies mismatched for HLADR or
HLADQ molecules. As CD4+ Tcells can recognize
HLAC molecules only if HLACderived peptides are
presented by MHC classII molecules, these results are
consistent with the possibility that, as in mice, Tcells in
pregnant women recognize conceptus-derived antigens
exclusively via indirect allorecognition. Recent work

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has also suggested that the CTL responses sometimes
observed in postpartum women to male HY antigens might in part be due to the presentation of these
antigens by maternal APCs during gestation itself 21.
Together, these results suggest that a reliance on indirect allorecognition which is a far less immunogenic
pathway than direct allorecognition is a conserved
mechanism that limits Tcell responses to the conceptus and that qualitatively distinguishes the fetal allograft
from surgical organ transplants.

Lymphoid tissue
Indirect allorecognition

Deletion

Maternal
MHC

TCR

Eector
T cell

T cell

Resident DC

Conceptus-derived
antigens

To spleen and
non-uterine
lymph nodes

To uterine
lymph nodes
Lymphatic
vessel

Uterine vein
Decidua

Spiral arteriole
Myometrium

Placenta

Endometrium

Uterine luminal
epithelial cells

Figure 2 | Restricted pathways mediate the presentation of conceptus-derived

antigens to maternal Tcells. Unlike surgical organ transplants,
mouse| Immunology
fetal
Naturethe
Reviews
allograft is recognized by Tcells exclusively via the indirect allorecognition pathway. This
means that the only responding Tcells are those that are reactive to conceptus-derived
antigens that have been processed and presented on maternal MHC molecules by
maternal antigen-presenting cells, such as dendritic cells (DCs). One probable reason for
the lack of direct recognition of paternal MHC molecules in mice is the fact that fetal DCs
only start appearing late in gestation. In addition, the maternal DCs that reside at the
maternalfetal interface are trapped within the decidua and so cannot reach the uterine
draining lymph nodes. As a result of these two restrictions, conceptus-derived antigens
probably reach the uterine lymph nodes in cell-free form, to be ingested and presented by
lymph node-resident DCs. The entrapment of DCs within the decidua would also be
expected to limit the homing of any fetal DCs arising in late gestation to the lymph nodes.
The shedding of trophoblast-derived material directly into maternal blood means that
placental antigens also have access to the spleen and non-uterine lymph nodes, where
again they are presented by the population of DCs resident in these secondary lymphoid
organs. The primary outcome following the presentation of conceptus-derived antigens
is the deletion of the responding Tcells. These events constitute the priming phase of
the Tcell response. In the effector phase, those Tcells that escape deletion and become
effector Tcells leave the spleen and lymph nodes to migrate via the blood to implantation
sites, where they might be able to induce fetal demise. TCR, Tcell receptor.

Dendritic cell entrapment in the decidua. A second

major difference between a surgical organ transplant
and the fetal allograft is the degree to which peripheraltissue DCs of host origin contribute to Tcell activation.
In the case of a surgical organ transplant, the DCs in
question are the ones that reside in the graft bed at the
time of transplantation or that differentiate within the
graft following the recruitment of DC precursors from
the blood. In the case of pregnancy, the cells in question are the maternal DCs that reside within the decidua.
In mice, these are likely to be, in large part, the same
DCs that populate the endometrium at the time of
implantation22.
Strikingly, decidual DCs do not contribute to the
presentation of conceptus-derived antigens in the uterine lymph nodes. Instead, they remain trapped in the
pregnant uterus, even when their maturation is induced
by an intravenous lipopolysaccharide (LPS) injection23.
As a result, conceptus-derived antigens reach the uterine lymph nodes in cell-free form, to be ingested and
presented solely by lymph node-resident APCs. This
removes a second major immunological threat to fetal
survival, as tissue-resident DCs are crucial initiators of
immunogenic Tcell responses to peripheral antigens24,25.
The entrapment of decidual DCs is likely to be due in
part to the absence of lymphatic vessels from the mouse
decidua. However, it is possible that the alterations in
extracellular matrix composition that occur during
decidualization also impede interstitial DC migration
or preclude the formation of the stable chemokine gradients necessary for DC homing to the lymphatics23.
Indeed, LPS injection induces the emigration of endometrial DCs from the non-pregnant uterus, even though
this tissue layer also lacks lymphatics23. Furthermore, the
tissue density of DCs dramatically declines in mice following the transformation of the endometrium into the
decidua, which probably serves to minimize the number
of migratory DCs that are exposed to conceptus-derived
antigens and that could ultimately reach the uterine
lymph nodes23. The human decidua also has a lower DC
density than the human endometrium26 and has been
reported to contain a paucity of lymphatic vessels27,
although this latter finding is controversial28. These
data suggest that curtailment of DCmediated immune
surveillance of the maternalfetal interface may be a
conserved mechanism of fetomaternal tolerance (for
a review, see REF.29).
Dominant immunosuppression and regulatory Tcells.
Given the lack of direct allorecognition of paternal
MHC molecules, the low levels of expression of these
molecules by the placenta and the inhibition of DC
emigration from the maternalfetal interface, we are
left with the conclusion that Tcell recognition of the
fetus and placenta is mediated largely, if not entirely,
by the resident DC subsets of maternal secondary
lymphoid organs presenting paternal minor histocompatibility antigens or placenta-specific tissue
antigens (FIG.2). Moreover, the use of the Act-mOVA
system demonstrated that conceptus-derived antigens
in addition to being presented in the uterine lymph

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Tetramer
A reagent comprised of a
fluorophore-conjugated core
surrounded by four peptide
MHC complexes or ligand
CD1d complexes. In reality,
these are much more than
tetramers, because each of
the four complexes involved
comprises multiple
components, and higher-order
associations can also occur.
Thus, oligomer might be a
more accurate term.

nodes as a result of their cell-free lymphatic transport

(see above) are presented in the spleen and in all
other lymph nodes as a result of their release into the
maternal circulation13,14. This results in a systemic
antigen-specific Tcell proliferative response starting
at mid-gestation, which coincides with the onset of
placental perfusion with maternalblood.
Strikingly, this proliferative response, although quite
robust, fails to generate substantially increased numbers
of Tcells13,30, and in the case of CD8+ Tcells it fails to
generate effector CTLs13. Instead, conceptus-specific
CD8+ Tcells are mostly deleted, as is typically seen
with TCR-stimulated Tcells lacking co-stimulation13.
It is likely that conceptus-specific CD4+ Tcells also fail
to undergo effector Tcell differentiation30. Thus, the
fact that spleen- and lymph node-resident DCs are not
spontaneously activated during pregnancy (C.-S. Tay
and A.E., unpublished observations, and REF.31) could
in itself be considered a mechanism of fetomaternal
tolerance.
An alternative possibility is that conceptus-specific
Tcells fail to become activated during pregnancy as a
result of dominant immunosuppression, as mediated in
an antigen-specific manner by regulatory T (TReg) cells.
TReg cells which are identified as CD4+ Tcells expressing the transcription factor forkhead box P3 (FOXP3)
can be divided into two subsets (for a review, see
REF.32). One subset is generated in the thymus, and
these cells are termed thymus-derived TReg cells. They
express TCRs with a high avidity for self-peptideMHC
complexes and are thought to maintain peripheral Tcell
tolerance to self antigens. By contrast, induced TReg cells
are generated in the periphery from naive CD4+ Tcells
and are thought to maintain tolerance to innocuous foreign antigens. The differentiation of induced TReg cells
occurs following TCR engagement in the absence of
strong costimulation and is enhanced by exposure to
transforming growth factor (TGF). The generation
of induced TReg cells also requires the CNS1 (conserved
non-coding sequence 1) enhancer element in the first
intron of the Foxp3 gene32,33.
A role for TReg cells in fetomaternal tolerance was initially suggested by several lines of evidence. First, it was
found that the replenishment of Tcell-deficient female
mice with TReg cell-depleted Tcell populations before
mating led to high levels of embryo resorption at midgestation when the females were mated to allogeneic males
but not after mating with syngeneic males34. Second, it
was found that the adoptive transfer of TReg cells attenuated the high rates of spontaneous fetal loss seen when
CBA/J females are mated with DBA/2J males, which is
a well-studied model of immune-mediated pregnancy
failure35. Furthermore, mouse pregnancy was found to be
associated with a systemic expansion of the total TReg cell
population34. A similar expansion has been described in
the blood of pregnant women, although this observation
has been controversial (REFS36,37 and references therein).
Decreased proportions of TReg cells in the blood have also
been linked to various pregnancy complications, but this
too has remained controversial (REFS38,39 and references
therein).

Evidence for a role specifically for induced TReg

cells in fetomaternal tolerance has come from two
recent studies30,40. One study 30 used two new tools:
Act2Wtransgenic mice, which express the variant
peptide 2W1S5568 derived from the MHC classII molecule IEd; and an MHC classII tetramer that detects
CD4+ Tcells specific for the 2W1S5568IAb peptide
MHC complex (referred to as 2W1Sspecific Tcells).
Importantly, the transgene used to express 2W1S5568 is
a variant of the Act-mOVA transgene, with sequences
encoding 2W1S5568 inserted into the construct expressing mOVA from the actin promoter 41. Thus, the
mating of non-transgenic C57BL/6 (H-2b) females to
Act2Wtransgenic males generates conceptuses that
express a 2W1SmOVA fusion protein as a surrogate
placental antigen. It is therefore likely that, in the pregnant mice, the 2W1SmOVA protein is shed systemically into the maternal circulation to be ingested and
presented by spleen- and lymph node-resident DCs, in
the same manner as the mOVA protein expressed by the
Act-mOVA transgene.
Using this mating system, Rowe etal.30 found that
the total population of 2W1Sspecific Tcells in nontransgenic females numerically expands following mating with Act2W-transgenic males. However, as expected
from the previously discussed studies on OVA-specific
Tcells13,14, this expansion was no greater than about
tenfold. Strikingly, however, the FOXP3+ 2W1Sspecific
Tcell population which constituted ~10% of the
total 2W1Sspecific Tcell population in virgin mice
expanded more than 100fold. Thus, by the end of gestation, ~60% of 2W1Sspecific cells were TReg cells. The
expanded population of FOXP3+ 2W1Sspecific TReg cells
was in part (~50%) derived from pre-existing FOXP3+
2W1Sspecific TReg cells and in part derived from the
conversion of FOXP3 2W1Sspecific Tcells. These
data suggest that conceptus-specific induced TReg cells
are generated during pregnancy.
The generation of conceptus-specific induced TReg
cells was also demonstrated in the second study, by
Samstein etal.40. In this study, female C57BL/6 mice with
a Tcell compartment completely comprised of TCRtransgenic CD4+ Tcells specific for the native I-Ed5568
peptide complexed to IA b were mated to BALB/c
males (which have an H-2d haplotype). This led to a low
level of TReg cell induction in the decidua and uterine
lymph nodes (such that ~13% of CD4+ T cells became
TRegcells). However, the physiological relevance of this
finding is complicated by the high frequency of antigenspecific naive Tcells available for TReg cell conversion
and the fact that MHC classII molecules including
IEd, which provides the required peptide for induced
TReg cell conversion in this model are not expressed by
the placenta19. More importantly, Samstein etal. found
that female mice lacking the CNS1 enhancer element,
and thus lacking induced TReg cells, had higher embryo
resorption rates when mated to allogeneic males than
when mated to syngeneic males (see below). This suggests a functional role for induced TReg cells in feto
maternal tolerance. Rowe etal. similarly inferred a role
for induced TReg cells in fetomaternal tolerance from

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their finding that second pregnancies which are
associated from the outset with higher numbers of conceptus-specific induced TReg cells are more resistant
than first pregnancies to embryo loss following partial
TReg cell depletion30. Intriguingly, the CNS1 enhancer element was found to exist in placental mammals but not in
monotremes or marsupials, thus linking the evolution of
induced TReg cells to the evolution of placentation.

Exosome
A small lipid-bilayer vesicle
that is released from activated
cells following the fusion of a
multivesicular body with the
plasma membrane.

Pathways of induced TReg cell generation during pregnancy. Although highly provocative, the papers mentioned above do not fully establish the role of induced
TReg cells in fetomaternal tolerance. One unresolved question concerns when, where and how conceptus-specific
induced TReg cells are generated during pregnancy. Rowe
etal. showed that the 2W1Sspecific Tcell population
only expands to a minor extent in female mice that are
lightly irradiated before mating with Act2W-transgenic
males30. As light irradiation renders the females sterile,
they are exposed to semen but not to conceptus-derived
products. One interpretation of this finding is that
2W1Sspecific induced TReg cells are primarily generated
from naive CD4+ Tcells later in gestation, in response
to the large amount of antigenic material released by the
placenta. This idea raises the question of why material
shed from the placenta would have properties that promote the generation of induced TReg cells. A major possibility stems from the observation that mOVA expressed
by the Act-mOVA transgene is not shed from the surface of trophoblast membranes as soluble monomers13.
Instead, mOVA probably remains membrane associated and is potentially incorporated into some kind
of microvesicle or exosome42. In humans, similar mat
erial is released in copious quantities into the maternal
blood during pregnancy and has been described to have
various immunosuppressive properties when assayed
invitro (for a review, see REF.43). Intriguingly, microvesicles shed from the human placenta also contain several
paternally inherited minor histocompatibility antigens44.
This suggests that there is a physical association between
these antigens and immunosuppressive factors, which,
following antigen ingestion by APCs, could promote
either conceptus-specific T cell anergy or induced
TReg cell differentiation.
The results of the aforementioned irradiation experiment are also consistent with the possibility that induced
TReg cells are generated from naive CD4+ Tcells by
antigens in semen, and that the release of these same
antigens from the placenta later in gestation mainly
serves to bolster the expansion of the induced TReg cell
population. The idea that seminal antigens can promote
induced TReg cell generation is supported by three additional findings. First, the OVA present in the semen
of Act-mOVA males induces a wave of OVA-specific
Tcell proliferation in the uterine lymph nodes immediately after copulation, thus demonstrating that seminal
antigens are presented in the regional lymph nodes14.
Second, there is an expansion of the TReg cell population
in these lymph nodes in response to semen45. And, third,
semen has been shown to contain high levels of TGF46.
Resolving the relative contributions of semen- versus

conceptus-derived antigens in induced TReg cell generation during pregnancy is likely to require experiments
based on reciprocal embryo transfers, which can generate
pregnancies in which semen but not the conceptus bears
the antigen of interest or vice versa. Not only would such
information have implications for the design of assisted
reproductive technologies, but a demonstration that
induced TReg cells are generated primarily in response to
semen would raise the question of how Tcell tolerance is
established to antigens unique to the conceptus.
TReg cell mechanisms of action. A second major unresolved issue is the exact role of TReg cells in pregnancy.
Given the demonstration that conceptus-specific induced
TReg cells are generated during pregnancy, it is tempting
to conclude that the primary function of these cells is
to suppress the activation of conventional CD4+ Tcells
with specificity for conceptus-derived antigens that
would otherwise induce fetal rejection (FIG.3a). Although
attractive, this idea is not yet fully supported by the current set of data. Indeed, the central idea that TReg cells act
in an antigen-specific manner to maintain pregnancy is
based solely on observations that their depletion elevates
fetal resorption rates in allogeneic mating combinations
but not in syngeneic mating combinations30,34,40,47,48.
Aside from the presence or absence of male-specific
HY antigens, all conceptuses in the allogeneic mating
combinations are antigenically identical, and the relevant antigens are likely to be both trophoblast-specific
antigens and paternal histocompatibility antigens. In
the case of syngeneic mating combinations, the antigens
can only be trophoblast-specific antigens. Importantly,
however, there has been no reproducible demonstration of antigen-specific fetal loss within a mixed litter of
antigen-positive and antigen-negative conceptuses,
which would conclusively exclude the possibility that
embryo resorption is due to the systemic effects of
TReg cell loss (see below).
This is a crucial issue because non-pregnant mice
acutely ablated of all TReg cells develop a systemic inflammatory syndrome associated with Tcell activation within
2days, which, if TReg cell ablation is continued, leads to
death after about 3weeks49. Elevated pro-inflammatory
cytokine levels in the blood might thus occur even after
partial TReg cell ablation. In turn, these cytokines might
be able to induce sporadic embryo loss through various
Tcell-independent pathways, including the promotion
of coagulation at the maternalfetal interface50,51 and/
or the inhibition of the ovarian progesterone synthesis52 or uterine progesterone responsiveness53 necessary
for maintaining decidual viability. Indeed, systemic
inflammation induced by intravenous LPS injection
leads to embryo loss in a Tcell-independent manner 54.
Conversely, evidence for robust Tcell infiltration into the
pregnant uterus as might be expected if the conceptus were being rejected in a similar manner to a surgical
organ transplant has not been forthcoming. In the
two studies with data relevant to this issue, infiltrating
Tcells either were only observed in the endometrium
after fetal loss had already occurred34 or were observed at
minimally increased numbers in the decidua40.

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REVIEWS
With these issues in mind, it is worthwhile considering additional reasons for why allogeneic mating combinations would have greater requirements for systemic
TReg cell-mediated immunosuppression than syngeneic
mating combinations (FIG.3b,c). One possibility is that
allogeneic mating combinations generate a greater antigenic burden than syngeneic mating combinations, and
thus induce greater levels of systemic inflammation
secondary to Tcell activation when TReg cell numbers
a

Induced TReg cells

specic for conceptusderived antigens

Fetal rejection
following inltration
of the maternalfetal
interface by antigenspecic eector T cells

Conceptusderived
antigens
Conventional T cells
specic for conceptusderived antigens

Antigen-specic
eector T cells

TReg cells

Fetal loss secondary to

systemic inammation

All conventional
T cells

TReg cells

Fetal loss secondary to

systemic inammation

General T cell
activation
Eector T cells

Figure 3 | Possible functions of TReg cells in fetomaternal tolerance. The figure

illustrates three possible reasons for why pregnancies resulting from allogeneic mating
Nature
combinations have higher levels of embryo resorption following
theReviews
depletion| Immunology
of
regulatoryT (TReg) cells than pregnancies resulting from syngeneic mating combinations.
a| Induced TReg cells that are generated in response to conceptus-derived antigens
suppress conventional Tcells that are responding to the same antigens and thus prevent
the conventional Tcells from mounting a typical graft-rejection response. These
conceptus-specific conventional Tcells, if activated, would induce fetal demise by
infiltrating the maternalfetal interface and attacking the conceptus in an antigen-specific
manner. b | TReg cells (both induced TReg cells and thymus-derived TReg cells, which may or
may not have specificity for conceptus-derived antigens) suppress the systemic activation
of conventional Tcells that are responding to conceptus-derived antigens. Without such
suppression, increased levels of systemic inflammation cause fetal demise in an antigennonspecific manner, for example by impairing placental function, by promoting the
formation of blood clots at the maternalfetal interface, or via endocrine disruption. In this
scenario, pregnancies resulting from allogeneic mating combinations are more sensitive to
TReg cell depletion because these combinations create a greater antigen burden and thus a
greater potential for conventional Tcell activation. c | TReg cells in pregnant mice function
to maintain immune homeostasis, as they do in non-pregnant mice, and their partial
depletion leads to low levels of systemic inflammation. Pregnancies resulting from
allogeneic mating combinations have higher levels of inflammation-mediated fetal loss
because the implantation sites formed are more sensitive to inflammation-induced
disruption. This sensitivity might be either a non-immune intrinsic feature of the
implantation site or secondary to allorecognition of the conceptus by natural killer cells.

are low. In this scenario, the expansion of conceptusspecific induced TReg cells might not be a crucial feature
of pregnancy, and fetal loss following TReg cell depletion
would be due to the various inflammatory mechanisms
listed above (FIG.3b). Alternatively, it is possible that the
mating strain combination influences the robustness of
developmental events at the maternalfetal interface that
render an implantation site more or less susceptible to
inflammation-induced disruption. Again, this scenario
would not require the expansion of induced TReg cell populations specific for conceptus-derived antigens. Instead,
the TReg cells would be performing the same function
as they do in non-pregnant mice: that is, maintaining
immune homeostasis (FIG.3c). This latter possibility is also
consistent with recent work showing that uterine vascular
remodelling can be influenced by decidual NK cell recognition of paternal MHC classI molecules expressed by
placental trophoblasts, with downstream effects on fetal
and placental growth17. It is also conceivable that the susceptibility of an implantation site to inflammation is influenced by the mating strain combination independently of
immune cell recognition of the conceptus.
Discerning the relative importance of these potential
mechanisms of fetal loss is likely to be a fruitful area of
future research. Such studies not only will provide fundamental insights into the phenomenon of fetomaternal
tolerance, but might also suggest new pathways that lead
to adverse pregnancy outcomes in humans. Conversely,
greater general insight into TReg cell physiology during
pregnancy might help to explain why certain auto
immune diseases (most notably rheumatoid arthritis
and multiple sclerosis) show marked clinical improvement in many pregnant women. Recent work in mice
has linked this otherwise mysterious observation to the
induction of TReg cells during pregnancy 4.
Importantly, however, progress on all of these fronts
will require the resolution of various contradictions that
currently exist in the literature. For example, Samstein
etal. showed that the specific lack of induced TReg cells
that exists in CNS1deficient mice increased embryo
resorption rates in allogeneic pregnancies to only ~10%,
from a baseline of ~5%, when assessed on embryonic
day 14.5 (E14.5)40. A similar increase was observed when
the entire TReg cell population was ablated starting on
E5.5, which suggests that induced TReg cells are the main
TReg cell subset responsible for maintaining fetomaternal tolerance. However, other studies have shown much
greater rates of fetal loss (>50%) in allogeneic mating
combinations when the total TReg cell population is either
depleted earlier in gestation or absent from the time of
mating 34,47,48. Furthermore, two studies gave discordant
results when TReg cell depletion was commenced at midgestation47,55. Indeed, the study that achieved greater TReg
cell loss reported no effect on litter sizes in allogeneic
pregnancies47, whereas the one with a partial loss of
induced TReg cells demonstrated a ~50% reduction in litter size30. As these studies all relied on the same mating
combinations (C57BL/6 females crossed to BALB/c or
C57BL/6 males), the discrepancies between the data are
presumably explained by the various methods used to
deplete the TReg cells, the extent of TReg cell depletion, the

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REVIEWS
timing of depletion and the days of gestation affected,
and/or the microbiota of the animals at different institutions. Also, as I have discussed elsewhere56, fetal loss
commencing before ~E12.5 has not been reconciled with
evidence that maternal Tcells only become aware of
placental antigens at around E10.5. Thus, the possibility that innate mechanisms of allorecognition dominate
pathways of early fetal loss also needs to be considered.
TReg cells and other immunosuppressive pathways. A final
major question regarding the role of TReg cells in pregnancy is how the function of these cells relates to other
pathways implicated in fetomaternal tolerance. In particular, tryptophan catabolism and kynurenine production
by indoleamine 2,3dioxygenase (IDO) and Tcell inhibition by the programmed cell death1 ligand1 (PDL1)
pathway have both been implicated in preventing fetal
loss, as well as in TReg cell generation5760. However, the
interrelationships between these pathways are quite
complex and potentially nonlinear 59,60. Moreover, work
on the roles of IDO in pregnancy has been stymied by
the failure of IDO-deficient females to show reproductive defects when mated with IDO-deficient allogeneic
males61, and only some studies have been able to document a role for PDL1 and its receptor in pregnancy success57,62,63. Interestingly, the induction and function of
TReg cells during pregnancy are unlikely to involve the
immunosuppressive cytokine interleukin10 (IL10), as
IL10deficient mice produce normal litter sizes64, even
though maternal IL10 deficiency renders females highly
sensitive to LPS-induced fetal loss65,66.

Indoleamine
2,3dioxygenase
(IDO). An intracellular
haem-containing enzyme that
catalyses the oxidative
catabolism of tryptophan. The
activity of IDO reduces the
availability of tryptophan,
which can lead to Tcell
apoptosis and anergy.

TC1 cells

CD8+ cytotoxic Tcells that

produce T helper 1type
cytokines, particularly
interferon.

Mechanisms relevant to effector Tcell function

The above discussion describes many key ways in which
the activation of naive maternal Tcells specific for paternal MHC molecules or conceptus-derived minor histocompatibility antigens is minimized during pregnancy.
However, given the imperfect nature of all biological
systems, it is unlikely that such mechanisms are 100%
effective. Furthermore, we might expect that pregnancy
should not be threatened by memory Tcells with specificity for conceptus-derived antigens, as the reactivation
of such cells would be less dependent on costimulation
and possibly less susceptible to TReg cell-mediated suppression. Such Tcells might be generated in the postpartum period of a prior pregnancy, or after infection by
organisms containing antigens that share some commonality with those of the fetus or placenta. Perhaps most
critical is the potential threat posed by Tcells lacking fetal
or placental specificity that are activated during pregnancy during the course of a non-uterine infection, even
one as benign as the common cold. Given the ability of
effector Tcells to migrate through peripheral tissues even
in the absence of a localized antigen source67,68, it is possible that such cells would gain access to the decidua, where
they might produce pro-inflammatory cytokines through
bystander, rather than antigen-specific, pathways.
Together, these considerations suggest that Tcellmediated harm to the fetus would best be prevented if
pathways also existed to minimize the accumulation or
function of effector Tcells at the maternalfetal interface.

Indeed, some of the mechanisms of fetomaternal tolerance discussed above might partly act in this manner.
For example, TReg cells can be detected in small numbers
within the pregnant mouse uterus35,69, and IDO and
PDL1 are respectively expressed by trophoblast giant
cells and decidual stromal cells in mice57,61. However,
intrauterine functions for TReg cells, IDO and PDL1 have
not been clearly defined, as the experiments performed
to date have not discriminated intrauterine from systemic
effects. Similarly, the glycan-binding lectin galectin1
which can attenuate immune-mediated fetal loss in some
mouse models and is expressed by decidual stromal cells
also has systemic immuneregulatory properties70.
Chemokine gene silencing in decidual stromal cells.
To clearly delineate mechanisms of fetomaternal tolerance that operate during the effector phase of the
Tcell response, we determined rates of fetal loss in
mice bearing conceptuses that express OVA as a surrogate antigen as well as large numbers of OVA-specific
effector Tcells 71. The pregnancies were generated
by mating wild-type females with syngeneic males
hemizygous for the Act-mOVA transgene, so that
~50% of the conceptuses would express OVA. The
effector Tcells were produced by first immunizing the
mice, 23weeks before mating, with OVA and adjuvants that promote T helper 1 (TH1) cell responses and
then by rechallenging the mice systemically with OVA
and the same adjuvants on E5.5. The pregnant mice
were also given daily injections of progesterone to
prevent fetal loss secondary to inflammation-induced
ovarian insufficiency 52. Remarkably, OVA-expressing
conceptuses remained viable, thus demonstrating the
existence of pathways that attenuate the effector phase
of the Tcell response71.
These observations led to the finding that the mouse
decidua actively resists being infiltrated by activated TH1
cells and TC1 cells. This is because the genes encoding
the key TH1 cell- and TC1 cell-attracting chemokines
CXCL9 (also known as MIG), CXCL10 (also known as
IP10), CXCL11 (also known as ITAC) and CCL5 (also
known as RANTES) become epigenetically silenced
in decidual stromal cells when these cells differentiate
from endometrial stromal cells71 (FIG.4). The silencing
of these genes prevents their transcription even after the
exposure of decidual stromal cells to pro-inflammatory
cytokines, and is associated with the accrual of histone
H3 trimethyl lysine 27 (H3K27me3) on the promoters
of these genes, a mark associated with gene repression
in a variety of developmental contexts72. The lack of
chemokine expression was shown to limit Tcell access to
the decidua, as ectopic, lentivirus-mediated expression
of CXCL9 and CCL5 in the decidua allowed for focal
Tcell accumulation71. Thus, inhibition of Tcell access
to the maternalfetal interface, as a consequence of the
epigenetic properties of decidual stromal cells, provides
a fail-safe mechanism for preventing fetal rejection. It is
currently unknown whether similar pathways are active
in human pregnancy, although it has been noted that
Tcell densities in the human decidua are lower than
those in the non-pregnant endometrium73.

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REVIEWS
H3K27me3 and H4 acetylation status
of chemokine gene promoters in
stromal cells

No pregnancy

Unmodied
Stromal cell

Pregnancy
Myometrium

Myometrium

T cell

Unmodied

Cxcl9

Endometrium
Unmodied

Repressive
H3K27me3

Decidua

Decidual
stromal cell

CXCL9, CXCL10,
CXCL11 and CCL5

Inammation

Inammation
Eector TH1
or TC1 cell
H4
acetylation

H4
acetylation

H4
acetylation

Repressive
H3K27me3

Figure 4 | Chemokine gene silencing in decidual stromal cells as a mechanism of fetomaternal tolerance. In the
non-pregnant uterus, the promoters of the chemokine genes Cxcl9 (shown), Cxcl10, Cxcl11 and Ccl5 in stromal cells are
Nature
Reviews
| Immunology
associated with chromatin configurations that allow for transcriptional induction following the
exposure
of the
cells to
pro-inflammatory cytokines, such as tumour necrosis factor (TNF) and interferon (IFN). Thus, in response to
inflammation, both the endometrium and the myometrium induce the expression of CXCL9, CXCL10, CXCL11 and CCL5,
which in turn promotes the extravasation and accumulation of activated Tcells in both tissue layers. Transcriptional
induction is associated with the accrual of acetylated histone H4 on the gene promoters. Following decidualization,
decidual stromal cells activate an epigenetic programme that targets all four chemokine genes for promoter accrual of
the repressive histoneH3 trimethyl lysine27 (H3K27me3) mark. Thus, at an implantation site, the myometrium becomes
the only tissue layer that can express Cxcl9, Cxcl10, Cxcl11 and Ccl5 in response to inflammation and thus the only tissue
layer that shows Tcell accumulation. Of note, this pathway limits the infiltration of all Thelper1 (TH1)- and TC1polarized
Tcells, even those lacking specificity for conceptus-derived antigens.

Perspective
By studying antigen-specific Tcell responses to the
fetus and placenta, recent work on the immunology
of pregnancy has uncovered a set of anatomical, cell
ular and molecular mechanisms that protect the fetus
from immune-mediated rejection. These mechanisms
either minimize the activation of maternal Tcells with
specificity for paternal MHC molecules or minor histo
compatibility antigens expressed by the conceptus, or
prevent such cells, if spuriously activated, from harming the fetus. Although these mechanisms provide an
overall framework for understanding how the fetus and
placenta avoid rejection, many details relevant to each
mechanism have yet to be determined. Furthermore,
their relative importance to true, antigen-specific fetal
rejection remains unresolved, and the possibility that
systemic inflammation is instead a cause of fetal loss in
many models has not been thoroughly addressed.
The relevance of these mechanisms to complications of human pregnancy is also an important area for
further exploration. The associations between altered

proportions of decidual and blood Tcell subsets and

recurrent spontaneous abortion and pre-eclampsia
remain correlative38, and the role of Tcells in preterm
birth that is not otherwise explained by infection is an
open question. Intriguingly, accumulations of Tcells at
the maternalfetal interface are characteristic of villitis
of unknown aetiology, chronic deciduitis and chronic
chorioamnionitis, three histopathological findings of the
third trimester decidua and placenta that are observed
at increased prevalence in cases of preterm birth and
intrauterine growth restriction7476. Although the antigenic specificity of the accumulating cells is unknown,
it is tempting to speculate that these lesions represent the
consequences of a failure of one of the mechanisms of
fetomaternal tolerance described here. Even if dysregulated Tcell responses during pregnancy have no direct
impact on pregnancy success perse, it is also important to consider how such responses might fit in with
the fetal origins hypothesis, which posits that inutero
events affecting fetal development have long-term effects
on human health77.

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Acknowledgements

Work in the authors laboratory has been supported by

grants from the US National Institutes of Health, the
American Cancer Society and the Leona M. and Harry B.
Helmsley Charitable Trust. The author would like to thank the
m e m b e rs o f h i s l a b o ra to r y fo r m a ny s t i m u l a t i n g
discussions.

Competing interests statement

The author declares no competing financial interests.

FURTHER INFORMATION
Adrian Erlebachers homepage: http://pathology.med.nyu.
edu/people/faculty/erlebacher-adrian
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