regulatory t cells: regulators of life

Regulatory T Cells: Regulators of LifeAnne Schumacher, Ana Claudia Zenclussen

Department of Experimental Obstetrics & Gynecology, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany

Keywords

Autoimmune disease, infection, pregnancy,

tolerance, Treg

Correspondence

Ana Claudia Zenclussen, Experimental

Obstetrics and Gynecology, Medical Faculty,

Otto-von-Guericke University, Gerhart-

Hauptmann-Straße 35, 39108 Magdeburg,

Germany.

E-mail: [email protected]

Submission January 30, 2014;

accepted February 25, 2014.

Citation

Schumacher A, Zenclussen AC. Regulatory T

cells: regulators of life. Am J Reprod Immunol

2014; 72: 158–170

doi:10.1111/aji.12238

Pregnancy still represents one of the most fascinating paradoxical phe-

nomena in science. Immediately after conception, the maternal immune

system is challenged by the presence of foreign paternal antigens in the

semen. This triggers mechanisms of recognition and tolerance that all

together allow the embryo to implant and later the fetus to develop.

Tolerance mechanisms to maintain pregnancy are of special interest as

they defy the classical immunology rules. Several cell types, soluble fac-

tors, and immune regulatory molecules have been proposed to contrib-

ute to fetal tolerance. Within these, regulatory T cells (Treg) are one of

the most studied immune cell populations lately. They are reportedly

involved in fetal acceptance. Here, we summarize several aspects of Treg

biology in normal and pathologic pregnancies focusing on Treg frequen-

cies, subtypes, antigen specificity, and activity as well as on factors influ-

encing Treg generation, recruitment, and function. This review also

highlights the contribution of fetal Treg in tolerance induction and

addresses the role of Treg in autoimmune diseases and infections during

gestation. Finally, the potential of Treg as a predictive marker for the

success of assisted reproductive techniques and for therapeutic interven-

tions is discussed.

Introduction

The nomenclature of regulatory T cells (Treg) was

first brought into light by Sakaguchi et al. in 1995,

although already in the 70s, the existence of ‘sup-

pressor T cells’ with regulatory properties was pro-

posed.1 Nowadays, this specialized T-cell

subpopulation is best known for their function in

suppressing autoreactive and alloreactive immune

responses, thereby preventing autoimmune diseases

and allograft rejection.2 Additionally, a crucial role

for these cells in the establishment and maintenance

of fetal tolerance has widely been reported in both

humans and mice.3–6 CD4+CD25+Foxp3+ Treg can be

either generated in the thymus as naturally occur-

ring Treg (nTreg) or in the periphery after encounter

of antigens as induced Treg (iTreg). iTreg can be fur-

ther subdivided into CD4+ T-cell subsets such as type

1 regulatory cells (Tr1), T-helper 3 cells (Th3), and

CD25+ Treg. Whereas Tr1 cells mainly produce IL-

10, TGF-b is the major cytokine of Th3 cells. Both

cytokines are fundamental for the function of each

Treg subtype.7,8 CD4+CD25+ Treg express surface

markers such as the alpha chain of the IL-2 receptor

CD25,2 CD127 (human only),9 glucocorticoid-

induced tumor necrosis factor receptor-related pro-

tein (GITR),10 cytotoxic T lymphocyte-associated

antigen 4 (CTLA-4)11, and a unique intracellular

marker, X-linked Forkhead box P3 (Foxp3) validated

as the ‘master switch’ for Treg development.12

Regulatory T cells in human and murine pregnancy

Origin and Generation

Thymus-derived Treg as well as peripherally induced

Treg seem to be involved in immune regulatory pro-

cesses toward fetal alloantigens.13,14 The peripheral

Treg pool is suggested to be maintained by a contin-

uous conversion of na€ıve T cells into Treg in the

presence of TGF-b15 or through T-cell stimulation by

suboptimal activated dendritic cells.16 Additionally,

American Journal of Reproductive Immunology 72 (2014) 158–170

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd158

REVIEW ARTICLE

molecules directly produced at the fetal–maternal

interface, such as the heme-degrading enzyme heme

oxygenase-1, may be involved in Treg generation.17

Recently, Samstein et al. proposed a conserved non-

coding sequence 1 (CNS1)-dependent mechanism of

extrathymic Treg differentiation that emerged in pla-

cental animals and enforce fetal–maternal tolerance.

In detail, the authors demonstrated that the Foxp3

enhancer CNS1 is essential for peripheral Treg but

dispensable for thymic Treg generation. Interestingly,

they proved that allogeneically paired CNS-1-defi-

cient females show slightly increased fetal resorption

rates,13 suggesting a role for peripheral Treg in fetal

tolerance induction. In agreement, we lately con-

firmed the existence of an extrathymic Treg popula-

tion at later pregnancy stages. Moreover, we found

an accumulation of thymus-derived Helios+ Treg in

thymus and draining lymph nodes at early preg-

nancy suggesting that Treg, predominantly of thymic

origin, are needed for pregnancy establishment.14

Kinetics of Treg during the reproductive cycle and

pregnancy

Treg frequency fluctuates during the human men-

strual cycle as well as during the murine estrus

cycle. In both reproductive cycles, Treg increase in

number every time a female becomes receptive and

decline afterward.18–20 This fluctuation of Treg dur-

ing the reproductive cycle is proposed to be hor-

mone driven.21

In human pregnancy, most studies observed a

Treg augmentation during the first and second tri-

mester in peripheral blood5,22 being this augmenta-

tion even more pronounced in decidual tissue.23 By

contrast, Mj€osberg et al.24 found diminished levels of

CD4+CD25high Treg in the second trimester and sug-

gested an re-evaluation of Treg frequencies during

pregnancy. In the third trimester, Treg levels start to

decrease25 and further decline with successive stages

of labor.26 These Treg changes may be related to a

brief activation of maternal immune system as labor

begins.27 Moreover, the suppressive activity of Treg

is decreased in term labor and preterm labor (PL)

suggesting that the change of Treg function

toward suppression may initiate labor. A distinct

subset of Foxp3+DR+ Treg among the total

CD4+CD127lowCD25+ Treg pool has been reported to

be critically involved in PL induction.28 In normal

pregnant mice, we observed an initial Treg augmen-

tation as early as day 2 of pregnancy, and then, Treg

decreased at implantation time and again raised on

day 10 of pregnancy.14,29

Treg Subtypes

The majority of studies evaluating Treg number and

functionality during normal and pathologic pregnan-

cies define Treg as CD4+CD25highFoxp3+ cells in

humans or as CD4+CD25+Foxp3+ cells in mice. How-

ever, some studies focus on Treg subtypes within the

so-called Treg population. In this regard, Andrea

Steinborn et al. evaluated the number of different

Treg subtypes within the total Treg pool during nor-

mal and pathologic pregnancies. The authors

observed between the 10th and 20th weeks of gesta-

tion a clear decrease in the percentage of DRhigh

CD45RA� and DRlow CD45RA� Treg and a clear

increase in the percentage of naive DR� CD45RA+

Treg. Compared with healthy pregnancies, the fre-

quency of naive DR� CD45RA+ Treg was signifi-

cantly reduced in the presence of pre-eclampsia (PE)

and after the onset of PL. Interestingly, they found

that the percentage of DRhigh CD45RA� and DRlow

CD45RA� Treg was increased significantly in preg-

nancies affected by PE, while PL was accompanied

by a significantly increased percentage of DRhigh

CD45RA� and DRlow CD45RA� Treg. The suppres-

sive activity of the total Treg pool was diminished in

both patient collectives. The authors suggest that PE

and PL are characterized by homeostatic changes in

the composition of the total Treg pool with distinct

Treg subsets that were accompanied by a significant

decrease in its suppressive activity.30 Additionally,

there are reports clearly identifying CD8+ T cells in

peripheral blood and decidual tissue with unique

regulatory properties and were suggested to be

involved in fetal tolerance.23,31,32 In addition, CD4�

CD8� gamma delta T cells with regulatory properties

have been also reported to function as immune reg-

ulators during pregnancy.33

Antigen Specificity

In 1953, Sir Peter Medawar hypothesized that the

recognition of foreign fetal antigens by maternal

immune cells is hampered by the placenta. He pro-

posed the placenta as a physiological barrier that

strictly separates fetal from maternal tissues. Half a

century later, the discovery of a bidirectional cell

transfer between the mother and her unborn child

revealed this hypothesis as not entirely true.


ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 159

REGULATORY T CELLS DURING PREGNANCY

Moreover, nowadays, there is a general consent that

the recognition of fetal antigens by maternal

immune cells is a necessary step that leads to an

active suppression of alloreactive maternal immune

responses and constitutes thereby the initial step of

fetal tolerance induction. Transplacental fetal–mater-

nal cell exchange has been proven to occur in both

species, human and mice.34–37 Tilburgs et al.38 nicely

showed that decidual T cells directly recognize fetal

HLA-C antigens and that this is associated with

increased Treg levels and diminished CD4+CD25dim

T-cell levels. In agreement with this observation,

there is a growing body of evidence that the recogni-

tion of foreign paternal/fetal alloantigens by Treg is

critically for their development and function.39–43

The first encounter of paternal alloantigens with

maternal immune cells, for example, antigen-pre-

senting cells (APCs), is supposed to take place imme-

diately after conception in vaginal lumen where

immune cells get in contact with paternal antigens

present in seminal fluid. These antigens can be pre-

sented by direct or indirect antigen-presenting path-

ways.44,45 Both seminal fluid and sperms have been

shown to foster Treg induction, expansion, and

immune regulatory activity.20,46–49 Furthermore,

TGF-b present in high amounts in seminal fluid may

contribute to Treg expansion.50 This early Treg

expansion may induce tolerance to paternal antigens

facilitating embryo implantation and fetal accep-

tance.

After implantation, the Treg pool is then further

maintained by the continuous release of fetal anti-

gens from the placenta guaranteeing fetal survival

until birth. Interestingly, post-partum fetal-specific

Treg may persist in the mother creating a memory

to paternal antigens51 and rapidly re-accumulate

during subsequent pregnancies.52

Treg Recruitment into the Fetal–Maternal Interface

Treg recruitment from the periphery into the fetal–maternal interface was proposed as an additional

pathway for local Treg accumulation.53–55 Here,

cytokines, chemokines, and hormones may serve as

attractors for a selective Treg migration into the

uterus. Cytokines and chemokine ligands such as

IL-8, MCP-1/3, RANTES, fractalkine, MIP-1a/b,IP-10, SLC, and CCL22 are widely expressed in uter-

ine tissue56,57, and the appropriate receptors CCR1,

CCR2, CCR5, CCR7, CXCR3, CX3CR1 have been

proven on T cells.58 We recently demonstrated that

mice genetically deficient for the homing receptor

CCR7 had diminished Treg numbers in the uterus.

Mating of these mice with syngeneic or allogeneic

males resulted in impaired implantation, even

though some animals did get implanted. Thus, CCR7

is partially involved in the homing of Treg to the

uterus.20 A study by Lin et al.59 revealed that

CXCL12 is dominantly expressed in trophoblast cells,

while its specific receptor CXCR4 is expressed on

Treg suggesting that CXCL12 can cause CXCR4+ Treg

migrating in the pregnant uterus. In a transplanta-

tion setting, Lee et al.60 showed that migration of

CCR4+ Treg in response to CCL22 was necessary to

promote tolerance toward the allograft. But not only

chemokines and their receptors are involved in the

mobilization of Treg to the uterus. Upon pregnancy

establishment, hormones dictate Treg migration into

the fetal–maternal interface. In particular, human

chorionic gonadotropin (hCG), the pregnancy hor-

mone per se, is of vital importance for Treg migration

and functionality. We confirmed the presence of the

luteinizing hormone/chorionic gonadotropin (LH/

CG) receptor on human and murine Treg and

showed an active Treg migration along a hormonal

gradient continuously maintained by human or

murine trophoblast cells that produce hCG or LH,

respectively.54,61,62

Normal Pregnancy versus Pathologic Pregnancy

Evidence for the relevance of Treg during pregnancy

can be derived from the finding by Andersen et al.63

who showed that a co-evolutionary process has

taken place between Foxp3 and the placenta. More-

over, since the first Treg pregnancy papers by Aluvi-

hare et al. as well as Heikkinen et al., both in

2004,3,5 several reports confirmed a fundamental

role of Treg for pregnancy success. In both, humans

and mice Treg have been shown to increase during

normal pregnancy and being diminished in their

number and activity in spontaneous abortion

cases.3–5,64–71 Consistently, Winger and Reed72 pro-

posed Treg as a superior pregnancy marker for

assessing miscarriage risk in newly pregnant women.

It is suggested that the impaired Treg augmentation

in patients suffering from recurrent spontaneous

abortions (RSA) is associated with an alteration in

the IL-6 trans-signaling pathway.73 Moreover,

diminished Treg numbers in unexplained RSA

(URSA) patients are accompanied with an increased

number of pro-inflammatory Th17 cells in peripheral


160 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

SCHUMACHER AND ZENCLUSSEN

blood and decidua. As suggested by the authors, the

balance between Th17 cells and Treg may be critical

to pregnancy outcomes.74 An impairment in Treg

augmentation is not only limited to RSA patients,

but can also be observed in patients suffering from

extra-uterine pregnancies54 or endometriosis.75

Interestingly, in the presence of hydatidiform moles,

decidual Foxp3+ Treg infiltration seems to be higher

compared to normal pregnancy.76 In PE patients,

Treg seem to be reduced in number and function,

while Th17 frequencies are elevated. Several studies

suggested a dysregulation of immune responses and

a shift to a more Th17-dominated inflammatory

response characteristic for this pregnancy disor-

der,77–84 while two studies were not able not find a

Treg alteration in PE patients.85,86 Hsu et al.87 pro-

posed that a special subtype of decidual CD14+DC-

SIGN+ APCs may play an important role in Treg

induction in healthy pregnancy and this is defective

in PE. In addition, an altered apoptosis signaling in

Treg might be associated with PE.88 All together,

these results clearly indicate the need for fully func-

tional Treg to ensure successful pregnancy.

Relevance of Treg at Different Pregnancy Stages

Although pregnancy complications have been associ-

ated with reduced Treg number and functionality, it

is still not defined whether Treg are absolutely

required at all pregnancy stages. Recent findings

suggest that Treg are critically involved in the estab-

lishment of fetal tolerance rather than in its mainte-

nance. Depleting Treg in two Foxp3.DTR-based

models prior to pairing drastically impaired implan-

tation. Treg deficiency before mating led to infiltra-

tion of activated effector T cells as well as to uterine

inflammation and fibrosis that later resulted in

reduced implantation success in both allogeneic and

syngeneic mating combinations.20 This suggests that

the presence of Treg in the uterus contributes to a

‘friendly’ microenvironment that will later facilitate

the implantation of the embryo, while their absence

converts the uterus in a hostile environment for

pregnancy to establish. This is consistent with obser-

vations carried out in infectious pathologies associ-

ated with infertility and worthy of further studies. In

an older study, Shima et al. depleted CD25+ cells by

injection of an anti-CD25 monoclonal antibody at

different pregnancy days. Based on their findings,

they concluded that Treg are important for the

implantation phase and tolerance during the early

stage of pregnancy, but might not be necessary for

maintenance of the late stage of allogeneic preg-

nancy.89

Mouse models to study Treg during pregnancy

One of the most prominent mouse models used to

study immune regulation during normal and patho-

logic pregnancies is the abortion-prone model first

described in 1980 by Clark et al.90 Abortion-prone

females show an impaired ability to increase their

peripheral and local Treg number as compared to

normal pregnant controls. A direct transfer of

Treg4,91 or the administration of substances provok-

ing Treg recruitment or expansion was successful in

preventing fetal resorption in abortion-prone

females as we and others showed.50,61,92–96 This

confirms the indispensability of Treg in this mouse

model. Furthermore, using models of selective and

transient ablation of cell populations (specifically

two Foxp3.DTR-based transgenic mouse models),

we could show that Treg ablation drastically impairs

the implantation process.20 In another transgenic

mouse model, D0Addio et al.97 showed that PDL-1

blockage resulted in diminished Treg numbers lead-

ing to increased embryo resorption and reduced lit-

ter size. In addition to these animal models where

Treg diminution can directly be correlated with

pregnancy success, other transgenic mouse models

may facilitate characterization of Treg in pregnancy.

Foxp3GFP animals previously described by Fontenot

et al.12 provide a good tool to easily localize Treg in

different organs, also in vivo by means of 2-photon

in vivo microscopy.20,61 Moreover, Treg induction

from CD4+Foxp3� T cells by Foxp3 upregulation

can be detected in recipient mice after adoptive

transfer of non-Treg by determining GFP expres-

sion.13,14

Hormonal influence of Treg function

Already, during the menstrual/estrus cycle and later

during pregnancy, hormones undergo profound

changes in their levels. The most prominent preg-

nancy-associated hormones are hCG, progesterone

(P4) and estrogen (E2). All three hormones have

been reported to influence Treg generation, migra-

tion, expansion and suppressive function. In the

last years, we focused our work on the highly

homologous hormones hCG and luteinizing hor-

mone (LH) and its influence on Treg. Both human




and murine Treg express the LH/CG receptor.54,61

Direct interactions between the hormones and their

receptor expressed on T cells had been shown

already in the 80s.98 We were able to prove that

hCG not only attracts Treg to trophoblast cells but

also positively influences Treg number and suppres-

sive activity contributing to fetal tolerance. More-

over, hCG retains DCs in a immature, and thus,

tolerogenic state supporting Treg induction via an

indirect pathway.61 Very recently, we showed that

LH similar to hCG modulates Treg and DCs suggest-

ing that both hormones are key players in regulat-

ing adaptive immune responses during pregnancy

(accepted for publication). In agreement with our

data, Shirshev et al. nicely showed that the addition

of hCG to human PBMCs significantly increased the

number of Treg. The same authors found similar

effects of E2 on Treg augmentation but could not

prove any statistically significant effect of P4 on

Treg.99 Other research groups confirmed a positive

effect of E2 on Treg induction, expansion and func-

tion,22,100–104 while data addressing a P4 impact on

Treg remain controversial. Holdstock et al. as well

as Mao et al. provide evidence that P4 positively

influences the proportion and function of

Treg.105,106 By contrast, Mj€osberg et al.24 found a

Treg reduction after P4 treatment. Interestingly, Lee

et al.107 showed that P4 drives allogeneic activa-

tion-induced differentiation of na€ıve T cells from

cord blood, but not from adult peripheral blood,

into Treg.

Mechanisms of immune regulation by Treg

Treg are central players in immune regulatory pro-

cesses and reportedly interact with every other

immune cell type regulating their number and activ-

ity. For instance, Treg suppress the proliferation and

cytokine production of CD4+ and CD8+ T cells108,109

and inhibit the cytotoxicity of NK cells.110 In addi-

tion, Treg dampen the proliferation of B cells and

their immunoglobulin production111 and act as effi-

cient suppressors of DCs and macrophages.112–114

These immune regulatory properties of Treg seem to

be executed mostly in a contact-dependent fashion

and potentiated by the action of IL-10, TGF-b and

CTLA-4.115–117 Recently, it has been suggested that

IL-9 expression by Treg recruit and activate immu-

nosuppressive mast cells.118,119 However, despite

this broad knowledge about immune regulatory

properties of Treg, little is known about the precise

mechanisms how Treg contribute to fetal tolerance.

In a murine model of disturbed fetal tolerance, it has

been suggested that Treg mediate their protective

function through PD-1, IL-10 and HO-1 rather than

through CTLA-4 and TGF-b.41,120,121 By contrast, in

a human study, Jin et al.67 proposed a role for

CTLA-4 in Treg function. Here, CTLA-4 expressed by

Treg may interact with B7 molecules on DCs and

monocytes enhancing IDO activity in those cells by

induction of IFN-c.122 IDO itself has been reported

to support fetal acceptance by suppressing T cell

activity.123 Moreover, we recently suggested an

important regulatory function for Treg in the pre-

implantation period. Here, Treg regulate the accu-

mulation of conventional CD8+ T cells as well as the

production of pro-inflammatory molecules in the

uterus and draining lymph nodes, thereby support-

ing embryo nidation by dampening inflammatory

processes occurring during the time of implanta-

tion.20

Fetal Treg – Immune regulation from the other site

of the placenta

Investigations on Treg contribution to fetal–maternal

tolerance were initially limited to the maternal side.

However, fetal immune cells and, in particular, fetal

Treg may be involved in fetal–maternal tolerance

mechanisms. More recent studies contributed to the

knowledge of the function of fetal Treg. According

to a study by Cupedo et al.,124 fetal Treg are gener-

ated in the thymus where they already gain the

potential to suppress the proliferation of CD25� cells.

After leaving the thymus, they enter fetal secondary

lymphoid organs and umbilical cord blood, where

they acquire a primed/memory phenotype and are

present in a high abundance.124–126 As the high

number of peripheral fetal Treg is not reflected in

the thymus, it is suggested that a significant propor-

tion of the peripheral Treg pool is expanded from

thymic Treg or is generated from conventional T

cells after antigen encounter.127 Encounter of non-

inherited maternal antigens (NIMAs) by fetal Treg

may occur in fetal lymph nodes and umbilical cord

blood where the presence of maternal cells has been

proven.128 Here, fetal immune responses against

maternal APCs bearing NIMAs can be dampened.

Thus, it can be assumed that both maternal and fetal

Treg are crucially involved in immune regulatory

processes that are indispensable for mother and child

to accept each other.




Role of Treg in autoimmune diseases during

pregnancy

It has been reported that pregnancy is associated

with alterations in the disease activity of some auto-

immune diseases depending on the predominant

immunological course of the disease. Whereas Th1-

dominated autoimmune diseases such as multiple

sclerosis (MS) improve, Th2-dominated autoim-

mune diseases such as systemic lupus erythema-

todes (SLE) aggravate.129 As Treg are best known

for their function in preventing autoimmune dis-

eases, they have to be involved in the reduction in

disease activity observed for some autoimmune dis-

eases during pregnancy. Reduced Treg numbers and

impaired functionality as demonstrated in humans

and mice suffering from SLE130–136 may influence

pregnancy outcome. The adoptive transfer of Treg

in lupus-prone mice could reduce disease progres-

sion and improve survival137 suggesting clinical pos-

sibilities for therapeutic interventions in the future.

Consistent with the situation in lupus patients,

asthmatic patients are unable to augment their Treg

number during pregnancy. These patients showed

an abnormal asthma-dependent Th17 elevation

together with blunted Treg increase that may play a

role in the compromised immune tolerance charac-

terizing asthmatic pregnancy.138 In contrast, it is

known that pregnancy has a beneficial effect on

disease progression of rheumatoid arthritis. In a

murine model of arthritis, Munoz-Suano et al.139

demonstrated that Treg were responsible for the

pregnancy-induced amelioration of arthritis. In

agreement, in humans, improvement in disease

activity in the third trimester corresponded to

increased Treg numbers that induced a pronounced

anti-inflammatory cytokine milieu.140 MS patients

experience a significant reduction in their disease

activity especially during the third trimester of preg-

nancy. However, contribution of Treg to disease

amelioration is questionable as Treg number seems

not to differ between healthy pregnant women and

pregnant patients with MS.141 Here, pregnancy hor-

mones were suggested to be responsible for disease

improvement, and some of them are known to

influence Treg. In this regard, Wang et al.142 nicely

proved that administration of E2 at pregnancy levels

in a murine model of MS, namely experimental

autoimmune encephalitis, enhanced Treg frequency

and suppressed the disease.

Role of Treg in infections during pregnancy

Systemic and local infections occurring during preg-

nancy are a big challenge for the maternal immune

system, as it has to attack pathogens while at the

same time tolerate foreign fetal antigens. When

the infection is overwhelming, as a consequence,

the fetus is often rejected. LPS administration, used

to mimic bacterial infection, to normal pregnant

mice at mid-gestation has been shown to result in

preterm delivery. In this murine model of LPS-

induced preterm delivery, CD4+ T cells are proposed

to play a protective but not indispensable role.143 In

infections with Tritrichomonas foetus, Foxp3, Th17

and Th1 cells are augmented and embryonic death is

suggested to result from an exacerbated effector

response of type 1 and 17. Additionally, increased

Treg number may contribute to worsen pregnancy

outcome by promoting persistence of infection.144

Pregnancy-associated Treg increase also causes

enhanced susceptibility to prenatal pathogens

including Listeria and Salmonella species. However,

expansion of Treg is essential to protect the fetus

from rejection. Thus, maternal Treg augmentation

required for maintaining pregnancy creates naturally

occurring holes in host defense that confer prenatal

bacterial susceptibility.145 Parasite infections of preg-

nant women with Toxoplasma gondii or Plasmodium

falciparum result in an imbalance of the Treg/Th17

ratio, with reduced Treg numbers and increased

Th17 frequencies.146–148 This suggests that embryo

loss caused by these parasites may be associated with

a reduced Treg/Th17 ratio.149 Finally, viral infection

with the human immunodeficiency virus (HIV) is

characterized by CD4+ T-cell depletion, chronic

immune activation and altered lymphocyte subsets

that may have importance for the induction of fetal–maternal tolerance and in part explain the increased

risk of abortion in HIV-infected women.150

Role of Treg in assisted reproductive techniques

and their therapeutic potential

Based on a growing body of knowledge, there is a

particular interest to employ Treg as a therapeutic

target to support women in getting pregnant and

treat patients with pregnancy complications. There is

evidence that the success of assisted reproductive

techniques (ART) such as artificial inseminations

and in vitro fertilization (IVF)/intracytoplasmic sperm




injection (ICSI) can be predicted by Treg frequencies.

In this regard, it has been shown that enhanced

pregnancy rates and live birth rates are associated

with increased Treg before and after ART.151,152

More precisely, Schlossberger et al. defined Treg

subpopulations after IVF/ICSI treatment. They found

that non-successfully IVF/ICSI-treated women have

a decreased percentage of naive CD45RA+ Treg and

an increased percentage of HLA-DR� memory Treg

within the total Treg pool. However, the percentage

of CD4+CD127lowCD25+Foxp3+ Treg within the

CD4+ T-cell pool did not differ between IVF/ICSI-

treated women who did or did not become

pregnant.153 During pregnancy, prevention of spon-

taneous abortion by adoptive Treg transfer has been

proven to work in the murine system4,91 and may

be applicable for RSA patients known to have

reduced Treg frequencies. A promising approach is

to immunize URSA patients with paternal leukocytes

shown to improve pregnancy outcome.154 Interest-

ingly, the proportion of CD4+CD25bright T cells in

peripheral blood from URSA patients increased sig-

nificantly after paternal or third-party lymphocyte

immunization therapy, whereas the percentage of

CD4+CD25dim T cells decreased significantly. Impor-

tantly, the percentage of CD4+CD25bright T cells was

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, chemokines

TregTolerogenic DCs

Peripheral blood

Maternal Treg

Teff Fetal Treg

Fetal Treg

hCGHO-1

MaternalTreg DCs

Tolerogenic DCs

TGF-β

Thymus

Non-pregnant uterus

Fibrosisinflammation

CCR7/CCL19

Fig. 1 This hypothetical cartoon depicts the issues discussed within this reviews about the pathways involved in the origin and expansion of

Treg. Thymus-originated Treg migrate to the uterus where they contribute to the generation of a friendly environment for the embryo to implant,

and this occurs independently of pregnancy and is mediated by CCL19/CCR7. 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 that could be harmful to fetal antigens present here as well as in several maternal tissues. 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 and HO-1 modulate the phenotype of function of immune cells.




significantly higher in successfully pregnant women

than in those with pregnancy loss after lymphocyte

therapy.155 A possible explanation for the success of

the immunotherapy is provided by Sugi et al. who

reported changes in the mixed lymphocyte reaction

(MLR), T-cell subsets and generation of anti-idiotyp-

ic antibodies after immunotherapy with paternal

leukocytes. The percentage of cytotoxic T cells was

significantly decreased, and the percentage of sup-

pressor T cells was significantly increased, suggesting

that a cell-mediated immune response was induced

by the immunotherapy.156

Conclusions

Treg are potent immunoregulators, and the exten-

sive revision of the literature highlights their func-

tion at every pregnancy stage as well as their

dysregulation during pregnancy pathologies. They

have a great potential as diagnostic and therapeutic

toll to monitor risk pregnancies. The concepts dis-

cussed in this review are summarized in Fig. 1.

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regulatory t cells: regulators of life

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