Human Chorionic Gonadotropin as a Central

Regulator of Pregnancy Immune Tolerance

This information is current as
of April 7, 2017.
Anne Schumacher, Kristina Heinze, Jeanette Witte, Eileen
Poloski, Nadja Linzke, Katja Woidacki and Ana C.
Zenclussen
J Immunol 2013; 190:2650-2658; Prepublished online 8
February 2013;
doi: 10.4049/jimmunol.1202698
http://www.jimmunol.org/content/190/6/2650

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 The Journal of Immunology

Human Chorionic Gonadotropin as a Central Regulator of

Pregnancy Immune Tolerance

Anne Schumacher, Kristina Heinze, Jeanette Witte, Eileen Poloski, Nadja Linzke,
Katja Woidacki, and Ana C. Zenclussen
Normal pregnancy is characterized by an early expansion of regulatory T cells (Tregs), which is known to contribute to fetal tol-
erance. However, mechanisms and factors behind Treg expansion are not yet defined. Recently, we proposed that the pregnancy
hormone human chorionic gonadotropin (hCG) efficiently attracts human Tregs to trophoblasts, favoring their accumulation lo-
cally. In this study, we hypothesized that hCG not only acts as a chemoattractant of Tregs but also plays a central role in pregnancy-
induced immune tolerance. Virgin, normal pregnant, and abortion-prone female mice were treated either with 10 IU/ml hCG or
PBS at days 0, 2, 4, and 6 of pregnancy. The hCG effect on Treg frequency and cytokine secretion was determined in Foxp3gfp
females. hCG impact on Treg suppressive capacity was studied in vitro. In vivo, we investigated whether hCG enhances Treg
suppressive capacity indirectly by modulating dendritic cell maturation in an established mouse model of disturbed fetal toler-

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ance. Application of hCG increased Treg frequency in vivo and their suppressive activity in vitro. In females having spontaneous
abortions, hCG provoked not only an augmentation of Treg numbers, but also normalized fetal abortion rates. hCG-generated
Tregs were fully functional and could confer tolerance when adoptively transferred. hCG also retained dendritic cells in a tolero-
genic state that is likely to contribute to both Treg expansion and prevention of abortion. Our results position hCG in a novel, so
far unknown role as modulator of immune tolerance during pregnancy. The Journal of Immunology, 2013, 190: 26502658.

F
etal survival within the maternal uterus is ensured by strong
hormonal changes and the regulation of maternal immune
responses toward the foreign fetal Ags. The interplay be-
tween hormonal and immunological factors contributes substan-
tially to fetal tolerance. Pregnancy-associated hormones like the
human chorionic gonadotropin (hCG), estrogen, and progesterone
(P) increase during pregnancy and are essential for successful
pregnancy outcome (1, 2). hCG is a primate-specific hormone; that
is, it is only produced by humans (3) and primates (4), and cannot
be detected in rodents (5). However, rodents produce the highly
homologous luteinizing hormone (LH) binding to the same re-
ceptor as hCG (namely, the LH/CG receptor) (6). Because of this
circumstance, hCG effects can be easily studied in the murine
system. Immediately after fertilization, hCG is produced by the
blastocyst and later by the syncytiotrophoblast (7, 8). After
reaching its maximum level between the 9th and 12th weeks of
pregnancy, hCG concentrations decline until birth (9). In addi-
tion to its main function in stimulating P production by the
corpus luteum, hCG has been described to facilitate trophoblast
invasion (1012), support angiogenesis, and ensure nourishment
Department of Experimental Obstetrics and Gynecology, Medical Faculty, Otto-von-
Guericke University, 39108 Magdeburg, Germany
Received for publication September 26, 2012. Accepted for publication January 11,
2013.
This work was supported by the Deutsche Forschungsgemeinschaft (Grant ZE526/7-1
to A.C.Z.) and the Medical Faculty of the Otto-von-Guericke University of Magdeburg
(Ph.D. grant to A.S.).
Address correspondence and reprint requests to Prof. Ana C. Zenclussen, Experi-
mental Obstetrics and Gynecology, Medical Faculty, Otto-von-Guericke University
Magdeburg, Gerhart-Hauptmann-Strasse 35, 39108 Magdeburg, Germany. E-mail
address: ana.zenclussen@med.ovgu.de
Abbreviations used in this article: AP, abortion-prone; DC, dendritic cell; hCG,
human chorionic gonadotropin; IVF, in vitro fertilization; LH, luteinizing hormone;
LH/CG, luteinizing hormone/chorionic gonadotropin; NP, normal pregnant; P, pro-
gesterone; Treg, regulatory T cell.
Copyright 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
of the fetus (1315). In the 1970s and 1980s, several studies
suggested hCG as an important factor modulating T and B cell
responses (16, 17) via the induction of the at that time called
suppressor T cells (18). This concept was no longer followed until
more recently when Khil and colleagues (19) showed an hCG-
mediated inhibitory effect on Th1 responses that was associated
with increased numbers of CD4+CD25+ regulatory T cells (Tregs)
in a murine model of autoimmune diabetes. During normal human
and murine pregnancy, Tregs expand systemically and locally at
the fetalmaternal interface (2022). Disturbed pregnancies are
associated with a diminished Treg number and activity (2325).
Zhou and colleagues (26) recently confirmed the positive associ-
ation of Treg expansion with pregnancy establishment as they
demonstrated that an increase of Tregs in peripheral blood was
a requisite for a successful in vitro fertilization (IVF). We first
introduced Tregs as a useful tool to control disturbed tolerance
during pregnancy in an abortion-prone (AP) model. We observed
that the transfer of Ag-specific Tregs at early pregnancy stages
restored tolerance and thereby prevented fetal rejection (24, 27),
which is in agreement with recent findings by Yin and colleagues
(28). Treg transfer induced a tolerant microenvironment directly at
the fetalmaternal interface (29), and Treg protective effects on
pregnancy were mediated at least in the studied model by IL-10
and programmed death-1, whereas TGF-b and CTLA-4 rather
played a secondary role (27, 30). In humans, we found a correla-
tion between hCG and Treg levels, which are both diminished
in patients suffering from spontaneous abortions or extrauterine
pregnancies when compared with normal pregnant (NP) women
(31). We also found hCG to attract Tregs, which express the LH/
CG receptor, into the fetalmaternal interface (31). Thus, hCG
acts as a chemoattractant to Tregs at early pregnancy stages. This
gives rise to a concept in which the very first hormone produced
by the trophoblast, hCG, contributes to the recruitment of immune
cells to the fetalmaternal interface that are pivotal for later tol-
erating the conceptus. In this work, we followed the hypothesis
that hCG would not only be a chemoattractant, but also an im-

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202698
The Journal of Immunology
portant modulator of Treg-mediated fetal tolerance by influencing
their numbers and/or suppressive capacity. To study whether hCG-
mediated effects are of direct or indirect nature, we also concen-
trated on the effects of hCG on the maturity state of dendritic cells
(DCs) that act as APCs at the very first step of an adaptive immune
response.
Materials and Methods
Animals
Wild type CBA/J female mice and BALB/c and DBA/2J male mice were
purchased from Charles River (Germany), maintained in our animal facility,
and treated according to the institutional guidelines with ministerial ap-
proval (Landesverwaltungsamt Sachsen-Anhalt AZ2/868 and AZ42502-2-
1125UniMD). The experiments were conducted by authorized persons
according to the Guide for Care and Use of Animals in Agriculture
Research and Teaching. DBA/2J-mated CBA/J females (AP combina-
tion) are known to spontaneously develop high abortion rates (24), whereas
BALB/c-mated CBA/J females (NP combination) represented the control
animals. Foxp3gfp transgenic females on a C57BL/6 background, kindly
provided by Dr. Rudensky, were allogeneically mated to BALB/c males.
In these animals, the complete coding sequence of the GFP (gfp) is
inserted in the first coding exon of the Foxp3 gene, which results in
a chimeric GFP-Foxp3 fusion protein (32) that enables the easy identifi-
cation of Foxp3+ Tregs. Animals were paired and checked twice a day for
vaginal plug, whose appearance indicated day 0 of pregnancy. Virgin and
pregnant animals were injected i.p. with 10 IU/ml hCG (Sigma Aldrich,
Steinheim, Germany) or PBS (PAA Laboratories, Colbe, Germany). hCG
and PBS injections were performed at days 0, 2, 4, and 6 of gestation or
experiment, and females were sacrificed on day 10 of pregnancy or 4 d
after the last injection in nonpregnant animals.
Sample collection and flow cytometry
Blood was obtained by retroorbital puncture under anesthesia. Females
were sacrificed by cervical dislocation; spleen, thymus, para-aortic lymph
nodes, and uterine tissue were removed, washed in ice-cold PBS, and kept in
RPMI 1640 medium at 4C. Pregnant uteri were opened longitudinally, and
the number of implantations and the abortion rate were determined.
Fetoplacental units were separated from their implantation sites; deciduae
were cut in small pieces and collected in HBSS without Ca2+ and Mg2+
(Sigma, Taufkirchen, Germany). Mononuclear cells from blood, spleen,
thymus, para-aortic lymph nodes, uterus, and decidua samples were iso-
lated using our established protocol (24). Thereafter, cells were stained for
extracellular and intracellular markers as described elsewhere (24) and
read on a FACSCalibur (Becton Dickinson, Heidelberg, Germany). The
following Abs were used: FITC-conjugated rat anti-mouse CD4 (clone
RM4-4), PE-labeled anti-mouse Foxp3 (clone FJK-16s), allophycocyanin-
FIGURE 1. hCG increased Treg number and their
capability to secrete suppressive cytokines. Application
of 10 IU/ml hCG in BALB/c-mated Foxp3gfp females
significantly increased the numbers of Foxp3-GFP+
cells (Tregs) in blood [(A); n = 7/group] and decidua
[(B); n = 79/group] when compared with PBS treat-
ment. Moreover, to assess cytokine secretion by Tregs
obtained from either hCG- or PBS-treated pregnant
Foxp3gfp females, we cultured Tregs for 24 h in the
presence of ionomycin and PMA. Tregs obtained from
hCG-treated females secreted increased amounts of
IL-10 [(C); n = 6 per group] and TGF-b [(D); n = 3 per
group] when compared with Tregs obtained from PBS-
treated females. (A and B) Each square represents one
single animal. Data are either presented as medians (A,
B) or means + SD (C, D), and statistical analysis was
carried out by the MannWhitney U test or Student
t test, respectively. *p , 0.05, **p , 0.01.
2651
conjugated anti-mouse CD11c (clone HL3), FITC-labeled anti-mouse
CD80 (clone 16-10A1), and PE-conjugated anti-mouse I-A/I-E (clone
M5/114.15.2). Foxp3 Ab was purchased from eBioscience (Kranenburg,
Germany). All other Abs were purchased from Becton Dickinson.
FACS and cell culture
For in vitro studies, Foxp3+ cells were sorted from spleen or draining
lymph nodes from BALB/c-mated Foxp3gfp transgenic females (treated
with either PBS or hCG), washed in ice-cold PBS, and kept in RPMI 1640
medium supplemented with 10% FBS (Biochrom, Berlin, Germany) and
100 ng/ml penicillin/streptomycin (Invitrogen, Karlsruhe, Germany) at 4
C. Mononuclear cells were isolated by disaggregating the tissue, filtering it
through a 100-mm cell strainer (Becton Dickinson), and lysing the eryth-
rocytes with an NHCl4/NaCl solution. After lysis, cells were stained with
PerCp-Cy5.5conjugated anti-mouse CD4 (clone RM4-5) from Becton
Dickinson. CD4 +GFP + (Foxp3 + ) Tregs were sorted by FACS using a
FACSDiva Flow Cytometer and Cell Sorter from Becton Dickinson.
Sorted Tregs were cultured for 24 h in RPMI medium supplemented with
FBS and penicillin/streptomycin. To induce cytokine secretion, we added
1 mg/ml ionomycin (Sigma-Aldrich, Steinheim, Germany) and 50 ng/ml
PMA (Sigma-Aldrich) to the culture. After 24 h, supernatants were taken
for analysis of IL-10 and TGF-b by ELISA.
ELISA
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The amounts of IL-10 and TGF-b were determined in the supernatants of
isolated Tregs from hCG- or PBS-treated pregnant Foxp3gfp females on day
10 of pregnancy by using the BD OptEIA ELISA Set for IL-10 provided
by Becton Dickinson and the Ready-Set-Go Kit for TGF-b provided by
eBioscience (Frankfurt, Germany). All steps were performed according to
the instructions of the manufacturer.
Magnetic cell isolation and adoptive transfer of Tregs
To investigate whether hCG induces the generation of pregnancy-protective
Tregs in vivo, we isolated CD4+CD25+ cells by magnetic beads (MACS;
Miltenyi Biotec, Bergisch Gladbach, Germany) from a mixture of spleen
and thymus from AP animals (day 12 of pregnancy) previously treated
with either 10 IU/ml hCG or PBS. The purity of the isolated Tregs was
95%. A total of 2 3 105 Tregs, diluted in 200 ml PBS, were adoptively
transferred into AP animals by i.v. injection on days 02 of pregnancy. The
used number of 2 3 105 Tregs, adoptively transferred into AP animals, was
proved to be the minimum cell number essential to confer fetal protection
in this model (24).
Suppression assay and conversion assay
To study the influence of hCG on Treg function, we isolated CD4+CD252
responder T cells and CD4+CD25+ Tregs by magnetic beads from a mix-
ture of spleen and draining lymph nodes (para-aortic plus inguinal lymph
2652
FIGURE 2. hCG application normalized Treg frequencies in AP
females. Application of 10 IU/ml hCG in AP females (n = 69/organ)
significantly increased the number of CD4+Foxp3+ cells (Tregs) within the
CD4+ cell population in thymus (A), para-aortic lymph nodes (B), blood
(C), and decidua (D) when compared with PBS-treated AP females (n = 6
7/organ). No significant differences in the Treg number could be detected
between hCG- (n = 56/organ) and PBS-treated (n = 56/organ) NP
females. Each square represents one single animal, and the lines show the
medians. Statistical analysis was carried out by KruskalWallis test fol-
lowed by MannWhitney U test. *p , 0.05, **p , 0.01.
nodes) from AP females previously treated with hCG or PBS. Responder
T cells were stained with CFSE (Vybrant CFDA-SE Cell Tracer Kit;
Molecular Probes, Leiden, The Netherlands) to determine proliferation and
cultured for 24 and 48 h with or without Tregs in a ratio of 1:1. This ratio
was determined in previous coculture experiments where we titrated the
number of Tregs to responder T cells and found best results in the ratio of
1:1 (24). Proliferation of responder T cells was stimulated by the presence
of mitomycin C (Sigma-Aldrich)inactivated splenocytes from DBA/2J
males. Proliferation rate was assessed by flow cytometry. Moreover,
CD11c+ DCs were isolated from spleen and decidua from hormone or
control-treated AP females to study the effect of hCG on DC capability
to promote Treg induction. For this, spleen and decidua tissue were cut
into small pieces and enzymatically digested for 20 min at 37C in
HBSS medium supplemented with 200 U/ml hyaluronidase (Sigma-Aldrich),
Table I. hCG increased the number of local Tregs in virgin females
Group n Tregs in Spleen (%)
CBA/J + PBS 4 4.08
CBA/J + hCG 4 4.52
Application of 10 IU/ml hCG in virgin CBA/J females increased the number of Tregs in uterus as compared with PBS
treatment but did not change the levels of Treg in spleen and blood. Data are presented as medians. Statistical analysis was
carried out by the MannWhitney U test. No statistical differences could be detected between hCG- and PBS-treated females.
hCG STIMULATES Treg AND SUPPORTS PREGNANCY
1 mg/ml collagenase type IV (Invitrogen, Karlsruhe, Germany), 0.2 mg/ml
DNase I (Roche Diagnostics, Mannheim, Germany), and 1 mg/ml BSA
(Sigma-Aldrich). Afterward, cell suspensions were filtered through a 100-
mm cell strainer (decidua) or 70- and 40-mm cell strainers (spleen).
Mononuclear cells were obtained by density gradient centrifugation. Iso-
lation of CD11c+ DCs was performed by MACS isolation. Then CD11c+
DCs were cultured for 24 and 48 h with CD4+ T cells from draining lymph
nodes (MACS isolation), and expression of Foxp3 was analyzed by flow
cytometry.
LH/CG receptor detection on Tregs and DCs by flow cytometry
To confirm the presence of the LH/CG receptor on both immune cell
populations, CD4+CD25+ Tregs were isolated from a mixture of spleen and
thymus, whereas CD11c+ DCs were isolated from spleen of virgin CBA/J
females by magnetic beads. Isolated Tregs and DCs were stained using the
polyclonal rabbit anti-mouse LH/CG receptor Ab (Acris, Hiddenhausen,
Germany) for 1 h at room temperature. Then cells were stained using an
FITC-conjugated goat anti-rabbit IgG Ab for 30 min at 4C. Afterward,
cells were analyzed by flow cytometry, and data are presented as histo-
grams using FlowJo 7.6.5 software (Tree Star).
Data analysis and statistics
Data obtained for in vitro assays and cytokine determinations by ELISA are
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presented as means + SD. Statistical analysis was realized by performing the
Student t test. All other data are presented as medians in graphs showing
individual values for each animal. Analysis of statistical differences among
all groups was performed using the nonparametric KruskalWallis test. For
analyzing differences between two particular groups, the MannWhitney
U test was applied. In all cases, p , 0.5 was considered to be statistically
significant.
Results
Application of hCG provoked an increase in Treg number and
their capability to secrete suppressive cytokines
In this study, we investigated whether hCG influences Treg expan-
sion and suppressive function in murine pregnancy. For this, we used
transgenic Foxp3gfp female mice allogeneically mated to BALB/c
males. Foxp3gfp females were treated with hCG or PBS at days 0, 2,
4, and 6 of gestation, and the frequency of Tregs was determined by
flow cytometry in several organs. We observed a significant ex-
pansion of Tregs in blood and decidua of hCG-treated females when
compared with PBS-treated controls (Fig. 1A, 1B). In lymph nodes,
spleen, and thymus, no significant differences among the groups
were found (data not shown). To further evaluate whether hCG
additionally influences the cytokine secretion pattern of Tregs, we
examined the amount of IL-10 and TGF-b, both known to mediate
Treg suppressive capacity (33, 34). hCG treatment increased, al-
though not significantly, the capability of Tregs to secrete IL-10
and TGF-b (Fig. 1C, 1D) when compared with PBS. Thus, hCG
provokes an expansion of Tregs and fosters their IL-10 and TGF-b
secretion.
hCG application restored tolerance and diminished fetal
rejection by normalizing Treg frequencies
We hypothesized that hCG enables fetal survival by promoting
Treg expansion. To prove this, we determined the systemic and
local number of Tregs in hCG- or PBS-treated NP or AP females.
hCG application at days 0, 2, 4, and 6 resulted in significantly in-
Tregs in Blood (%) Tregs in Uterus (%)
1.63 12.30
1.06 17.08
The Journal of Immunology
creased Treg numbers in thymus, para-aortic lymph nodes, blood,
and decidua (Fig. 2). The levels of Tregs in control pregnant mice
were only slightly augmented (Fig. 2). In virgin CBA/J females,
hCG application increased the number of Tregs in uterus as
compared with PBS but did not change the levels of Tregs in spleen
and blood (Table I). Thus, hCG seems to expand Tregs in the uterus
regardless of an ongoing pregnancy, whereas systemic expansion
of Tregs has been observed only in pregnant female mice.
To study the effect of hCG-mediated Treg augmentation on
pregnancy, we analyzed the abortion and implantation rates after
hCG or PBS treatment. Negative side effects of hCG on pregnancy
were excluded after observing no differences in the pregnancy
outcome when compared with PBS-treated NP females and no
anomalies in any organ (Fig. 3A). hCG application could impres-
sively prevent fetal rejection in AP females when compared with
AP females treated with PBS (Fig. 3A), and this was not dependent
on the number of total implantations (Fig. 3B). As shown in Fig.
3C, AP females displayed fetal resorptions together with healthy
embryos, whereas hCG-treated AP females had no resorptions.
These results clearly confirm a positive influence of hCG on preg-
nancy outcome in this model by restoring the levels of Tregs to the
values observed in the normal pregnancy combination.
hCG application generated fully functional Tregs that can
confer tolerance when adoptively transferred
To know whether Tregs generated after hCG injection are fully
functional, we next isolated Tregs from hCG- or PBS-treated, DBA/
FIGURE 3. hCG prevented fetal rejection in AP females. (A) PBS-
treated AP females (n = 10) showed an increased abortion rate when
compared with PBS-treated NP females (n = 6). Fetal rejection in AP
females could be completely prevented by application of 10 IU/ml hCG
(n = 12). No differences have been observed between hCG- (n = 6) and
PBS-treated NP females. (B) The number of implantations was comparable
among all groups. (C) Representative pictures of whole implantation sites
revealed that PBS-treated AP females showed fetal resorptions among
healthy fetuses, whereas hCG-treated AP females, as well as hCG- and
PBS-treated NP females, displayed only healthy implantations. Each
square represents one single animal, and the lines show the medians.
Statistical analysis was carried out by KruskalWallis test followed by
MannWhitney U test. ***p , 0.001.
2653
2J-mated AP CBA/J females and adoptively transferred them into
AP animals at days 02 of pregnancy. As expected, Tregs obtained
from PBS-treated, DBA/2J-mated CBA/J females were not able to
confer fetal protection to AP females because of their impaired
functionality (Fig. 4A). By contrast, Tregs obtained from hCG-
treated, DBA/2J-mated CBA/J females completely rescued fetuses
from immune rejection after adoptive transfer in AP females (Fig.
4A). This was not dependent on the number of implantations
because they were all comparable among the groups (Fig. 4B).
The adoptive transfer of Tregs from hCG-treated females resulted
in a significant increase in systemic and local Treg levels when
compared with the transfer of Tregs from PBS-treated females
(Fig. 4CF).
Tregs generated in vivo after hCG application retained their Ag
specificity toward paternal Ags
We next wondered whether Tregs isolated from hCG- or PBS-
treated AP females would suppress proliferation of autologous
responder T cells in an MLR in vitro and whether this effect is Ag
specific. As expected, Tregs from both hCG- and PBS-treated
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animals had a strong suppressive impact on the proliferation rate
of responder T cells when compared with proliferation of responder
T cells without Tregs (Fig. 5A). Interestingly, we found that Tregs
from hCG-treated female mice had a significantly increased sup-
pressive capacity compared with Tregs obtained from PBS-treated
females (Fig. 5A), which confirms that hCG contributes to Treg
suppressive capacity. To further examine whether the effect of
hCG on Treg function is specific for paternal Ags, we determined
the proliferation rate of responder T cells obtained from a third-
party combination (CBA/J 3 C57BL/6) in the presence of Tregs
from hCG- or PBS-treated AP females. Tregs from both sources
had no effect on the proliferation rate of responder T cells when
compared with their proliferation without Tregs (Fig. 5B). These
results reveal the important influence of hCG on Treg function-
ality: hCG applied to pregnant animals fosters the expansion of
Tregs that are specific against paternal Ags and play, therefore, an
important role in tolerating these Ags.
Tregs and DCs expressed the LH/CG receptor
After we proved an influence of hCG on Treg expansion and
functionality, we wondered whether this effect is mediated di-
rectly by hCG binding to the LH/CG receptor on Treg cell surface
or indirectly by modulation of APCs, that is, DCs, which then
may induce Treg generation. To test both possibilities, we
checked for the presence of the LH/CG receptor on both murine
Tregs and DCs. Interestingly, 97% of Tregs and 69.4% of DCs
(Fig. 6) isolated from virgin CBA/J females expressed the LH/
CG receptor on their surface, making them susceptible to hCG
signaling.
hCG retained DCs in an immature state, and in vitro these cells
favor Treg generation/expansion
To evaluate the possibility that the influence of hCG on Treg
number and function is mediated indirectly via altering the phe-
notype and activity of other immune cells, especially cells of the
innate immune system presenting Ags, we investigated the influ-
ence of hCG on the phenotype of DCs. According to their mat-
uration state, DCs support activation of the immune system or
suppress immune responses. Mature DCs have been shown to
activate T cell responses (35, 36) that might result in fetal rejection
whereas immature tolerogenic DCs induce the generation of Tregs
favoring fetal protection (37). DCs from hCG-treated AP females
were characterized for their maturation state and compared with
DCs obtained from PBS-treated control females. We observed that
2654
FIGURE 4. hCG induced fully functional Tregs
that conferred tolerance and further Treg augmen-
tation after their adoptive transfer. (A) Adoptive
transfer of Tregs isolated from hCG-treated AP
females (n = 12) in AP females (n = 11) rescued
females from abortion. By contrast, adoptively
transferred Tregs from PBS-treated AP females (n =
10) in AP females (n = 7) had no effect on the
abortion rate. (B) The number of implantations was
comparable among all groups. AP females (n = 68/
organ) adoptively transferred with Tregs from hCG-
treated AP females (n = 69/organ) showed in-
creased numbers of CD4+Foxp3+ cells (Tregs)
within the CD4+ cell population in thymus (C),
para-aortic lymph nodes (D), blood (E), and decidua
(F) when compared with AP females (n = 7/organ)
adoptively transferred with Tregs from PBS-treated
AP females (n = 67/organ). Each square represents
one single animal, and the lines show the medians.
Statistical analysis was carried out by Kruskal
Wallis test followed by MannWhitney U test. *p ,
0.05, **p , 0.01, ***p , 0.001.
hCG significantly decreased the total number of CD11c+ cells at
the fetalmaternal interface but not in the spleen (Fig. 7A, 7B).
Moreover, the number of mature DCs determined by the expres-
sion of CD80 and MHC class II was significantly reduced in de-
cidua but not spleen after hCG treatment as compared with PBS
treatment (Fig. 7CF). In virgin CBA/J females, hCG slightly
decreased the number of total CD11c+ DCs and mature CD11c+
MHCII+ DCs in spleen and uterus when compared with PBS, but
did not alter the levels of CD11c+CD80+ DCs in both organs
(Tables II, III).
To test whether uterine DCs from hCG-treated animals favor the
generation or expansion of Tregs, we cocultured DCs obtained
from spleen or decidua of either hCG- or PBS-treated AP females
with CD4+ T cells and determined the number of CD4+ cells
expressing Foxp3. Indeed, we observed an upregulation of Foxp3
expression in CD4+ T cells when decidual DCs from hCG-treated
AP females were present in the culture (Fig. 8A). This effect was
not observed when DCs were of splenic origin (Fig. 8B). Alto-
gether, our results confirm an effect of hCG on DC phenotype
and at least in vitro, these cells contribute to augmented Treg
frequency.
Discussion
Although it is well-known that both the endocrine and the immune
systems substantially contribute to successful pregnancy outcome,
the interplay between both systems and the major players at
molecular and cellular levels are still under investigation. We had
already reported that hCG produced mainly by the trophoblast is
one of the factors attracting human Tregs efficiently to the fetal
maternal interface contributing to their local accumulation. This
local accumulation of Tregs might also be favored by other im-
mune cells secreting hCG at the fetalmaternal interface. In this
hCG STIMULATES Treg AND SUPPORTS PREGNANCY
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context, it was already demonstrated that stimulation of mono-
cytes with hCG augmented the production of IL-8 (38) known to
attract leukocytes (39). Moreover, hCG is a potent attractor of
neutrophils, monocytes, and lymphocytes at very low doses (40).
In this work, we hypothesized that hCG may also be of central
importance in promoting tolerance by directly influencing Treg
number and functionality. Because this may also occur indirectly
by interaction with, for example, DCs, we extended our study to
these cells as well.
We tested our hypothesis in the murine system knowing that hCG
is commonly used in superovulation protocols for genetic engi-
neering of mouse strains and efficiently binds to the endogenous
murine LH/CG receptor (41). We first took advantage of the
Foxp3gfp transgenic animals that allowed us to easily isolate and
characterize Foxp3+ Tregs after hormonal treatment in an allo-
geneic pregnancy setting. We observed that hCG significantly
increased the number of Tregs in the periphery, as well as locally,
suggesting an accumulation of Tregs at the fetalmaternal inter-
face according to our observations in human pregnancy. Further-
more, hCG application contributed to Treg suppressive capacity
by elevating the secretion of IL-10 and TGF-b. Having these
results, we wondered whether the positive effect of hCG on Treg
number and function may favor fetal survival. For this, we next
used a model of disturbed tolerance, in which we previously
demonstrated diminished Treg number and activity, and proved
that Treg reconstitution can diminish the abortion rates to normal
levels (24, 27). hCG not only restored the Treg levels to normality,
but also completely prevented abortion. In particular, we proved
a significant increase of thymic Tregs after hCG application,
suggesting that this hormone supports the expansion of naturally
occurring Tregs produced in the thymus. Because more Tregs
were also found in para-aortic lymph nodes, blood, and decidua,
The Journal of Immunology
FIGURE 5. hCG promoted the in vivo generation of Treg that retained
their Ag-specificity in vitro. (A) Both, Treg isolated from hCG- and PBS-
treated AP females significantly suppressed proliferation of autologous
responder T cells (Tresp). Interestingly, Treg from hCG-treated females
showed a significant increased suppressive capacity when compared with
Treg from PBS-treated females. (B) Neither Tregs from hCG- nor PBS-
treated AP females were able to suppress the proliferation of allologous
responder T cells from a third-party combination (Tthird party). In addition,
no differences could be observed between the suppressive capacities from
hCG- or PBS-treated females. Suppression assays were performed in
duplicates and repeated at least three times to ensure reproducibility. Data
are presented as means plus SD. Statistical analysis between two different
groups was performed using the Student t test. *p , 0.05, **p , 0.01,
***p . 0.001, ****p . 0.0001.
and knowing that hCG also fosters their migration (37), we pro-
pose that Tregs previously activated by specific paternal Ags in
draining lymph nodes actively migrate to the fetalmaternal in-
terface to support fetal survival. The positive effect of hCG on
FIGURE 6. Tregs and DCs expressed the LH/CG
receptor. Ninety-seven percent of CD4+CD25+ Tregs
and 69.4% of CD11c+ DCs isolated from virgin CBA/J
females expressed the LH/CG receptor on their cell
surface. Negative controls are included to exclude un-
specific binding of the second Ab. Data are presented as
histograms using FlowJo software.
2655
pregnancy is further underlined by a study of Mansour and col-
leagues (42), who lately showed that intrauterine injection of
hCG before embryo transfer in patients undergoing IVF/
intracytoplasmic sperm injection significantly improved implan-
tation and pregnancy rates. Unfortunately, in that study, the
authors did not analyze the number and activity of Tregs (42).
However, increased pregnancy rates after IVF have been shown to
be associated with increased Treg numbers in peripheral blood
(26). Moreover, based on our human data showing hCG as a po-
tent chemoattractor for Tregs (31), the Egyptian IVF-ET Center
conducted a clinical trial investigating the effect of uterine in-
jection of hCG on endometrial Tregs. Upcoming results will prove
whether the intrauterine application of hCG around implantation
time may increase endogenous endometrial Treg levels, and
thereby favor the implantation process. This would support our
assumption that normal progressing pregnancies with normal hCG
levels ensure the generation and/or expansion of cells that are
fundamental for pregnancy maintenance (43).
To prove the generation of functionally active, pregnancy-
protective Tregs after hCG application, we adoptively trans-
Downloaded from http://www.jimmunol.org/ by guest on April 7, 2017
ferred Tregs isolated from hCG-treated AP females (having oth-
erwise dysfunctional Tregs) into AP mice. These cells had the
capacity to completely protect from fetal rejection, whereas Tregs
isolated from PBS-treated AP females did not. Our data raise the
possibility that transferred Tregs induce the generation of new
Tregs by infectious tolerance or by conversion from naive T cells,
as it has been described by others (4446). In vitro, Tregs from
hCG-treated females had a significantly increased capacity to in-
hibit responder T cell proliferation when compared with Tregs
from PBS-treated females. This confirms our in vivo data on hCG-
mediated generation of pregnancy-protective Tregs. In agreement
with this, Khil and colleagues (19) showed that application of
hCG in a murine model for autoimmune diabetes prevented the
onset of the disease. In this model, hCG application resulted in an
increase of Tregs in spleen and pancreatic lymph nodes, and re-
duced the number of effector T cells. Moreover, hCG elevated IL-
10 and TGF-b expression in splenic cells, which indicates an
augmented suppressive Treg function (19). In our model, hCG
also provoked Tregs to secrete more IL-10 and TGF-b. Fuchs and
colleagues (18, 47) demonstrated an hCG-dependent generation of
suppressor T cells, leading to inhibited mitogen-induced activation
of B cells. Previous data from our group confirmed an Ag-specific
Treg function in the AP model (27). Thus, we wondered whether
activation of Tregs can be mediated by hCG alone or is also de-
pendent on the presence of specific paternal Ags. We could clearly
show that neither Tregs obtained from PBS- nor from hCG-treated
AP females significantly suppressed proliferation of responder
T cells from a third-party combination, confirming that there is
a need for specific Ag stimulation of Tregs in addition to their
2656 hCG STIMULATES Treg AND SUPPORTS PREGNANCY

FIGURE 7. hCG reduced the number of DCs and

retained them in an immature state. Application of
10 IU/ml hCG in AP females (n = 57/organ)
resulted in a significant reduction of total CD11c+
DCs in decidua (B), but had no effect on the number
of splenic CD11c+ DCs (A) when compared with
PBS-treated females (n = 57/organ). The number
of mature CD11c+CD80+ and CD11c+MHCII+ DCs
in decidua was significantly decreased after hCG
treatment (n = 78) as compared with PBS treat-
ment (n = 7) (D, F). By contrast, hCG had no impact
on the number of mature DCs in spleen (n = 45/

Downloaded from http://www.jimmunol.org/ by guest on April 7, 2017

treatment) (C, E). Each square represents one single
animal, and the lines show the medians. Statistical
analysis was carried out by the MannWhitney U
test. *p , 0.05, **p , 0.01.

activation by hCG. According to these data, we did not observe
a significant increase in the Treg number in uterine tissue of virgin
CBA/J females injected with hCG when compared with CBA/J
females treated with PBS. Therefore, we assume that although
hCG has a strong impact on Treg expansion and functionality, the
presence of specific paternal Ags is essential to ensure complete
Treg induction during pregnancy.
Next, we focused on learning whether hCG mediates Treg in-
duction directly or indirectly via DCs. Because both Tregs and DCs
expressed the LH/CG receptor, both ways are possible. According
to our murine data, the presence of the LH/CG receptor has been
proved on human DCs (48) and human Tregs (31). Several studies
suggest an impact of tolerogenic DCs on Treg generation (49, 50).
We recently demonstrated an involvement of the pregnancy-
protective enzyme heme oxygenase-1 for DC-mediated Treg in-
duction and function during murine pregnancy (37). In addition,
it has been shown that P induces an immature phenotype in bone
marrowderived DCs (51) and estrogen reduces the production
of inflammatory cytokines by mature DCs (52), suggesting that
pregnancy hormones may also influence DC phenotype and ac-
tivity. In terms of an hCG-mediated effect on DCs, in vitro data
are quite controversial (48, 53, 54) and in vivo data are not
available yet.
Because the maturation state of DCs is important for their ca-
pability to induce immunity or tolerance (35), we were interested
to study the influence of hCG on DC phenotype and function.
Blois and colleagues (55) showed that during pregnancy, the
number of CD11c+ DCs in decidua strongly increases when
compared with the peripheral blood. The majority of murine DCs
at the fetalmaternal interface are of myeloid origin, having an
immature tolerogenic phenotype, and produce high amounts of IL-
10 (56). In this regard, the secretion of IL-10 seems to be an es-
sential mechanism for DC function at the fetalmaternal interface
as IL-10 directly inhibits effector T cells (57) and promotes Treg
generation (36). Segerer and colleagues (54), as well as Wan and
colleagues (53), nicely showed in vitro that hCG inhibits upreg-
ulation of MHC class II molecules on DCs, reduces their T cell
stimulatory capacity, and induces IL-10 production. By contrast,

Table II. hCG decreases the number of total and CD11c+MHCII+ DCs in spleen

CD11c+ in Spleen CD11c+CD80+ in Spleen CD11c+MHCII+ in

Group n (%) (%) Spleen (%)

CBA/J + PBS 4 3.38 0.97 1.69

CBA/J + hCG 4 2.81 1.13 1.18
Application of 10 IU/ml hCG in virgin CBA/J females reduces the number of total CD11c+ and mature CD11c+MHCII+ DCs
in spleen as compared with PBS but did not change the levels of CD11c+CD80+ DCs. Data are presented as medians. Statistical
analysis was carried out by the MannWhitney U test. No statistical differences could be detected between hCG- and PBS-
treated females.
The Journal of Immunology
Table III. hCG decreases the number of total and CD11c1 MHCII1 DCs in uterus
CD11c1
Group n in Uterus (%)
CBA/J 1 PBS 4 1.07
CBA/J 1 hCG 4 0.89
Application of 10 IU/ml hCG in virgin CBA/J females reduces the number of total CD11c1 and mature CD11c1MHCII1
DCs in uterus as compared to PBS but did not change the levels of CD11c1CD801 DCs. Data are presented as medians.
Statistical analysis was carried out by the MannWhitney U test. No statistical differences could be detected between hCG- and
PBS-treated females.
Yoshimura and colleagues (48) revealed that hCG induces the
maturation of myeloid and lymphoid DCs in conjunction with
increases in the expression of adhesion/costimulatory molecules,
their stimulatory activities in MLR, and cytokine secretion. In this
article, we provide in vivo evidence for a strong impact of hCG
that reduces the number of total and mature DCs directly at the
fetalmaternal interface, but not in the periphery. Regarding the
impact of a reduction of total DCs for pregnancy outcome, Plaks
and colleagues (58) nicely showed that a depletion of uterine DCs
by applying diphtheria toxin to CD11cDTR mice resulted in an
impaired decidualization and implantation of embryos. However,
DC depletion in this model took place either before pregnancy or
at very early gestation days. In this study, we can only make
conclusions about the number of total CD11c+ DCs on day 10 of
pregnancy after hCG or PBS treatment. Moreover, although hCG
reduced the number of total CD11c+ DCs, there are still plenty of
DCs present at the fetalmaternal interface that can do their job.
Furthermore, we could prove that hCG especially reduced the
number of mature CD11c+ DCs within the whole CD11c+ DC
population. Thus, we would assume that hCG favors the presence
of a population consisting of immature tolerogenic DCs that have
been shown to promote fetal survival by reducing alloreactive
FIGURE 8. hCG provoked the in vivo generation of tolerogenic DCs
that induced Tregs in vitro. CD11c+ DCs isolated from the decidua of
hCG-treated AP females significantly induced the generation/expansion of
CD4+Foxp3+ cells within the CD4+ population when compared with
CD11c+ DCs isolated from PBS-treated females (B). By contrast, CD11c+
DCs isolated from the spleen of hCG-treated females had no influence on
Treg generation/expansion when compared with CD11c+ DCs isolated
from PBS-treated AP females (A). Conversion assays were performed in
duplicates and repeated at least three times to ensure reproducibility. Data
are presented as means + SD. Statistical analysis between two different
groups was performed using the Student t test. *p , 0.05.
2657
CD11c1CD801 in CD11c1MHCII1
Uterus (%) in Uterus (%)
0.09 0.62
0.09 0.38
maternal T cell responses toward the fetus and supporting gen-
eration of Tregs. Coculture of decidual CD11c+ cells and CD4+
T cells further reveal that DCs from hCG-treated AP females in-
deed provoked the generation or expansion of Tregs by upregu-
lating Foxp3 expression in CD4+ T cells. According to our
observation that hCG seems to have no influence on the phenotype
of splenic DCs, we could not detect Foxp3 upregulation in CD4+
T cells in the presence of splenic DCs from hCG-treated AP
females. Thus, we suppose that hCG directly modulates DCs at the
Downloaded from http://www.jimmunol.org/ by guest on April 7, 2017
fetalmaternal interface, and this favors a local Treg expansion.
Our work provides strong evidence that hCG, the most important
pregnancy hormone, secreted by the trophoblast, contributes to
fetal tolerance not only by attracting Tregs to the fetalmaternal
interface, but also by augmenting the number and suppressive
capacity of Tregs. hCG has a direct effect on Tregs but also acts
indirectly by maintaining DCs in an immature, tolerogenic state.
Our observations offer a possible explanation for improved
pregnancy outcomes of IVF patients treated with hCG after em-
bryo transfer. This contributes to the basic knowledge of Treg
generation and functionality during pregnancy and may help to
establish therapies to prevent pregnancy failures.
Disclosures
The authors have no financial conflicts of interest.
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