Modulating impact of human chorionic gonadotropin hormone on the maturation

Epub ahead of print August 30, 2011 - doi:10.1189/jlb.0910520
Article
Modulating impact of human chorionic
gonadotropin hormone on the maturation
and function of hematopoietic cells
Michael Koldehoff,*,1 Thomas Katzorke,† Natalie C. Wisbrun,‡ Dirk Propping,†
Susanne Wohlers,† Peter Bielfeld,† Nina K. Steckel,* Dietrich W. Beelen,* and
Ahmet H. Elmaagacli*
*Department of Bone Marrow Transplantation, West German Cancer Center, Medical School of University of Duisburg-Essen,
and ‡Central Animal Laboratories, University of Duisburg-Essen, Essen, Germany; and †Novum, Center for Reproductive
Medicine, Essen, Germany
RECEIVED SEPTEMBER 21, 2010; REVISED JULY 11, 2011; ACCEPTED JULY 14, 2011. DOI: 10.1189/jlb.0910520
ABSTRACT
hCG hormone is a naturally occurring, immune-modulating agent, which is highly expressed during pregnancy
and causes improvements of some autoimmune diseases such as multiple sclerosis and Crohn’s disease.
Little is known about its immune-modulating effects. This
study in MNCs of women who received hCG as preconditioning prior to IVF demonstrates that hCG increases antiinflammatory IL-27 expression and reduces inflammatory
IL-17 expression. In addition, we found increased IL-10
levels and elevated numbers of Tregs in peripheral blood
of women after hCG application. Rejection of allogeneic
skin grafts was delayed in female mice receiving hCG.
We conclude that hCG may be useful for the induction of
immune tolerance in solid organ transplantation. J. Leukoc. Biol. 90: 000 – 000; 2011.
Introduction
Higher eukaryotes are capable of discriminating self from nonself and thus, mount an immune response to protect themselves against foreign organisms and fight infections and to
reject allogeneic cells while tolerating self-antigens. In pregnant mammals, this response allows for maternal and fetal immune competence while enabling fetal (allograft) survival [1].
The implanting embryo and the maternal uterine decidua require adaptation of the maternal immune system to prevent
rejection of the allogeneic fetus without compromising the
ability of the mother to fend off infection. It is well established
that the innate immune system at the feto-maternal interface
undergoes less-stringent, selective pressure to ensure quick
Abbreviations: CsA⫽cyclosporin A, EBI3⫽EBV-induced gene 3, Egr1⫽early
growth response 1, FOXP3⫽forkhead box P3, FSH⫽follicle-stimulating hormone, G0S2⫽putative lymphocyte G0G1 switch gene 2, Gadd45⫽growth
arrest and DNA damage-inducible gene 45, GITR⫽glucocorticoid-induced
TNF family-related, GvHD⫽graft-versus-host-disease, HSC⫽hematopoietic
stem cell, IVF⫽in vitro fertilization, MNC⫽mononuclear cell, Nr4a1⫽nuclear
receptor subfamily 4 group A member 1, Treg⫽regulatory T cells
0741-5400/11/0090-0001 © Society for Leukocyte Biology
and efficient local protection against infection, while the adaptive immune system has to be restrained to prevent rejection
of the semiallogeneic fetus [2, 3]. The mechanism of allogeneic immune-tolerance induction during pregnancy is poorly
understood, and its uncovering may help to develop strategies
to improve long-term allogeneic graft function after solid organ transplantation or to control GvHD after allogeneic HSC
transplantation [4, 5]. One of the initial hormonal signals
postconception is hCG, detected on Days 7–9 after the LH
surge, which corresponds to Days 19 –21 of the menstrual cycle. hCG is a heterodimeric placental glycoprotein and is excessively expressed during pregnancy, besides the hormones
estrogene and progesterone. The ␣-subunit is identical to that
of other glycoprotein hormones, such as thyroid-stimulating
hormone, FSH, and LH, whereas the ␤-subunit is unique to
hCG [6]. Human endometrium contains membrane-bound
hCG/LH receptors, and hCG has direct actions on the decidualization of human endometrial stromal cells. During pregnancy, hCG is produced initially by the blastocyst, 6 – 8 days
after fertilization and later by the syncytiotrophoblast [7, 8].
The structure of hCG has similarities to the LH and is capable
of inducing ovulation. hCG administration helps the follicle to
burst and release the egg, 36 – 48 h after its application. As a
result of these features of hCG, it is widely used as preconditioning in women undergoing IVF. It has been suggested to be
involved in the induction of allogeneic tolerance in peritrophoblastic cells and possibly also in immune-competent cells
of the maternal system. Receptors for hCG have been detected
in macrophages and monocytes, which might be involved in
immune-modulating effects of hCG [9, 10]. The most abundant maternal immune cells in the decidualized endometrium
are NK cells and macrophages. There is some evidence for a
wide spectrum of cell targets and biological properties, by
which hCG plays a crucial role in the implantation and growth
1. Correspondence: Department of Bone Marrow Transplantation, West German
Cancer Center, University Hospital of Duisburg-Essen, Hufelandstrasse 55, 45122
Essen, Germany. E-mail: [email protected]
Volume 90, November 2011
Journal of Leukocyte Biology
Copyright 2011 by The Society for Leukocyte Biology.
1
of the human embryo [11]. In a normal pregnancy, an appropriate balance between proinflammatory (Th1) and anti-inflammatory (Th2) cytokines is thought to be crucial for determining pregnancy outcome. Several lines of evidence point to
a pivotal role of decidual APCs in shaping the cytokine profile
toward the establishment of a more immunologically tolerant
microenvironment at the maternal-fetal interface [12, 13].
There is no doubt that the maternal immune system is able to
recognize and react to fetus-derived antigens. However, the
fetus is recognized in such a way that the MHC-specific, acquired arm of the maternal immunity is suppressed [14].
Tregs expand during pregnancy and are present at the fetalmaternal interface at very early stages of pregnancy. The migration mechanisms of Tregs to the pregnant uterus are still
unclear. However, with the discovery of Tregs and more-elaborate cytokine profiling of T cell subsets, it became clear that
the Th1/Th2 paradigm is not as straightforward as previously
thought [15, 16]. In general, Tregs have been detected in any
circumstance where self-tolerance plays a determining role in
fetal maintenance. Moreover, many study groups regard Tregs
to be actively engaged, not only in the prevention of autoimmunity but also in facilitating transplantation tolerance; targeting them therapeutically may help to potentiate tumor immunotherapy [17, 18]. Tregs constitutively express CD25 (IL-2R␣)
and CD4, as well as the forkhead family transcription factor
FOXP3, a key control gene in their development and function.
In addition, Tregs express the GITR gene, the CTLA-4, and
TGF-␤ on their cell surface. They secrete TGF-␤ and IL-10,
both thought to contribute to their suppressor activity. Moreover, strong immune-modulating effects have been reported
recently from Th-17 cells, a subset of CD4⫹ T cells that produce IL-17A, IL-17F, TNF, and IL-6 in response to IL-23 [19 –
21]. These cytokines have been suggested to be mediators of
inflammation associated with several autoimmune diseases,
including experimental autoimmune encephalitis and collagen-induced arthritis. As a counterpart of IL-17, IL-27 has
been discussed as a possible physiological antagonist of Th-17
cells, mediating suppressive effects on T cell subsets and inhibiting function of IL-17 [22, 23].
The aim of this study was to evaluate possible effects of hCG
on maturation and functional alterations of hematopoietic
cells in women receiving hCG as preconditioning for IVF, postulating an immunoregulatory role besides its classical role in
maintaining pregnancy. In addition, we investigated the immune-modulating effects of hCG in skin transplants with nonpregnant mice designed to express a tolerogenic phenotype.
MATERIALS AND METHODS
Patients
We included a total of 34 women in this prospective study who underwent
IVF treatment at Novum, Center of Reproductive Medicine (Essen, Germany). Pituitary desensitization was obtained by s.c injection of 3.75 mg
leuprorelinacetate in the midluteal phase (Days 20 –22) of the menstrual
cycle preceding treatment. At the onset of menses (Day 3 of a cycle), all
women began gonadotropin stimulation by daily s.c. injection of 150 –300
IU human menopausal gonadotropin or rFSH. The dose was adjusted to
the individual response, as recorded by serum 17␤-estradiol measurements
2 Journal of Leukocyte Biology
Volume 90, November 2011
and transvaginal ultrasound scanning, performed every other day until the
day of ovulation induction. All patients received the first dose of 10,000 IU
hCG s.c. on the day of ovulation induction. Transvaginal follicle aspiration
was performed 35–36 h later, and again, 5000 IU hCGs were injected s.c.
Two days after oocyte retrieval, two to three embryos were placed into the
uterine cavity via the transcervical route, and again, 5000 IU hCGs were
injected. Luteal-phase support was sustained with 5000 IU hCG s.c. (on Day
7 after embryo transfer) and 3 ⫻ 200 mg natural progesterone intravaginally. Blood samples from all women (median age 35, range 25– 45) were
examined at cycle Days 8 –9 (i.e., before the first application of hCG),
35–36 h after the first application of hCG (i.e., at follicle aspiration), and
another 48 h after the second hCG application (i.e., at embryo transfer).
Pregnancy testing for serum hCG and progesterone was done on Day 15
after embryo transfer. In addition, blood samples of six normal, pregnant,
healthy women were analyzed in the first trimester as control. All women
gave their written, informed consent to be included in this study. Physical
and biochemical findings of all women were within normal limits. None of
the women had a history or clinical findings suggestive of chronic infections, rheumatic disorders, or autoimmune disease. Use of drugs (excluding the procedures of IVF) or conditions affecting lymphokine production
(fever, infection, or another inflammatory process) were excluded. All aspects of this study involving human subjects were approved by the Human
Research Ethics Committee at the University Hospital of Duisburg-Essen
(Essen, Germany).
Mice
We obtained female C57BL/6 (H-2b) mice and female BALB/c (H-2d)
mice from the Central Animal Laboratory, University Hospital of DuisburgEssen. Mice used for experiments were 8 –12 weeks old. To condition immune-competent cells, we treated donor (BALB/c) mice and recipient
(C56BL/6) mice i.p. with 25 IE hCG (Sigma Chemical, Germany), 2 days
prior to skin transplantations. Skin transplants were performed as described previously by Markees et al. [24]. In brief, syngeneic (n⫽2) and
allogeneic (n⫽20) skin transplants were transferred on the back of
C56BL/6 recipients. The graft was fixed with fibrin glue, covered with
Vaseline gauze, and fixed by a bandage. The bandage was removed on the
sixth postoperative day. After transplant, the recipients were treated with 25
IE hCG i.p. or placebo (0.1 ml NaCl 0.9% i.p. or heated-inactivated hCG
i.p.) at Day 0 and every other day until the skin graft was rejected. Mice
were housed in sterilized microisolator cages and received normal chow
and autoclaved hyperchlorinated drinking water (pH 3.0). Graft rejection
was evaluated by a “blinded” assessor daily and documented when the
whole graft (⬎95%) was necrotized. All animal studies were approved by
the state Animal Ethics Committee.
Isolation of MNCs from mouse spleens and
treatment with hCG
Spleens from C57BL/6 mice (n⫽6) were removed aseptically, placed in 60 ⫻
15-mm Petri dishes (Costar, Germany) containing 3 ml cold PBS, and finely
minced with scalpels. MNC suspensions were prepared from the meshed
spleens after RBC lysis by hypo-osmolaric [NH4Cl 1.66% (w/v)] treatment for
5 min and passed through a 50-␮m filter to remove debris. The MNCs were
treated with three different hCG doses (10 IE, 20 IE, and 50 IE hCG i.p.) every other day for 1 week or placebo (0.1 ml NaCL 0.9% i.p.). The treated
MNCs (1⫻105) were washed with DPBS (Gibco, Invitrogen, Karlsruhe, Germany; at a ratio of 1:1) and harvested for real-time PCR.
Isolation of human peripheral blood MNCs and cell
culture
Peripheral heparinized blood was diluted with DPBS, and MNCs were obtained by standard Ficoll-Hypaque separating solution (Seromed, Biochrom
KG, Berlin, Germany) gradient centrifugation. MNCs were harvested from the
interface, washed three times in HEPES-buffered HBSS, and resuspended in
RPMI-1640 medium (Invitrogen), supplemented with 10% FBS and incubated
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Koldehoff et al. hCG modulating maturation and function of hematopoietic cells
at 37°C in a 5% CO2 humidified incubator. To obtain monocytes, MNCs were
layered on Petri dishes (5 ml; 35⫻10 mm; Costar) and incubated at 37°C in a
5% CO2 humidified incubator for 1 h. Nonadherent cells were discarded from
the Petri dishes, and the remaining adherent cells (monocytes) were collected
by rinsing with cold HBSS and mechanical scraping. Adherent monocytes were
washed in HBSS and were divided into aliquots for tissue culture (resuspended
in RPMI 1640, supplemented with 10% FCS, 100 U/ml penicillin, and 100
␮g/ml streptomycin; Sigma Chemical), flow cytometric analysis, and real-time
PCRs. Cell viability, as determined by staining with acridine orange, was ⬎98%
after monocyte isolation. Cell suspension contained a median of 92.4% (range
90.7–95.1%) monocytes.
Flow cytometry
Cells were phenotypically analyzed by a direct, one-step, triple-labeling procedure. Briefly, 1 ⫻ 106 cells were labeled with antibodies for multicolor
flow cytometry using FITC-, PE-, PerCP-, or biotin (along with steptavidinPerCP)-conjugated mAb directed against CD3 (UCHT1), CD4 (13B8.2),
CD5 (BL1a), CD8 (B9.11), CD14 (RM052), CD16 (3G8), CD19 (J4.119),
CD25 (B1.49.9), CD38 (LS198-4-3), CD45 (J.33), CD45RA (ALB11),
CD45RO (UCHL1), CD56 (N901), ␣/␤-TCR (WT31), ␥/␦-TRC (11F2),
HLA-DR (Immu357), and 7-amino-actinomycin D. For intracellular IL-4
(4D9) and IFN-␥ (45.15) staining, cells were stimulated with 0.01 ␮g/ml
PMA and 0.5 ␮g/ml ionomycin in the presence of 5 ␮g/ml brefeldin A.
After 4 h, cells were fixed with 2% paraformaldehyde for 15 min and permeabilized with 0.1% Nonidet P-40 for 4 min before intracellular staining.
All antibodies were obtained from Beckman Coulter (Krefeld, Germany)
and Becton Dickinson (Heidelberg, Germany). Nonspecific binding was
corrected with isotype-matched controls. Flow cytometric data were acquired using a four-color Epics XL AF 14075 flow cytometer with Expo 32
Advanced Digital Compensation software (Beckman Coulter).
Cytokine ELISA
Quantification of cytokines in serum or culture supernatants was measured by
sandwich ELISA using the BD OptEIA kit (BD Biosciences, Heidelberg, Germany), according to the manufacturer’s instructions. The mAb pairs used were
as follows, listed as capture-biotinylated detection mAb: IL-2, IL-4, IL-8, IL-10,
IL-12, TNF-␣, IFN-␥, and TGF-␤. The intra- and interassay coefficients of variation were ⬍5%, and these reagents are sufficiently sensitive to measure concentrations of at least 5 pg/ml for the above-mentioned cytokines.
RNA purification
RNA was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany),
according to the manufacturer’s instructions.
Real-time RT-PCR
The expression for G0S2 was analyzed by a sensitive, real-time RT-PCR assay published by Zandbergen et al. [25]. The expression for FOXP3,
CLTA-4, and GITR was quantified by real-time RT PCR with a detection
system (LightCycler) using hybridization probes and primers published by
Miura et al. [26]. The expression of IL-17 and IL-27 was also measured
with a LightCycler device. For IL-17A-RT-PCR, we used the following primers and hybridization probes: primers 5⬘-AAC gTg gAC TAC CACA-3⬘ and
5⬘-ggg TCg gCT CTC CATA-3⬘; hybridization probes 5⬘-CCA ACT CCT
TCC ggC Tgg-x (x⫽3⬘Fluoescin) and 5⬘-LC-Red 640-AAg ATA CTg gTG
TCC gTg gg-p (p⫽3⬘Phosphat). For IL-27-RT-PCR, we used the following
primers and hybridization probes: primers 5⬘-gCT CgT CTT ATC TCg gg-3⬘
and 5⬘-CAg TTA CTg ggT AgA gCC-3⬘; hybridization probes 5⬘- CCA CCC
TTT AgA ACT TTA ggA CTg g-x and 5⬘-LC-Red 640-TCT Tgg CAT CAg
ggC AgC-p. The PCR reaction was performed in a volume of 10 ␮L containing 50 mmol/L KCl, 10 mmol/L "Tris", pH 8.3, 0.25 ␮l BSA (20 mg/
ml), 1 mmol/L each dNTP, 20 pmol each primer, and 15 pm each probe,
2.5 U Sigma Taq DNA polymerase (Sigma Chemical), 3.0 mmol/L MgCl2,
and 100 ng blood DNA template. To control if mRNA amplification was
successfully done, a coamplification of the housekeeping gene GAPDH (or
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G6PDH) was performed with primers for GAPDH (G6PDH), as published
previously [27]. Real-time PCR conditions were as following: DNA was denatured for 2 min at 95°C, followed by 45 cycles at 95°C (3 s), 55°C (10 s),
and 72°C (25 s). After the last cycle, samples were cooled to 4°C. The fluorescence-labeled hybridization probes were purchased from TIB MOLBIOL
(Berlin, Germany) and primers from Invitrogen. Quantification was performed by normalizing the value of the target to the housekeeping genes.
Statistics
Values are presented as mean ⫾ sem. Differences in data between groups
were tested by two-tailed unpaired t test or Mann-Whitney U-test using Statistical Package for the Social Science software, Version 14 (SPSS, Chicago,
IL, USA). The differences were considered to be significant at P ⬍ 0.05.
For in vivo data regarding skin-transplant survival, mice were randomly assigned to the treatment groups. The log rank test was used to compare survival curves between groups.
RESULTS
Application of hCG increased cell counts of maternal
peripheral blood
Blood samples from all 34 women were examined at Cycle
Days 8 –9 (i.e., before first application of hCG), 35–36 h after
the first application of hCG (i.e., at follicle aspiration), and
another 48 h after the second hCG application (i.e., at embryo
transfer). We first evaluated the number of leukocytes and
their subsets (monocytes, granulocytes, and lymphocytes) before and after each hCG application by flow cytometry. After
two applications of hCG in women undergoing IVF, total leukocyte counts were increased significantly up to 24.5%
(P⬍0.002) from 7550/␮l ⫾ 2097 to 9402/␮l ⫾ 2755, as well as
granulocyte counts from 5140/␮l ⫾ 1700 to 6616/␮l ⫾ 2204
and monocyte counts from 447/␮l ⫾ 207 to 601/␮l ⫾ 214
(28.7%, P⬍0.003, and 34.4%, P⬍0.004, respectively). However,
lymphocyte counts increased only slightly from 1963/␮l ⫾ 755
to 2179/␮l ⫾ 823 (11% at mean; NS). Next, we evaluated leukocytes and their subsets in a small group of women (n⫽4)
after successful IVF. Interestingly, we observed no significant
difference in leukocyte counts (8450/␮l⫾1353), granulocyte
counts (5898/␮l⫾578), monocyte counts (430/␮l⫾300), and
lymphocyte counts (2115/␮l⫾748) at the median of the 41
cycle days comparing the subsets before and after each hCG
application, as shown in Table 1.
Influence of hCG on maternal cellular immunity
As expected, median levels of B- and T-lymphocyte subsets in
peripheral blood showed no clear trend toward suppression or
enhancement of maternal systemic immune function during
hCG application. There was no difference in CD3⫹ subsets,
CD19⫹ subsets, and CD3–/CD8⫹ cells between the two hCG
application groups and before hCG treatment was started. We
found that the number of CD3⫹/CD16/56⫹ NKT cells was
increased significantly from 69/␮l ⫾ 45 to 89/␮l ⫾ 72, as well
as the number of CD3–/CD16/56⫹ NK cells from 873/␮l ⫾
492 to 1182/␮l ⫾ 795 after hCG application (29%, P⬍0.05,
and 35.4%, P⬍0.05, respectively). The percentage of CD25⫹
cells increased after the first hCG application from 1.7% ⫾ 1.6
to 2.1% ⫾ 1.5, possibly as a result of shifts in the different
CD25⫹ subsets. The median cell number of CD25⫹ cells inVolume 90, November 2011
Journal of Leukocyte Biology 3
TABLE 1. Application of hCG Increased the Counts of the Maternal Peripheral Blood Cells
Before hCG (cycle days ⬎8)
1. hCG application
2. hCG application
IVF pregnant, (cycle days median 41)
Normal pregnant, 1. trimester
Leukocytes (/␮l)
Granulocytes (/␮l)
Monocytes (/␮l)
Lymphocytes (/␮l)
7550 ⫾ 2097a,b
8118 ⫾ 2595
9403 ⫾ 2755a
8450 ⫾ 1353
9322 ⫾ 757b
5140 ⫾ 1700c,d
5713 ⫾ 2107
6616 ⫾ 2204c
5898 ⫾ 578
6463 ⫾ 733d
447 ⫾ 207e–h
480 ⫾ 169f
601 ⫾ 214e
430 ⫾ 300
792 ⫾ 172g,h
1963 ⫾ 755
1912 ⫾ 687
2179 ⫾ 823
2115 ⫾ 748
2065 ⫾ 614
Two applications of hCG in women undergoing IVF increased total leukocyte counts significantly (aP⬍0.002) by up to 25%, as well as granulocyte
counts (29%; cP⬍0.003) and monocyte counts (34%; eP⬍0.004). Lymphocyte counts increased only slightly (11% at mean; n.s.). Only for monocytes did
we notice a significant increase up to 7% (fP⬍0.01) after the first hCG application. Comparing leukocytes and their subsets before and after each hCG
application with IVF-induced pregnant women, we noticed no significant differences between the groups. As expected, the leukocyte, granulocyte, and
monocyte counts showed significant differences between normal pregnant women in 1st trimester and women before hCG application and after the first
hCG application: leukocytes bP ⬍ 0.03, granulocytes dP ⬍ 0.04, and monocytes gP ⬍ 0.0001 and hP ⬍ 0.002.
creased from 139/␮l ⫾ 146 to 164/␮l ⫾ 129 after early hCG
treatment (increased to 18%) and held a steady state at 175/
␮l ⫾ 180 after the second hCG application (increased to
25.9%, P⬍0.08), as shown in Fig. 1.
Influence of hCG on the maternal adaptive immune
system
To assess whether hCG treatment for IVF affects the feto-maternal tolerance, we studied the CD3⫹ and CD4⫹ subsets. We
noticed that hCG treatment resulted in a more-pronounced T
cell response toward Th2 differentiation, as illustrated by a
shift of CD3⫹/␥-IFN⫹ and CD3⫹/IL-4⫹ subsets. The amount
of CD3⫹/IL-4⫹ cells increased significantly from 29/␮l ⫾ 8 to
60/␮l ⫾ 16 (207% induction; P⬍0.036), and the number of
CD3⫹/␥-IFN⫹ cells decreased moderately from 178/␮l ⫾ 57
to 160/␮l ⫾ 55 (10% reduction; NS). The CD3⫹/CD4⫹ subsets showed a similar shift toward Th2 cells (CD4⫹/IL-4⫹)
from 31/␮l ⫾ 8 to 56/␮l ⫾ 16 (181% induction; P⬍0.045)
and a decrease of Th1 cells (CD4⫹/␥-IFN⫹) from 136/␮l ⫾
32 to 114/␮l ⫾ 35 (16% reduction; NS), as shown in Fig. 2.
Next, we evaluated T cell subsets responsible for maintaining peripheral tolerance. CD3⫹CD4⫹ CD16/56⫹bright cells
were moderately elevated from 10.1/␮l ⫾ 6.2 to 14.3/␮l ⫾ 7.4
(P⬍0.048) after the second hCG application. We noticed that
the number of Tregs (CD4⫹CD25⫹) increased significantly
from 22.0/␮l ⫾ 18.0 to 27.0/␮l ⫾ 16.0 after the first hCG application and reached to 32.0/␮l ⫾ 20.0, demonstrating an
induction of up to 45% after the second hCG application
(P⬍0.04). Further analyses showed that other subpopulations
such as Th3 cells, CD4⫹CD45RO⫹, CD4⫹CD45RA⫹, and the
CD8⫹CD25⫹ cells were not affected by hCG. The increase of
Tregs in the peripheral blood after hCG application prompted
us to measure the expression of FOXP3 by real-time RT-PCR.
Concordantly, with the increased Tregs, the CD4⫹CD25⫹/
FOXP3 Tregs also increased significantly from 6.6/␮l ⫾ 4.2 to
13.6.0/␮l ⫾ 6.4 after the first hCG application and increased
to 16.0/␮l ⫾ 7.2, indicating up to a 2.5-fold increase after the
second hCG application (P⬍0.05), as shown in Fig. 3.
Comparison of maternal immune system of normal
pregnant women and women who underwent IVF
treatment
As expected, the mean number of leukocytes, granulocytes,
monocytes, lymphocytes, and many lymphocyte subsets in peripheral blood showed no significant difference between
women after the second hCG application and women with
2000
1750
$ = p<0.03 ; & = p<0.05
$
1500
&
Figure 1. Two applications of hCG in women undergoing IVF significantly increased the amount of
CD3ⴙCD16/56ⴙ (P<0.05) and CD3–CD16/56ⴙ cells
(P<0.03). Counts of CD25⫹ cells increased only slightly
(26% at mean; NS).
Ce
ell counts/µll
1250
1000
750
500
&
250
&
0
3+
4+
8+
6+
R+
O+
A+
56 +
CD
6/5
CD
CD
AD
45R
16/
45R
D1
3+ /
3+ /
/HL
C
CD
CD
CD
9
/
/
/
/
1
CD
CD
+
+
+
3
3
3
3
CD
CD
CD
CD
CD
= before HCG
4 Journal of Leukocyte Biology
Volume 90, November 2011
= after 1. HCG
25+
25+
CD
CD
4+/
D
C
= after 2. HCG application
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Koldehoff et al. hCG modulating maturation and function of hematopoietic cells
200
& = p<0.05
175
Cell countts/µl
150
125
100
Figure 2. Polarization of the Th1/Th2 balance after
hCG treatment. The number of Th1 cells (CD4⫹IFN␥⫹) did not change significantly, whereas the number
of Th2 cells (CD4⫹IL-4⫹) increased significantly
(P⬍0.048).
&
75
50
25
0
CD4+/γ-IFN+
= before HCG
CD4+/IL-4+
= after 1. HCG
= after 2. HCG application
normal pregnancy in the first trimester (see Table 1). Surprisingly, we detected a significant difference in the cell subsets of
␥/␦-T cells (55/␮l⫾58 vs. 28/␮l⫾10; P⬍0.04), CD3–/CD8⫹
(110/␮l⫾97 vs. 56/␮l⫾22; P⬍0.02), CD3–/CD16/56⫹ (1182/
␮l⫾795 vs. 279/␮l⫾63; P⬍0.03), and CD4⫹/CD25⫹ (28/
␮l⫾24 vs. 45/␮l⫾12; P⬍0.03) for women after IVF and
women with normal pregnancy. Comparing cell subsets of
pregnant women after IVF and normal pregnant women only
indicated a difference in the cell levels of ␥/␦-T cells (9/␮l⫾8
vs. 28/␮l⫾10; P⬍0.02), CD25⫹ cells (134/␮l⫾32 vs. 267/
␮l⫾90; P⬍0.03), and CD4⫹/CD25⫹ cells (28/␮l⫾13 vs. 45/
␮l⫾12; P⬍0.03). Among immune cell types, Tregs and other
specialized immune subsets play an important role in protecting
the fetus by dampening a harmful inflammatory immune response at the maternal-fetal interface. In addition, these data
show in humans that the number of Tregs increases early in normal pregnancy, and the lower number of Tregs observed after
hCG stimulation may contribute to impaired immune tolerance
at the maternal-fetal interface after IVF-induced pregnancy.
Variant gene expression measured by real-time RTPCR in hematopoietic cells or monocytes of women
who underwent hCG treatment
In preliminary investigations of molecular effects of hCG, we
analyzed blood cells from pregnant and nonpregnant women
by oligonucleotide microarray technology, and after rigorous
statistical analysis, relevant genes were chosen for confirmation
by RT-PCR, including the G0S2 and signaling cascade of immune cells (data not shown). We found a marked decrease of
G0S2 expression by real-time RT-PCR in MNCs of women who
underwent IVF treatment, measured each time after both hCG
applications, from 358 ⫾ 922% to 65 ⫾ 68% G0S2/GAPDH
expression (82% reduction; P⬍0.045). To verify the findings
described above, we further studied the effect of hCG stimula-
60
& = p<0.05
&
Cell counts
s/µl
50
Figure 3. hCG administration significantly increased
the number of Tregs (CD4ⴙCD25ⴙ) by up to 45%
(P<0.05) after the first and second hCG application,
respectively. We found a twofold increase of
CD4⫹CD25⫹FOXP3⫹ cells after hCG application
(P⬍0.05), whereas at the CD4⫹/TGF-␤⫹ cells did not
increase significantly after the first hCG application
and remained at this level. Remarkably, the number of
CD3⫹CD4⫹CD16/56bright⫹ cells increased after the
second hCG application, whereas the number of
⫹
CD8 CD25⫹ cells was not affected by hCG expression.
40
30
&
&
20
10
0
CD4+/CD25+
= before HCG
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CD8+/CD25+
CD4+/CD25+/FOXP3+
= after 1. HCG
CD4+/TGF-β+
CD3+/CD4+/CD16/56bright+
= after 2. HCG application
Volume 90, November 2011
Journal of Leukocyte Biology 5
TABLE 2. Variant Gene Expression Measured by Real-Time RT-PCR in MNCs of Women Who Underwent hCG Treatment
Gene expression
Before hCG
1. hCG application
2. hCG application
G0S2
IL-17
IL-27
FOXP3
CTLA-4
GITR
IDO
TGF-␤
358 ⫾ 922a
486 ⫾ 238b
368 ⫾ 260c
151 ⫾ 39d,e
212 ⫾ 175f
424 ⫾ 307
14 ⫾ 19
23 ⫾ 8
223 ⫾ 461
350 ⫾ 255
497 ⫾ 249
464 ⫾ 367d
648 ⫾ 775f
401 ⫾ 738
35 ⫾ 31
27 ⫾ 8
65 ⫾ 68a
227 ⫾ 226b
546 ⫾ 384c
533 ⫾ 198e
364 ⫾ 352
277 ⫾ 172
28 ⫾ 25
27 ⫾ 7
P values: aP ⬍ 0.05; bP ⬍ 0.03; cP ⬍ 0.04; dP ⬍ 0.03; eP ⬍ 0.02; fP ⬍ 0.05. G0S2, IL-17, IL-27, FOXP3, CTLA-4, GITR, IDO, and TGF-␤ expression in MNCs of women who received hCG for IVF. Gene expression was detected by real-time RT-PCR and normalized to GAPDH expression.
Controls were set to 100%.
tion on MNCs or monocytes in vitro. In these cells, we confirmed the reduction of G0S2 expression after hCG administration, as seen in Table 2. Next, we measured IL-17 and IL-27
expression by real-time RT-PCR in MNCs of women who received
hCG, and we found that IL-17 expression dropped continuously
from 486 ⫾ 239% to 277 ⫾ 238% IL-17/GAPDH expression
(43% reduction; P⬍0.03) after both hCG applications, whereas
IL-27 expression increased at the same time from 368 ⫾ 260% to
546 ⫾ 384% IL-27/GAPDH expression (48% induction; P⬍0.04).
The FOXP3 gene expression in MNCs of women who received
hCG showed a threefold induction to 464 ⫾ 367% (P⬍0.03) after the first hCG application and increased to 533 ⫾ 198%
(350% induction; P⬍0.02) of the spontaneous level, as shown in
Table 2. Taken together, these data demonstrated that hCG induces FOXP3 and IL-27 expression and at the same time, reduces G0S2 and IL-17 expression in MNCs.
The increase of Tregs in the peripheral blood prompted us
to investigate the influence of CTLA-4 and GITR in MNCs by
real-time RT-PCR. We found a threefold increased CTLA-4 expression after the first hCG application (P⬍0.05), and remark-
ably, CTLA-4 levels decreased after the second hCG application. GITR expression was not altered by the first hCG application and decreased after the second hCG application by 35%.
IDO expression distribution showed a similar trend as GITR
expression after both hCG applications. TGF-␤ was not affected by hCG. IDO and CTLA expression and increased numbers of Tregs are often associated with the induction of tolerance in the setting of autoimmunity, as well as alloimmunity.
Our data suggest significant differences between women undergoing IVF and normal, fertile women.
Influence of hCG on serum cytokine levels
We investigated serum levels of various cytokines in women
before and after each hCG application by ELISA. We found
that two applications of hCG increased serum levels of IL-8
and IL-10 significantly (P⬍0.02 and P⬍0.04, respectively),
whereas serum levels for IFN-␥, Il-1␤, IL-2, IL-4, IL-6, TNF-␣,
and TGF-␤1 (see Fig. 4) were not altered significantly by hCG.
IL-8 is secreted by several cell types as chemoattractant and
potent angiogenic factor. IL-10 is a Th2 cytokine associated
80
# = p<0.02
& = p<0.04
Figure 4. We found that two applications of hCG significantly increased IL-8 (P<0.02) and IL-10 serum levels (P<0.04) measured by ELISA, whereas the serum
levels for IFN-␥, IL-1␤, IL-2, IL-4, IL-6, TNF-␣, and
TGF-␤1 were not changed significantly by hCG. A nonsignificant induction was observed for IL-12.
Protein concentration (pg/ml)
70
&
#
60
50
40
&
30
20
10
0
IFN-γγ
IL-1ββ
IL-2
= before HCG
6 Journal of Leukocyte Biology
Volume 90, November 2011
IL-4
IL-6
IL-8
= after 1. HCG
IL-10
IL-12
TNF-ά TGF-β1
β
= after 2. HCG application
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Koldehoff et al. hCG modulating maturation and function of hematopoietic cells
with an immune-modulating response. Levels of IL-12, a cytokine responsible for Th1 differentiation, increased slightly in
response to hCG treatment.
Skin transplants after hCG applications in female,
nonpregnant mice
To further evaluate the immune modulation-inducing effects
of hCG, we performed skin transplantations in nonpregnant
mice. Syngeneic (BALB/C3 BALB/C; n⫽2) transplants served
as control for the skin graft technique. As shown in Fig. 5, not
all transplants were rejected during the entire observation period of 30 days. In allogeneic (BALB/C3 C57BL/6; n⫽20)
transplants, rejection times of 7–23 days were observed. In our
experiments, the median graft survival time of 16 days was prolonged in mice treated with hCG (n⫽10) compared with controls treated with placebo (n⫽10), in which median graft survival was 10 days (P⬍0.001). In summary, these data show that
hCG i.p. induced prolonged survival of skin grafts in female,
nonpregnant mice.
To correlate the allogeneic graft results with the IVF findings, changes of G0S2 were assessed by real-time PCR analysis
in nonpregnant C57BL/6 mice treated in a dose-dependent
manner with or without hCG for 1 week (every other day,
threefold). Comparing spleen MNCs of C57BL/6 mice without
hCG (control set to 100%) and with MNCs treated with different hCG doses (10 IE hCG/mouse, 20 IE hCG/mouse, up to
50 IU hCG/mouse), we found a significant reduction in
G0S2/GAPDH expression from 100 ⫾ 29% (control), to
54.5 ⫾ 9% (10 IE hCG/mouse; P⬍0.02), to 31.5 ⫾ 12% (20
IE hCG/mouse; P⬍0.005), and to 15.4 ⫾ 15% (50 IE hCG/
mouse; P⬍0.002), respectively. Taken together, these data
show that hCG inhibits the G0S2 gene in mouse MNCs and in
MNCs of women who underwent IVF treatment.
DISCUSSION
Successful pregnancy depends on a complex interplay between
the immune and the endocrine system to tightly regulate immune responses at the feto-maternal interface [3, 28]. The pregnancy hormone hCG has been shown to be indispensable for the
establishment of a successful pregnancy and has been described
to have immunoregulatory properties supporting the implantation process of the fetus in the maternal endometrium [10, 29].
Treatment of peripheral blood MNCs with hCG and their subsequent re-infusion 2 days after oocyte retrieval have shown to increase the implantation rates of blastocytes in women suffering
from repeated IVF failure [30]. We first performed a descriptive
evaluation, in which we analyzed the effects of hCG on maternal
blood cells of women who received hCG as preconditioning for
IVF. Our data confirm increased cell recruitment in these
women, particularly involving increased granulocytes and monocytes but without significant changes in lymphocyte subpopulations. Of note, effects at the feto-maternal interface were hCG
dose-dependent. hCG is considered to transmit its hormonal signal to target cells through the LH/hCG receptor or distinct variants by partial degradation of the hCG molecule interacting with
TGF-␤Rs [31]. Differential expression of carbohydrates needed
for glycosylation is associated with inhibition of E-selectin-mediated homing of leukocytes and may contribute to various physiological processes, such as cell– cell adhesion [32]. In addition to
this role, hCG has been implicated as an intracrine, autocrine,
paracrine, and endocrine regulator of human feto-placental function and as a regulator in various nongonadal tissues [7]. Further
global gene expression analyses indicated that multiple cell signaling regulators and transcription factors were up-regulated in
response to hCG, especially Gadd45, Egr1, Nr4a1, and other
genes [33]. Gadd45 proteins modulate signaling in response to
Figure 5. Syngeneic (BALB/C3 BALB/C; nⴝ2) and
allogeneic (BALB/C3 C57BL/6; nⴝ20) skin transplants were transferred onto the back of female recipients. C56BL/6 mice treated with repeated hCG i.p.
tolerated their grafts longer than controls, which were
treated with repeated placebo i.p. The median graft
survival time was prolonged to 16 days in mice treated
with hCG (n⫽10) compared with controls treated with
placebo (n⫽10), in which it was 10 days (P⬍0.001).
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Volume 90, November 2011
Journal of Leukocyte Biology 7
physiological and environmental stressors, involving activation of
the Gadd45a-p38-NF-␬B survival pathway in myeloid cells. Egr1 is
involved in homeostasis of HSCs by coordinating proliferation
and migration. The Nr4a transcription factors transduce diverse
extracellular signals into altered gene transcription to coordinate
apoptosis, proliferation, cell cycle arrest, and inflammatory cytokines [34]. In addition, more findings suggested that an immuneendocrine network involving hCG and blood immune cells exists
and plays an important role in early pregnancy. Kosaka and coworkers [9] demonstrated that peripheral blood monocytes are
able to respond to hCG at high concentrations by enhancing
their production of IL-8. This study also showed that system(s)
different from the LH/hCG receptor system are responsible for
this effect of hCG on immune cells [9]. Recently, novel results
suggested that cell surface lectins have the ability to recognize carbohydrate moieties in glycosylated proteins and that
hCG-carbohydrate-side-chain recognition by the mannose
receptor (CD 206) is fundamental in propagating noncanonical hCG signaling [35].
During normal pregnancy, the decidua is populated by a variety of leukocytes and macrophages that constitutes 20 –30% of
the decidual cells at the site of implantation. Macrophages are
believed to protect the embryo against infection and to play an
important role in maternal tolerance and maintenance of pregnancy [36]. The systemic responses are characterized by leukocytosis, increased monocyte priming, increased phagocytic activity,
and production of proinflammatory cytokines. Animal studies
have shown that systemic or circulating cells can influence implantation. Transfer of spleen cells from pregnant mice to pseudopregnant mice receiving donated embryos enhances implantation rates [37]. In addition, we found that hCG application leads
to increased cell recruitment of CD3⫹/CD16/56⫹ NKT cells and
CD3–/CD16/56⫹ NK cells without significant modulation of distinct, mature T or B cells. NK cells are the dominant lymphocyte
population in the decidua during pregnancy and are reported to
mediate a delicate balance between placental trophoblast invasion and sufficient access to maternal blood flow. Another cell
that requires regulation to protect the fetus is the NKT cell.
These cells are present in large numbers at the maternal-fetal
interface during pregnancy [38, 39]. NKT cells express the NK
receptors NK1.1 or NKR-P1A, as well as the TCR, and have important regulatory functions in pregnancy [40]. Activation of NK
cells is tightly regulated by a set of activating and inhibitory receptors on the cell surface. These receptors interact with HLA
molecules expressed on the invading trophoblast and thereby
affect cytokine production and cytolytic activity of maternal uterine NK cells [41]. During the menstrual cycle, as well as through
pregnancy, the leukocyte numbers vary, suggesting a sex steroidmediated mechanism. Lukassen et al. [42] showed that hormonal
stimulation for IVF treatment positively affects the CD56bright/
CD56dim ratio of endometrium during the window of implantation by a relative decrease in the number of cytotoxic
CD56dimCD16⫹ NK cells.
Concerning specific maternal T or B cells, we determined the
influence of hCG in the IVF setting on the modulation of these
cell patterns. The transient tolerance during gestation is at least
partially achieved via the presence of immunoregulatory properties. Tregs were described to play an important role in the main8 Journal of Leukocyte Biology
Volume 90, November 2011
tenance of the tolerant state during pregnancy and to allow the
acceptance of allografts [43]. Interestingly, we found an increased recruitment of CD3⫹/IL-4⫹ and CD3⫹/CD4⫹/IL-4⫹
Th2 cells after hCG application. IFN-␥-secreting Th1 subsets were
reduced slightly. hCG treatment resulted in shifts of the Th1/
Th2 balance in a dose-dependent manner. In humans, Th1 activity is required at several stages of pregnancy, in particular, during
the early implantation period. In addition, Th1 environment
stimulates the production of the Th2 cytokines [44]. More evidence is provided by our finding of significantly increased recruitment of Tregs after the hCG application. CD4⫹/CD25⫹ cells
increased ⬃1.5-fold, and CD4⫹/CD25⫹/FOXP3⫹ cells increased
2.5-fold after hCG application. Tregs may play a role in implantation and are essential for the establishment of peripheral tolerance by suppressing (auto-) reactive T cells [15]. Recent evidence
indicates that DCs are capable of expanding the Treg population
and control T cell maturation and phenotype switching in general. Treg levels are highest in the peripheral blood during the
first trimester of pregnancy [45, 46]. Our study provides evidence
that during IVF or pregnancy, abundantly expressed hCG has
remarkable immune-regulating features that contribute to immune-tolerance induction at the feto-maternal interface. Furthermore, we demonstrate increased IL-27 mRNA expression and
simultaneously decreased IL-17 mRNA expression in MNCs of
women receiving hCG prior to a scheduled IVF. IL-27 is a heterodimeric cytokine consisting of EBI3 and p28, which along with
IL-12, IL-23, and IL-35, belongs to the IL-12 cytokine family. The
main sources of IL-27 appear to be activated APCs. IL-27 signals
via its heterodimeric receptor (IL-27R), which consists of the receptor subunits gp130 and WSX-1 [47]. The two IL-27R subunits
are expressed by a variety of immune cells, including T cells, NK
cells, mast cells, B cells, and activated DCs. Consistent with the
idea that the innate immune system regulates many aspects of
the adaptive immune system, EBI3 expression can be up-regulated by pathogen- and host-derived inflammatory stimuli, including LPS, CD40 ligation, or exposure to inflammatory cytokines
[48]. Although IL-27 is one of the most potent inhibitors of Th17
differentiation, little is known about how IL-27 regulates committed Th17 cells. IL-27 was also found to suppress induction of a
subset of Tregs (induced Tregs), which has been differentiated
and expanded upon stimulation with TGF-␤. Moreover, we show
that hCG induces a transient increase of Tregs in vivo and an
increase of CTLA-4 expression. In addition, we noticed that the
expression of IDO was increased simultaneously after the first
hCG application, as seen in Table 2. There is a link between IDO
and Tregs, whereas CD4⫹CD25⫹ T cells induce tryptophan catabolism by DCs and a tolerogenic phenotype in a CTLA-4-dependent manner [49, 50]. Similarly, expression of IDO by DCs and
macrophages in the maternal decidua is up-regulated via CTLA-4,
and this has been suggested to indicate that CTLA-4-expressing
CD4⫹CD25⫹ Tregs, which infiltrate the decidua in early pregnancy, induce IDO expression by decidual DCs/macrophages
expressing the appropriate counter-ligand (B7 family members
CD80 and CD86) [51]. Support for our hypothesis of immunemodulating effects of hCG was seen in the increase of anti-inflammatory cytokine IL-10 serum levels in women after hCG application, which is associated with tolerance induction in the allogeneic transplant setting [52]. In accordance with this, we observed
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Koldehoff et al. hCG modulating maturation and function of hematopoietic cells
no significant changes in proinflammatory cytokine serum levels
of IL-1B, IL-2, and TNF in women receiving hCG. Only IL-8 expression was markedly increased after hCG application, as previously reported elsewhere. IL-8 is a chemokine that serves as a
chemical signal, which attracts neutrophils to the site of inflammation and is therefore also known as the neutrophil chemotactic factor. Recently, De Oliveira et al. [53] implied that IL-8, produced by uterine NK cells, regulated the extravillous trophoblast
cell invasion. Many studies conducted in murine and human
models have established that a correct balance of cytokines
at the maternal-fetal interface is an essential requirement
for proper placental development and therefore, reproductive success [54, 55].
Another indication for induction of tolerance by hCG is the
inhibition of the G0S2 in monocytes of our studied women after
hCG application. G0S2 expression is required to commit cells to
enter the G1 phase of the cell cycle and may therefore be necessary for lymphocyte proliferation. The G0S2 gene has a NF of
activated T cells binding site in the 5⬘ flank and encodes for a
small, basic, potential phosphoprotein of unknown function. Expression of G0S2 mRNA is increased in response to Con A or to
the combination of TPA and the calcium ionophore, ionomycin,
but inhibited by CsA, a potent and widely used immunosuppressive agent. Early inhibition of G0S2 expression by CsA may be
important in achieving immunosuppression [56]. Our study demonstrates a marked decrease of G0S2 by hCG, suggesting that
hCG might be a naturally occurring, immunosuppressive agent in
pregnancy. G0S2 was recently found to be markedly increased in
serum of patients with rheumatoid arthritis, indicating that G0S2
gene expression might have proinflammatory features in this autoimmune disease [57]. In addition, we assessed the possible immunomodulating features of hCG in a nonpregnancy skin-transplant model in female mice. Indeed, we found that skin graft
rejection was delayed significantly by i.p. hCG treatment, suggesting that hCG induces immunosuppression. Also, in our mice experiments with dose-dependent hCG application, we found a significant reduction of the G0S2 gene expression by very high
hCG. Confirming our hypothesis, skin graft rejection was not prevented for an unlimited period of time, as required in pregnancy,
suggesting that the immunosuppressive features of hCG alone
may not be sufficient to prevent rejection of the fetus. Its immunosuppressive potential is probably not as strong as that of other
immunosuppressive agents such as CsA. Importantly, during pregnancy, an anatomic barrier exists at the maternal-fetal interface,
which reduces cell traffic between maternal and fetal tissue in
both directions, contributing to the prevention of graft rejection
[1, 3]. The reduction of cell traffic by the placenta may therefore
result in a constellation similar to the T cell-depleted, allogeneic
transplantation setting. In this transplant setting, effective T cell
depletion (4 –5 log-fold) reduces the number of immune-competent T cells in the graft below the number of 5 ⫻ 104/kg/body
weight of the patient, which allows the omission of any immunosuppressive agents, as GvHD prophylaxis post-transplant, as in
pregnancy [58]. The number of maternal cells that pass the placenta and invade the fetal blood circulation remains below the
critical number of T cells in the graft in the allogeneic transplant
setting and is therefore too low to lead to an immune rejection
of the fetus.
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In conclusion, our results demonstrate immunosuppressive features of hCG, which may be of clinical interest. Although it is
well-known that in pregnancy, some autoimmune diseases, such
as Crohn’s disease, multiple sclerosis, or rheumatoid arthritis,
may improve, we do not know the underlying mechanisms mediating these improvements of autoimmune diseases. It has often
been speculated that some unknown substances might induce
tolerance. Our findings suggest a possible role for hCG in the
improvement of autoimmune diseases in pregnancy. hCG can be
used as a safe medication for IVF and many other applications.
Its use as an immunosuppressive agent may be of benefit in various areas of clinical immunology, including autoimmune diseases
and the solid organ or allogeneic transplant setting. More studies
about the possible immune tolerance-inducing effects of hCG are
necessary.
AUTHORSHIP
M.K. designed, performed, and analyzed research and wrote the
manuscript. N.C.W., N.K.S., D.P., S.W., and P.B. contributed patient samples and analyzed data. T.K., D.W.B., and A.H.E. participated in coordination of the study and funded the study.
ACKNOWLEDGMENTS
This work was supported by a grant from the Kulturstiftung
Essen (Germany). The authors thank Silke Gottwald, Melanie
Kroll, and Christiana Schary for their excellent technical performance of the PCR analyses, ELISA analyses, and mice skin
transplantations. Special thanks also go to Martina Franke and
Ursula Hill for the cytometry analyses.
DISCLOSURE
All authors had no financial support to disclose.
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KEY WORDS:
hCG 䡠 immune tolerance 䡠 pregnancy 䡠 transplant
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