Evidence for a Selective Migration of Fetus-Specific
CD4?CD25brightRegulatory T Cells from the Peripheral
Blood to the Decidua in Human Pregnancy
Tamara Tilburgs,1*†Dave L. Roelen,* Barbara J. van der Mast,†Godelieve M. de Groot-Swings,†
Carin Kleijburg,†Sicco A. Scherjon,†and Frans H. Claas*
During pregnancy, the maternal immune system has to tolerate the persistence of fetal alloantigens. Many mechanisms contribute
to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic
fetus. Murine studies suggest that CD4?CD25?T cells provide mechanisms of specific immune tolerance to fetal alloantigens
during pregnancy. Previous studies by our group demonstrate that a significantly higher percentage of activated T cells and
CD4?CD25brightT cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy. In this
study, we examined the phenotypic and functional properties of CD4?CD25brightT cells derived from maternal peripheral blood
and decidual tissue. Depletion of CD4?CD25brightT cells from maternal peripheral blood demonstrates regulation to third party
umbilical cord blood cells comparable to nonpregnant controls, whereas the suppressive capacity to umbilical cord blood cells of
her own child is absent. Furthermore, maternal peripheral blood shows a reduced percentage of CD4?CD25brightFOXP3?and
CD4?CD25brightHLA-DR?cells compared with peripheral blood of nonpregnant controls. In contrast, decidual lymphocyte
isolates contain high percentages of CD4?CD25brightT cells with a regulatory phenotype that is able to down-regulate fetus-
specific and fetus-nonspecific immune responses. These data suggest a preferential recruitment of fetus-specific regulatory T cells
from maternal peripheral blood to the fetal-maternal interface, where they may contribute to the local regulation of fetus-specific
responses. The Journal of Immunology, 2008, 180: 5737–5745.
blasts play a crucial role in circumventing a destructive maternal
immune response in different ways. Fetal tissue can inhibit allo-
geneic immune responses by expressing IDO (that inhibits rapid
proliferation of cells) (1, 2), FAS ligand (that can cause apoptosis
of activated cells that express FAS) (3), and complement inhibitory
proteins to prevent complement activation (4). These mechanisms
can inhibit immune responses at the fetal-maternal interface in an
Ag-nonspecific manner (5).
Trophoblasts do not express the classical HLA-A, HLA-B,
HLA-DR, HLA-DQ, and HLA-DP molecules that are the main
targets for alloreactive T cells in transplantation. However, tro-
phoblasts do express HLA-C, HLA-E, and HLA-G molecules by
which they can avoid NK cell-mediated cytotoxicity. HLA-G-ex-
pressing cells have been shown to induce regulatory T (Treg)2
cells (6). In contrast, the highly polymorphic HLA-C can induce
any mechanisms are suggested to be involved in ma-
ternal immune tolerance and immunologic acceptance
of the allogeneic fetus during pregnancy. Fetal tropho-
NK cell tolerance, but can also be a target for allogeneic T cells.
Data from bone marrow transplantation patients have shown that a
single HLA-C allele mismatch can elicit a cytotoxic T cell re-
sponse (7) and is associated with a lower patient survival. In ad-
dition, HLA-E can decrease NK and CTL cytotoxicity (8), but has
also been shown to exhibit alloantigenic properties that are indis-
tinguishable from classical MHC class I molecules (9). Neutral-
ization of possible CTLs with direct specificity for HLA-C,
HLA-E, or indirectly presented minor histocompatibility Ags
seems essential for the immunologic acceptance of the allogeneic
Maternal leukocytes present at the fetal-maternal interface in-
clude decidua-specific CD16?CD56brightNK cells and T cells,
whereas B cells are virtually absent. Decidual NK cells have been
shown to regulate trophoblast invasion by expression of NK cell
receptors and the secretion of cytokines (10). Incompatibility of
maternal killer Ig-like receptor (KIR) genotype and the fetal
HLA-C allotype leads to increased risk of pregnancy complica-
tions like pre-eclampsia (11), suggesting that NK cells play a role
in fetus-specific immune regulation.
Murine studies have shown that depletion of peripheral blood
CD4?CD25?cells leads to gestation failure in allogeneic, but not
in syngeneic pregnancy (12). These data suggest that T cells play
a role in specific immune tolerance to fetal alloantigens in murine
pregnancy. Recent studies have shown that high percentages of T
cells are present in decidual tissue in human term pregnancy and
that peripheral blood T cell subsets change during pregnancy (13–
16). In addition, a significantly higher percentage of CD4?
CD25brightT cells is present in decidual tissue compared with ma-
ternal PBL (mPBL) (13, 17). CD4?CD25?T cells are extensively
studied by many groups for their regulatory capacities. Expression
of CD25 is not exclusive for Treg cells. Effector T cells can also
*Department of Immunohematology and Blood Transfusion and†Department of Ob-
stetrics, Leiden University Medical Centre, Leiden, The Netherlands
Received for publication May 25, 2007. Accepted for publication February 6, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1Address correspondence and reprint requests to Dr. Tamara Tilburgs, Leiden Uni-
versity Medical Centre, Department of Immunohematology and Blood Transfusion
E3Q, PO Box 9600, 2300 RC Leiden, The Netherlands. E-mail address:
2Abbreviations used in this paper: Treg, T regulatory; cPBL, control PBL; d.basalis,
decidua basalis; d.parietalis, decidua parietalis; mPBL, maternal PBL; S.I., suppres-
sion index; UCB, umbilical cord blood.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
express high levels of CD25, whereas Treg cells can be found in
the CD25?or CD25dimfraction (18, 19). Additional markers
like CTLA-4 and FOXP3, and activation markers like HLA-DR
and CD69 can help to distinguish effector from regulatory cells.
However, conclusions based on phenotypic characterization re-
main controversial (20, 21). Until a specific marker for Treg
cellular CTLA-4, CD69, and HLA-DR expression in d.basalis (a) and d.parietalis (b) after term pregnancy. All plots are gated for CD3?CD4?T cells within
the lymphocyte gate. Percentage of FOXP3?(c), CTLA-4?(d), CD69?(e), and HLA-DR?(f) cells within CD4?CD25dimor CD4?CD25brightT cell
fraction of d.basalis and d.parietalis in early pregnancy (17–24 wk) and after term (?37 wk) pregnancy. Lines indicate median percentages; ?, p ? 0.05;
??, p ? 0.01.
Characteristics of decidual CD4?CD25dimand CD4?CD25brightT cells. Representative dot plots of CD25 and intracellular FOXP3, intra-
5738 MIGRATION OF FETUS-SPECIFIC CD4?CD25brightTreg CELLS
cells is found, functional tests are required to identify and study
The aim of this study is to analyze phenotypic and functional
properties of CD4?CD25brightT cells during pregnancy in tissue
isolates from decidua basalis (d.basalis), the maternal part of the
placenta at the implantation site connected to invading fetal tro-
phoblasts; the decidua parietalis (d.parietalis), the maternal part of
the membranes connected to the fetal trophoblasts of the chorion;
and mPBL samples.
Materials and Methods
Blood and tissue samples
Paired samples of d.basalis, d.parietalis, heparinized mPBL, and heparin-
ized umbilical cord blood (UCB) were obtained from healthy women after
uncomplicated term pregnancy (gestational age range: 37–42 wk). Tissue
samples were obtained after delivery by elective caesarean section or un-
complicated spontaneous vaginal delivery. Early pregnancy samples were
obtained from healthy women undergoing surgical termination of preg-
nancy for social reasons (gestational age range: 17–23 wk). From the early
decidua samples, in not all cases could paired d.basalis and d.parietalis be
obtained. mPBL samples were obtained either directly before or directly
after delivery or surgical curettage. UCB cells were obtained directly after
delivery from the umbilical cord veins. Control PBL (cPBL) samples were
obtained from healthy nonpregnant female volunteer donors (age range:
22–43 years). Tissue samples used for phenotypic analysis are partly sim-
ilar to those described previously (13). Signed informed consent was ob-
tained from all women, and the study received approval by the Leiden
University Medical Centre Ethics Committee (P02-200).
Lymphocyte isolation from decidua was done, as described previously
(13). Shortly, d.basalis was macroscopically dissected from the maternal
side of the placenta. D.parietalis was collected by removing the amnion and
delicately scraping the d.parietalis from the chorion. The obtained tissue
was washed thoroughly with PBS and thereafter finely minced between
two scalpel blades in PBS. Decidual fragments were incubated with 0.2%
collagenase I (Invitrogen Life Technologies) and 0.02% DNase I (Invitro-
gen Life Technologies) in RPMI 1640 medium, gently shaking in a wa-
terbath at 37°C for 60 min, and thereafter washed once with RPMI 1640.
The resultant suspensions were filtered through a 70-?m sieve (BD Dis-
covery Labware) and washed once in RPMI 1640. For phenotypic analysis,
the isolates were layered on Ficoll-Hypaque (Leiden University Medical
Centre pharmacy) for density gradient centrifugation (20 min/800 ? g).
PBL and UCB samples were directly layered on a Ficoll-Hypaque gradient.
Mononuclear cells were collected and washed twice with PBS containing
1% FCS, and all cells were fixed with 1% paraformaldehyde and stored at
4°C until cell staining and flow cytometric analysis. For functional anal-
ysis, the decidual lymphocyte isolates were layered on a Percoll gradient of
(7.5 ml 1.08 g/ml; 12.5 ml 1.053 g/ml; 20 ml 1.034 g/ml) for density
gradient centrifugation (30 min/800 ? g) to minimize contaminating cell
debris and nonlymphocyte cell types. Lymphocytes were isolated from the
1.08–1.053 g/ml interface. Comparison of the cell suspension obtained
after Ficoll gradient and Percoll gradient isolation did not show any sig-
nificant difference in composition of lymphocyte and T cell subsets (data
The following directly conjugated mouse anti-human mAb were used for
four-color immunofluorescence surface staining: CD45 allophycocyanin,
CD14 PE, CD25 PE, CD3 PerCP, CD4 allophycocyanin, CD69 FITC, and
HLA-DR FITC (BD Biosciences), and used in concentrations according to
manufacturer’s instructions. For intracellular expression of CTLA-4, cells
were stained for surface expression of CD3, CD4, and CD25; treated with
permeabilizing solution buffer (containing: 0.1% saponine, 5% FCS, and
0.05% sodium-azide in PBS) for 10 min; and thereafter stained with anti-
CTLA-4 allophycocyanin mAb (BD Biosciences). Intracellular expression
of FOXP3 was determined using an allophycocyanin anti-human FOXP3
staining set (eBioscience), according to manufacturer’s instructions. Flow
cytometry was performed on a FACSCalibur using CellQuest-pro software
(BD Biosciences), as described previously (13). The percentages of CD4?
CD25dimand CD4?CD25brightT cells were calculated within the CD3?
CD4?cell fraction, and the percentages of FOXP3-, CTLA-4-, CD69-, and
HLA-DR-positive cells were calculated within the CD3?CD4?CD25dimor
CD3?CD4?CD25brightcell fractions. FACS analysis of all decidua and
CD25brightcells. Percentage of FOXP3?(a), CTLA-4?(b), CD69?(c),
and HLA-DR?(d) cells within CD4?CD25brightT cell fraction of non-
pregnant (np) cPBL and mPBL, d.basalis, and d.parietalis in early (17–24
wk) and after term (?37 wk) pregnancy. Lines indicate median percent-
ages; ?, p ? 0.05; ??, p ? 0.01.
Characteristics of decidual and peripheral blood CD4?
5739The Journal of Immunology
PBL samples was done using the same CellQuest-pro template; the fluo-
rescence intensity to distinguish CD4?CD25dimand CD4?CD25brightcells
was determined on decidual samples and exactly copied to PBL samples.
For functional analysis, the decidual and peripheral blood isolates were
FACS sorted on a flow sorter ARIA (BD Biosciences) with DIVA soft-
ware. Isolates were stained for surface CD4 FITC, CD25 PE, and CD45
allophycocyanin, and thereafter sorted for viable CD45?cells or CD45?
cells without CD4?CD25brightcells. All cells were sorted within the lym-
phocyte gate set around the viable lymphocytes, avoiding granulocytes,
macrophages, and other contaminating cell types. Cells were washed once
in RPMI 1640 and thereafter incubated in RPMI 1640 supplemented with
2 mM L-glutamine, 50 U/ml penicillin, and 50 ?g/ml streptomycin (all
obtained from Invitrogen Life Technologies), and 10% human serum in a
round-bottom 96-well plate (Costar) at a density of 50,000 cells/well in
triplicate. For anti-CD3 stimulation, wells were precoated with 10 ?g/ml,
2 ?g/ml OKT-3 (Orthoclone) or 5 ?g/ml, 1 ?g/ml UCHT-1 (BD Pharm-
ingen) for 2 h at 37°C. For stimulation with UCB, 50,000 irradiated (3000
rad) UCB cells were added. All responders and stimulator cells were DNA
typed for HLA-A, -B, -C, -DR, and -DQ. Cells were incubated at 37°C with
5% CO2. At day 4, 50 ?l of supernatant was collected and stored at ?20°C
until the time of analysis. Supernatants were analyzed with a Th1-Th2
Bio-plex premixed human cytokine panel Th1/Th2 (containing IL-2, IL-4,
IL-5, IL-10, IL-13, GM-CSF, IFN-?, and TNF-?; Bio-Rad), according to
manufacturer’s description. After the collection of the supernatants, pro-
liferation was measured as [3H]thymidine (1 ?Ci) incorporation for an-
other 16 h by liquid scintillation spectroscopy using a betaplate
(PerkinElmer-Wallac). Results were expressed as the median cpm for each
triplicate culture. The suppression index (S.I.) (2) of CD4?CD25brightT
cells is depicted as the ratio between paired proliferation (cpm) or cytokine
production (pg/ml) of the CD45?fraction depleted for CD4?CD25bright
cells and the CD45?fraction. All samples below the background of 700
cpm or 7 pg/ml IFN-? are excluded to calculate a S.I., and samples with a
negative S.I. are depicted as 0.
Paired d.basalis and d.parietalis isolates of early and term pregnancies were
embedded in paraffin for immunohistochemical analysis. Serial 4-?m-thick
tissue sections were deparaffinized using xylene and 100% ethanol and
rehydrated with 70 and 50% ethanol. Endogenous peroxidase activity was
blocked with methanol containing 0.3% H2O2. Ag retrieval was performed
by microwaving the sections for 12 min in boiling citrate buffer (10
mMol/L (pH 6.0)). The tissue sections were incubated with the primary Ab
diluted in PBS containing 1% BSA overnight in a moist chamber. Sections
were washed three times and incubated with secondary Ab for 60 min in a
CD25brightcells to OKT-3 stimula-
tion. Representative dot plots of CD4
and CD25 expression within the
CD45?fraction (a) and the CD45?
fraction depleted for CD4?CD25bright
cells (b) of cPBL, mPBL, d.basalis,
and d.parietalis isolates after FACS
sorting. All plots are gated for CD45?
cells within the lymphocyte gate. Pro-
liferation (c) and IFN-? production
(e) of CD45?cells (?) and CD45?
cells depleted for CD4?CD25bright
cells (?) after OKT-3 stimulation.
Isolates of cPBL, mPBL, d.basalis,
and d.parietalis are shown. The S.I. of
proliferation (d) and IFN-? produc-
tion (f) of all samples is depicted.
5740 MIGRATION OF FETUS-SPECIFIC CD4?CD25brightTreg CELLS
moist chamber. Following three washes in PBS, the sections were embed-
ded in Mowiol (Calbiochem). The Abs used are as follows: rabbit poly-
clonal CD4 (Santa Cruz Biotechnology); rabbit polyclonal CD3 (Abcam);
goat anti-rabbit IgG Texas Red (Abcam); mouse monoclonal to FOXP3
(236A/E7; Abcam); and goat anti-mouse IgG1-FITC (BD Biosciences).
The localization of CD4?FOXP3?or CD3?FOXP3?cells was determined
using fluorescence microscopy.
To determine differences between ?2 groups, a nonparametric Kruskal-
Wallis one-way ANOVA was performed. If p ? 0.05, a Dunn’s multiple
comparison posttest was performed to compare all pairs of columns. The
Wilcoxon signed rank test was performed to determine differences between
paired groups. The Mann-Whitney U test was used to determine differences
between nonpaired groups. Values of p ? 0.05 are considered to denote
Characterization of decidual CD4?CD25dimand CD4?
Consistent with a previous report by our group (13), we observed
a significantly higher percentage of CD4?CD25brightT cells in all
decidual samples compared with nonpregnant control PBL sam-
ples and mPBL samples. In addition, a significantly higher per-
centage of CD4?CD25brightT cells is observed in d.parietalis com-
pared with d.basalis both in early (17–24 wk) and term pregnancy
(?37 wk) (data not shown). To further characterize decidual CD4?
CD25dimand CD4?CD25brightT cells, we performed flow cyto-
metric analysis for the Treg markers FOXP3 and CTLA-4 (both
intracellular), and surface expression of the T cell activation mark-
ers CD69 and HLA-DR. Representative FACS plots and the gating
strategy for determining CD4?CD25dimand CD4?CD25brightT
cells and FOXP3?, CTLA-4?, CD69?, and HLA-DR?cells are
shown in Fig. 1, a and b.
The decidual CD4?CD25dimand CD4?CD25brightT cell pop-
CD25brightT cells show a regulatory phenotype with high percent-
ages of FOXP3?, CTLA-4?, and HLA-DR?cells and low
percentages of CD69?cells. In contrast, the CD4?CD25dimT cell
fraction of all decidual isolates shows an activated phenotype con-
taining low percentages of FOXP3?, CTLA-4?, and HLA-DR?
cells and high percentages of CD69?cells. The decidual CD4?
CD25brightT cell population is a small, but homogeneous cell pop-
ulation with no significant differences in percentage of FOXP3?,
CTLA-4?, CD69?, and HLA-DR?cells between d.basalis and
d.parietalis samples and no differences between early (17–24 wk)
and term (?37 wk) pregnancy samples (Fig. 1, c–f). The decidual
CD4?CD25dimT cell population contains minor differences in
percentages of CTLA-4?cells and CD69?cells between d.basalis
and d.parietalis samples and early (17–24 wk) and term (?37 wk)
pregnancy samples (Fig. 1, c–f).
Different phenotype of CD4?CD25brightT cells in decidua
compared with peripheral blood
To compare the phenotype of decidual and peripheral blood CD4?
CD25brightT cells, peripheral blood samples from healthy non-
pregnant female donors and the mPBL samples from early and
term pregnancy were analyzed similar to the decidual isolates. All
decidual CD4?CD25brightT cell fractions contain a significantly
higher percentage of CTLA-4?, CD69?, and HLA-DR?cells
compared with nonpregnant control PBL (p ? 0.0001; p ?
0.0001; p ? 0.008, respectively), and mPBL both in early and term
pregnancy (all p values ?0.0001) (Fig. 2, b–d). In addition, a
significantly higher percentage of FOXP3?cells is observed in
decidual CD4?CD25brightT cells compared with CD4?CD25bright
T cells from mPBL (p ? 0.0001). However, no significant differ-
ence in percentage of FOXP3?cells in the decidual CD4?
CD25brightT cell fractions compared with the CD4?CD25brightT
cell fractions nonpregnant control PBL is observed (Fig. 2a).
Comparison of the CD4?CD25brightT cell fraction from mPBL
and nonpregnant control PBL shows a significantly lower percent-
age of FOXP3?in the CD4?CD25brightT cell fraction in mPBL in
early (52%) and term (53%) pregnancy, compared with the CD4?
CD25brightT cell fractions of nonpregnant controls (79%) (p ?
0.05; p ? 0.05; Fig. 2a). In addition, the CD4?CD25brightT cell
fraction in mPBL in early (35%) and term (37%) pregnancy con-
tains significantly less HLA-DR?cells compared with nonpreg-
nant controls (50%) (p ? 0.01; p ? 0.05; Fig. 2d).
Functional analysis of CD4?CD25brightT cells
To examine the suppressive capacity of decidual and peripheral
blood CD4?CD25brightT cells, we isolated a lymphocyte fraction
containing all CD45?cells and a CD45?fraction depleted for
CD4?CD25brightT cells by FACSort. Representative FACS plots
and the gating strategy are shown in Fig. 3, a and b. Both fractions
were stimulated with plate-bound OKT-3 (10 and 2 ?g/ml) and
plate-bound UCHT-1 (5 and 1 ?g/ml) and examined for prolifer-
ation capacity by tritium incorporation, whereas the supernatants
were examined for cytokine production by a Bio-plex bead array.
The proliferative capacity and IFN-? production of peripheral
blood isolates were not affected by depletion of the CD4?
CD25brightcells using OKT-3 or UCHT-1 stimulation. In contrast,
the d.basalis isolate shows a significant increase in IFN-? produc-
tion after depletion of the CD4?CD25brightcells (p ? 0.027) and
a slight, but not significant increase in proliferation (p ? 0.064)
using OKT-3 stimulation (Fig. 3, c and e). UCHT-1 stimulation
induces high proliferative responses (range 61,000–240,000 cpm)
ual CD4?CD25brightT cells. a, Shows proliferation of CD45?cells (?)
and CD45?cells depleted for CD4?CD25brightcells (?) after fetus-spe-
cific UCB (left) and third party UCB stimulation (right). Isolates of mPBL,
d.basalis, and d.parietalis are shown. b, Shows the S.I. of proliferation of
mPBL, d.basalis, and d.parietalis after fetus-specific UCB (left) and third
party UCB stimulation (right) (?, p ? 0.05; ??, p ? 0.01).
Fetus-specific and fetus-nonspecific suppression by decid-
5741 The Journal of Immunology
and IFN-? production (range 175–6,400 pg/ml) in all decidual
isolates. However, proliferation and IFN-? production after
UCHT-1 stimulation were not affected by depletion of CD4?
CD25brightcells in all isolates. In d.parietalis, a group of high re-
sponders (proliferation ? 30,000 cpm and IFN-? ? 400 pg/ml)
and group of low responders (proliferation ? 10,000 cpm and
IFN-? ? 100 pg/ml) can be identified (Fig. 3, c and e). Both
groups were checked for differences in clinical parameters (birth
order, time of membrane rupture, maternal age, etc.) that could
have led to an increased immune activation. There was no differ-
ence in any of these parameters, except for gender of the child: the
high responders carried all female children (n ? 5) and the low
responders all male children, except for 1 female (n ? 5 ? 1). To
compare the suppressive capacity of the four different isolates, a
S.I. was determined, but no significant differences were observed
with regard to proliferation (Fig. 3d) and IFN-? production (Fig.
3f). Besides IFN-?, all other cytokines (IL-2, IL-4, IL-5, IL-10,
IL-13, GM-CSF, and TNF-?) were analyzed, but no significant
differences in these cytokine concentrations were observed in the
CD45?fraction compared with the CD4?CD25brightdepleted
Fetus-specific suppression capacity of CD4?CD25bright
To determine whether there is a fetus-specific component in the
suppressive capacity of mPBL and decidual CD4?CD25brightT
cells, CD45?cells and a CD45?fraction depleted for CD4?
CD25brightT cells were stimulated with UCB cells of the fetus and
with a third party UCB. In both d.basalis and d.parietalis isolates,
the depletion of CD4?CD25brightT cells leads to a significant in-
crease in proliferation to UCB cells (p ? 0.034; p ? 0.027) and a
third party UCB (p ? 0.001; p ? 0.039) (Fig. 4a). To compare the
suppressive capacity of mPBL and the decidual isolates, a S.I. was
determined. CD4?CD25brightT cells from d.basalis and d.pari-
etalis contain a significantly higher suppressive capacity to regu-
late fetus-specific UCB cells compared with mPBL CD4?
CD25brightT cells (p ? 0.05; p ? 0.0.05) (Fig. 4b). However, no
difference in suppressive capacity between decidual and mPBL
CD4?CD25brightT cells to third party UCB cells is observed (Fig.
4b). Interestingly, mPBL shows a reduced suppressive capacity to
UCB of her own fetus (median SI ? 1.0) compared with UCB of a
third party fetus (median SI ? 1.29) (p ? 0.052) (data not shown).
All mother-child combinations are haploidentical for HLA-A, -B, -C,
-DR, and -DQ. No difference is observed between fully mismatched
or haploidentical third party UCB stimulator cells using maternal and
nonpregnant control responder cells. The capacity of mPBL to sup-
press third party UCB is similar to the capacity of nonpregnant con-
trols to suppress UCB (data not shown).
Percentage of CD4?CD25brightT cells
To investigate whether the observed difference in suppressive ca-
pacity is caused by differences in percentages of CD4?CD25bright
cells in the isolates, all fractions obtained after FACS sorting were
reanalyzed on a FACSCalibur. The percentages of CD4?
CD25brightT cells within the CD4?T cell population and within
the CD45?populations were determined. In line with previous
studies, d.basalis and d.parietalis lymphocyte isolates contain
higher percentages of CD4?CD25brightT cells within the CD4?T
cell population compared with peripheral blood isolates (Fig. 5).
Within the CD45?fraction, the cPBL, mPBL, and d.basalis con-
tain similar percentages of CD4?CD25brightT cells (median per-
centages: 1.0, 1.1, and 1.4%, respectively), resulting in Treg-lym-
phocyte ratio of ?1:100. D.parietalis contains a higher percentage
of CD4?CD25brightT cells (2.8%) within the CD45?fraction,
resulting in a ratio of 1:36. No correlation between the Treg-lym-
phocyte ratios and the suppression index of all individual experi-
ments was observed. The percentage of CD4?CD25dimT cells did
not differ in the CD45?fraction and the CD4?CD25brightdepleted
fraction (data not shown).
Localization of decidual CD3?FOXP3?cells at the
In order to confirm the localization of CD3?Treg cells in decidual
tissue, we analyzed paraffin-embedded tissue sections of the pla-
centa (containing d.basalis and villi) and the membranes (contain-
ing amnion, chorion, and d.parietalis) in early and term pregnancy.
The sections were stained for CD4 in combination with FOXP3 or
CD3 in combination with FOXP3. All sections show a preferential
localization of CD4?FOXP3?and CD3?FOXP3?cells in mater-
nal tissue (i.e., present in d.basalis, but not in villous tissue (Fig.
6a), and in d.parietalis, but not in chorion and amnion (Fig. 6b)).
In addition, a high variation in numbers of CD3?FOXP3?and
CD3?FOXP3?is observed between individual patients (Fig. 6, b
CD25brightT cells in CD45?fraction (?) and CD45?fraction depleted for
CD4?CD25brightT cells (?) of cPBL, mPBL, d.basalis, and d.parietalis
isolates after FACS sorting. Percentages of CD4?CD25brightT cells within
the CD45?T cell population (a) and within the CD4?population (b) are
depicted. c, Indicates the median percentages of CD4?CD25brightT cells of
Percentage of CD4?CD25brightT cells. Percentage of CD4?
5742 MIGRATION OF FETUS-SPECIFIC CD4?CD25brightTreg CELLS
In this study, we investigated the phenotypic and functional prop-
erties of decidual and peripheral blood CD4?CD25brightT cells.
CD25brightT cell subsets were found in all decidual isolates. CD4?
CD25dimT cells show an activated phenotype containing high per-
centages of HLA-DR?and CD69?cells and low percentages of
FOXP3?and CTLA-4?cells. In contrast, decidual CD4?
CD25brightT cells show a regulatory phenotype containing high
percentages of FOXP3?, CTLA-4?, and HLA-DR?cells. Decid-
ual CD4?CD25brightT cells are a homogeneous cell population
with no significant differences in phenotype between d.basalis and
d.parietalis isolates or between second and third trimester preg-
nancies. Decidual CD4?CD25brightT cells show an increased ex-
pression of CTLA-4, HLA-DR, CD69, and CD25 compared with
peripheral blood CD4?CD25brightT cells. Understanding the func-
tional significance of the phenotypic differences in peripheral and
decidual CD4?CD25brightTreg cells is limited by the lack of true
Treg-specific surface markers and therefore the inability to define
mechanisms of suppression. Identification of Treg cells based upon
their phenotypic characterization is controversial (20, 21), and
functional tests are required to identify Treg cells. The identifica-
tion of novel Treg-specific markers CD39 and CD73 that are func-
tionally involved in immunosuppressive activity in mice (22) is
promising for future studies, but their relevance remains to be con-
firmed in the human system. In addition, mechanistic studies on
FOXP3 function or signaling of immunoregulatory molecules such
as TGF-? show the dynamics of Treg generation (23, 24) and may
eventually lead to elucidation of the differences between peripheral
and decidual CD4?CD25brightT cells.
Many studies have shown that CD4?CD25brightT cells can sup-
press specific and nonspecific immune responses in a dose-depen-
dent manner. Similar results were obtained in functional assays,
which differed with regard to experimental setup, effector cell pop-
ulations (total CD3?cells, CD4?CD25?T cells), Treg-T effector
ratios, sources of APCs, and readout systems (proliferation, cyto-
kines) (25, 26). The aim of our study was to compare the contri-
bution of CD4?CD25brightT cells in regulating maternal lympho-
cyte responses at the fetal-maternal interface and in mPBL. For
this, we isolated the complete lymphocyte fraction and compared
proliferation and cytokine responses of the total lymphocyte frac-
tion with the CD4?CD25brightdepleted lymphocyte fraction. In
contrast to other studies in which the modulating effect of an iso-
lated subpopulation of responder cells is tested, we measured the
potential of CD4?CD25brightcells to suppress the reactivity of the
different lymphocyte populations, including CD4?CD25?, CD8?
T cells, and NK cells, present in the blood or in the decidua, which
is compatible to the in vivo situation. In addition, we used CD3
stimulation and stimulation with UCB cells to determine whether
there is a fetus-specific component in CD4?CD25brightT cell-me-
Upon CD3 stimulation, we found a variable increase in prolif-
eration or IFN-? production after depletion of CD4?CD25brightT
cells. In the d.basalis, a significant increase in IFN-? production
after depletion of CD4?CD25brightT cells was observed in all
individuals. In the d.parietalis, a group of high responders with a
clear increase in proliferation and IFN-? production after depletion
of CD4?CD25brightT cells was found next to a group of low
responders without a clear regulatory capacity of the CD4?
CD25brightcells. Between these two groups, the gender of the child
differed, the high responders being all female (n ? 5) and the low
responders all male except for 1 female (n ? 5 ? 1). The numbers
in this group are too small to state significance, but in further
studies this difference should be further elucidated. These data are
suggestive for an individual variation in the contribution of CD4?
CD25brightT cells in the regulation of the local immune response.
In this study, we did not observe differences in the suppression
capacity of PBL isolates and decidual lymphocyte isolates to CD3
stimulation using the OKT-3 and UCHT-1 clone. The type of sup-
pression assay we used, lacking APCs and the low ratio CD4?
CD25brightcells that are depleted from the total lymphocyte iso-
late, might lead to a low sensitivity to detect regulation. It does,
however, provide the best reflection of the in vivo activation status
of all lymphocytes and capacity of Treg cells to regulate their
response. Nevertheless, future experiments should elucidate pos-
sible differences in regulatory capacity of decidual and peripheral
CD4?CD25brightT cells by mixing Treg cells and lymphocytes in
higher ratios and testing the influence of APCs.
The dynamics of immune regulation during pregnancy are
shown by the fact that depletion of CD4?CD25brightT cells from
mPBL does not affect the immune response to her own child,
whereas immune regulation to a third party UCB is comparable to
nonpregnant controls. In addition, mPBL samples show a reduced
percentage of CD4?CD25brightFOXP3?and CD4?CD25bright
HLA-DR?cells compared with peripheral blood of nonpregnant
controls. In contrast, decidual tissue contains a high proportion of
CD4?CD25brightT cells with a regulatory phenotype and despite
the individual variation between the patients; the decidual CD4?
CD25brightT cells contain the capacity to regulate fetus-specific
interface. Immunohistochemical staining of CD3-Texas Red and FOXP3-
alexa488 in placenta sections (a and b) and sections of the membranes
(c–f). a, Shows an overview of placental tissue containing villi and d.basa-
lis; b, shows the localization of CD3?FOXP3?and CD3?FOXP3?cells in
d.basalis. c, Shows an overview of membranes containing amnion, chorion,
and d.parietalis tissue; d, shows the localization of a CD3?FOXP3?cell in
d.parietalis. e, Shows an overview of membranes from a second individual
containing chorion and d.parietalis tissue; f, shows the localization of
CD3?FOXP3?and CD3?FOXP3?cells in d.parietalis.
Localization of CD3?FOXP3?cells at the fetal-maternal
5743 The Journal of Immunology
and fetus-nonspecific responses. These data suggest that fetus-spe-
cific Treg cells are specifically recruited from the periphery to the
A recent study examining the dynamics of CD4?CD25brightT
cells during the menstrual cycle has demonstrated an expansion
of CD4?CD25brightFOXP3?T cells just before ovulation (27).
In addition, reduced numbers of Treg cells and a diminished
suppressive capacity of these cells were observed in women
with recurrent spontaneous abortions (17). Besides the impair-
ment of expansion of functional Treg populations, defects in
recruitment of CD4?CD25brightTreg cells to the fetal-maternal
interface may play a role in development of pathology during
The leukocyte composition of decidual isolates is highly vari-
able among individuals (data not shown). Analysis of 14 uncom-
plicated term deliveries show an average T cell percentage of 51 ?
13% in d.basalis, 64 ? 11% in d.parietalis, and 71 ? 11% in
mPBL (all calculated within the CD45?lymphocyte fraction),
compared with 75 ? 3% in peripheral blood of nonpregnant con-
trols. In addition, there is high variation in percentage of CD4?
CD25brightT cells in d.basalis and d.parietalis isolates (13). We did
not find a correlation between the percentage of depleted CD4?
CD25brightT cells and the observed suppression capacity. How-
ever, the variation in suppression capacity between the samples
might be due to a different leukocyte composition of the isolates.
Decidual T cells comprise a very heterogeneous subset of T cells
containing CD4?and CD8?cells with highly activated pheno-
types as well as cells with a merely regulatory phenotype (13, 16).
The activated decidual T cells might be more difficult to suppress
in comparison with peripheral blood T cells, resulting in similar
suppression indexes. The decidual isolates also contain variable
percentages of decidual NK cells, and although studies have shown
that CD4?CD25?T cells can inhibit NK cell functions (28), future
studies should examine the potential inhibitory effect of CD4?
CD25brightT cells on decidual NK cells.
Based on the high variation between lymphocyte properties in
individual pregnancies, including lymphocyte gain, lymphocyte
composition, and the variable contribution of CD4?CD25brightT
cells to suppress decidual lymphocyte responses, we hypothesize
that each pregnancy generates a unique combination of regulatory
mechanisms to result in a successful pregnancy. These regulatory
mechanisms can include nonspecific suppression mechanisms me-
diated by the expression of IDO, FAS, complement inhibitor pro-
teins, or more specific mechanisms mediated by HLA-expression
patterns (1–5), NK cell-trophoblast interactions (10, 11), decidual
macrophages, or Treg cells (1, 13, 14). Maternal genotype (such as
HLA genotype, killer Ig-like receptor genotype, or cytokine poly-
morphisms), or maternal history (regarding birth order, infection
history) and the combination of fetal HLA matches and mis-
matches may determine which regulatory mechanisms are most
The mechanisms by which Treg cells can inhibit fetus-spe-
cific responses at the fetal-maternal interface remain to be elu-
cidated. Examining the functional differences between decidual
and peripheral blood CD4?CD25brightT cells might identify
factors that can induce CD4?CD25brightTreg cells at the fetal-
maternal interface and may help to understand conditions of
placental pathology in which Treg cells are reduced (17, 29). In
this study, we demonstrate that fetus-specific Treg cells are ab-
sent in mPBL at term pregnancy. In addition, we demonstrate
that decidual CD4?CD25brightT cells suppress fetus-specific
and fetus-nonspecific responses. Our data suggest a preferential
recruitment of fetus-specific Treg cells from mPBL to the fetal-
maternal interface and suggest that CD4?CD25brightT cells
contribute to the regulation of fetus-specific responses in human
We thank Cees van Kooten and Anneke Brand for critically reviewing the
manuscript; Reinier van der Linden en Guido de Roo for cell sorting; Clara
Kolster, Yvonne Beuger, and the midwives and residents of the Depart-
ment of Obstetrics for collecting all term pregnancy samples; and Willem
Beekhuizen of the Center of Human Reproduction, Leiden, for collecting
the early pregnancy samples.
The authors have no financial conflict of interest.
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5745The Journal of Immunology