ARTICLES

Regulatory T cells mediate

maternal tolerance to the fetus
1,2,3 1 1,3
Varuna R Aluvihare , Marinos Kallikourdis & Alexander G Betz
http://www.nature.com/natureimmunology

Pregnancy constitutes a major challenge to the maternal immune system, as it has to tolerate the persistence of paternal
alloantigen. Although localized mechanisms contribute to fetal evasion from immune attack, maternal alloreactive
+
lymphocytes persist. We demonstrate here an alloantigen-independent, systemic expansion of the maternal CD25 T cell
pool during pregnancy and show that this population contains dominant regulatory T cell activity. In addition to their
function in suppressing autoimmune responses, maternal regulatory T cells suppressed an aggressive allogeneic response
directed against the fetus. Their absence led to a failure of gestation due to immunological rejection of the fetus.

The immunological principles that govern immune responses 14 + +

responses . Here we show that the maternal CD4 CD25 regulatory T
directed against nonself major histocompatibility complex (MHC)
1 cell pool is systemically expanded, that this expansion is not driven by
seem to be circumvented during pregnancy . Several specialized alloantigen and that this population is essential for the suppres-sion of
mechanisms have evolved to help the fetus, which expresses paternal maternal immune aggression directed against the fetus.
antigens, evade maternal immune attack. Fetal tissue depletes tryp-
tophan, an essential amino acid for rapidly dividing cells, and RESULTS
© 2004 Nature Publishing Group

2,3
thereby inhibits T cell proliferation . The specialized fetal tissue + +
Expansion of the maternal CD4 CD25 T cell pool
(trophoblast) that invades maternal uterine tissue at the site of Naturally occurring regulatory T cells are characterized by the
implantation expresses human leukocyte antigen G. This nonpoly- surface expression of CD4 and CD25 (ref. 12). We quantified the
4 + +
morphic MHC inhibits natural killer cell activation . The maternal- proportion of CD4 CD25 T cells in tissues from pregnant
fetal interface has a highly organized structure and provides an b b b
5 C57BL/6 (H-2K , H-2D , H2L ) mice at gestational day 10.5
anatomical barrier . Furthermore, the expression of Fas ligand by k
6 (E10.5) that had been suc-cessfully mated with CBA (H-2K , H-
trophoblast cells may cause apoptosis of activated maternal lym- k k
2D , H2L ) males, and com-pared these with tissues from
phocytes, which express its ligand (Fas). Finally, complement
activa-tion, which promotes inflammatory and immune responses, is nonpregnant, age-matched female C57BL/6 mice. We obtained
lymphocytes from spleen, iliac lymph nodes, inguinal lymph nodes
inhibited by expression of the Crry protein and thereby promotes and blood of nonpregnant control mice. In accordance with
7 12 +
maternal-fetal tolerance . All of these mechanisms act locally at the published data , less than 6% of all CD4 cells coexpressed CD25
site of fetal antigen exposure and may operate in parallel to sustain within all tissues: 5% in spleen, 6% in inguinal lymph nodes, 4% in
gestation. Although some of the mechanisms described above may iliac lymph nodes and 3% in blood (Fig. 1). In contrast, we found a
contribute to the deletion of antifetal effector cells, maternal lym- + +
phocytes capable of attacking the fetus do persist throughout gesta- notable increase in the proportion of CD4 CD25 cells in all tissues
1,8 of pregnant C57BL/6 mice that had mated with CBA males (Fig. 1).
tion, although they seem to be hyporesponsive . In addition, the + +
CD25 cells constituted 17% of all CD4 cells in spleen, 12% in
maternal immune system can specifically tolerate the engraftment of
1 inguinal lymph nodes, 9% in iliac lymph nodes and 11% in blood.
paternally derived tumor cells . Furthermore, pregnancy can amelio- + +
The expansion of the CD4 CD25 cell pool was consistent in all
rate the course of autoimmune disease, which often relapses after mice analyzed, with only minor variation. In contrast, we noted less
9,10 – +
delivery . Thus, in addition to locally acting mechanisms, more consistent changes in the percentage of CD4 CD25 T cells during
global changes of the maternal immune system must occur that facil- pregnancy, which have not been charac-terized in detail.
1,11
itate fetal tolerance . Naturally occurring regulatory T cells are Although regulatory T cells are characterized by the surface
+ + +
characteristically CD4 CD25 (ref. 12) and are pivotal in inhibiting expression of CD25, activated CD4 T cells also express this marker.
+ +
inappropriate activation of cell- and antibody-mediated immune The expansion of the CD4 CD25 T cell pool could therefore be due to
12,13 an expansion of the alloreactive effector T cell population
responses against self antigen as well as innate immune

1 2
Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, United Kingdom. Addenbrooke’s NHS Trust, Department
3
of Medicine, Hills Road, Cambridge, CB2 2YH, United Kingdom. These authors contributed equally to this work. Correspondence should be addressed to
V.R.A. (vra1000@cus.cam.ac.uk) or A.G.B. (betz@mrc-lmb.cam.ac.uk).

Published online 1 February 2004; doi:10.1038/ni1037

266 VOLUME 5 NUMBER 3 MARCH 2004 NATURE IMMUNOLOGY


a b
http://www.nature.com/natureimmunology
© 2004 Nature Publishing Group
responding to paternal antigens expressed by the fetus and/or an
expansion of the regulatory T cell population suppressing antifetal
+ +
responses. We therefore investigated the proportion of CD4 CD25
T cells during pregnancy in a syngeneic context, in which alloanti-
genic stimulation is minimal.
+ +
Alloantigen-independent expansion of the CD4 CD25 T cell pool
We purified lymphocytes from syngeneically mated C57BL/6 female
mice on the day of detection of the vaginal plug, marking successful
mating (E0), as well as on E2, E4 and E8. In spleen, inguinal lymph
+ +
nodes, iliac lymph nodes and blood, the percentage of CD4 CD25 cells
increased with gestation. This increase was similar to that observed for
allogeneic matings (Fig. 2; means at E8: spleen, 14%; inguinal lymph
nodes, 9%; iliac lymph nodes, 13%; blood, 9%).
NATURE IMMUNOLOGY VOLUME 5 NUMBER 3 MARCH 2004
ARTICLES
+ +
Figure 1 CD4 CD25 T cell pools of pregnant and nonpregnant mice.
Surface expression of CD4 and CD25 on lymphocytes prepared from
lymphoid tissues of nonpregnant (NP) and pregnant (P) mice at E10.5 of an
allogeneic pregnancy. (a) Representative dual plots of lymphocytes prepared
from spleen, inguinal lymph nodes, iliac lymph nodes and blood (pooled cells
+ +
from three mice). (b) Percentage of CD25 cells within the CD4 pool of cells
in nonpregnant mice (n = 8) and pregnant mice (n = 6).
Each tissue examined showed a statistically significant trend in the
+ +
increase of CD4 CD25 T cells from E0 to E8 (spleen, P < 0.0001;
inguinal lymph nodes, P = 0.01; iliac lymph nodes, P = 0.03; blood,
P = 0.005). Even at E2, we noted a small but consistent increase in
+ +
CD4 CD25 T cells compared with E0. These data indicate that the
presence of fetal alloantigen is not necessary to drive the expansion
+ +
of the CD4 CD25 T cell pool within secondary lymphoid compart-
ments during pregnancy. Although the expression of male minor
15
histocompatibility antigens such as H-Y may contribute to such an
+ +
expansion, comparison of the scatter profiles of CD4 CD25 cells
showed no differences in the proportion of activated lymphocytes,
whether they originated from nonpregnant mice or syngeneic or
allogeneic pregnant mice (data not shown). Thus, our results are
+ +
more consistent with the expansion of the CD4 CD25 T cell pool’s
being driven by pregnancy itself.
+ +
The increase in CD4 CD25 cells in blood at E8 was mirrored by
a simultaneous decrease in these cells in inguinal lymph nodes.
Although we have not undertaken a formal study of lymphocyte
migration, this suggests an alteration in the recirculation of
+ +
CD4 CD25 cells at mid-gestation, favoring relocation to the spleen
and iliac nodes.
+
Maternal CD25 T cells suppress alloresponses
+ +
The unusually high proportion of CD4 CD25 and in some cases
+ +
also CD8 CD25 T cells (data not shown) indicates that the
maternal immune system undergoes a systemic change during
+
pregnancy. The alloantigen-independent expansion of the CD25
cell pool indicates that pregnancy promotes an expansion of the
regulatory T cell population. We sought to formally test whether
these cells have a regulatory function by investigating the effect of
+
CD25 cell depletion using a mixed lymphocyte reaction. We
obtained lymphocytes from either spleen or iliac and inguinal lymph
nodes from pregnant C57BL/6 mice (E10.5–E12.5) that had been
mated with CBA males. We stimulated these in vitro with irradiated
splenocytes from CBA males (allogeneic) or C57BL/6 males
(syngeneic). In all experiments, we tested the same respon-ders
(total or CD25 depleted) against both allogeneic and syn-geneic
stimulator cells (Fig. 3).
Figure 2 Kinetics of the expansion of the
+ +
CD4 CD25 T cell pool in syngeneic
pregnancies. Surface expression of CD4 and
CD25 on lymphocytes prepared from spleen,
inguinal lymph nodes, iliac lymph nodes and
blood of C57BL/6 mice mated with C57BL/6
males 0 d (E0), 2 d (E2), 4 d (E4) and 8 d (E8)
after detection of vaginal plugs. For the E8 time
point, the pregnancies were confirmed by visual
examination of the gravid uterus. Each dot
represents the result from an individual mouse.
A single outlier (P = 0.0005; Grubb’s test) in the
blood E0 data is enclosed in parenthesis.
267
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by CD25 depletion would be expected to reduce the proliferation in

response to alloantigen, indicating that the dominant functional
activity of this pool of lymphocytes is suppressive.
+ +
Uterine CD4 CD25 T cells express Foxp3
+ +
We have shown a systemic expansion of the CD4 CD25 T cell
pool, which contains regulatory activity capable of suppressing
allore-sponses. Although suppression of antifetal responses might
occur within the secondary lymphoid organs, we sought to determine
whether these cells access the maternal-fetal interface, which is the
main site of alloantigen exposure. Using immunohistochemistry, we
+
found CD4 cells to be diffusely scattered at the maternal-fetal inter-
face of allogeneically mated pregnant mice at E10.5 (Fig. 4a). The
+ +
proportion of uterine CD4 CD25 T cells in the same mice was 30%
+
of all CD4 T cells (Fig. 4b).
As the lineage marker Foxp3 is expressed exclusively by regulatory T
16–18
http://www.nature.com/natureimmunology

cells within the lymphoid system , we used it to distinguish between

+ regulatory T cells and activated T cells within the uterine lym-phocyte
Figure 3 Suppression of alloreactive proliferation by CD25 regulatory cells.
pool. Using quantitative PCR, we determined the amounts of uterine
Mixed lymphocyte reaction with responder cells prepared from spleens and
Foxp3 mRNA in E14.5 pregnant BALB/c mice that had mated with
lymph nodes of pregnant C57BL/6 females mated with CBA males. Cells were
obtained at E10.5. Responses against irradiated splenocytes from allogeneic
allogeneic (C57BL/6) or syngeneic (BALB/c) males. In both cases, the
(CBA) or syngeneic (C57BL/6) male mice were measured abundance of Foxp3 mRNA was 1,000-fold higher than that in uterine
3 tissue from age-matched nonpregnant mice in diestrus (Fig. 4c). As
by [ H]thymidine incorporation. Unmanipulated responders (Total) were
– – pregnant uterine tissue contains a relatively small number of
compared with CD25-depleted (CD25 ) and CD25-reconstituted (CD25 +
+ lymphocytes, the amount of Foxp3 mRNA found in the purified
CD25 ) cell samples. n.d., not done. The experiment was done in + +
quadruplicate and the results are representative of five experiments. CD4 CD25 T cells we used as a positive control is higher. The amounts
+
of Foxp3 mRNA in purified uterine CD4 lymphocytes from
Before CD25 depletion, total responder cells showed increased pro- syngeneically and allogeneically mated mice were similar (data not
+
liferation when activated by allogeneic stimulators, compared with shown) and consistent with 30% of uterine CD4 T cells’ being regula-
background proliferation induced by syngeneic stimulators. In the tory T cells. Our data further support the conclusion that changes in the
allogeneic context, CD25 depletion led to a notable increase in prolif- size of the regulatory T cell pool are driven by pregnancy rather than by
© 2004 Nature Publishing Group

eration, which was entirely suppressed by readdition of CD25-enriched alloantigen stimulation.

cell samples. As expected, CD25 depletion also resulted in a smaller
increase in proliferation in the syngeneic context, which may reflect
activation by autoantigens. The considerable difference between
proliferation induced by allogeneic and syngeneic stimulators after
+ 13
CD25 depletion demonstrates a regulatory activity within the CD25
population that suppresses alloresponses beyond that induced by
15
autoantigens and minor male histocompatibility antigens .
Our findings demonstrate that the population of splenic and lymph
+
node–derived CD25 T cells from pregnant female mice con-tains
suppressor activity. Incidental removal of activated lymphocytes
+
CD25 T cell depletion leads to gestation failure
We tested whether regulatory T cells are necessary for toleration of the
fetal allograft by the maternal immune system in vivo using a standard
adoptive transfer system . We chose this model rather than in vivo
+
antibody-mediated CD25 cell depletion because the latter also leads to
19
newly activated effector T cell depletion . The persis-tence of antibody
activity beyond the initial depletion would com-promise our ability to
study regulatory T cell function in isolation. Furthermore, the onset and
duration of regulatory T cell depletion

a b

Figure 4 Regulatory T cells in the gravid uterus. (a) Uterus of a pregnant

(E10.5) C57BL/6 female mated with a CBA male. Immunohistochemical
+
staining of CD4 cells (black) and detection of placental alkaline
+
phosphatase activity (red). Black arrows, CD4 cells. M, maternal uterine
wall; P, placenta. Scale bar, 50 µm. (b) Uterine lymphocyte preparations
from pregnant (E10.5) C57BL/6 females mated with CBA males were
+
c
enriched for CD4 cells by positive MACS selection and analyzed for
coexpression of CD25. The results are representative of three
experiments each done with cells pooled from three mice. The vertical
+ –
dashed line separates the CD25 and CD25 populations as determined
in Figure 1. (c) Relative expression of Foxp3 and Hprt, compared using
quantitative PCR. Foxp3 expression in the uterus of a nonpregnant
mouse is compared with that in pregnant (E14.5) mice mated with either
syngeneic or allogeneic males. The relative expression of Foxp3 in
+ +
CD4 CD25 lymphocytes purified from the spleen was used as a positive
control. The experiment was done in triplicate.

268 VOLUME 5 NUMBER 3 MARCH 2004 NATURE IMMUNOLOGY

 ARTICLES

a b

c d
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7
Figure 5 Regulatory T cells prevent rejection of the fetal allograft. (a) BALB/c nu/nu mice were injected with 2 × 10 lymphocytes (Total) or an equal number of
–
cells from CD25-depleted cell preparations (CD25 ). The day after the adoptive transfer, the mice were mated with C57BL/6 (allogeneic) or BALB/c (syngeneic)
males. The numbers of normal fetuses or pups (fetal number) were determined between E11.5 and full term for each mouse successfully mated within 4 d of
adoptive transfer. In all cases, the donor cells were obtained from female BALB/c mice. Symbols indicate the source of transferred cells: filled circles, females
fertilized by C57BL/6 males; open circles, females fertilized by BALB/c males; filled boxes, nonpregnant females. (b) Gross morphology of maternal-fetal tissues
–
(E11.5) from pregnant BALB/c nu/nu mice mated with C57BL/6 males after adoptive transfer of total lymphocytes (left) or CD25 lymphocytes (right). Black arrows
indicate positions of resorbing fetuses. (c) Hemotoxylin and eosin stain of resorbing fetus with arrow indicating region
+
of hemorrhage. (d) Invasion of CD3 cells (black) at the maternal-fetal interface. M, uterine wall; F, fetal tissue. Scale bars (c,d), 50 µm.

20,21 interpretation is further supported by the fact that incidental removal

after antibody treatment is not precisely defined and would affect
of alloreactive effector cells would be expected to lessen the effect
© 2004 Nature Publishing Group

the narrow time frame of our studies, which is dictated by the short
gestation of mice. We prepared lymphocytes and pooled them from
spleens, inguinal and iliac lymph nodes. We used as donor mice pregnant
BALB/c mice (E10.5–E12.5) that had mated with C57BL/6 males. We
adoptively transferred the cells into T cell–deficient BALB/c nude
+ characterized by grossly abnormal fetal architecture, hemorrhages at
(nu/nu) females as a whole-lymphocyte preparations or after CD25 cell
depletion. Because of homeostatic mechanisms, lymphoproliferation can
occur when low lymphocyte numbers are adoptively transferred into
22,23
lymphopenic mice . The cell numbers we used to reconstitute the
7
lymphoid compartment in our experi-ments, 2 × 10 lymphocytes, which
consitutes approximately 20–30% of the full complement of
22–
lymphocytes in wild-type mice, minimize such nonspecific expansion
24
. All recipient BALB/c nu/nu females were mated with C57BL/6
males on the day after adoptive transfer, and those carrying a vaginal
plug within 4 d were analyzed between E10.5 and completed gestation.
As the estrus cycle in mice is 3 d, this ensured that all females had the
opportunity to mate successfully. Breeding efficiency within nude mice
colonies is estimated at approximately 50% (M. Bunce, Harlan UK,
Blackthorn, UK, personal communication); as expected, 9 of 15 mice
(60%) that received total lymphocytes became pregnant (Fig. 5a). This
indicates that lymphoid reconstitution with total lymphocytes does not
influ-ence the outcome of a subsequent pregnancy. In those mice
analyzed before they gave birth (E11.5–E19.5), the number of
morphologi-cally normal fetuses was within the expected range. The
average litter size for this group was 5.4. In distinct contrast, 0 of the 15
mice receiving lymphocyte samples depleted of CD25 cells sustained
nor-mal pregnancies (E10.5–E19.5; Fig. 5a). These results were highly
statistically significant (P = 0.0003, Fisher’s test) and demonstrate that
in an allogeneic context, regulatory T cells are necessary for the
prevention of a maternal immune response against the fetus. Our
of regulatory T cell depletion and therefore bias against the observed
outcome. Consistent with our interpretation, at E10.5, the earliest
gestational stage analyzed, we found fetal resorption in recip-ients of
lymphocyte preparations depleted of CD25 lymphocytes,
+
the maternal-fetal interface and overwhelming infiltration by CD3
T cells (Fig. 5b–d). Although these findings suggest that maternal
regulatory T cells act in the postimplantation phase, a large-scale
investigation focusing on the immediate postfertilization period is
needed to formally exclude the possibility of any involvement
during implantation.
As the donor mice were pregnant with semiallogeneic fetuses,
enhanced allogeneic priming of the maternal immune system may have
contributed to the fetal loss we noted after CD25 depletion. We therefore
repeated the adoptive transfer experiment with one modifi-cation: we
transferred cells isolated from female donor BALB/c mice that had
mated with syngeneic males. As before, the recipient BALB/c nu/nu
females were mated with C57BL/6 males. None of the mice that
received preparations depleted of CD25 cells sustained normal preg-
+
nancies, indicating that the presence of CD25 lymphocytes is
neces-sary for successful gestation (Fig. 5a), independent of prior
exposure of the maternal immune system to alloantigen.
Regulatory T cells suppress antifetal alloresponses
Although our data indicate the involvement of regulatory T cells in
mediating maternal-fetal tolerance, they do not exclude the possibil-
ity of indirect effects of regulatory T cell depletion. Indeed, ovarian
failure through autoimmune ovarian syndrome and generalized
autoimmunity associated with lymphoid activation occur after
+ 12,13
CD25 cell depletion .

NATURE IMMUNOLOGY VOLUME 5 NUMBER 3 MARCH 2004 269

 ARTICLES
To test these possibilities, we analyzed the effect of CD25 depletion
on the outcome of pregnancy in a syngeneic context. We transferred
lymphocyte preparations depleted of CD25 lymphocytes from BALB/c
female mice into BALB/c nu/nu recipients, which we then mated with
BALB/c males. In marked contrast to the dependence of semiallo-geneic
fetuses on regulatory T cells for survival, 50% of mice that received
lymphocyte preparations depleted of CD25 lymphocytes became
pregnant (Fig. 5a). The average litter size for this group was five, and
the fetuses were developmentally normal when examined at E11–E12.5.
These results are statistically significant (P = 0.0385, Fisher’s test)
compared with the outcome predicted if all fetal losses resulted from
indirect effects of CD25 depletion. Thus, within the time scale of our
experiments, autoimmune ovarian failure and generalized autoimmunity
were not chief determinants of outcome. Furthermore, whereas donor
mice in this case were not pregnant, these data never-theless strongly
indicate that regulatory T cell function is essential only when the fetus
expresses paternally derived alloantigen.
http://www.nature.com/natureimmunology
DISCUSSION
Our data indicate that regulatory T cells are required for the maternal
immune system to tolerate the fetal allograft. The fact that fetal out-
come is normal in syngeneic pregnancies, when regulatory T cells
are absent, supports the conclusion that these cells suppress maternal
immune responses directed against fetal alloantigen rather than
responses against male-specific minor histocompatibility antigens.
Our findings indicate a more general adaptation of the maternal
immune system during pregnancy. However, regulatory T cells may
also affect the well-described fetal evasion mechanisms that act
11
locally at the maternal-fetal interface to suppress antifetal
+ +
responses. The unusually high proportion of CD4 CD25 regula-
© 2004 Nature Publishing Group
tory T cells we found in the pregnant uterus indicates that these cells
are actively recruited to and/or their population is expanded at this
site. Regulatory T cells express the cell surface marker CTLA-4 (ref.
25) and can induce dendritic cells to express indoleamine 2,3-
dioxygenase by an interaction with its ligand CD80-CD86 (refs.
26,27). In turn, expression of indoleamine 2,3-dioxygenase pro-
2 CD25 cells were added at the same density. All samples that did not receive
motes maternal-fetal tolerance .
Given the increase in spleen and lymph node size during preg-
28
nancy , indicating a systemic expansion of the total lymphocyte pop-
ulation, our data indicate an increase in total regulatory T cell number.
As the expansion is not driven by alloantigen, this might be due to a
9 3,000 rad and cultured at a density of 5 × 10 cells/well. Cells were pulsed with 1
hormonal modulation of the immune system . Furthermore, the gen-
eralized expansion of the regulatory T cell population indicates that
suppression of antifetal responses may also occur within the draining
lymph nodes through which potentially aggressive effector cells move.
Although enhancement of regulatory T cell function may con-
tribute to systemic changes that occur in the maternal immune sys-
9,10
tem, such as suppression of some autoimmune diseases , this does
not necessarily indicate a deleterious suppression of protective
immune responses. Naturally occurring regulatory T cells seem to be
29,30
activated by self antigen–MHC complexes and therefore are
potent suppressors of autoimmune and, in this context, semiallo-
12
geneic responses rather than responses to exogenous antigens .
Pregnancy-induced expansion of the regulatory T cell population
may also contribute to the phenomenon of specific maternal toler-
1
ance to some paternal grafts during gestation . The ability of the
maternal immune system to reject more immunogenic grafts while
maintaining tolerance to the fetus may reflect differences in fetal
immunogenicity, maternal-fetal anatomy and the interplay of regula-
tory T cells with additional tolerance-promoting mechanisms that
5
function during pregnancy .
270
The endogenous, natural suppression of alloresponses during
preg-nancy that we have shown here defines a previously unknown
func-tion of regulatory T cells. Understanding how they work in this
context may permit manipulation of the immune system to suppress
rejection of organ transplants and autoimmune responses. As an
+
analogous population of CD25 regulatory T cells exists in
31,32
humans , it is likely that these cells have a similar function in
human gestation. Our observations would indicate that recruitment
or function of regulatory T cells may be impaired in certain
patholog-ical conditions, such as premature termination syndromes,
pre-eclampsia and infertility, with which a failure of immunological
33
tolerance has been linked .
METHODS
Animals and cell preparations. All animal care was provided by expert animal
technicians in compliance with the relevant laws and institutional guidelines.
BALB/c, C57BL/6, CBA and BALB/c nu/nu mice 6–12 weeks old were bred and
maintained in specific pathogen–free conditions. Lymphocytes were prepared
from the spleen, lymph nodes and uterus with Lympholyte M or from the blood
with Lympholyte Mammal (Cedarlane Laboratories). Cells used for flow
cytometry were obtained from pregnant (E0–E12.5) C57BL/6 mice that had mated
with CBA or C57BL/6 males and were stained with either fluores-cein
isothiocyanate (FITC)-conjugated antibody to CD4 (anti-CD4; GK1.5) or FITC-
conjugated anti-CD8 (53-6.7) and were costained with phycoery-thrin-conjugated
anti-CD25 (3C7; all from PharMingen). For uterine lym-phocytes, the CD4- or
CD8-stained cells were obtained by magnetic-activated cell separation (MACS)
selection with anti-FITC beads and MACS reagents (Miltenyi Biotech), according
to the manufacturer’s instructions. Cell samples for adoptive transfer experiments
+
and mixed lymphocyte reactions were depleted of CD25 cells with anti-FITC
beads and MACS reagents with an AutoMACS (Miltenyi Biotech). In all cases the
+
preparations contained less than 0.3% CD25 cells after several rounds of
depletion.
Mixed lymphocyte reactions. Lymphocytes were obtained from pregnant (E10.5–
E12.5) C57BL/6 mice fertilized by CBA males. Total or CD25-depleted cell
samples from spleen, iliac and inguinal lymph nodes, blood or uterus were
cultured in RPMI medium with 10% fetal calf serum in round-bottomed 96-well
5
plates (Corning B.V.) at a density of 1 × 10 cells/well. Preparations enriched in
+
CD25 cells were corrected by the addition of CD25-depleted cell samples (cal-
– +
culated from the percentage of CD25 cells present in CD25 -enriched cell sam-
ples) to ensure an identical number of potential responder cells. Stimulator cells
(splenocytes) were obtained from CBA or C57BL/6 male mice, irradiated with
5
3
µCi [ H]thymidine (Amersham) at 24 h and were collected at 36 h with a
Filtermate Harvester and analyzed with a TopCount microplate scintillation
counter (Packard BioScience) according to the manufacturer’s instructions.
Immunohistochemistry. Cryogenic sections 10 µm in thickness were mounted on
Vectabond-coated slides (Vector Laboratories), incubated at 56 °C for 2 h and
stored at –80 °C. Frozen slides were thawed, fixed in acetone for 10 min, air-dried
and rehydrated in PBS for 10 min. Sections were submersed in H 2O2 for 5 min
followed by PBS for 10 min. Next, the sections were blocked with the Biotin
Blocking Kit (Vector Laboratories) followed by blocking with Tris-NaCl Blocking
Buffer (Perkin Elmer Life Sciences) and then incubated for 1 h at 20 °C with
biotinylated anti-CD3 (145-2CH) or CD4 (GK1.5; both from Pharmingen) in Tris-
NaCl Blocking Buffer. After sections were washed three times in PBS, the bound
antibodies were detected with a TSA-Biotin System (Perkin Elmer Life Sciences)
followed by a Vector ABC Glucose Oxidase Kit (Vector Laboratories) and were
developed with tetranitroblue tetrazolium substrate (Vector Laboratories). All
sequential stains were done on the same day. Before being mounted with
Vectamount (Vector Laboratories), the sec-tions were cleared with 100% ethanol
and left to air dry. All Vector Laboratories and Perkin Elmer Life Sciences
products were used according to the manufacturers’ instructions.
VOLUME 5 NUMBER 3 MARCH 2004 NATURE IMMUNOLOGY

Real-time RT-PCR. Total RNA was prepared with an RNeasy kit (Qiagen) fol-
lowed by DNaseI treatment (Invitrogen). cDNA was synthesized with Superscript
II (Invitrogen) with random hexamer primers (Amersham Biosciences). Real-time
PCR used Taqman Universal PCR master mix (Applied Biosystems) and primers
and a fluorescent TaqMan probe specific for Foxp3 or Hprt. The sequences were:
Hprt primers, 5′-TTAAGCAGTACAGCCCCAAAA TG-3′ and 5′-
CAAACTTGTCTGGAATTTCAAATCC-3′; Hprt probe, 5′-VIC-C
CTTTTCACCAGCAAGCTTGCAACCTTA-3′; Foxp3 primers, 5′-CCCAGGA
AAGACAGCAACCTT-3′ and 5′-TTCTCACAACCAGGCCACTTG-3′; Foxp3
probe, 5′-FAM-ATCCTACCCACTGCTGGCAAATGGAGTC-3′. An ABI prism
7700 sequence detection system (Applied Biosystems) was used for 45 cycles of
PCR. All PCRs were set up at least in triplicate. Uterine RNA from mice in
diestrus was prepared after identification of the estrus cycle stage by a vaginal
smear stained with Giemsa (Sigma).
Adoptive transfer experiments. Spleens and lymph nodes from pregnant
(E10.5–E12.5) BALB/c mice fertilized by C57BL/6 or BALB/c males or
from 6-to 12-week-old nonpregnant female BALB/c mice were used to
+ 16. Fontenot, J.D., Gavin, M.A. & Rudensky, A.Y. Foxp3 programs the development
http://www.nature.com/natureimmunology
prepare lym-phocytes. Preparations were depleted of CD25 cells as outlined
7 17. Hori, S., Nomura, T. & Sakaguchi, S. Control of regulatory T cell development by
above. BALB/c nu/nu female mice were intravenously injected with 2 × 10
cells per mouse of total lymphocytes or of lymphocyte preparations depleted
of CD25 lymphocytes. Matings with C57BL/6 or BALB/c males were set up
24 h after transfer. Only mice that had received a vaginal plug within 4 d of
adoptive transfer were analyzed between E11.5 and full-term gestation.
Fetuses were analyzed before birth by gross morphology and developmental
landmarks as well as by immunohistochemistry.
ACKNOWLEDGMENTS
We thank R. Bystry for help with the immunohistochemistry; M.S. Neuberger,
D.T. Fearon, K.J. Patel and F. Randow for critical reading of the manuscript;
M. Bradley (Centre for Applied Medical Statistics, University of Cambridge) for
statistical advice; and T. Langford, L. Stuckey, V. Smith, M. Brown and G. Ledger
for help with technical animal procedures. All work was done at the Laboratory of
Molecular Biology and funded by the Medical Research Council.
© 2004 Nature Publishing Group
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Received 20 October; accepted 2 December 2003
Published online at http://www.nature.com/natureimmunology/
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