2590?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 119? ? ? Number 9? ? ? September 2009
Maternal alloantibodies induce a postnatal
immune response that limits engraftment
following in utero hematopoietic cell
transplantation in mice
Demetri J. Merianos,1 Eleonor Tiblad,1,2 Matthew T. Santore,1 Carlyn A. Todorow,1
Pablo Laje,1 Masayuki Endo,1 Philip W. Zoltick,1 and Alan W. Flake1
1Children’s Center for Fetal Research, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA. 2CLINTEC, Karolinska Institute, Stockholm, Sweden.
One of the predictions of Burnet and Fenner’s theory of immunity
(1) is that prenatal exposure to foreign antigens prior to the devel-
opment of the immune system should lead to tolerance rather than
immunization. Billingham, Brent, and Medawar experimentally
confirmed this prediction by inoculation of murine fetuses with
cellular material from another mouse strain, which led to what
they termed “actively acquired tolerance” (2). Additional support
for the concept was provided by observations in numerous species
of hematopoietic chimerism and associated tolerance in dizygotic
twins that share placental circulation (3–8). Finally, mechanistic
insight into tolerance for self-antigens (and, by inference, foreign
antigens), and the central role of the thymus in this process, has
been provided by numerous studies, primarily in TCR transgenic
mice, over the past 2 decades (9, 10).
The potential for strategies based on actively acquired toler-
ance to facilitate organ or cellular transplantation was immedi-
ately appreciated (2) but has not been clinically achieved. One such
strategy is in utero hematopoietic cell transplantation (IUHCT),
an approach that has, as of yet, unfulfilled promise for the treat-
ment of congenital hematologic disorders (11). The assumption
that fetal tolerance will be permissive of allogeneic IUHCT is a
primary rationale for this strategy and follows naturally from the
classic observations outlined above. The primary events required
for tolerance of self-antigen occur in the developing thymus and
consist of positive- and negative-selection events that result in the
clonal deletion of developing T cells with high-affinity recognition
of self-antigen as well as the maintenance of a repertoire of T cells
reactive to foreign antigen. The assumption has been that intro-
duction of allogeneic cells by IUHCT, prior to completion of the
thymic processing of self-antigen, would mimic self-antigen and
result in clonal deletion of alloreactive lymphocytes and secondary
permanent donor-specific tolerance.
We recently demonstrated in a murine model of IUHCT that
there is an unequivocal and dramatic difference in the frequency
of engraftment in allogeneic compared with congenic recipients
(12). This observation strongly suggests the presence of an adap-
tive immune response as a barrier to engraftment after IUHCT
and challenges the assumption of fetal tolerance as a facilitator
of IUHCT. If the observed difference in frequency of chimerism is
due to an adaptive immune response, we hypothesized that chi-
meric and non-chimeric recipients of allogeneic IUHCT would
have quantitative differences in their allospecific humoral and
effector T cell response. In the present study, we confirm the
presence of an adaptive immune response in murine allogeneic
recipients of IUHCT that lose their chimerism after IUHCT and
the absence of that response in animals that maintain hemato-
poietic chimerism. Unexpectedly, we also demonstrate a mater-
nal immune response after IUHCT that appears after delivery of
the pups. Furthermore, we show that the immune response in
the recipients is entirely dependent on breast feeding from the
immunized mother, and that the period of loss of chimerism
corresponds to the appearance of maternal alloantibodies. We
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 119:2590–2600 (2009). doi:10.1172/JCI38979.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 9 September 2009
further show that transfer of maternal serum to fostered pups
is sufficient to induce loss of chimerism, supporting an indi-
rect mechanism by which transfer of maternal alloantibodies in
breast milk induces a postnatal, allospecific immune response in
the chimeric pup. Finally, we show that non-fostered pups that
maintain their chimerism have higher levels of Tregs, as well as a
more suppressive Treg phenotype, compared with their non-chi-
meric, non-fostered siblings. These findings explain the appar-
ent contradiction of activation of an immune response in the
pre-immune fetal recipient and confirm, in the absence of mater-
nal immunization, the potential efficacy of strategies based on
actively acquired tolerance for facilitation of allogeneic cellular
or organ transplantation.
Non-chimeric pups exhibit an increased frequency of alloreactive T cells. To
elucidate the role of an effector T cell response in the loss of allo-
geneic chimerism after IUHCT, we performed allogeneic IUHCT
at E14 from MHC-H2Kb+GFP+ C57BL/6TgN(act-EGFP)OsbY01
mouse donors (referred to as “B6GFP mice”) into MHC-H2Kd+
BALB/c recipient fetuses. In this model, we have previously dem-
onstrated that while all recipients are initially engrafted, 70% of
recipients lose their donor chimerism between 2 and 4 weeks of
age (5 weeks after IUHCT) (12). We confirmed our previous find-
ings of 100% chimerism at 2 weeks of age. In a separate cohort of
recipients, peripheral blood hematopoietic chimerism was deter-
mined by flow cytometry at 4 weeks of age, and the pups were
divided into 2 groups, chimeric and non-chimeric (Figure 1). The
effector T cell response to donor cells was measured in each group
using the in vivo mixed lymphocyte reaction (MLR) (13). While
quantitative, this method does not provide a true frequency of
alloreactive cells, but rather allows a sensitive and quantitative
assessment of the relative frequency of allospecific T cells between
2 comparable groups. After determination of chimerism, lympho-
cytes from BALB/c recipient mice were harvested, stained with
CFSE dye, and adoptively transferred to CB6F1 (H2Kb+/d+) recipi-
ents, generating a parent into F1 MHC mismatch in which BALB/c
T cells allospecific for MHC-H2Kb+GFP– C57BL/6 (referred to as
“B6”) donor cells would proliferate against the F1 host. F1 recipi-
ents were euthanized at 24, 48, 72, and 96 hours after the adoptive
transfer, and CFSE-stained CD4+ H2Kb– lymphocytes were ana-
lyzed by flow cytometry. Cell division began after 24 hours fol-
lowing adoptive transfer, and maximal discernible proliferation
consisting of 8 rounds of cell division was present by 96 hours
(Figure 2, A and B). To evaluate background proliferation, BALB/c
lymphocytes were adoptively transferred into syngeneic BALB/c
recipients and demonstrated negligible proliferation (0.02%) in
this system (data not shown). The relative frequency of alloreac-
tive T cells was calculated at 96 hours (13) (see Methods), and
the frequency of alloreactive T cells in non-chimeric mice was
6.32% ± 0.92% as compared with 1.63% ± 0.66% in chimeric mice
(P = 0.006), indicating the presence of an allospecific cellular
response in the non-chimeric pups (Figure 2C). Lymphocytes
from naive and B6-immunized BALB/c mice were also assayed
as negative (4.47% ± 0.45%; n = 5) and positive (8.57% ± 0.33%;
n = 5) controls, respectively. These data are consistent with the
expected frequency of between 1% and 10% of peripheral T cells
that recognize an alloantigen (14). Alloreactive lymphocytes from
chimeric pups were also present at a significantly lower frequency
than were alloreactive lymphocytes from naive pups (P < 0.001),
suggesting a mechanism of partial clonal deletion of allospecific
lymphocytes with associated donor-specific tolerance.
Peripheral blood chimerism levels at 2 and 4 weeks of age. Donor cell
chimerism was assessed as the percentage of CD45+ cells that were
GFP+ by flow cytometry, with chimerism being defined as more than
1% GFP+. Data are mean ± SEM.
T cell alloreactivity in vivo. Flow
cytometry of adoptively transferred
CD4+H2Kb– CFSE+ lymphocytes
from (A) chimeric pups and (B)
non-chimeric pups. Each tracing
is representative of 5 independent
experiments. (C) Frequency of
alloreactive T cells in naive (nega-
tive control), immunized (positive
control), chimeric, and non-chime-
ric BALB/c pups 5 weeks after B6
IUHCT. Data are mean ± SEM.
2592? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 9 September 2009
Analysis of CD8+ lymphocytes revealed a higher frequency of
alloreactive lymphocytes with a CD8+/CD4+ cell ratio of 2:1 but a
similar pattern of proliferation on in vivo MLR. To confirm that
our analysis included all of the transferred alloreactive cells, we
analyzed all of the hematopoietic compartments of the assay mice
and found that the CFSE+ cells were restricted to the spleen and
lymph nodes, with no CFSE+ lymphocytes detected in the periph-
eral blood, bone marrow, and thymus (Supplemental Figure 1; sup-
plemental material available online with this article; doi:10.1172/
JCI38979DS1). We also measured serum cytokine levels in non-
chimeric pups relative to those in naive pups and IUHCT moth-
ers. While levels of IL-4 were undetectable in all groups, levels of
IL-2, IL-10, and IFN-γ were elevated in the non-chimeric pups and
immunized mothers compared with naive controls (P < 0.01), with
the greatest difference seen in IFN-γ, thus indicating a predomi-
nantly Th1 profile in the non-chimeric
pups (Supplemental Figure 1). To assess
the potential contribution of anergy or
suppression to our findings of toler-
ance in fostered and non-fostered chi-
meric pups, we assessed reactivity to
third party donors and the effect of the
addition of IL-2 to in vitro MLR. Both
groups demonstrated normal reactivity
to third party cells, excluding anergy as
a mechanism for tolerance. However,
donor-specific tolerance to B6 cells was
partially overcome with the addition of
IL-2, supporting the presence of sup-
pression by regulatory populations (15)
(Supplemental Figure 2).
Non-chimeric pups have circulating alloan-
tibodies. For assessment of the humoral
immune response, serum was isolated
from 4-week-old chimeric and non-chi-
meric BALB/c pups after IUHCT. After
incubation of serum with B6 target cells
and subsequent staining with anti-IgG
secondary antibody, the magnitude of
the humoral response was determined
by the mean anti-IgG fluorescence
intensity using flow cytometry. Naive
BALB/c serum and serum from B6
immunized BALB/c mice were used as negative and positive con-
trols, respectively, and showed a significant difference (P < 0.01) at
all serum to target cell ratios (Figure 3A). At a 1:1 ratio of serum to
target cells, the fold increase in anti-IgG immunofluorescence was
9.50 ± 3.02 (n = 20) in non-chimeric mice as compared with 1.00 ± 0.04
(n = 10) in chimeric mice (P < 0.001), demonstrating clear evidence
of anti-donor alloantibody formation in the non-chimeric pups
compared with the chimeric pups (Figure 3B).
Injected dams are sensitized against allogeneic donor cells. The pres-
ence of an immune response in non-chimeric pups suggested
that either the fetus was not tolerant or the maternal immune
system had been sensitized and had directly or indirectly caused
the loss of chimerism in the pups. We therefore analyzed injected
dams for evidence of sensitization to donor cells after IUHCT.
We utilized the in vivo MLR assay described above to assess the
Alloantibody assay in chimeric and non-chimeric
mice. (A) Control mice showed a significant dif-
ference in alloantibody formation at all ratios of
serum to target cells. (*P < 0.01). (B) Non-chime-
ric BALB/c pups (n = 20) showed evidence of allo-
antibody formation as compared to chimeric pups
(n = 10) at ratios of 1:1, 1:2, and 1:10 (**P < 0.001).
Data are mean ± SEM.
Maternal T cell alloreactivity in vivo and frequency of alloreactive T cells. (A) Flow cytometry of
CD4+H2Kb– CFSE+ lymphocytes. (A and B) Histogram overlay representing CFSE profile at the
24-hour time point (gray histogram) and 96-hour time point (white histogram) for (A) naive (nega-
tive control) and (B) immunized (positive control). (C) Histogram overlay for injected dams. (D)
Frequency of maternal alloreactive T cells (11.13% ± 1.74%) compared with positive and negative
controls. Data are mean ± SEM. Each tracing represents at least 5 independent experiments.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 9 September 2009
frequency of alloreactive maternal T cells and found that there
was no statistically significant difference in the frequency of allo-
reactive T cells between injected dams and immunized positive
controls (Figure 4), which was consistent with maternal sensiti-
zation by donor cells after IUHCT.
We next analyzed the maternal humoral response. Alloantibod-
ies were found in maternal serum but did not appear until approx-
imately 2 weeks after IUHCT, when the pups were 1 week of age
(Figure 5). This suggested that if in fact the maternal immune sys-
tem was causative in the adaptive immune response in the pups,
this response would occur during the postnatal period, implicat-
ing maternal breast milk as a likely mode of transmission. Inter-
estingly, antibody levels increased dramatically between 2 and
5 weeks after IUHCT, which corresponds with the time period
when chimerism is lost (12). Upon analysis of anti-B6–specific
maternal alloantibody by class and subclass, we saw a predomi-
nance of IgG2a and, to a lesser extent, IgM and IgG2b (Figure
6). This correlated with the anti-B6–specific alloantibody profile
in non-chimeric pups and suggested a mechanism of maternal-
fetal antibody transmission, as IgG2a and IgG2b are known to be
preferentially transferred in rodent breast milk (16). Additionally,
we found that the magnitude of the maternal humoral response
strongly influenced the loss of chimerism in pups, as non-chi-
meric pups, on average, were found to have been exposed to sig-
nificantly higher levels of maternal alloantibodies than chimeric
pups (P = 0.0002) (Supplemental Figure 3).
The 2 most likely sources of maternal sensitization are leakage of
donor cells into the maternal peritoneal cavity during injection and
exposure to injected cells upon re-absorption of aborted fetuses. By
keeping all injected dams in separate cages and counting the num-
ber of fetuses that were injected and then subsequently born, we
were able to gain some insight into the mechanism of maternal sen-
sitization. Statistically significant correlations were noted between
the magnitude of the maternal humoral response and both the
number of aborted fetuses per litter (n = 24, r = 0.547, P = 0.0057)
and the total number of fetuses injected per litter (n = 23, r = 0.546,
P = 0.0070) (Supplemental Figure 4). Only 3 of 24 litters were born
without fetal loss, and 2 of those 3 injected dams still developed
alloantibodies, suggesting that while fetal loss is strongly associ-
ated with maternal immunization, it is not required.
To better define this relationship, we next performed a series of
experiments designed to differentiate between the effects of i.p.
leakage of donor cells and re-absorption of donor cells within
aborted fetuses. To explore the effect of i.p. leakage, we performed
a midline laparotomy and then injected an appropriate volume of
B6 donor cells onto the uterine horns to mimic leakage of either
10% or 25% of the volume normally injected during our standard
IUHCT. To test exposure to fetal antigens during fetal loss, we
utilized an F1 model (BALB/c female crossed with B6 male), in
which we performed a midline laparotomy without IUHCT, with
transient occlusion of the uterine blood supply, resulting in mis-
carriage of 10 of 10 fetuses. Upon analyzing maternal serum for
evidence of anti-B6 alloantibodies, we found that both i.p. leakage
of donor cells and exposure to fetal antigens during fetal loss were
sufficient to generate maternal alloantibodies (Figure 7). There-
fore, the source of maternal immunization appeared to be the
combined effect of leakage of cells into the maternal peritoneal
cavity during IUHCT and reabsorption of cells following fetal loss.
This mechanism is consistent with previously described mecha-
nisms of reproductive immunology in which paternal antigens are
able to induce a pathologic immune response (17, 18).
Postnatal exposure to maternal breast milk results in loss of chimerism.
Having shown that the injected dams are sensitized against donor
cells during IUHCT, we next attempted to ascertain whether the
immune response in pups was in fact induced by maternal influ-
ence or whether both mother and fetus had been independently
sensitized. The timing of the maternal humoral response allowed
isolation of the pups from postnatal maternal influence through
the use of non-injected foster dams. Upon substitution of non-
injected foster dams, we found that 100% (22/22) of fostered pups
maintained their chimerism (Table 1), with stable levels of chime-
rism 6 months after IUHCT (Supplemental Figure 5). In contrast,
only 30.4% (21/69) of pups nursed by dams injected on E14 main-
tained their chimerism at 5 weeks after IUHCT, consistent with
previous data (12). This striking result demonstrates that in the
absence of a maternal immune response, fetal immunologic toler-
ance is uniformly permissive of hematopoietic engraftment across
MHC barriers after IUHCT and definitively identifies postnatal
exposure to maternal breast milk as a requirement for loss of chi-
merism. Immunologic analysis of the fostered pups confirmed an
Maternal alloantibody assay. Median anti-IgG immunofluorescence
representing maternal alloantibodies at 1, 2, and 5 weeks after IUHCT.
Data are median ± SEM.
Alloantibody class and subclass. Serum from injected BALB/c dams
and non-chimeric BALB/c pups was analyzed for anti-B6 alloanti-
body at the time of loss of chimerism (5 weeks after IUHCT). Data
are median ± SEM.
2594?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 9 September 2009
absence of alloantibodies as well as donor-specific tolerance, as
represented by a frequency of alloreactive T cells of 0.81% ± 0.33%
(n = 5), which was significantly lower than that seen in naive mice
(P = 0.0002), again consistent with at least partial clonal deletion
of donor-reactive cells. The mean level of chimerism in fostered
pups was 30.96% (range, 10.24%–73.96%), which was not signifi-
cantly different from the level of chimerism in chimeric pups
nursed by dams injected on E14.
To further confirm the findings of the fostering experiment, we
bred BALB/c males with B6 females and performed E14 IUHCT
using B6GFP donor cells. Because donor cells were of maternal ori-
gin, we hypothesized that there would be no maternal sensitization
and that, similar to the fostering experiment, all of the pups would
maintain their chimerism. On analysis, we found no evidence of
maternal antibody formation or T cell alloreactivity, and all pups
(20/20) were chimeric (Supplemental Figure 5). This finding fur-
ther supported our conclusion that the donor cells were responsible
for maternal immunization and that it was the maternal immune
response that caused loss of chimerism in the pups.
Maternal lymphocytes are not required for loss of chimerism. There are
2 possible explanations for our data: the maternal immune cells
might be directly involved in ablation of chimerism, or transfer of
maternal antibodies might indirectly induce the immune response
in the pups. We hypothesized that if maternal cells are directly par-
ticipating in donor cell rejection, then maternal cells should be
present in the lymphocyte population from non-chimeric pups and
should proliferate in an in vitro MLR assay. To allow discrimina-
tion of maternal cells in this assay, we bred BALB/c females with B6
males and performed IUHCT on E14 using B6GFP donor cells. We
then isolated lymphocytes from the spleen, lymph nodes, periph-
eral blood, bone marrow, and thymuses of non-chimeric 4-week-old
pups after IUHCT and performed an in vitro MLR assay using CFSE
labeling to assess proliferation against irradiated B6 stimulator cells.
The utilization of the F1 model allowed us to gate on only H2Kb–
cells, excluding all cells except those of maternal origin. Congenic
B6 cells were used as a negative control and showed no evidence of
bystander proliferation (Figure 8A). Our data show proliferation by
CD4+H2Kb+/d+ T cells from non-chimeric pups (Figure 8B) as well as
proliferation by CD4+H2Kb– T cells from dams injected on E14 (Fig-
ure 8C). However, we found no proliferation of H2Kb– cells in the
non-chimeric pups (Figure 8D), showing that maternal-fetal cellular
trafficking is minimal and direct maternal cell-mediated ablation of
donor cells is not responsible for loss of chimerism. Examination of
the bone marrow, peripheral blood, lymph node, and thymus com-
partments excluded sequestration of maternal cells, as these tissues
also lacked H2Kb– lymphocytes (Supplemental Figure 6).
Although this experiment suggested that primary ablation of
donor cells by maternally derived lymphocytes was unlikely, it
did not rule out the possibility that a low frequency of maternally
derived lymphocytes were serving in a costimulatory or antigen-
presenting capacity. Therefore, in order to prove that transfer of
maternal lymphocytes was not required for immune activation, we
isolated serum from injected dams and administered it to fostered
pups after IUHCT. Interestingly, i.p. administration of maternal
serum resulted in a 50% frequency of chimerism (3/6 pups chi-
meric), while oral administration of maternal serum resulted in
complete loss of chimerism (0/3 pups chimeric) (Table 2). These
experiments established the fact that serum alone was capable
of transferring the maternal immune response to pups and that
transfer of maternal cells was not required.
CD4+CD25+ Tregs are more prevalent in non-fostered chimeric pups and
exhibit a more suppressive phenotype. The existence of a maternally
derived immune response still failed to explain the fact that many
litters contained both chimeric and non-chimeric pups, despite
ingestion of the same breast milk. Although previous studies had
shown a correlation between the level of maternal humoral response
and a loss of chimerism, this pattern alone does not explain how
some pups in a litter can be chimeric while others in the same lit-
ter are non-chimeric. We hypothesized that enhanced peripheral
regulatory mechanisms controlled by Treg populations resulted in
suppression of the alloimmune response in these mice. To investi-
gate this hypothesis, we first measured the levels of Tregs in naive,
fostered, and non-fostered chimeric and non-chimeric pups, and
we found that the non-fostered chimeric pups had higher levels of
CD4+CD25+ Tregs (P < 0.001) in all tissue compartments except the
spleen (P = 0.06) (Figure 9). These cells were confirmed by FACS to
be more than 85% FoxP3+. We next analyzed the functional sup-
pressive ability of these cells in vitro, and we found that the non-
fostered chimeric Tregs exhibited an enhanced ability to suppress
effector T cell alloimmune reactivity (P = 0.001), as compared with
naive, fostered, and non-fostered non-chimeric Tregs (Figure 10).
Given the current understanding of the mechanistic basis of
fetal tolerance, one would anticipate that with appropriate tim-
ing and mode of antigen administration, consistent donor-spe-
cific tolerance would be achievable by a primary mechanism of
clonal deletion of donor-reactive lymphocytes. However, histori-
cally that has not been the case. Even in Billingham, Brent, and
Maternal immunization. Evidence of anti-B6 alloantibodies in BALB/c
dams that did not undergo IUHCT, after i.p. injection of 10% or 25% of the
standard B6 IUHCT cell dose, as well as one F1 dam (BALB/c female ×
B6 male) following fetal loss and re-absorption of 10 F1 fetuses.
Effect of maternal breast feeding on the frequency of chimerism
Non-injected foster dam
Mice were analyzed 5 weeks after IUHCT.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 9 September 2009
Medawar’s original report (2), only 3 of the 5 CBA strain mice
born after prenatal injection of cells at E15 from A strain mice
demonstrated evidence of tolerance. They concluded from that
experiment and their larger experience with many injections in
fetal and neonatal mice that “the conferment of tolerance is
not of an all-or-nothing character; every degree is represented.”
Subsequent studies by many investigators on fetal and neona-
tal tolerance have documented the entire spectrum of immune
response from donor-specific tolerance to immunization. Part
of that confusion can historically be explained by the lack of rec-
ognition of mechanistic differences between fetal and neonatal
tolerance in mice. Thus, observations made in neonatal mice
demonstrating the ability to mount mature immune responses
given an appropriate presentation of antigen have been used as
an argument against the validity of actively acquired tolerance
(19–21). These findings, as well as observations of immunization
in large animal models (22, 23) and humans (24) after relatively
late administration of antigen, can be easily attributed to miss-
ing the window of opportunity for central tolerance induction.
More difficult to explain are failures of engraftment and associ-
ated donor-specific tolerance induction after IUHCT when the
procedure has been performed at an early developmental time
point (prior to the emergence of mature lymphocytes in the
thymus and peripheral circulation), when one would anticipate
appropriate thymic processing of antigen. In the mouse, this
period exists prior to E17 (25). In the murine model of alloge-
neic IUHCT, when transplants have been performed at E14–E15,
numerous studies have reported failure of engraftment or of only
microchimerism (26–34), inconsistent or absent tolerance induc-
tion (26, 31, 35), or immunization to alloantigen after IUHCT
(30, 33). Similarly, studies of organ transplantation after IUHCT
with minimal levels of chimerism in large animal studies have
shown an absence of tolerance (36), incomplete tolerance (37), or
donor-specific tolerance (38). Interpretation of the results from
both the murine and large animal studies has been complicated
by marginal levels of engraftment or concerns about inconsistent
delivery of donor cells to the fetus.
The most convincing argument that engraftment in the fetus is
not limited by an immune barrier was the early observation in the
murine model by ourselves and others that there was no signifi-
cant engraftment advantage for congenic versus allogeneic cells
(28, 31, 34, 39). However, in retrospect, those studies were mislead-
ing because of the minimal levels of chimerism present and a low,
and sometimes transient, frequency of chimerism, which made
interpretation difficult. We were able to overcome the low levels of
chimerism in the model by increasing the number of donor cells
to achieve consistent levels of chimerism easily measurable by flow
cytometry. With higher levels of chimerism we demonstrated con-
sistent association of donor-specific tolerance with levels of donor
hematopoietic chimerism of greater than 1%–2% as determined by
skin grafting or the ability to boost postnatal engraftment with a
minimal conditioning bone marrow transplant from the donor
strain (40–42). Chimerism and tolerance were associated with
reduced frequencies of donor-specific lymphocytes, consistent with
a mechanism of partial clonal deletion, supporting the absence of
an adaptive immune barrier to IUHCT (26, 41). However, although
Analysis of the origin of alloreactive lymphocytes in non-chimeric pups. (A) CD4+H2Kb+ lymphocytes were harvested from congenic B6 mice.
(B) CD4+H2Kb+ lymphocytes were harvested from F1 neonatal recipients. (C) CD4+H2Kb– lymphocytes were harvested from E14 injected
BALB/c dams. (D) A negligible number of H2Kb– maternal cells were derived from F1 recipients. Each histogram is representative of at least
8 independent experiments.
2596? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 9 September 2009
higher cell doses increased the level of chimerism in chimeric pups,
it did not increase the frequency of chimerism, and we could not
explain why, despite consistent injection techniques, a minority of
the recipients were chimeric. We therefore reexamined congenic
versus allogeneic engraftment using much higher cell doses, which
were permitted by an intravascular injection technique (43), and
performed tracking experiments following engraftment at early
and late time points (12). This study revealed a marked differ-
ence in the frequency (but not the level) of chimerism in congenic
versus allogeneic recipients and made the clear observation that
all animals were initially engrafted (confirming equal delivery of
donor cells), but that engraftment was lost between 2 and 4 weeks
of age in most of the allogeneic recipients, which strongly suggests
that there was in fact an immune barrier to IUHCT, contradicting
the assumption that actively acquired tolerance could facilitate
The results of this study explain this apparent contradiction
and provide a mechanism for the inconsistencies observed in
murine studies of IUHCT. Specifically, the ability to achieve a
100% frequency of chimerism through the substitution of non-
injected foster dams or the use of maternally derived donor cells
confirms that it is maternal immunization, rather than a fetal
immune barrier, that results in loss of engraftment after IUHCT.
Furthermore, this effect can be reproduced by postnatal oral or
i.p. administration of maternal serum alone and is mediated by
recipient effector T cells rather than those of the mother. These
observations support a mechanism whereby passive transfer of
maternal alloantibodies via breast milk induces a postnatal cel-
lular and humoral immune response in the recipient.
Traditionally, maternal-fetal antibody transmission has been
thought to act transiently, through direct binding of maternal
antibody to fetal antigen, however there is increasing evidence that
maternal antibody is capable of inducing a durable and pathogenic
T cell response. In the mouse, the bulk of passively acquired mater-
nal antibody is derived from breast milk (44). Antibody transport
occurs at the level of the neonatal duodenum and jejunum, where
enterocytes expressing a surface membrane receptor (FcγR) bind
the Fc region of IgG and facilitate transcytosis of immunoglobu-
lins (16).There have been a number of recent studies documenting
de novo T cell–mediated immune responses triggered by maternal
antibodies. Greeley et al. (45) demonstrated prevention of diabetes
in NOD mice through the elimination of maternal autoantibodies,
establishing a direct connection between maternal-fetal antibody
transmission and T cell–mediated autoimmune disease in prog-
eny. Setiady et al. (46) more recently demonstrated transmission of
autoimmune ovarian disease via the same pathway, whereby mater-
nal autoantibodies induce a pathogenic neonatal T cell response.
A mechanism connecting humoral and cellular responses
involves immune effector cells that express FcγRs (47). This family
of receptors has activating and inhibitory functions and varies in
its distribution within monocytes, macrophages, and neutrophils,
which may display activating or inhibitory Fcγ receptors. NK cells
only express the activating FcγIIIaR. In vitro studies have shown
that antibody can facilitate the uptake of its cognate antigen into
APCs and that antibodies are also capable of activating antigen-
specific T cells through the interaction of the immune complex
and FcγR on dendritic cells (48–50). Additionally, the epitope
specificity of a given antibody has been shown to influence the
specificity and magnitude of the T cell response induced by that
antibody (51). Finally, an allospecific antibody can directly activate
NK cells via the mechanism of antibody-dependent cell-mediated
cytotoxicity (ADCC). Therefore, the possible consequences of the
passive transfer of maternal allospecific antibody may include
direct antibody cytotoxicity, ADCC, antigen-antibody complex
processing by APCs with immune activation of T cells, and inflam-
mation, which would enhance antigen presentation and a cascade
of other signals driving an adaptive immune response.
In this study we have not formally ruled out the innate immune
system as a potential barrier to engraftment. Recent studies of both
autoimmune disease (52) and allogeneic IUHCT (53) have estab-
lished that murine neonatal NK cells are not only functional but
also important for modulation of T cell reactivity. In the context
of IUHCT, NK cells have been implicated in loss of minimal chime-
Ability of immunized maternal serum to induce loss of chimerism
Non-injected foster dam
Non-injected foster dam
plus i.p. serum from
Non-injected foster dam
plus oral serum from
0% (0/3) 100% (3/3)
Mice were analyzed 5 weeks after IUHCT.
Treg distribution. (A) CD4+CD25+ lymphocytes were
shown to be more than 85% FoxP3+. (B) The per-
centage of CD4+CD25+ lymphocytes was measured
in peripheral blood (PB), bone marrow, lymph nodes
(LN), spleen, and thymus. The percentage of chimeric
cells was significantly higher (P < 0.01) in all compart-
ments except the spleen (P = 0.06). The histogram is
representative of at least 5 independent experiments.
Data are mean ± SEM.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 9 September 2009
rism (<1.8%) despite the presence of T cell tolerance. Our findings
of transfer of maternal alloantibodies would suggest that a possible
mechanism for this observation is activation of donor MHC class
I–specific NK cells by maternal alloantibody via an ADCC mecha-
nism in a milieu of low frequencies of donor cells. Levels of chime-
rism in our model of intravascular injection far exceed the thresh-
old level of initial chimerism postulated to be required for host NK
cell tolerance and subsequent durable engraftment (53). The fact
that 100% of our foster-reared pups maintained stable engraftment
demonstrates that fetal NK cells are not a barrier to the levels of
engraftment seen in this study and supports previous data (54, 55)
suggesting that the milieu of high levels of donor cells during NK
cell development may modify their receptor profile and reduce the
frequency of donor-reactive NK cells, negating their effect.
An intriguing initial observation in this study was the fact that
pups not reared by foster mothers that lose their chimerism were
often exposed to the same breast milk as pups that remained chi-
meric. One explanation is that the magnitude of the maternal
immune response, and therefore the dose of alloantibody trans-
ferred, determines the likelihood of chimerism. At the extremes
this appears to hold true. Two dams that underwent IUHCT
and had minimal humoral response delivered pups that main-
tained chimerism, whereas high levels of humoral response were
uniformly associated with no chimeric pups. Statistically, there
appears to be a negative correlation between level of maternal
antibody and chimerism in pups, whether analyzed by individual
pup or by comparing litters with either 0 or at least 1 chimeric
pup. However, the magnitude of an individual mother’s response
was not an explanation for how individual pups within the same
litter could be chimeric or non-chimeric.
Self-reactive T cells are known to escape thymic deletion in sig-
nificant numbers due to inadequate or late presentation of anti-
gen in the thymus, and to be controlled by regulatory mechanisms,
including Treg populations, which are essential for the prevention
of autoimmune disease (56, 57). It is also known that maternal-fetal
cell trafficking in humans results in the generation of tolerogenic
fetal Tregs (58). This suggested that donor cells would induce Tregs
in our chimeric pups and that these would potentially counteract
a low-level alloimmune response. Therefore, we examined the level
and suppressive capacity of CD4+CD25+ Tregs in each group of
pups and found that there does appear to be a more robust Treg
response in the non-fostered chimeric pups. Our data support the
hypothesis that after IUHCT, tolerance occurs by a primary mecha-
nism of clonal deletion that is supplemented by the generation of
Tregs to suppress donor-reactive cells that escape thymic deletion.
In our fostered pups, this mechanism is uniformly successful in
maintaining a tolerant state. However, the transfer of allospecific
antibodies induces an allogeneic response that may overwhelm
Treg suppression, resulting in a loss of engraftment. We speculate
that in the context of maternal immunization and breast-feeding, it
is the balance of immune-activating and regulatory influences that
determines whether a given pup remains chimeric.
Finally, although we view the identification and characterization
of this immune response to be potentially critically important to
overcoming the barriers to successful IUHCT, the importance of
host cellular competition is not to be overlooked. Indeed, despite
the delivery of what would be considered massive doses of donor
cells (2 × 1011 cells/kg fetal weight) in fostered allogeneic recipi-
ents or in the congenic model, levels of donor chimerism can be
variable and, in many animals, low (12). In fact, our current view
from studies in the murine model is that the level of engraftment
is limited by donor cell dose, donor cell competitive capacity, and
host cell competition. However, given an adequate dose of donor
cells, it appears that the frequency of allogeneic engraftment (or
the corollary, loss of engraftment) is a function of the adaptive
immune barrier as characterized in this study.
The applicability of these findings to other species and specifi-
cally humans remains a major question. We recognize that there are
species-specific differences in gestational length and maternal-fetal
lymphocyte and antibody trafficking that could result in an entirely
different sequence of events after clinical IUHCT. If maternal sen-
sitization is in fact relevant to the outcome of human IUHCT, it
likely occurs via a different mechanism. In humans, antibodies in
breast milk do not enter the neonatal circulation because gut clo-
sure occurs precociously (16). In light of the longer gestation period
and the fact that the murine FcγR is similar to the placental recep-
tor responsible for active placental transfer of IgG in humans, the
more likely route of transmission in large animal models is via the
placenta during the late second and third trimesters of pregnancy.
Whether maternal sensitization occurs in humans and whether this
would result in pre- or perinatal rejection of donor cells via direct or
indirect mechanisms needs to be investigated in relevant large ani-
Treg suppression assay. (A) CD4+CD25+
Tregs and CD4+CD25– effector T cells
were sorted. (B and C) Controls con-
sisted of (B) unstimulated and (C)
stimulated effector T cells. (D and E)
Suppression was measured after a 1:1
addition of (D) non-chimeric and (E)
chimeric Tregs. Each histogram is rep-
resentative of at least 5 independent
experiments. (F) The percentage sup-
pression at a 1:1 Treg/effector T cell
ratio. Data are mean ± SEM.
2598? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 9 September 2009
mal models. In any case, an obvious strategy to avoid any potential
maternally derived immune barrier would be the use of maternal
donor cells when appropriate.
To our knowledge, this is the first study documenting mater-
nal immunization and its consequences after IUHCT. Multiple
mechanisms have been implicated that contribute to maternal-
fetal tolerance, both at the placental interface (59) and systemati-
cally (60). The important observation from this study is that, at
least in the context of IUHCT, one cannot assume that the normal
mechanisms responsible for maternal-fetal tolerance will prevent
a maternal immune response against donor cells. Future experi-
mental and clinical studies of allogeneic IUHCT need to consider
and assess the importance of the maternal immune response. Our
results may also have implications for prenatal gene therapy, in
which potentially immunogenic transgene or viral proteins are
injected by various routes into the fetus.
A second and equally important observation of this study is
that, in the absence of maternal influence, engraftment and long-
term chimerism uniformly occur across full MHC barriers. This
confirms the absence of an adaptive immune barrier in the pre-
immune fetus and validates the potential for practical applica-
tion of actively acquired tolerance to facilitate allogeneic cellular
and/or organ transplantation. Our findings may account for
much of the inconsistency in previous studies, including perhaps
the inconsistent tolerance observed in the classic study of Billing-
ham, Brent, and Medawar (2).
Mice. BALB/c (H2Kd+) mice time-dated at 14 days of gestation were used
as fetal IUHCT recipients. Six- to 8-week-old MHC-H2Kb+GFP+ C57BL/
6TgN(act-EGFP)OsbY01 mice were used as bone marrow donors (provided
by M. Okabe, Genome Information Research Center, Osaka University,
Osaka, Japan). Adult CB6F1 (H2Kb+/d+) mice were used as in vivo MLR hosts.
C57BL/6 (referred to as B6 - H2Kb+, GFP–) cells were used as MLR stimula-
tors. Except as noted, animals were purchased from Charles River Labora-
tories and bred in our colony as previously described (12). The experimental
protocols were approved by the Institutional Animal Care and Use Commit-
tee at The Children’s Hospital of Philadelphia and followed guidelines set
forth in the NIH Guide for the Care and Use of Laboratory Animals.
Flow cytometry. Fluorochrome-labeled mAbs and isotype controls were
purchased from BD — Pharmingen. Anti-mouse Ig secondary antibodies
were purchased from Jackson ImmunoResearch Laboratories Inc. Nonspe-
cific FcγR binding was blocked by the mAb against mouse FcγR 2.4G2.
Conjugated mAbs with irrelevant specificities served as negative controls.
Propidium iodide staining was used to exclude dead cells in dual color flow
cytometry. Flow cytometry was performed on a FACSCalibur, and cell sort-
ing was performed on a FACSAria (Becton Dickinson).
In utero transplantation. Whole donor bone marrow was harvested from
6- to 8-week-old B6GFP mice. Low-density mononuclear cells were sepa-
rated by Ficoll gradient centrifugation and resuspended at a concentra-
tion of 1 × 106 cells/μl. On day E14 of gestation, a midline laparotomy
was performed under isoflurane anesthesia and the uterine horns were
exposed. The vitelline vein was identified with a dissecting microscope,
and each fetus was injected with 2 × 107 whole bone marrow cells (20 μl
of cells at a concentration of 1 × 106 cells/μl). A successful intravenous
injection was confirmed by visualization of clearance of the blood in the
vein by the injectate and the absence of extravasation at the site of injec-
tion. The uterus was returned to the maternal peritoneal cavity and the
abdomen closed with 2 layers of absorbable 4-0 Vicryl suture. To determine
chimerism status, peripheral blood from E14 injected mice was obtained at
4 weeks of age by retroorbital puncture. Mononuclear cells were isolated
by Ficoll gradient centrifugation. Donor cell chimerism was assessed as the
percentage of CD45+ cells that were GFP+ by flow cytometry, with chime-
rism being defined as more than 1% GFP+. Analysis of allogeneic donor cell
chimerism using the H2Kb marker after IUHCT of B6GFP (H2Kb+) cells
into BALB/c (H2Kd+) fetuses demonstrated good correlation with GFP
expression, confirming that GFP reliably represented all donor cells pres-
ent in chimeric mice.
T cell alloreactivity in vivo. Spleen and lymph nodes (axillary, inguinal, cer-
vical, para-aortic, and mesenteric) were harvested from 4-week-old, E14
injected chimeric and non-chimeric BALB/c mice, and mononuclear cells
were isolated using ACK lysing buffer. Positive control BALB/c mice were
immunized with an i.p. injection of approximately 2 × 107 B6 cells at days
0 and 7, and cells were harvested on day 14. Mononuclear cells from each
BALB/c mouse were stained with CFSE dye and injected into the tail vein
of a CB6F1 mouse (H2Kb+/d+). The spleen, lymph nodes, peripheral blood,
bone marrow, and thymus of the CB6F1 mouse was harvested at 24, 48, 72,
or 96 hours, and mononuclear cells were isolated and stained with anti-CD4
(L3T4, APC), anti-CD8 (Ly-2, PerCP), and anti-H2Kb (AF6-88.5, PE) anti-
bodies. The CD4+H2Kb– or CD8+H2Kb– cells were then analyzed for CFSE
fluorescence by flow cytometry, and the frequency of alloreactive T cells in
vivo was quantified as described by Suchin et al. (13). Briefly, the frequency
of alloreactive T cells (F) was defined as the number of cells that had divided
(Pdiv), divided by the total number of cells (Ptot). Ptot was further defined as
the number of cells that had successfully engrafted at 24 hours, and Pdiv
was further defined as ΣMn/2n–1, where Mn represents the number of cells
(M) in a given CFSE peak n, and n–1 represents the number of cell divisions
that those cells have undergone. For example, cells in the second CFSE peak
(n = 2) had, by definition, undergone 1 cell division, and therefore 100 cells
in this peak were derived from 100 / 22-1 original cells, or 50 cells. The total
number of original cells that had divided (Pdiv), divided by the total number
of original cells that have engrafted (Ptot), yielded the frequency (F).
T cell anergy assays and cytokine ELISAs. Lymphocytes were harvested from
non-fostered chimeric and fostered chimeric BALB/c spleens, CFSE stained,
and resuspended in RPMI-10 media in the presence of irradiated splenocytes
from BALB/c (congenic), B6 (donor allogeneic), or Swiss Webster (third-
party allogeneic) mice. Anti-CD3ε and anti-CD28 were added at concentra-
tions of 1.0 μg/ml to each tube, and recombinant mouse IL-2 was added at a
concentration of 10 ng/ml. For cytokine ELISAs, serum was obtained from
peripheral blood and analyzed for IL-2, IL-4, IL-10, and IFN-γ using mouse
Ready-SET-Go! ELISA kits (eBioscience).
Alloantibody assay. Peripheral blood from E14 injected chimeric and non-
chimeric BALB/c mice was obtained at 4 weeks of age by retroorbital punc-
ture, and serum was isolated via centrifugation. Positive control BALB/c
mice were immunized with an i.p. injection of approximately 2 × 107 B6 cells
at days 0 and 7, and serum was collected on day 14. Serum was also obtained
from maternal blood on P1, P8, and P28. Splenocytes were isolated from
adult B6 mice for use as allogeneic target cells, and 1 × 106 spleen cells were
pre-incubated with 5 μl of anti-CD16/32b Fc block and then incubated for
45 minutes with BALB/c serum at serum/splenocyte ratios of 1:100, 1:50,
1:10, 1:2, and 1:1. The cells were washed twice to remove excess serum and
then incubated for 45 minutes with secondary antibody against mouse IgG,
IgM, IgA, IgG1, IgG2a, IgG2b, or IgG3 to detect the presence of bound anti-
bodies. B cells were stained with anti-CD19 and excluded by gating because
these cells exhibit a large amount of nonspecific binding. Cells were analyzed
by flow cytometry for median fluorescence. Median fluorescence, represent-
ing the relative concentration of alloantibodies in serum, was compared with
negative control (no serum) and expressed as a fold increase.
Materno-fetal cell trafficking (in vitro MLR). BALB/c females were bred
with B6 males, and fetuses were injected with B6 donor cells according to
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 9 September 2009
standard IUHCT protocol. Spleens, lymph nodes, peripheral blood, bone
marrow, and thymuses were harvested from F1 pups at 4 weeks of age.
Lymphocytes were isolated by ACK lysis, CFSE stained, and resuspended in
RPMI-10 media at a concentration of 1 × 106/ml (responder cells). B6 lym-
phocytes were isolated from the spleen by ACK lysis, irradiated (2,000 rad),
and resuspended in RPMI-10 media at a concentration of 1 × 106/ml (stim-
ulator cells). From each population of cells, 500 μl was placed into a 5-cc
FACS tube and incubated for 72 hours. Anti-CD3ε (145-2C11, purified)
and anti-CD28 (37.51, purified) were added at a concentration of 1.0 μg/ml
to each tube. H2Kb–CD4+ cells were then harvested and analyzed by flow
cytometry for a CFSE profile.
Transfer of maternal serum to fostered pups. Standard IUHCT with intravenous
injection of B6GFP cells into E14 BALB/c fetuses was performed, and pups
were fostered at birth. Fostered pups then received maternal serum from dams
injected on E14 via either oral (100 μl pure serum twice per week for 4 weeks)
or i.p. (100 μl pure serum 3 times per week for 4 weeks) administration, and
they were analyzed for peripheral blood chimerism at 4 weeks of age.
Treg distribution and suppression. Peripheral blood, bone marrow, lymph
nodes, spleen, and thymuses were harvested from 4-week-old naive, chi-
meric, non-chimeric, and fostered BALB/c pups. Lymphocytes were iso-
lated and stained for Tregs with the Mouse Regulatory T Cell Staining
Kit 2 (eBioscience). For suppression assays, spleen and lymph nodes were
harvested and lymphocytes were sorted on a FACSAria for CD4+CD25+
Tregs and CD4+CD25– effector T cells. These cells were resuspended at a
concentration of 1 × 106 cells/ml in RPMI-10 media and then combined
at Treg/effector T cell ratios of 1:8, 1:4, 1:2, and 1:1. Effector T cells were
stimulated with irradiated B6 lymphocytes, and anti-CD3ε and anti-
CD28 were added at concentrations of 1.0 μg/ml to each tube. Effector T
cells were analyzed for a CFSE profile on a FACSCalibur. The percentage
suppression was calculated using the precursor frequency (PF) method
described by Brusko et al. (61). Briefly, percentage suppression = 100 ×
(1 – [PF (Treg + Teff) / PF (Teff)]). The precursor frequency was calculated
as described above, by analyzing the CFSE profile; PF (Treg + Teff) is the
effector T cell PF in the presence of Tregs; and PF (Teff) is the effector T
cell PF in the absence of Tregs.
Statistics. One-way ANOVA was used for analysis of the frequency of allore-
active T cells (Figure 2), Treg distribution (Figure 9), Treg suppression (Fig-
ure 10), and serum cytokine levels (Supplemental Figure 1). The Mann-Whit-
ney U test was used for analysis of the alloantibody assay because population
variance was not equal (Levene’s test, <0.05) (Figure 3). The Fisher’s exact
test was used for analysis of the frequency of chimerism (Tables 1 and 2)
because the data were categorical. The correlation between maternal humor-
al response and the numbers of injected and aborted fetuses was performed
by calculating the Pearson’s correlation coefficient (Supplemental Figure 4).
In the remaining experiments, the significance of differences among groups
was determined by use of the Student’s t test. Results with a P value less than
0.01 were considered to be significant. Calculations were performed using
SPSS version 15.0 (SPSS Inc.).
This work was supported by grant RO1 HL64715 from the NIH (to
A.W. Flake) and by funds from the Ruth and Tristram C. Colket Jr.
Chair of Pediatric Surgery and the Albert M. Greenfield Foundation
(to A.W. Flake). We thank Marcus Davies for his assistance with
the statistical analyses and Keith Alcorn and Christina Hughes for
assistance with breeding and technical procedures. We also thank
David Stitelman, Courtney Quinn, Aimee Kim, Todd Heaton, and
Jessica Roybal for their assistance with bone marrow harvests.
Received for publication February 19, 2009, and accepted in revised
form June 3, 2009.
Address correspondence to: Alan W. Flake, Department of Surgery,
Abramson Research Center, Room 1116B, 3615 Civic Center Blvd.,
Philadelphia, Pennsylvania 19104-4318, USA. Phone: (215) 590-
3671; Fax: (215) 590-3324; E-mail: firstname.lastname@example.org.
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