582? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 121? ? ? Number 2? ? ? February 2011
Maternal T cells limit engraftment after in utero
hematopoietic cell transplantation in mice
Amar Nijagal,1,2 Marta Wegorzewska,1,2 Erin Jarvis,1,2
Tom Le,1,2 Qizhi Tang,2 and Tippi C. MacKenzie1,2
1Eli and Edythe Broad Center of Regeneration Medicine and 2Department of Surgery, UCSF, San Francisco, California, USA.
Stem cell transplantation is a promising treatment strategy for
many genetic disorders such as hemoglobinopathies, immuno-
deficiencies, or inborn errors of metabolism, but clinical appli-
cations are limited by graft rejection and toxic immunosup-
pression. Transplantation of allogeneic stem cells into the early
gestational fetus offers an avenue to overcome this limitation.
Theoretically, introduction of allogeneic cells prior to the matu-
ration of the developing immune system may result in donor-
specific tolerance. Since the seminal experiments of Billingham,
Brent, and Medawar (1), animal models of in utero hematopoiet-
ic cell transplantation (IUHCTx) have shown that the fetal envi-
ronment offers considerable advantages for the success of stem
cell transplantation (reviewed in ref. 2). In the mouse model,
fetal mice can be tolerized to fully allogeneic stem cells without
any immunosuppression (3, 4). The treatment of hemoglobin-
opathies has also been achieved in mice using this approach (5).
These results have been confirmed in large animal models: fetal
lambs can accept xenogeneic human HSCs with long-term hema-
topoietic chimerism (6), and we have observed long-term engraft-
ment and tissue-specific differentiation of human mesenchymal
stem cells in sheep using this approach (7).
In spite of the results from animal models, the clinical success
of IUHCTx for congenital disorders has been hampered by poor
donor cell engraftment except in severe combined immunodefi-
ciency (8, 9), in which there is a clear survival advantage for trans-
planted cells without a host adaptive immune response. While
there are many potential barriers to engraftment, such as cell dose,
technique of transplantation, and lack of competitive advantage of
transplanted cells (10), the host immune system, even in the fetus,
may be an important barrier to consider. Even in the mouse model,
it has been described that not all transplanted fetuses ultimately
engraft with allogeneic cells, possibly secondary to a host immune
response (11). This observation has created a conundrum in the
field, since the fetus can also be tolerized to foreign antigens such
as non-inherited maternal antigens (NIMAs) during gestation
(reviewed in refs. 12, 13). One potential explanation, then, is that
it is the maternal, not fetal, immune response which is the real bar-
rier. In fact, it was recently reported that maternal alloantibodies,
transmitted postnatally via breast milk, impede engraftment after
transplantation of adult BM cells in fetal mice (14).
In addition to antibodies, there is considerable evidence that
maternal leukocytes cross into the fetus during gestation. Mater-
nal-fetal cellular trafficking (MFCT) is the bidirectional passage of
cells across the placenta, resulting in long-lived fetal cells in moth-
ers (15) and maternal cells in children (16). Although the func-
tional significance of this phenomenon is unclear, there is specula-
tion that microchimerism resulting from this trafficking may be
involved in the pathogenesis of autoimmune disease or in the tissue
response to injury (17, 18). In humans, the presence of microchi-
meric maternal cells in fetuses was recently shown to induce fetal
Tregs against NIMAs (19), suggesting that the fetus can develop
dominant tolerance to foreign antigens encountered during devel-
opment. However, the role of trafficking maternal leukocytes in
fetal transplantation has not, to our knowledge, been studied. It is
possible that they could instead induce an immune response that
limits the engraftment of cells transplanted into the fetus.
In this study, we examined the trafficking of maternal leukocytes
into the fetus and the role of the maternal immune system in limit-
ing the engraftment of in utero transplanted cells in mice. We report
that maternal T cells pose a significant barrier to engraftment after
IUHCTx and suggest that the clinical success of fetal transplanta-
tion may be improved by transplanting stem cells harvested from the
mother or by HLA-matching the transplanted cells to the mother.
Authorship?note: Qizhi Tang and Tippi C. MacKenzie are co–senior authors.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J Clin Invest. 2011;121(2):582–592. doi:10.1172/JCI44907.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 2 February 2011
The adaptive immune system limits engraftment after IUHCTx. We chose to
use a mouse model of in utero fetal liver (FL) transplantation, based
on previous publications showing increased engraftment with FL
compared with adult BM in the fetal environment (20). To validate
our model of IUHCTx, we first performed FL transplants in con-
genic fetuses by using C57BL/6.CD45.1 (designated CD45.1) mice
as donors and C57BL/6.CD45.2 (designated B6) mice as recipients.
Donor-derived cells were detected in the blood of transplant recipi-
ents by flow cytometry (Supplemental Figure 1A; supplemental mate-
rial available online with this article; doi:10.1172/JCI44907DS1).
The frequency of engraftment (chimerism level, >1%) at 4 weeks
after transplantation in congenic recipients was 82% (n = 17),
similar to what has been previously reported (11). Chimerism levels
were stable for 28 weeks, the last time point tested. Analysis of donor
and host T cell, B cell, and granulocyte composition in lymphoid
organs confirmed multilineage engraftment of donor cells (Figure 1),
indicating that using this stem cell source, injection technique, and
dose, engraftment is not limited by the availability of hematopoietic
niches in the recipient. Given that this method of IUHCTx generates
stable, mixed chimeras, we then used our model to focus on the role
of the immune system on engraftment.
Previous studies have suggested that the adaptive immune
response plays a role in limiting the engraftment of adult BM-
derived hematopoietic cells after in utero transplantation (11). To
determine whether the adaptive immune system also limits engraft-
ment of FL-derived hematopoietic cells, we compared the frequen-
cies of engraftment in congenic (CD45.1 into B6) and allogeneic
recipients (B6 into BALB/c or BALB/c into B6). Donor-derived cells
were detected in the blood of transplanted hosts using flow cytom-
etry (Supplemental Figure 1B). The frequency of engraftment after
transplantation of B6 FL into BALB/c fetuses was 42% (n = 43),
significantly lower than the 82% achieved in the congenic setting
(Figure 2A, c2 test, P < 0.005 compared with congenic). Similarly,
the rate of chimerism after transplantation of BALB/c FL into B6
fetuses was 53% (n = 40, c2 test < 0.05 compared with congenic).
Lineage analysis of donor- and host-derived leukocytes in congenic recipients. Multilineage engraftment of FL-derived hematopoietic cells was
seen in primary (thymus and BM) and secondary (spleen and lymph node [LN]) lymphoid organs and peripheral blood at 43–57 weeks after in
utero transplantation (n ≥ 5 mice per group). Donor- and host-derived double-positive (DP: CD4+CD8+) and single-positive (SP: CD4+CD8– or
CD4–CD8+) thymocytes, T lymphocytes (T, CD4, and CD8), B lymphocytes (B), and granulocytes (Gr) are shown as a percentage of their respec-
tive CD45+ leukocyte gate (donor, CD45.1; host, B6; CD45.2). No significant differences were observed between the percentages of donor and
host leukocyte subpopulations, with the exception of a decreased percentage of donor-derived B cells in spleen. *P < 0.05 by t test.
The adaptive immune response limits engraftment after allogeneic IUHCTx. (A) Frequency of chimerism (number of chimeric animals/number
of surviving animals) after in utero transplantation of B6 FL cells into congenic (CD45.1 into B6 [CD45.1/B6], n = 17), allogeneic (B6 into BALB/c
[B6/BALB/c], n = 43), or immunodeficient (B6 into BABL/c.Rag1–/– [B6/Rag1–/–], n = 9) recipients. *P < 0.005, c2 test for allogeneic versus con-
genic. (B) Levels of chimerism in individual engrafted animals at 4 weeks after in utero transplantation (CD45.1/B6, n = 18; B6/BALB/c, n = 16;
B6/Rag1–/–, n = 10). **P < 0.05, ANOVA with Tukey’s multiple comparison test. (C) Change in levels of chimerism over time when normalized to
the initial level of chimerism at 4 weeks after transplantation (CD45.1/B6, n = 18; B6/BALB/c, n = 10; B6/Rag1–/–, n = 10). *P < 0.05 comparing
CD45.1/B6 and B6/BALB/c, and CD45.1/B6 and B6/Rag1–/– using ANOVA with Tukey’s multiple comparison test.
584?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 2 February 2011
These data indicate that the alloimmune response is a barrier to
early engraftment after IUHCTx with FL. To determine whether the
barrier is due to an innate immune response, such as NK cell activa-
tion, or an adaptive immune response, we performed allogeneic in
utero FL transplants into BALB/c.Rag1–/– (Rag1–/–) recipients which
lack T and B cells but have an intact innate immune system (21).
In this instance, the frequency of engraftment of B6 FL in Rag1–/–
recipients was 89% (Figure 2A, n = 9, c2 test, P = 0.83 compared with
congenic controls), indicating that the barrier to IUHCTx in our
model was likely due to an adaptive alloimmune response. While
NK cells have been shown to be important in engraftment after
IUHCTx (22, 23), their effect can be overcome using high-cell doses,
as we are doing in this model (24). As expected, the overall levels
of chimerism in these animals were much higher (59.2% ± 7.2%
compared with 22.2% ± 4.5% for allogeneic wild-type recipients,
P = 0.0001), likely secondary to the competitive advantage of
transplanted cells in the Rag1–/– hosts (ref. 25 and Figure 2B). Lin-
eage analysis of chimeric cells in these animals indicated that the
increase in chimerism levels in the Rag1–/– hosts was limited to the
lymphoid component, while granulocyte chimerism levels were low
and similar to those obtained in wild-type hosts (Supplemental
Figure 2). Among wild-type recipients of IUHCTx, the levels of chi-
merism were not different in any of the experiments in this report
and are detailed in Supplemental Figure 3.
When we analyzed our chimeric animals over time, we observed
a gradual loss of engraftment in allogeneic, but not congenic or
immunodeficient, chimeras, suggesting that the adaptive immune
system also contributes to a late-phase graft loss.
There was a significant difference (P < 0.05) between
the levels of chimerism in congenic and allogeneic
recipients that remained chimeric 16 and 20 weeks
after transplantation (Figure 2C).
Engrafted mice are tolerant to donor antigen. To con-
firm that chimeric animals exhibited allospecific
tolerance, we stimulated lymphocytes from chime-
ric mice with the donor alloantigen using an in vivo
mixed lymphocyte reaction (MLR) (26). We injected
CFSE-labeled lymphocytes from naive (uninjected),
chimeric (adult animals that were stably chimeric as
a result of their in utero injection), or non-chimeric
animals (adult animals that were never engrafted after their in utero
injection) into B6 × BALB/c F1 recipients and examined the prolif-
eration of injected CD4+ (Figure 3A) and CD8+ (Figure 3B) T cells
3 days later. Using this assay, the frequencies of donor-reactive
T cells can be calculated based on their proliferative histories record-
ed in the CFSE fluorescence intensities. In naive animals, the fre-
quency of alloreactive CD4+ T cells was 15.3% ± 1.1% and that of CD8+
T cells was 5.3% ± 0.9%, which was in concordance with previously
described results (26). All chimeric mice exhibited marked reduc-
tion in response to donor alloantigens compared with both naive
animals and injected non-chimeras (Figure 3C) while retaining the
ability to respond to a third-party antigen (Figure 3D). We observed
decreased frequencies of both alloreactive CD4+ (2.6% ± 0.2%,
P < 0.0001) and CD8+ T cells (1.2% ± 0.2%, P = 0.0005) in chimeras
when compared with naive mice. These results indicate that chi-
meric mice exhibit donor-specific tolerance, although this assay
does not distinguish between deletion and anergy.
Maternal leukocytes are present in the circulation of normal fetuses and
increase after in utero transplantation. The immune system of fetal
mice at E14.5 is still immature, and antigenic encounter at this
age should lead to tolerance, which is at odds with our observa-
tion that the adaptive immune response limits engraftment in half
of the recipients. We hypothesized that maternal leukocytes may
instead be responsible for limiting engraftment in our model. We
first asked whether maternal leukocytes are present in the fetus at
the time of in utero transplantation using a flow cytometry–based
method to detect maternal cells in fetuses. For these experiments,
Chimeras are tolerant to donor alloantigen. Lym-
phocytes harvested from spleens and lymph nodes
of naive mice, injected non-chimeras, and chime-
ras were labeled with CFSE and injected into (A–C)
B6 × BALB/c or (D) C3H × DBA/2 (third-party control)
F1 recipients. Representative flow cytometric histo-
grams showing CFSE profiles after gating on donor-
derived (A) CD4+ or (B) CD8+ T lymphocytes are
shown. (C) Percentage of alloreactive T cells in naive
mice, non-chimeras, and chimeras. The data shown
are representative of at least 4 independent experi-
ments (naive, n = 10; non-chimera, n = 7; chimera,
n = 10). *P < 0.05 comparing chimeras with naive
mice and non-chimeras using ANOVA with Tukey’s
multiple comparison test. (D) Percentage of donor-
derived proliferating cells (%CFSElow) from naive and
chimeric mice in response to a third-party antigen.
Data are representative of at least 2 independent
experiments (naive, n = 3; chimera, n = 5).
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 2 February 2011
we bred B6 mothers with CD45.1 fathers, such that the fetuses
were heterozygous (CD45.1+CD45.2+) and maternal cells present
in the fetuses could be identified by their lack of CD45.1 (Figure 4,
A and B). We analyzed blood and tissues from the fetuses of these
matings by flow cytometry after staining for CD45.1, CD45.2, and
leukocyte lineage markers. Using this strategy, we found that there
is a sizeable population of maternal cells in the blood of mid-ges-
tational fetuses (Figure 4B). At E13.5–E14.5, the age at which we
performed in utero transplantations, the CD45+ population in the
fetal blood contained 8.9% ± 1.4% maternal cells (n = 12).
We further analyzed the lineage composition of the trafficking
maternal cells using markers for T cells (CD3, CD4, CD8), B cells
(CD19, B220), NK cells (NK1.1), macrophages (F4/80), granu-
locytes (Gr-1), and dendritic cells (CD11c). At E12.5–E15.5, we
detected populations of maternal Gr-1+, F4/80+, NK 1.1+, CD11c+,
CD19+, B220+, and CD3+ leukocytes (Figure 4, C and D). While
CD3+ T cells were not observed in all fetuses at baseline, when they
were present, both CD4+ and CD8+ populations were detected (Fig-
ure 4D). When we compared the lineage composition of maternal
cells in the fetus with that in the mother, we found significant dif-
ferences in the lineage distribution of maternal cells in fetal blood
compared with those in maternal blood (Figure 4E). The mater-
nal leukocytes in fetal circulation contained a significantly higher
proportion of Gr-1+ cells and a significantly lower proportion of
T cells and NK cells when compared with the cells in maternal
circulation (Figure 4E). These results confirm that the observed
maternal cells are not secondary to contamination of maternal
blood during harvesting and further suggest that trafficking of
maternal leukocytes to the fetus is selective.
Maternal cells were not detectable by flow cytometry in the liver,
spleen, or thymus at any time point tested, likely secondary to dilu-
tion of small numbers of maternal cells by the higher concentra-
tion of fetal CD45+ cells in these lymphoid organs. When we ana-
lyzed fetuses at later gestational ages, we found a strong negative
correlation between peripheral blood maternal macrochimerism
and gestational age (Figure 4F, Pearson r = –0.94, P = 0.002), such
that maternal cells were rarely detected late in gestation and not
detectable after birth.
We next examined whether fetal intervention leads to changes
in maternal-fetal cellular trafficking. We compared the number of
maternal cells in fetuses from the B6 female and CD45.1 male mat-
ings after in utero transplantation with allogeneic NOD.CD45.1
Maternal macrochimerism in mid-gestation fetal blood. (A) The breeding scheme used to identify maternal leukocytes in fetal blood. (B) Representa-
tive flow cytometric plots depicting the profile of CD45.2+ (maternal, left panel), CD45.1+/CD45.2+ (adult control, middle panel), and E15.5 fetal blood
(right panel; F, fetal; M, maternal). Lineage analysis of maternal leukocytes found in fetal blood (gate M in B) was performed using cell surface mark-
ers for (C) innate and (D) adaptive immune cells. (C) The gating strategy for identifying innate immune cells involved first detecting Gr-1+ or F4/80+
leukocytes. NK cells were identified among the Gr-1–F4/80– cells. Gr1–F4/80–NK1.1– cells were further divided into CD11c+ and B220+ leukocytes.
(D) Adaptive immune cells were characterized by identifying CD3+ and CD19+ maternal leukocytes. The CD3+ subpopulation was further character-
ized based on CD4 and CD8 expression. (E) Percentages of various leukocyte subsets found in the mother (maternal) and in the fetus (trafficked)
at E12.5–E15.5 (n ≥ 3; *P < 0.01, **P < 1 × 10–8 by t test). (F) Percentage of maternal leukocytes (number of CD45.2+ cells/total CD45+ cells) in fetal
circulation at various embryonic days of gestation (E12.5, n = 1; E13.5, n = 4; E14.5, n = 8; E15.5, n = 5; E18.5, n = 14; E20, n = 12; E22, n = 3).
There was a significant negative correlation between maternal macrochimerism and gestational age (Pearson r = –0.94, P = 0.002).
586?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 2 February 2011
FL at E14.5. Control fetuses received PBS injection or no injection,
and all animals were harvested on E18.5–E19.5. This experimen-
tal scheme allowed us to detect the presence of the transplanted
NOD cells (CD45.1+CD45.2–) and trafficking maternal leukocytes
(CD45.1–CD45.2+) among fetal cells (CD45.1+CD45.2+) (Figure
5A). As expected, NOD donor cells were found in 95% (n = 21) of
fetal recipients at this early time point, supporting our interpreta-
tion that graft loss occurs gradually and is not due to technical
variations in the injection method.
Analysis of maternal cells in these animals showed no maternal
cell trafficking in unmanipulated fetuses (n = 21), while 33% (n = 33)
of PBS-injected and 57% (n = 21) of FL-injected pups had greater
than 0.5% maternal cells (Figure 5B, c2 test, P = 0.15 between FL
and PBS), indicating that nonspecific tissue injury after fetal intra-
hepatic injection alters trafficking, with further increases in mater-
nal cells after FL transplantation. Analysis of the percentage of
maternal leukocytes in fetal blood in each animal revealed a trend
toward increased maternal cell chimerism with FL transplantation
compared with PBS (Figure 5C, 1.1% ± 0.3% PBS vs. 2.2% ± 0.7% FL,
P = 0.21). These results indicate either increased maternal cell traf-
ficking into the fetus or increased survival of trafficking maternal
cells after fetal intervention.
Although the overall percentage of maternal cells found in the
fetus was not significantly different between the two groups, we
detected significant increases in both maternal T and B cells found
in the fetus after allogeneic FL transplantation compared with PBS
injection (Figure 5D, T cell: 12.1% ± 2.9% PBS vs. 33.8% ± 2.3% FL,
P < 0.0001; B cell: 13.7 ± 1.9 PBS vs. 25.7% ± 3.0% FL, P < 0.005).
Since the fetus at this gestational age has very few circulating
T cells, we then determined whether the trafficked maternal
cells contributed to a significant population of the T cell pool.
We found that maternal T cells represented 15.5% ± 4.3% of the
total circulating T cell pool in the host after FL transplantation,
whereas they represented 3.5% ± 0.8% of the total T cell population
after PBS injection (Figure 5E, P < 0.05). Thus, IUHCTx leads to
a significant increase in the levels of maternal T cells in the fetal
circulation, suggesting that these cells may play a functional role
in the engraftment of allogeneic cells.
Maternal B cells and antibodies are not the critical component in limiting
engraftment after in utero transplantation. To determine whether traf-
ficking maternal B cells contribute to an immune response against
the cells transplanted into the fetus, we bred B cell–deficient JHD
mothers (27) with wild-type BALB/c fathers (such that the fetuses
were heterozygous and immunocompetent) and transplanted
Maternal-fetal cellular trafficking after fetal intervention. B6 mothers were mated with CD45.1 fathers, and the CD45.1+/CD45.2+ fetuses were
injected with allogeneic NOD.CD45.1 FL cells or PBS on E14.5. Injected (and uninjected control) fetuses were sacrificed on E18.5–E19.5, and
the number of maternal leukocytes (CD45.2+) in fetal blood was quantified. (A) Flow cytometric analysis of donor (gate D), maternal (gate M),
and fetal (gate F) leukocytes. (B) Frequency of fetuses with circulating maternal leukocytes (number of fetuses with circulating maternal leuko-
cytes/total number of fetuses) after PBS injection (n = 11/33, 33%) and allogeneic FL injection (n = 12/21, 57%) and in age-matched uninjected
controls (n = 0/21, 0%). (C) Percentage of maternal leukocytes (CD45.2+ maternal leukocytes/total CD45.2+ cells) in fetal circulation after PBS
(n = 11) and allogeneic FL injection (n = 12). (D) Lineage analysis of trafficking maternal cells shown as percentage of maternal leukocytes (e.g.,
trafficked maternal Gr-1+ cells/total trafficked maternal leukocytes). *P < 0.005, **P < 0.0001 by t test. (E) Lineage analysis of trafficking maternal
cells shown as the percentage of maternal cells contributing to each of the leukocyte subsets in fetal circulation (e.g., trafficked maternal Gr-1+
cells/total number of fetal and trafficked maternal Gr-1+ cells). *P < 0.05 by t test.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 2 February 2011
allogeneic B6 FL into the fetuses. The frequency of chimerism in this
setting was 56% (n = 9, Figure 6A), which is not different from what
is seen when the mother is immunocompetent (c2 test, P = 0.11).
The chimerism levels over time were similar compared with ani-
mals born to wild-type mothers (Figure 6B).
In the course of our investigation, it was reported that maternal
alloantibodies passing through breast milk limit engraftment after
IUHCTx in mice (14). In this model, the authors transplanted 20 × 106
mature BM cells using a vitelline vein injection method and deter-
mined that this approach led to maternal alloantibody formation and
engraftment in only 30% of the recipients; fostering of animals born
after IUHCTx with naive mothers led to 100% engraftment. We com-
pared these results with those obtained using our method of intrahe-
patic injection of FL cells. When we fostered BALB/c pups born after
allogeneic IUHCTx with naive mothers, the frequency of engraftment
was 65% (n = 20, Figure 6C), which was slightly increased but not
significantly different (c2 test, P = 0.11) from that of non-fostered
animals. We also analyzed mothers for the presence of donor-specific
antibodies (IgG and IgM) after allogeneic IUHCTx (Figure 6, D and E).
We determined a modest and transient increase in donor-specific IgM
only at 1 week after IUHCTx, while the overall levels of IgM and IgG
were much lower than those seen among sensitized positive controls.
Taken together with the results of the experiments using JHD moth-
ers, these data indicate that in our model, maternal B cells are not the
critical component in limiting engraftment.
Maternal T cells limit engraftment after in utero transplantation. We
next determined whether the maternal cellular immune system
plays a role in the engraftment of in utero transplanted alloge-
neic hematopoietic cells. We bred Rag1–/– mothers to wild-type
BALB/c fathers, such that the fetuses were immunocompetent,
The rejection of in utero transplanted allogeneic hematopoietic cells occurs independent of maternal B cells and maternal alloantibodies. (A)
Frequency of chimerism after IUHCTx of B6 FL cells into fetuses born to a wild-type BALB/c father and either a wild-type BALB/c mother (n = 43)
or a B cell–deficient (JHD) mother (n = 9). (B) Change in levels of chimerism over time in engrafted animals when normalized to the initial level
of chimerism at 4 weeks after transplantation (BALB/c mother, n = 10; JHD mother, n = 4). (C) Frequency of chimerism in pups fostered by
naive mothers (BALB/c fostered, n = 20) and in non-fostered pups (BALB/c, n = 43) after IUHCTx with B6 FL. (D) Serum from BALB/c mothers
whose fetuses received allogeneic IUHCTx (n ≥ 10) was analyzed by flow cytometry to quantify total serum IgM (left panels) and IgG (right
panels) alloantibody. Comparison groups include naive (n = 7) and sensitized (Sens., n ≥ 3) mice. (E) Total IgM (left panel) and IgG (right panel)
alloantibody production at 1, 2, 4, and 6 weeks after sensitization is shown as the MFI relative to a no-serum sample (relative MFI). *P < 0.05
comparing IUHCTx with naive by ANOVA with Tukey’s multiple comparison test.
588?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 2 February 2011
and performed allogeneic IUHCTx with B6 FL. We compared the
rate of chimerism in these animals to those born to wild-type
BALB/c mothers after IUHCTx with B6 FL. All surviving pups
demonstrated engraftment of donor hematopoietic cells (Figure
7A, n = 10, c2 test, P = 0.004), indicating that maternal lympho-
cytes limit donor cell engraftment after IUHCTx.
We next analyzed the effect of selectively removing T cells from
mothers by breeding Tcra–/– mothers (28) to wild-type BALB/c
fathers and transplanting the fetuses (immunologically replete)
with B6 FL cells. In these experiments, 91% of the recipients became
chimeric (Figure 7A, n = 21, c2 test, P < 0.001 compared with ani-
mals born to wild-type mothers after IUHCTx with B6 FL), indicat-
ing that maternal T cells are the key limiting factor in engraftment.
Interestingly, when we analyzed the chimerism levels over time in
pups born to Rag1–/– or Tcra–/– mothers, none of the engrafted ani-
mals demonstrated a loss of chimerism compared with the ongoing
losses seen in pups born to wild-type BALB/c mothers, suggesting
that maternal T cells also contribute to late-phase graft losses in
the recipients (Figure 7B). This result, combined with our observa-
tion of increased maternal T cell trafficking after fetal intervention,
indicates that maternal T cells play a critical role in the rejection of
allogeneic hematopoietic cells after in utero transplantation.
MHC matching of the graft to the mother improves engraftment. The
obvious clinical implication of the above observations is that MHC
matching the graft to the mother, instead of the fetus, should
result in improved engraftment. To determine this experimentally,
we used the F1 backcross model described by Zhang and Miller
(29) to generate crosses in which the transplanted B6 cells would
be matched to the mother and allogeneic to the fetuses. We crossed
B6 × BALB/c F1 (H-2b/d) mothers to BALB/c (H-2d/d) fathers, such
that half of the fetuses in each litter were H-2b/d and the other half
were H-2d/d. We transplanted all of the fetuses with B6 (H-2b/b) FL,
such that the grafts were non-immunogenic to the mother and the
H-2b/d fetuses and were allogeneic to the H-2d/d fetuses, creating
an internal control for assessing the fetal host immune response
in each litter (Figure 8A). The frequency of engraftment in allo-
geneic H-2d/d fetuses was 83% (Figure 8B, n = 12), comparable to
that found in H-2b/d fetuses (100%, n = 11, c2 test, P = 0.564). These
results indicate that if the graft is matched to the mother, the fetal
host immune response does not limit engraftment.
Transplantation of stem cells into the preimmune fetal environ-
ment can be an innovative strategy to treat congenital stem cell
disorders and establish donor-specific tolerance. However, the
lack of success of clinical IUHCTx for all diseases except severe
combined immunodeficiency has decreased enthusiasm for this
field. We investigated the cellular mechanisms of graft loss after
allogeneic IUHCTx using a mouse model. We report that the fetal
circulation harbors a previously unrecognized level of maternal
leukocytes and that there is a particular increase in maternal
T cells found in the fetus after fetal hematopoietic cell transplan-
tation. Furthermore, by selectively removing T or B cells from the
mother using knockout mice, we show that the maternal immune
system, particularly T cells, plays an important role in limiting
engraftment following IUHCTx. Finally, we demonstrate that
MHC matching of the graft to the mother results in comparable
engraftment in allogeneic and syngeneic fetal recipients, support-
ing a clinical application for these observations.
It is important to note that even in immunocompetent moth-
ers, half of fetal recipients of hematopoietic cell transplants dem-
onstrate engraftment without any conditioning. These results are
consistent with numerous previous reports that hematopoietic chi-
merism observed naturally in twin gestations (30) and following
Maternal T cells limit engraftment and contribute to ongoing losses in
chimerism after allogeneic IUHCTx. Engraftment after transplantation of
B6 FL cells into fetuses born to wild-type BALB/c fathers and either wild-
type BALB/c or immunodeficient (Rag1–/– or Tcra–/–) mothers. (A) Fre-
quency of chimerism (BALB/c, n = 43; Rag1–/–, n = 10; Tcra–/–, n = 21).
*P < 0.005 comparing Rag1–/– with BALB/c, c2 test; **P < 0.001 com-
paring Tcra–/– with BALB/c, c2 test. (B) Change in levels of chimerism
over time when normalized to the initial level of chimerism at 4 weeks
after transplantation (BALB/c, n = 10; Rag1–/–, n = 12; Tcra–/–, n = 13).
*P < 0.05 comparing BALB/c and Tcra–/–; **P < 0.05 comparing both
BALB/c and Tcra–/–, and BALB/c and Rag1–/–. Comparisons were per-
formed using ANOVA with Tukey’s multiple comparison test.
MHC matching between mother and graft improves engraftment in
MHC-mismatched fetuses. (A) Breeding scheme used to MHC match
the mother with donor cells. B6 × BALB/c F1 (H-2b/d) female mice were
mated to BALB/c (H-2d/d) males, and fetuses received B6 H-2b/b FL cells.
(B) Frequency of chimerism among fetuses that were MHC matched
(H-2b/d, n = 11) or mismatched (H-2d/d, n = 12) to the donor graft.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 2 February 2011
in utero transplantation in multiple animal models (3, 5, 14, 31,
32) and in humans (33) can lead to donor-specific tolerance. Thus,
fetal tolerance induction for stem cell transplantation remains an
important and approachable clinical goal. Since immune reactions
have been observed after postnatal embryonic stem cell transplan-
tation (34), the fetal environment may even offer an important
potential avenue to overcome the existing barriers to some clini-
cal applications of developing stem cell technologies. Our finding
of near-complete absence of donor reactivity measured by in vivo
MLR in chimeric mice suggests that deletional mechanisms may
be important, as has been reported previously both in a fetal model
(35) and for postnatal transplantation (36). However, our results do
not rule out contribution of anergy and Tregs in tolerance induc-
tion, as has been reported after fetal BM transplantation (14).
A recent study by Merianos et al. has also demonstrated the
importance of the maternal immune response to engraftment
after IUHCTx (14). The authors showed that only one-third of
pups engraft after allogeneic IUHCTx, but the frequency of engraft-
ment increases to 100% if they are fostered by naive dams, indicat-
ing that maternal alloantibodies transmitted through breast milk
limit engraftment. Our experiments indicate that maternal T cells
are the primary barrier, although it is possible that the improved
engraftment in the Tcra–/– mice in our experiments may be second-
ary to diminished B cell responses as a result of deficient T cell help.
However, in our model, the mothers did not develop high levels of
alloantibodies following IUHCTx, and pups born to B cell–deficient
JHD mothers had similar rates of engraftment when compared
with pups born to wild-type mothers, suggesting that rejection is
independent of maternal B cells. One potential explanation for our
discrepant findings is that Merianos et al. transplanted a high dose
of adult BM cells containing mature APCs, while we transplant-
ed a 10-fold-lower dose of FL cells, which do not contain mature
APCs. The presence of mature donor APCs can make the graft more
immunogenic in several ways. First, mature APCs express higher
levels of MHC molecules than stem cells and are therefore more
potent stimulators of alloreactive T cells. Second, mature APCs
express MHC class II molecules that can directly stimulate allore-
active CD4+ T cells, which can, in turn, help B cells to produce allo-
antibodies. Third, the breakdown product of MHC proteins, which
are highly expressed on mature donor APCs, can be presented by
host APCs to stimulate T cells that recognize alloantigens through
the indirect pathway of allorecognition. Therefore, it is highly likely
that the HSC grafts used by Merianos et al. are more immunogenic,
leading to the sensitization of the mother. Additional experiments
to directly compare these models may distinguish between these
possibilities. Nonetheless, our independent conclusion that the
maternal immune response is an important variable should lead
to changes in the way clinical transplants are conducted, if these
findings are confirmed in large animal models.
We have demonstrated a two-phase immune response to alloge-
neic grafts in this model. The first phase of graft rejection occurs
early, leading to loss of engraftment in half of transplanted mice
within 4 weeks. It is important to note that when the mice are sac-
rificed in the first week after transplantation, donor cells are found
in 95% of recipients (similar to what was reported by Peranteau et al;
ref. 11), indicating that the loss of chimerism seen in transplanted
animals at 4 weeks is secondary to rejection and not to technical
errors in transplantation. In the second phase, there are ongoing
losses in chimerism even in engrafted animals, with a plateau at later
time points. The surprising finding that chimerism levels decline
in animals born to wild-type mothers whereas they are steady in
those born to Rag1–/– or Tcra–/– mothers suggests that the maternal
immune response contributes to this second phase of graft loss as
well. There are two potential mechanisms by which maternal cells
may exert such an influence: maternal cells may themselves persist
in chimeras and cause an ongoing immune response; or they may
prime the host immune system (for example, by inducing earlier
maturation of APCs) to reject the transplanted cells. A previous
study reported detection of maternal T cells in 6-week-old animals
by flow cytometry (29). However, we have not been able to consis-
tently detect maternal T cells in offspring after their birth.
In this article, we demonstrate high levels of maternal leukocytes
in fetal blood in mid-gestation. Maternal cells have been detected
in fetal mice using PCR, immunohistochemistry, and flow cytom-
etry (29, 37–41), although we have not found a previous analysis of
maternal leukocytes in the blood of mouse fetuses. Our particular
breeding scheme allowed us to focus on CD45+ leukocytes and to
fully characterize these maternal cells by flow cytometry. The fact
that the composition of cells found in the fetal blood is distinct from
that found in maternal circulation argues against contamination of
fetal samples with maternal blood and suggests that maternal traf-
ficking across the placenta is active and selective, rather than a result
of general “leakiness” of the maternal-fetal interface. The functional
significance of these maternal leukocytes during normal gestation
is presently unclear. In humans, maternal-fetal cellular trafficking
may contribute to fetal immune development and maternal-fetal
tolerance, inducing the fetus to develop Tregs against maternal anti-
gens (19). Changes in the levels of maternal-fetal cellular trafficking
have been reported to correspond with maternal-fetal histocompat-
ibility in the mouse model, suggesting that cellular trafficking has
implications for maternal-fetal tolerance (37, 40).
In addition to baseline trafficking, we have determined that
there are key changes in the composition of maternal cells in fetal
blood after fetal intervention, with particular increases in the lev-
els of maternal T cells following in utero transplantation. Several
mechanisms may result in such a finding: there may be selective
recruitment of T cells across the placenta, increased prolifera-
tion or decreased turnover of T cells that have already crossed, or
decreased homing of maternal T cells to fetal tissues with resultant
increases in the circulation. The fact that trafficking was variable
among the fetuses in each litter is intriguing and may explain why
some fetuses in a litter engraft while littermates do not: the per-
centage of fetuses with detectable maternal-fetal cellular traffick-
ing is similar to the percentage that ultimately fails to engraft after
allogeneic FL transplantation.
The finding that fetal PBS injection alone leads to maternal
cell trafficking may have clinical implications in the field of fetal
intervention. The amount of fetal trauma from the intrahepatic
injection in our model is likely more than would be expected for
human IUHCTx, which uses minimally invasive methods. There-
fore, it is not known whether human fetal interventions will lead
to similar alterations in trafficking of maternal cells into the fetus,
although changes in the amount of fetal DNA in the mother have
been described (42). If cellular trafficking is related to maternal-
fetal tolerance, alterations in trafficking may correlate with the
onset of preterm labor, an idea that has been defined in sponta-
neous preterm labor (43, 44) but not in fetal intervention. Given
the growing interest in clinical fetal surgery for various anatomic
anomalies, our findings may have important implications for the
pathogenesis of preterm labor following fetal intervention.
590? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 2 February 2011
If the maternal immune response limits engraftment, a potential
clinical solution is to transplant cells that are harvested from (or
HLA-matched to) the mother, as we have demonstrated in our F1
experiment (Figure 8). Such a strategy is also supported by observa-
tions that tolerance to NIMAs may improve transplant outcomes
in some settings (45, 46). In our experiment, it is possible that there
is some improvement in the engraftment of H-2b FL cells in H-2d/d
fetuses because these fetuses are exposed to the NIMA, H-2b. Interest-
ingly, it has been reported that there are strain-dependent variations
in tolerance versus sensitization to NIMAs, and in utero exposure to
H-2b may instead be sensitizing (47). Thus, it is especially striking
that this possible sensitization was overcome with IUHCTx.
In summary, we have demonstrated a critical role for maternal
T cells in limiting fetal engraftment after allogeneic IUHCTx. Our
finding of increased T cell trafficking with fetal intervention indi-
cates a mechanism by which maternal cells respond to the trans-
planted cells. The observation that almost all fetuses engraft once
maternal T cells are removed or once the graft is matched to the
mother validates the promise of using the fetal environment for
inducing donor-specific tolerance.
Reagents and antibodies. The following reagents were used: ACK Lysing Buffer
(Lonza), Invitrogen Vybrant CFDA SE Cell Tracer Kit (CFSE, Invitrogen),
Histopaque 1077 Ficoll (Sigma-Aldrich), Ficoll-Paque Plus (GE Health-
care). The following antibodies for flow cytometry were purchased from
BD: CD3 (145-2C11), CD8 (clone 53-6.7), CD11c (HL-3), CD19 (1D3),
CD45 (30-F11), CD45.1 (A20), CD45R/B220 (RA3-6B2), H-2Kb (AF6-88.5),
H-2Kd (SF1-1.1), H-2Kk (clone 36-7-5), NK1.1 (PK136); eBioscience: CD4
(RM4-5), CD8 (clone 53-6.7), CD45.1 (A20), CD45.2 (104), F4/80 (BM8),
anti-IgG, anti-IgM (II/41); from SouthernBiotech: CD8 (clone 53-6.7);
UCSF Hybridoma Core: Gr-1 (RB6-8C5), Fc receptor (2.4G2).
Mice. The inbred strains, BALB/c, B6, and CD45.1, and F1 hybrid strains
B6 × BALB/c and C3H × DBA/2 were obtained from either NCI or The
Jackson Laboratory. BALB/c.Tcra–/– (Tcra–/–), and BALB/c.JHD (JHD), mice
were obtained from A. Abbas (UCSF). NOD.CD45.1.uGFP (NOD.CD45.1)
mice were generated by backcrossing B6.uGFP transgenic mice (strain
004353, The Jackson Laboratory) at least 6 times to NOD (The Jackson
Laboratory) mice. BALB/c.Rag1–/– mice were obtained from The Jackson
Laboratory. All mice were bred and maintained in a specific pathogen–free
facility at UCSF. All mouse experiments were performed according to a
UCSF Institutional Animal Care and Use Committee–approved protocol.
In utero FL transplantation. B6 or BALB/c fetal livers were harvested from
E13.5–E14.5 donor fetuses in PBS. Single-cell suspensions were made by
gently pipetting the fetal livers and filtering through a 70-μm Nitex filter.
FL mononuclear cells (FLMCs) were isolated by density gradient separation
using Histopaque 1077 or Ficoll-Paque Plus. The FLMCs were washed twice
and adjusted to a concentration of 2.5 × 106 cells/5 μl. To prepare the recipi-
ent fetuses, we anesthetized pregnant dams at E13.5–E14.5 using isoflu-
rane. After a midline laparotomy was made, the uterus was exteriorized, and
5 μl of the FL cell suspension was injected into the fetal livers of recipients
using pulled glass micropipettes fabricated in our laboratory. The uterus was
returned to the abdominal cavity, and the wound was closed in layers. Surviv-
ing pups were counted on the day of birth and at the time of weaning.
Determination of chimerism levels. Blood was collected from the maxillary
vein into heparinized tubes and washed, and red blood cells were lysed
using ACK Lysing Buffer. The cells were then stained with antibodies to
CD45, H-2Kd, and H-2Kb for chimeras of B6 and BALB/c strain combina-
tions (Supplemental Figure 1B). Chimerism in the congenic control group
(CD45.1 into B6) and in the B6 and NOD strain combination was deter-
mined by staining with antibodies to CD45.1 and CD45.2 (Supplemental
Figure 1A). Samples were analyzed on a FACSCalibur cytometer (BD), and
the data were analyzed using FACSDiva software (BD). Levels of chimerism
in each animal were calculated by dividing the number of donor leukocytes
by the total number of CD45+ leukocytes. A chimerism threshold of 1% was
used, similar to that reported in ref. 14, although analysis of the data with a
chimerism threshold of 0.1% did not alter any of the conclusions. Animals
were first analyzed at 4 weeks after transplantation to determine the fre-
quency of engraftment (number of chimeric animals/surviving animals).
Levels of chimerism were also determined every 4 weeks and normalized to
the value obtained at the initial analysis. The results for each strain combi-
nation reported represent at least 3 independent litters.
In vivo MLR. Lymphocytes were collected from spleens and inguinal, cer-
vical, axillary, brachial, and mesenteric lymph nodes from chimeric (>1%
chimerism), injected non-chimeric (<0.1% chimerism), or naive animals
and labeled with CFSE. Cells (25 × 106 to 50 × 106) were injected into the
retroorbital plexus of B6 × BALB/c F1 or C3H × DBA/2 F1 (third-party con-
trols) hybrid animals. Recipient F1 mice were sacrificed between 60 and 72
hours after injection. Lymphocytes from spleens were stained with anti-
bodies to H-2Kb, H-2Kd, H-2Kk, CD4, and CD8, and the proliferation of
donor-derived CD4+ and CD8+ cells was analyzed on an LSRII flow cytom-
eter (BD). To estimate the frequency of alloreactive T cells, the number
of precursor cells that proliferated was divided by the total (proliferated
and non-proliferated) number of precursor cells. The number of precursor
cells that proliferated was calculated as described in ref. 26, by quantifying
the numbers of cellular events within a given CFSE peak and dividing the
number of events by 2n, where n is the division cycle number. The number
of non-proliferated precursors was determined by quantifying the number
of events in the peak with the highest CFSE intensity (26).
Detection of maternal cells in fetal mice. B6 mothers were bred to CD45.1
fathers, and the resulting pups were harvested at indicated time points.
Pups were washed twice in PBS prior to decapitation in heparinized HBSS
to minimize contamination with maternal blood. At the earlier gestational
ages (E12.5–E15.5), blood from 2–3 pups was pooled to obtain enough sam-
ple for flow cytometry. After blood collection, pups were dissected under
a dissecting microscope, individual organs were collected and dissociated
using collagenase and DNase, and single-cell suspensions were prepared.
Red blood cells in blood and spleen samples were lysed using ACK Lysing
Buffer. The cells were stained for CD45.1, CD45.2, CD3, B220 (or CD19),
and Gr-1 to detect maternal cells and analyze their lineage composition. The
presence of maternal leukocytes was quantified by calculating the percentage
of CD45.2+CD45.1– cells over the total CD45+ pool. A clear population of
maternal leukocytes that was greater than 0.5% and more than 50 events was
considered significant. In some experiments, a more detailed lineage analysis
was performed using two panels of antibodies to detect lymphocytes of the
adaptive (CD3, CD4, CD8, CD19) and innate (CD11c, Gr-1, NK1.1, F4/80)
immune systems. To determine the effect of IUHCTx on maternal cell traf-
ficking, we sacrificed fetuses 4–5 days after in utero transplantation of NOD.
CD45.1 FL and analyzed them for the presence of donor and maternal lym-
phocytes by flow cytometry (LSRII, BD). Dead cells were gated out using
DAPI. The results for uninjected, PBS-injected, and FL-injected groups were
compiled from at least 3 independent litters per group.
Detection of donor-specific antibody responses. To detect circulating alloanti-
bodies that might be present in mothers that underwent IUHCTx, serum
was collected weekly for 6 weeks after in utero transplantation and ana-
lyzed as reported in Merianos et al (14). Serum from naive BALB/c mice
was used as a negative control, and serum from sensitized BALB/c mice
was used as a positive control. Sensitized mice were generated by injec-
tion of BALB/c animals with 20 × 106 B6 splenocytes intraperitoneally,
followed by a repeat injection 7 days later. To quantify the amount of
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 2 February 2011
alloantibodies in the serum, lymphocytes harvested from the spleen
and lymph nodes of naive B6 mice were incubated with antibodies to
Fc receptor for 15 minutes and then incubated with serum samples for
45 minutes. Cells were then washed twice in FACS buffer and incubated
with anti-FcR for 5 minutes, followed by the addition of anti-IgM, anti-
IgG, and CD19 antibodies. The stained cells were analyzed using flow
cytometry to determine the MFI of IgM and IgG staining on CD19-nega-
tive cells. The relative MFI was normalized to that of B6 lymphocytes that
were not exposed to serum.
Statistics. Frequencies of engraftment were compared using c2 test. Com-
parisons involving 2 groups were evaluated using Student’s t test, and those
involving multiple groups were evaluated using ANOVA with Tukey’s mul-
tiple comparison test. A P value of less than 0.05 was considered to be sig-
nificant. Data represent mean ± SEM.
We acknowledge Abul Abbas, Mike McCune, Susan Fisher,
Jeff Bluestone, and Sang-Mo Kang for numerous helpful dis-
cussions. We would like to thank the members of the Tang
and Abbas laboratories for their advice and expertise and
Greg Emmanuel and Catherine Tsai for technical assistance.
Funding support includes the Irene Perstein Award (to T.C.
MacKenzie), UCSF Sandler Funds (to T.C. MacKenzie and
Q. Tang), an American Pediatric Surgical Association (APSA)
Foundation Scholarship (to T.C. MacKenzie), NIH/NIAID
grant K08 AI085042 (to T.C. MacKenzie), a California Institute
for Regenerative Medicine Postdoctoral Training Grant (to A.
Nijagal), a National Science Foundation Training Grant (to
M. Wegorzewska), and the Joslin Diabetes and Endocrinology
Research Center (DERC) flow cytometry core.
Received for publication August 26, 2010, and accepted in revised
form November 23, 2010.
Address correspondence to: Tippi C. MacKenzie, Campus Box
0570, University of California, San Francisco, 513 Parnassus
Avenue, San Francisco, California 94143-0570, USA. Phone:
415.476.4086; Fax: 415.476.2314; E-mail: Tippi.Mackenzie@
ucsfmedctr.org. Or to: Qizhi Tang, Campus Box 0780, Univer-
sity of California, San Francisco, 513 Parnassus Avenue, San
Francisco, California 94143-0780, USA. Phone: 415.476.1739;
Fax: 415.502.8326; E-mail: Qizhi.Tang@ucsfmedctr.org.
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