Transplantation of stem cells into the preimmune fetal environment 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 transplantation. 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, supporting a clinical application for these observations.
It is important to note that even in immunocompetent mothers, half of fetal recipients of hematopoietic cell transplants demonstrate engraftment without any conditioning. These results are consistent with numerous previous reports that hematopoietic chimerism observed naturally in twin gestations (30
) and following in utero transplantation in multiple animal models (3
) 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 transplantation (34
), the fetal environment may even offer an important potential avenue to overcome the existing barriers to some clinical 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 induction, 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 engraftment increases to 100% if they are fostered by naive dams, indicating 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 secondary 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 transplanted 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 alloreactive CD4+
T cells, which can, in turn, help B cells to produce alloantibodies. 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 allogeneic 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 sacrificed 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–/–
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 consistently 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 cytometry (29
), 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 trafficking 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 antigens (19
). Changes in the levels of maternal-fetal cellular trafficking have been reported to correspond with maternal-fetal histocompatibility in the mouse model, suggesting that cellular trafficking has implications for maternal-fetal tolerance (37
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 levels 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 proliferation 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 percentage of fetuses with detectable maternal-fetal cellular trafficking 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. Therefore, 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 spontaneous preterm labor (43
) 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.
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 ). Such a strategy is also supported by observations that tolerance to NIMAs may improve transplant outcomes in some settings (45
). 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
. Interestingly, 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 indicates a mechanism by which maternal cells respond to the transplanted 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.