Given the current understanding of the mechanistic basis of fetal tolerance, one would anticipate that with appropriate timing and mode of antigen administration, consistent donor-specific tolerance would be achievable by a primary mechanism of clonal deletion of donor-reactive lymphocytes. However, historically that has not been the case. Even in Billingham, Brent, and 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 neonatal 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 recognition 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
). These findings, as well as observations of immunization in large animal models (22
) and humans (24
) after relatively late administration of antigen, can be easily attributed to missing the window of opportunity for central tolerance induction. More difficult to explain are failures of engraftment and associated 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 allogeneic IUHCT, when transplants have been performed at E14–E15, numerous studies have reported failure of engraftment or of only microchimerism (26
), inconsistent or absent tolerance induction (26
), or immunization to alloantigen after IUHCT (30
). 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 significant engraftment advantage for congenic versus allogeneic cells (28
). However, in retrospect, those studies were misleading 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 consistent 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
). 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
). However, although 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 difference 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 allogeneic engraftment.
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 cellular 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 maternal 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 immunoglobulins (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 progeny. Setiady et al. (46
) more recently demonstrated transmission of autoimmune ovarian disease via the same pathway, whereby maternal 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
). 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 inflammation, 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 established 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 chimerism (<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 mechanism in a milieu of low frequencies of donor cells. Levels of chimerism in our model of intravascular injection far exceed the threshold 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
) 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 chimeric. One explanation is that the magnitude of the maternal immune response, and therefore the dose of alloantibody transferred, 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 maintained 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 significant numbers due to inadequate or late presentation of antigen in the thymus, and to be controlled by regulatory mechanisms, including Treg populations, which are essential for the prevention of autoimmune disease (56
). 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+
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 mechanism 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 recipients 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 specifically 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 sensitization 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 closure occurs precociously (16
). In light of the longer gestation period and the fact that the murine FcγR is similar to the placental receptor 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 animal 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 maternal immunization and its consequences after IUHCT. Multiple mechanisms have been implicated that contribute to maternal-fetal tolerance, both at the placental interface (59
) and systematically (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 experimental 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 application 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 Billingham, Brent, and Medawar (2