Maternal alloantibodies induce a postnatal immune response that limits engraftment following in utero hematopoietic cell transplantation in mice

The lack of fetal immune responses to foreign antigens, i.e., fetal immunologic tolerance, is the most compelling rationale for prenatal stem cell and gene therapy. However, the frequency of engraftment following in utero hematopoietic cell transplantation (IUHCT) in the murine model is reduced in allogeneic, compared with congenic, recipients. This observation supports the existence of an immune barrier to fetal transplantation and challenges the classic assumptions of fetal tolerance. Here, we present evidence that supports the presence of an adaptive immune response in murine recipients of IUHCT that failed to maintain engraftment. However, when IUHCT recipients were fostered by surrogate mothers, they all maintained long-term chimerism. Furthermore, we have demonstrated that the cells responsible for rejection of the graft were recipient in origin. Our observations suggest a mechanism by which IUHCT-dependent sensitization of the maternal immune system and the subsequent transmission of maternal alloantibodies to pups through breast milk induces a postnatal adaptive immune response in the recipient, which, in turn, results in the ablation of engraftment after IUHCT. Finally, we showed that non-fostered pups that maintained their chimerism had higher levels of Tregs as well as a more suppressive Treg phenotype than their non-chimeric, non-fostered siblings. This study resolves the apparent contradiction of induction of an adaptive immune response in the pre-immune fetus and confirms the potential of actively acquired tolerance to facilitate prenatal therapeutic applications.

One of the predictions of Burnet and Fenner’s theory of immunity (1) is that prenatal exposure to foreign antigens prior to the development 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 tolerance to facilitate organ or cellular transplantation was immediately 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 treatment 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 introduction 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 adaptive 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 chimeric 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 hematopoietic chimerism. Unexpectedly, we also demonstrate a maternal 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 further show that transfer of maternal serum to fostered pups is sufficient to induce loss of chimerism, supporting an indirect 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-chimeric, non-fostered siblings. These findings explain the apparent contradiction of activation of an immune response in the pre-immune fetal recipient and confirm, in the absence of maternal 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 allogeneic chimerism after IUHCT, we performed allogeneic IUHCT at E14 from MHC-H2K^b+GFP⁺ C57BL/6TgN(act-EGFP)OsbY01 mouse donors (referred to as “B6GFP mice”) into MHC-H2K^d+ BALB/c recipient fetuses. In this model, we have previously demonstrated 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 findings of 100% chimerism at 2 weeks of age. In a separate cohort of recipients, peripheral blood hematopoietic chimerism was determined 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, lymphocytes from BALB/c recipient mice were harvested, stained with CFSE dye, and adoptively transferred to CB6F1 (H2K^b+/d+) recipients, generating a parent into F1 MHC mismatch in which BALB/c T cells allospecific for MHC-H2K^b+GFP^– C57BL/6 (referred to as “B6”) donor cells would proliferate against the F1 host. F1 recipients were euthanized at 24, 48, 72, and 96 hours after the adoptive transfer, and CFSE-stained CD4⁺ H2K^b– lymphocytes were analyzed by flow cytometry. Cell division began after 24 hours following 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 alloreactive 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.

Figure 1

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.

Figure 2

T cell alloreactivity in vivo. Flow cytometry of adoptively transferred CD4⁺H2K^b– 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 (negative control), immunized (positive control), chimeric, and non-chimeric BALB/c pups 5 weeks after B6 IUHCT. Data are mean ± SEM.

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 peripheral blood, bone marrow, and thymus (Supplemental Figure 1; supplemental 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 mothers. 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 predominantly Th1 profile in the non-chimeric pups (Supplemental Figure 1). To assess the potential contribution of anergy or suppression to our findings of tolerance in fostered and non-fostered chimeric 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 suppression by regulatory populations (15) (Supplemental Figure 2).

Non-chimeric pups have circulating alloantibodies. For assessment of the humoral immune response, serum was isolated from 4-week-old chimeric and non-chimeric 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 controls, 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).

Figure 3

Alloantibody assay in chimeric and non-chimeric mice. (A) Control mice showed a significant difference in alloantibody formation at all ratios of serum to target cells. (*P < 0.01). (B) Non-chimeric BALB/c pups (n = 20) showed evidence of alloantibody 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.

Injected dams are sensitized against allogeneic donor cells. The presence 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 frequency of alloreactive maternal T cells and found that there was no statistically significant difference in the frequency of alloreactive T cells between injected dams and immunized positive controls (Figure 4), which was consistent with maternal sensitization by donor cells after IUHCT.

Figure 4

Maternal T cell alloreactivity in vivo and frequency of alloreactive T cells. (A) Flow cytometry of CD4⁺H2K^b– 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 (negative 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.

We next analyzed the maternal humoral response. Alloantibodies were found in maternal serum but did not appear until approximately 2 weeks after IUHCT, when the pups were 1 week of age (Figure 5). This suggested that if in fact the maternal immune system was causative in the adaptive immune response in the pups, this response would occur during the postnatal period, implicating maternal breast milk as a likely mode of transmission. Interestingly, 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 predominance 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-chimeric pups, on average, were found to have been exposed to significantly higher levels of maternal alloantibodies than chimeric pups (P = 0.0002) (Supplemental Figure 3).

Figure 5

Maternal alloantibody assay. Median anti-IgG immunofluorescence representing maternal alloantibodies at 1, 2, and 5 weeks after IUHCT. Data are median ± SEM.

Figure 6

Alloantibody class and subclass. Serum from injected BALB/c dams and non-chimeric BALB/c pups was analyzed for anti-B6 alloantibody at the time of loss of chimerism (5 weeks after IUHCT). Data are median ± SEM.

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 number of fetuses that were injected and then subsequently born, we were able to gain some insight into the mechanism of maternal sensitization. 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 associated 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 miscarriage 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). Therefore, 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 mechanisms of reproductive immunology in which paternal antigens are able to induce a pathologic immune response (17, 18).

Figure 7

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.

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 influence 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 chimerism 6 months after IUHCT (Supplemental Figure 5). In contrast, only 30.4% (21/69) of pups nursed by dams injected on E14 maintained 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 tolerance 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 chimerism. Immunologic analysis of the fostered pups confirmed an 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 significantly different from the level of chimerism in chimeric pups nursed by dams injected on E14.

Table 1

Effect of maternal breast feeding on the frequency of chimerism after IUHCT

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 origin, 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 further 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 participating 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 discrimination 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, peripheral 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 H2K^b– 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⁺H2K^b+/d+ T cells from non-chimeric pups (Figure 8B) as well as proliferation by CD4⁺H2K^b– T cells from dams injected on E14 (Figure 8C). However, we found no proliferation of H2K^b– 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 compartments excluded sequestration of maternal cells, as these tissues also lacked H2K^b– lymphocytes (Supplemental Figure 6).

Figure 8

Analysis of the origin of alloreactive lymphocytes in non-chimeric pups. (A) CD4⁺H2K^b+ lymphocytes were harvested from congenic B6 mice. (B) CD4⁺H2K^b+ lymphocytes were harvested from F1 neonatal recipients. (C) CD4⁺H2K^b– lymphocytes were harvested from E14 injected BALB/c dams. (D) A negligible number of H2K^b– maternal cells were derived from F1 recipients. Each histogram is representative of at least 8 independent experiments.

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 chimeric), 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.

Table 2

Ability of immunized maternal serum to induce loss of chimerism

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 litter 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 investigate 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 suppressive 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).

Figure 9

Treg distribution. (A) CD4⁺CD25⁺ lymphocytes were shown to be more than 85% FoxP3⁺. (B) The percentage 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 compartments except the spleen (P = 0.06). The histogram is representative of at least 5 independent experiments. Data are mean ± SEM.

Figure 10

Treg suppression assay. (A) CD4⁺CD25⁺ Tregs and CD4⁺CD25^– effector T cells were sorted. (B and C) Controls consisted 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 representative of at least 5 independent experiments. (F) The percentage suppression at a 1:1 Treg/effector T cell ratio. Data are mean ± SEM.

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 (19–21). 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 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 (26–34), inconsistent or absent tolerance induction (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 significant engraftment advantage for congenic versus allogeneic cells (28, 31, 34, 39). 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 (40–42). 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 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 (48–50). 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 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, 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 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, 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 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).

View Supplemental data

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.

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: flake@email.chop.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.119:2590–2600 (2009). doi:10.1172/JCI38979.

1. Burnet, F.M., and Fenner, F. 1949. The production of antibodies. 2nd edition. Macmillan and Co., Ltd. Melbourne, Victoria, Australia. 142 pp.
2. Billingham, R., Brent, L., Medawar, P.B. 1953. Actively acquired tolerance of foreign cells. Nature. 172:603-607.
   View this article via: CrossRef PubMed Google Scholar
3. Owen, R.D. 1945. Immunogenetic consequences of vascular anastomoses between bovine cattle twins. Science. 102:400-401.
   View this article via: CrossRef PubMed Google Scholar
4. Anderson, D., Billingham, R., Lampkin, G., Medawar, P. 1951. The use of skin grafting to distinguish between monozygotic and dizygotic twins in cattle. Heredity. 5:379-397.
   View this article via: CrossRef Google Scholar
5. Gill, T. 1977. Chimerism in humans. Transplant Proc. 9:1423-1431.
   View this article via: PubMed Google Scholar
6. Hansen, H.E., Niebuhr, E., Lomas, C. 1984. Chimeric twins. T.S. and M.R. reexamined. Hum. Hered. 34:127-130.
   View this article via: CrossRef PubMed Google Scholar
7. Picus, J., Aldrich, W.R., Letvin, N.L. 1985. A naturally occurring bone-marrow-chimeric primate. I. Integrity of its immune system. Transplantation. 39:297-303.
   View this article via: PubMed Google Scholar
8. Picus, J., Holley, K., Aldrich, W.R., Griffin, J.D., Letvin, N.L. 1985. A naturally occurring bone marrow-chimeric primate. II. Environment dictates restriction on cytolytic T lymphocyte-target cell interactions. J. Exp. Med. 162:2035-2052.
   View this article via: CrossRef PubMed Google Scholar
9. Palmer, E. 2003. Negative selection--clearing out the bad apples from the T-cell repertoire. Nat. Rev. Immunol. 3:383-391.
   View this article via: CrossRef PubMed Google Scholar
10. Takahama, Y. 2006. Journey through the thymus: stromal guides for T-cell development and selection. Nat. Rev. Immunol. 6:127-135.
    View this article via: CrossRef PubMed Google Scholar
11. Merianos, D., Heaton, T., Flake, A.W. 2008. In utero hematopoietic stem cell transplantation: progress toward clinical application. Biol. Blood. Marrow Transplant. 14:729-740.
    View this article via: CrossRef PubMed Google Scholar
12. Peranteau, W.H., Endo, M., Adibe, O.O., Flake, A.W. 2007. Evidence for an immune barrier after in utero hematopoietic-cell transplantation. Blood. 109:1331-1333.
    View this article via: CrossRef PubMed Google Scholar
13. Suchin, E.J., et al. 2001. Quantifying the frequency of alloreactive T cells in vivo: new answers to an old question. J. Immunol. 166:973-981.
    View this article via: PubMed Google Scholar
14. Sherman, L.A., Chattopadhyay, S. 1993. The molecular basis of allorecognition. Annu. Rev. Immunol. 11:385-402.
    View this article via: CrossRef PubMed Google Scholar
15. Thornton, A.M., Shevach, E.M. 1998. CD4⁺CD25⁺ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J. Exp. Med. 188:287-296.
    View this article via: CrossRef PubMed Google Scholar
16. Van de Perre, P. 2003. Transfer of antibody via mother’s milk. Vaccine. 21:3374-3376.
    View this article via: CrossRef PubMed Google Scholar
17. Kotlan, B., et al. 2001. High anti-paternal cytotoxic T-lymphocyte precursor frequencies in women with unexplained recurrent spontaneous abortions. Hum. Reprod. 16:1278-1285.
    View this article via: CrossRef PubMed Google Scholar
18. Gurka, G., Rocklin, R.E. 1987. Reproductive immunology. JAMA. 258:2983-2987.
    View this article via: CrossRef PubMed Google Scholar
19. Forsthuber, T., Yip, H.C., Lehmann, P.V. 1996. Induction of TH1 and TH2 immunity in neonatal mice. Science. 271:1728-1730.
    View this article via: CrossRef PubMed Google Scholar
20. Pennisi, E. 1996. Teetering on the brink of danger. Science. 271:1665-1667.
    View this article via: CrossRef PubMed Google Scholar
21. Ridge, J.P., Fuchs, E.J., Matzinger, P. 1996. Neonatal tolerance revisited: turning on newborn T cells with dendritic cells. Science. 271:1723-1726.
    View this article via: CrossRef PubMed Google Scholar
22. Gerdts, V., Babiuk, L.A., van Drunen Littel-van den, H., Griebel, P.J. 2000. Fetal immunization by a DNA vaccine delivered into the oral cavity. Nat. Med. 6:929-932.
    View this article via: CrossRef PubMed Google Scholar
23. Otsyula, M.G., et al. 1996. Fetal or neonatal infection with attenuated simian immunodeficiency virus results in protective immunity against oral challenge with pathogenic SIVmac251. Virology. 222:275-278.
    View this article via: CrossRef PubMed Google Scholar
24. Orlandi, F., et al. 1996. Evidence of induced non-tolerance in HLA-identical twins with hemoglobinopathy after in utero fetal transplantation. Bone Marrow Transplant. 18:637-639.
    View this article via: PubMed Google Scholar
25. Strominger, J.L. 1989. Developmental biology of T cell receptors. Science. 244:943-950.
    View this article via: CrossRef PubMed Google Scholar
26. Kim, H.B., Shaaban, A.F., Milner, R., Fichter, C., Flake, A.W. 1999. In utero bone marrow transplantation induces tolerance by a combination of clonal deletion and anergy. J. Pediatr. Surg. 34:726-730.
    View this article via: CrossRef PubMed Google Scholar
27. Shaaban, A.F., Kim, H.B., Gaur, L., Liechty, K.W., Flake, A.W. 2006. Prenatal transplantation of cytokine-stimulated marrow improves early chimerism in a resistant strain combination but results in poor long-term engraftment. Exp. Hematol. 34:1278-1287.
    View this article via: PubMed Google Scholar
28. Howson-Jan, K., Matloub, Y.H., Vallera, D.A., Blazar, B.R. 1993. In utero engraftment of fully H-2-incompatible versus congenic adult bone marrow transferred into nonanemic or anemic murine fetal recipients. Transplantation. 56:709-716.
    View this article via: PubMed Google Scholar
29. Taylor, P.A., McElmurry, R.T., Lees, C.J., Harrison, D.E., Blazar, B.R. 2002. Allogenic fetal liver cells have a distinct competitive engraftment advantage over adult bone marrow cells when infused into fetal as compared with adult severe combined immunodeficient recipients. Blood. 99:1870-1872.
    View this article via: CrossRef PubMed Google Scholar
30. Carrier, E., Gilpin, E., Lee, T.H., Busch, M.P., Zanetti, M. 2000. Microchimerism does not induce tolerance after in utero transplantation and may lead to the development of alloreactivity. J. Lab. Clin. Med. 136:224-235.
    View this article via: CrossRef PubMed Google Scholar
31. Carrier, E., Lee, T.H., Busch, M.P., Cowan, M.J. 1995. Induction of tolerance in nondefective mice after in utero transplantation of major histocompatibility complex-mismatched fetal hematopoietic stem cells. Blood. 86:4681-4690.
    View this article via: PubMed Google Scholar
32. Carrier, E., Lee, T.H., Busch, M.P., Cowan, M.J. 1997. Recruitment of engrafted donor cells postnatally into the blood with cytokines after in utero transplantation in mice. Transplantation. 64:627-633.
    View this article via: CrossRef PubMed Google Scholar
33. Donahue, J., et al. 2001. Microchimerism does not induce tolerance and sustains immunity after in utero transplantation. Transplantation. 71:359-368.
    View this article via: CrossRef PubMed Google Scholar
34. Barker, J.E., et al. 2003. Donor cell replacement in mice transplanted in utero is limited by immune-independent mechanisms. Blood Cells Mol. Dis. 31:291-297.
    View this article via: CrossRef PubMed Google Scholar
35. Chen, J.C., et al. 2008. Prenatal tolerance induction: relationship between cell dose, marrow T-cells, chimerism, and tolerance. Cell Transplant. 17:495-506.
    View this article via: CrossRef PubMed Google Scholar
36. Hedrick, M.H., et al. 1994. Hematopoietic chimerism achieved by in utero hematopoietic stem cell injection does not induce donor-specific tolerance for renal allografts in sheep. Transplantation. 58:110-111.
    View this article via: PubMed Google Scholar
37. Mychaliska, G.B., et al. 1997. In utero hematopoietic stem cell transplants prolong survival of postnatal kidney transplantation in monkeys. J. Pediatr. Surg. 32:976-981.
    View this article via: CrossRef PubMed Google Scholar
38. Lee, P.W., et al. 2005. In utero bone marrow transplantation induces kidney allograft tolerance across a full major histocompatibility complex barrier in swine. Transplantation. 79:1084-1090.
    View this article via: CrossRef PubMed Google Scholar
39. Shaaban, A.F., Milner, R., Kim, H.B., Fichter, C., Flake, A.W. 1999. Absence of MHC restriction facilitates equal engraftment of prenatally transplanted congenic or allogeneic hematopoietic cells with predictable donor-specific tolerance [abstract]. Blood. 94:320a.
40. Ashizuka, S., Peranteau, W.H., Hayashi, S., Flake, A.W. 2006. Busulfan-conditioned bone marrow transplantation results in high-level allogeneic chimerism in mice made tolerant by in utero hematopoietic cell transplantation. Exp. Hematol. 34:359-368.
    View this article via: CrossRef PubMed Google Scholar
41. Hayashi, S., Peranteau, W.H., Shaaban, A.F., Flake, A.W. 2002. Complete allogeneic hematopoietic chimerism achieved by a combined strategy of in utero hematopoietic stem cell transplantation and postnatal donor lymphocyte infusion. Blood. 100:804-812.
    View this article via: CrossRef PubMed Google Scholar
42. Peranteau, W.F., Hayashi, S., Hsieh, M., Shaaban, A.F., Flake, A.W. 2002. High level allogeneic chimerism achieved by prenatal tolerance induction and postnatal non-myeloablative bone marrow transplantation. Blood. 100:2225-2234.
    View this article via: CrossRef PubMed Google Scholar
43. Waddington, S.N., et al. 2003. Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice. Gene Ther. 10:1234-1240.
    View this article via: CrossRef PubMed Google Scholar
44. Appleby, P., Catty, D. 1983. Transmission of immunoglobulin to foetal and neonatal mice. J. Reprod. Immunol. 5:203-213.
    View this article via: CrossRef PubMed Google Scholar
45. Greeley, S.A., et al. 2002. Elimination of maternally transmitted autoantibodies prevents diabetes in nonobese diabetic mice. Nat. Med. 8:399-402.
    View this article via: CrossRef PubMed Google Scholar
46. Setiady, Y.Y., Samy, E.T., Tung, K.S. 2003. Maternal autoantibody triggers de novo T cell–mediated neonatal autoimmune disease. J. Immunol. 170:4656-4664.
    View this article via: PubMed Google Scholar
47. Cohen-Solal, J.F., Cassard, L., Fridman, W.H., Sautes-Fridman, C. 2004. Fc gamma receptors. Immunol. Lett. 92:199-205.
    View this article via: CrossRef PubMed Google Scholar
48. Amigorena, S., Bonnerot, C. 1999. Fc receptor signaling and trafficking: a connection for antigen processing. Immunol. Rev. 172:279-284.
    View this article via: CrossRef PubMed Google Scholar
49. Sallusto, F., Lanzavecchia, A. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179:1109-1118.
    View this article via: CrossRef PubMed Google Scholar
50. Schuurhuis, D.H., et al. 2002. Antigen-antibody immune complexes empower dendritic cells to efficiently prime specific CD8+ CTL responses in vivo. J. Immunol. 168:2240-2246.
    View this article via: PubMed Google Scholar
51. Antoniou, A.N., Watts, C. 2002. Antibody modulation of antigen presentation: positive and negative effects on presentation of the tetanus toxin antigen via the murine B cell isoform of FcgammaRII. Eur. J. Immunol. 32:530-540.
    View this article via: CrossRef PubMed Google Scholar
52. Setiady, Y.Y., Pramoonjago, P., Tung, K.S. 2004. Requirements of NK cells and proinflammatory cytokines in T cell-dependent neonatal autoimmune ovarian disease triggered by immune complex. J. Immunol. 173:1051-1058.
    View this article via: PubMed Google Scholar
53. Durkin, E.T., Jones, K.A., Rajesh, D., Shaaban, A.F. 2008. Early chimerism threshold predicts sustained engraftment and NK-cell tolerance in prenatal allogeneic chimeras. Blood. 112:5245-5253.
    View this article via: CrossRef PubMed Google Scholar
54. Shaaban, A.F., Milner, R., Kim, H.B., Fichter, C., Flake, A.W. 1999. Donor natural killer cell Ly49 inhibitory profile is altered by development in an MHC mismatched recipient environment following in utero hematopoietic stem cell transplantation [abstract]. Transplantation. 67:573a.
55. Westerhuis, G., Maas, W.G., Willemze, R., Toes, R.E., Fibbe, W.E. 2005. Long-term mixed chimerism after immunologic conditioning and MHC-mismatched stem-cell transplantation is dependent on NK-cell tolerance. Blood. 106:2215-2220.
    View this article via: CrossRef PubMed Google Scholar
56. Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M., Toda, M. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J. Immunol. 155:1151-1164.
    View this article via: PubMed Google Scholar
57. Takahashi, T., et al. 2000. Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192:303-310.
    View this article via: CrossRef PubMed Google Scholar
58. Mold, J.E., et al. 2008. Maternal alloantigens promote the development of tolerogenic fetal regulatory T cells in utero. Science. 322:1562-1565.
    View this article via: CrossRef PubMed Google Scholar
59. Guleria, I., Sayegh, M.H. 2007. Maternal acceptance of the fetus: true human tolerance. J. Immunol. 178:3345-3351.
    View this article via: PubMed Google Scholar
60. Aluvihare, V.R., Kallikourdis, M., Betz, A.G. 2004. Regulatory T cells mediate maternal tolerance to the fetus. Nat. Immunol. 5:266-271.
    View this article via: CrossRef PubMed Google Scholar
61. Brusko, T.M., Hulme, M.A., Myhr, C.B., Haller, M.J., Atkinson, M.A. 2007. Assessing the in vitro suppressive capacity of regulatory T cells. Immunol. Invest. 36:607-628.
    View this article via: CrossRef PubMed Google Scholar