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Regular Article |
a Equine Genetics Center, James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
ABSTRACT
A system for transplanting invasive equine trophoblast (i.e., chorionic girdle) to ectopic sites has been developed as a means to study the differentiation of this tissue and to assess maternal immune responses to the conceptus tissue in a site outside the uterus. Chorionic girdle was isolated from Day 33 to 34 conceptuses and surgically placed into the vulvar mucosa or subdermal skin of recipient mares. Biopsy specimens of the graft sites for immunohistochemical staining were taken at weekly or biweekly intervals after grafting. Serum samples were collected from each recipient and tested for antibody to donor major histocompatibility complex (MHC) class I antigens using the lymphocyte microcytotoxicity assay. Transplanted trophoblast cells expressed differentiation markers associated with invading chorionic girdle and endometrial cup cells. The transplanted trophoblast cells were also labeled by an antibody to eCG. Strong cellular and humoral immune responses to the transplanted tissue were mounted by the recipients, similar to those occurring during normal equine pregnancy. Despite these responses, the invasive trophoblast transplants survived for at least 28 days after grafting and downregulated MHC class I antigens, as do the mature endometrial cup cells in equine pregnancy. These findings suggest that invasive equine trophoblast has the capacity to differentiate fully in equine nonuterine tissues, and that it can evade maternal immune responses independent of the physiological state of pregnancy and in sites other than the uterus.
conceptus, developmental biology, implantation/early development, placenta, pregnancy, reproductive immunology, trophoblast
INTRODUCTION
The complex mechanisms by which the fetal-placental unit evades maternal immune attack in a potentially hostile uterine environment appear to revolve around three major branches: 1) the immune capacity of the uterus, 2) the influence of pregnancy on the maternal immune system, and 3) the immune regulatory nature of trophoblast, the tissue that forms the primary barrier between mother and fetus. The relative importance of each branch remains to be elucidated. However, research continues to reveal insights regarding new mechanisms underlying maternal sensitization to the fetal-placental unit and the routes of fetal evasion associated with these immune responses.
The pregnant uterus has often been described as an immune-privileged site, in which the antigenically foreign fetus is protected from maternal immune responses. It was once thought that immune privilege in the uterus primarily resulted from the anatomic separation of mother and fetus [1]. Evidence is accumulating, however, that the maternal immune response is actually modified by active mechanisms associated with the fetal allograft. Maternal T and B lymphocytes acquire a transient state of tolerance toward paternal alloantigens during mouse pregnancy [2, 3], and other studies have proposed that pregnancy causes the maternal immune system to be biased toward a T helper 2 (TH2) antibody-mediated immune response rather than toward a T helper 1 (TH1) cell-mediated immune response [4]. Cytokines of the TH1 response are generally thought to be harmful to maintenance of pregnancy, and when TH1 cytokines predominate, excessive induction of natural killer (NK) cells by interleukin (IL)-2 and interferon-
and an associated increase in fetal resorptions occur in mice [5]. This maternal bias toward a TH2 response appears to originate from TH2-specific cytokines, including IL-4, IL-5, and IL-10, produced at the maternal-fetal interface [6]. Therefore, this apparent TH2 shift of the maternal immune response during pregnancy may function to protect or accommodate the integrity of the fetus and placenta.
Because major histocompatibility complex (MHC) antigens are the principal immunogenetic barriers to allograft survival, regulation of fetal and placental MHC antigen expression is considered to be an important component in fetal evasion of local maternal immune recognition and destruction during pregnancy. In the majority of species, most trophoblast populations do not express either MHC class I or II antigens, and the absence of these molecules makes it more difficult for the maternal immune system to recognize foreign paternal MHC antigens expressed by the fetus [7, 8]. Despite widespread down-regulation of MHC genes and antigens, some trophoblast subpopulations can express MHC class I antigens. For example, human extravillous cytotrophoblast cells express the relatively nonpolymorphic MHC class I antigens, HLA-C, -E, and -G, and evidence is mounting that these molecules are involved in regulating decidual NK cell-mediated lysis [9–11].
Recently, a tryptophan-catabolizing enzyme from trophoblast, indoleamine 2,3-dioxygenase (IDO), has been found to suppress maternal T lymphocyte activity against fetal allografts [12]. In these experiments, rapid T lymphocyte-mediated rejection of all allogeneic, but not syngeneic, conceptuses occurred when pregnant mice were treated with an IDO inhibitor.
Fas ligand (FasL) expression at the fetal-maternal interface may also be important for maintenance of fetal immune privilege during pregnancy. Murine studies have shown that FasL expression at the decidual-placental interface could protect the fetus from maternal leukocyte infiltration by promoting apoptosis of these Fas+ cells [13]. Similarly, human placental tissues express high amounts of FasL mRNA and protein, which could be responsible for apoptosis of Fas+ leukocytes in the decidua during the first trimester of pregnancy [14, 15].
Complement regulatory proteins have been identified on trophoblast that may be responsible for protecting the placenta from complement-mediated lysis. Inhibition of complement regulatory proteins, CD46 and CD59, on human trophoblast results in increased susceptibility of these cells to maternal complement lysis [16]. Most recently, a murine complement regulator, Crry, has been found to prevent complement deposition at the fetal-maternal interface and appears to be critical for fetal survival during pregnancy [17].
A bidirectional interaction between the maternal immune system and the fetal-placental unit thus appears to be evident. Many overlapping factors are involved in determining the successful outcome of pregnancy, but the relative contributions of the uterine site, the physiological state of pregnancy, and the nature of the trophoblast tissue to this complex interaction are difficult to study independently.
A series of events during early equine pregnancy provides a unique opportunity to study trophoblast differentiation and the immunological interactions that occur between mother and fetus. Equine invasive trophoblast cells are distinguished by the expression of high levels of paternally inherited, polymorphic MHC class I antigens. The invasive form of horse trophoblast, called the chorionic girdle, invades the endometrium by Day 36–38 of pregnancy and terminally differentiates into binucleate, eCG-secreting endometrial cup cells. By Day 45 of pregnancy, these trophoblast cells express low or undetectable levels of MHC class I antigens [18].
For nearly all histoincompatible matings, a reproducible cytotoxic alloantibody response to paternal MHC class I antigens occurs within 3 wk after chorionic girdle cell invasion of the endometrium [19]. This equine maternal anti-fetal MHC antibody response is stronger and occurs relatively earlier during gestation than in any other species studied and provides strong evidence for maternal immunological recognition of the conceptus [20].
On invasion of the endometrium, the chorionic girdle cells also evoke a dense accumulation of maternal CD4+ and CD8+ T lymphocytes around them, which appears to result in the eventual destruction of the endometrial cups by Day 120 of pregnancy [21]. During the 60- to 80-day life span of the endometrial cups as well as throughout the 340-day gestation of the mare, no such accumulations of maternal lymphoid cells occur at the interface between the noninvasive trophoblast of the allantochorion and the endometrium [22]. Surprisingly, the humoral and cellular immune responses that are mounted against the developing horse conceptus do not appear to compromise the successful outcome of pregnancy.
A system for transplanting pure populations of invasive trophoblast cells to ectopic sites has been developed as a means to study the differentiation of these tissues and to assess the maternal immune responses to the developing conceptus independent of other fetal and placental tissues and in sites distant from the uterine environment during pregnancy.
MATERIALS AND METHODS
Animals
Horses of various breeds and ages were used for these studies. These animals were housed at the Cornell Equine Genetics Center (Ithaca, NY), and all animal care was performed in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee of Cornell University. The MHC types of mares and stallions were determined using a panel of alloantisera that had been previously characterized from a series of international workshops [23]. Stallions homozygous for either the ELA-A2 or -A3 haplotypes were used as semen donors. The MHC class I compatible and incompatible pregnancies were established using artificial insemination of mares during estrus. Pregnancy was later confirmed by transrectal ultrasonography.
Trophoblast Transplant Model
The trophoblast transplant model is summarized in Figure 1. To assure primary sensitization to the trophoblast transplants, recipients of allogeneic and MHC class I compatible trophoblast transplants were normally cycling, postpubertal mares that had never been pregnant. Recipients of semiallogeneic trophoblast transplants were pregnant until the day of the transplant. These pregnancies were terminated at Day 33–34 of gestation, corresponding to the stage of pregnancy before chorionic girdle cell invasion. The vulvar mucosa was chosen as an easily accessible mucosal site. Subdermal skin was selected as a nonmucosal site. Chorionic girdles from Day 33 to 34 conceptuses were recovered and dissected as previously described [24]. Dissected chorionic girdles were then sectioned into 2-cm pieces before transplantation into fully allogeneic, semiallogeneic (i.e., autotransplant), or MHC compatible recipient mares (Table 1). For the procedure, the recipients were lightly sedated with i.v. doses of xylazine (0.2–0.4 mg/kg; Bayer Corporation, Shawnee Mission, KS) and butorphanol tartrate (0.01–0.02 mg/kg; Fort Dodge, Fort Dodge, IA). Sites of transplantation were aseptically prepared, and a local nerve block was performed with a 2% lidocaine injection (Butler Company, Columbus, OH). A small, 1- to 2-cm incision was made into the blocked sites in which 2-cm strips of dissected chorionic girdle were placed into the wound before closure using sterile suture material. The transplantation sites were monitored daily for signs of inflammation and infection. Controls included biopsies of undisturbed tissues, sutured incision sites without tissue transplants (incision-plus-suture), and endometrium from nonpregnant and pregnant mares at various stages of gestation.
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The recipient horses were sedated, and the biopsy sites were aseptically prepared and blocked as before. Sutures were removed from the sites of transplantation, and biopsies were taken weekly or biweekly between Days 7 and 28 after transplantation. Each surgical site was closed and monitored as described above.
Tissues
Blood samples for serum collection and peripheral blood lymphocyte (PBL) isolation were taken from the external jugular vein of the recipient and donors (i.e., sire and dam) of the conceptus, respectively. Serum samples were collected one to three times per week for 3 mo after transplantation and stored at -20°C. Fresh PBLs were isolated for all lymphocyte microcytotoxicity assays.
Biopsy samples were placed immediately into ice-cold PBS solution supplemented with 2x penicillin-streptomycin (Gibco BRL, Grand Island, NY). Samples for immunohistochemistry were trimmed and embedded in O.C.T. compound (VWR Scientific Products, Willard, OH), snap frozen in an isopentane bath in liquid nitrogen, and stored at -70°C. Tissue sections of 5 µm in thickness were cut using a cryostat, placed on uncoated glass slides, and fixed for 10 min at 4°C in acetone.
Immunohistochemistry and Monoclonal Antibodies
Indirect immunoperoxidase assays were performed on test and control tissue samples as described previously [25]. A panel of monoclonal antibodies specific for horse trophoblast markers [26, 27], eCG [27], and "cluster of differentiation" (CD) molecules [28, 29] were used as first-stage reagents (Table 2). Peroxidase-conjugated goat anti-mouse or anti-rat antisera (Jackson ImmunoResearch Laboratories, West Grove, PA) were used as second-stage reagents and reacted with amino-ethyl-carbazol substrate. Controls included the use of irrelevant monoclonal antibodies as first-stage reagents [30]. All sections were counterstained with hematoxylin (Fisher Scientific, Pittsburgh, PA).
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Morphometric Analysis
A morphometric analysis was performed on immunolabeled sections to determine the ratio of CD4+ T cells to CD8+ T cells surrounding the trophoblast transplants as previously described [21]. Three to four nonoverlapping fields of view were analyzed for each section. The data obtained from each section were averaged, and the ratio of CD4+ cells to CD8+ cells was calculated based on the averages obtained from each section.
Microcytotoxicity Assay
A standard lymphocyte microcytotoxicity dye exclusion assay was used to measure the antibody response to the transplanted trophoblast [31]. Briefly, fresh PBLs from the donor sire and dam of the transplanted trophoblast were isolated and tested against serially diluted antisera from the trophoblast recipient. The cytotoxic antibody titer was determined by the highest dilution of each serum that resulted in killing of at least 80% of the cells from each of the lymphocyte donors. With this technique, the titer, onset, and duration of the antibody response was determined and compared to the antibody responses during pregnancy.
RESULTS
Equine Invasive Trophoblast Cells (Chorionic Girdle) Differentiate and Function in Sites Other than the Uterus
By Day 47 of pregnancy (
10 days after chorionic girdle cell invasion of the endometrium), the invasive trophoblast cells of the chorionic girdle have terminally differentiated into binucleate, eCG-secreting endometrial cup cells. Grossly, endometrial cups appear as ulcer-like protuberances (1–2 cm in diameter) on the surface of the endometrium which encircle the conceptus at the base of the gravid uterine horn. Trophoblast transplanted to the vulvar mucosa or subdermal skin was difficult to identify grossly; however, a small (1–2 mm in diameter), red discoloration could be seen on the cut surface of some Day 7 and 14 biopsy samples.
An indirect immunohistochemistry assay was performed on each biopsy sample using a panel of monoclonal antibodies to equine trophoblast (Table 2). These results were compared to those of invading trophoblast cells at an equivalent stage of pregnancy.
Figure 2 (left panels) demonstrates the typical immunohistochemical staining pattern of endometrial cup cells at Day 47 of pregnancy when labeled with monoclonal antibodies to trophoblast differentiation markers or to eCG. Trophoblast cells of the endometrial cup aggregate at the site of chorionic girdle cell invasion and surround maternal endometrial glands (Fig. 2A). Chorionic girdle cells transplanted to the vulvar mucosa (Fig. 2B, right panels) or subdermal skin (data not shown) exhibited less cell-to-cell contact with other trophoblast cells at the site of transplantation; however, they clearly differentiated into binucleate endometrial cup cells (Fig. 2B, arrow). The intracellular staining pattern for eCG was similar for Day 47 endometrial cup cells and chorionic girdle cell transplants at Day 14 after transplantation (Fig. 2, C and D, arrow). These findings offer supporting evidence that ectopic chorionic girdle cells differentiate into functional endometrial cup cells.
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Monoclonal antibodies to noninvasive and invasive trophoblast markers were used to compare Day 47 endometrial cup cells with Day 14 chorionic girdle cell transplants. Figure 2, E and G, shows the typical staining pattern of Day 47 endometrial cup cells and surrounding tissues. The monoclonal antibody 71.7, which is specific for noninvasive trophoblast, labeled allantochorion trophoblast and some maternal tissues (Fig. 2E, arrowhead) but failed to label the mature endometrial cup cells (Fig. 2E, arrow). Monoclonal antibody 71.3, which is specific for invasive trophoblast, labeled the endometrial cup cells (Fig. 2G, arrow) but failed to label the noninvasive allantochorion trophoblast cells (Fig. 2G, arrowhead). A comparable staining pattern was identified with Day 14 chorionic girdle cell transplants. Antibody 71.7 failed to label the chorionic girdle cell transplants (Fig. 2F, arrow), whereas antibody 71.3 strongly labeled the chorionic girdle cell transplants (Fig. 2H, arrow). These findings indicate that transplanted chorionic girdle cells differentiate autonomously in sites other than the uterus.
Equine Invasive Trophoblast Cells (Chorionic Girdle) Evoke Cellular and Humoral Immune Responses Similar to Those in the Uterus During Normal Pregnancy
An indirect immunohistochemistry assay was performed on each biopsy sample using a panel of monoclonal antibody reagents specific for horse CD molecules (Table 2). Transplanted trophoblast cells in the vulvar mucosa (Fig. 3) and subdermal skin (data not shown) induced a vigorous leukocytic response similar to that seen during early equine pregnancy. As seen during developmental stages of endometrial cup cells, transplanted trophoblast cells were surrounded predominantly by CD4+ T lymphocytes, which also expressed CD3 molecules (Fig. 3, B and C). Morphometric analysis was performed on these sections using a technique developed previously for horse endometrium and endometrial cup tissues [21]. Classical CD4+ cells outnumbered CD8+ cells by a ratio of approximately 2:1 throughout the sampling duration of four recipient horses on four different days after trophoblast transplantation (Table 3). Figure 3, C and D, shows a serial section of transplanted chorionic girdle cells (arrow points to the same trophoblast cell) and the relative distribution of CD4+ and CD8+ T lymphocytes surrounding the transplanted trophoblast cells.
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To determine whether the surgical incision and suture material influenced the cellular response to transplanted trophoblast, immunohistochemical assays were performed on Day 14 biopsy specimens of undisturbed tissues (data not shown), incision-plus-suture controls (Fig. 4, right panels), and transplanted trophoblast (Fig. 4, left panels). Monoclonal antibodies to equine trophoblast, leukocyte, and MHC class II antigens were included in this assay (Table 2). Expression of MHC class II antigen was not increased in the control tissues (Fig. 4D) but was very high in the transplants (Fig. 4C). No leukocyte aggregations were associated with the incision-plus-suture control (Fig. 4F) compared to the transplanted trophoblast in the subdermal skin (Fig. 4E) and vulvar mucosa (data not shown). The surgical incision and suture material, therefore, did not appear to play an influential role in the cellular immune or inflammatory responses to transplanted trophoblast.
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The onset, duration, and titer of the cytotoxic antibody to each allogeneic, semiallogeneic, and MHC class I compatible trophoblast transplant were determined using a lymphocyte microcytotoxicity assay. Antibodies appeared approximately 14 days after transplantation of allogeneic and semiallogeneic trophoblast (Fig. 5, A–D), but no antibody was detected after transplantation of MHC class I compatible trophoblast (Fig. 5E). Testing of the antisera on a panel of MHC-typed horses revealed that the antibodies were directed against equine MHC class I antigens (data not shown).
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A stronger cytotoxic antibody response against the MHC class I antigens of the sire of the trophoblast (Fig. 5, solid circles) was observed compared to the response to MHC class I antigens of the dam of the trophoblast (Fig. 5, open circles). The peak cytotoxic antibody titer to the MHC class I antigens of the sire ranged between 1:2 and 1:64, whereas the peak cytotoxic antibody titer to the MHC class I antigens of the dam ranged between 0 and 1:8.
Semiallogeneic trophoblast transplants elicited a weaker cytotoxic antibody response against the MHC class I antigens of the donor sire compared to allogeneic trophoblast transplants. In these experiments, the highest cytotoxic antibody titer to MHC class I antigens of two different MHC homozygous sires was 1:4.
Equine Invasive Trophoblast Cells (Chorionic Girdle) Down-Regulate MHC Class I Antigens as They Do after Invading the Uterus During Normal Pregnancy
To characterize MHC class I antigen expression by the transplanted trophoblast, an indirect immunohistochemistry assay was performed on all biopsy samples using monoclonal antibodies to equine MHC class I and II molecules (Table 2). Transplanted trophoblast cells slowly began to lose MHC class I antigen expression between 14 and 28 days after transplantation (Fig. 6, A–C), similar to the loss of expression observed in endometrial cups in situ [18]. As seen during normal pregnancy, the chorionic girdle cells also remained MHC class II negative after transplantation to the vulvar mucosa (Fig. 6D) and subdermal skin (data not shown).
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Equine Invasive Trophoblast cells (Chorionic Girdle) Survive for 28 Days or Longer after Transplantation to Sites Other than the Uterus
Biopsies of transplanted trophoblast were performed at weekly or biweekly intervals up to 4 wk after transplantation to the vulvar mucosa or subdermal skin. To identify transplanted trophoblast cells and to determine the survival duration of these cells, the indirect immunohistochemistry assay was performed on sections of all biopsy samples using monoclonal antibody 102.1 (specific for equine trophoblast). Transplanted trophoblast cells were identified in 79% (11/14) of the biopsy samples. Transplanted trophoblast cells were also found to survive for at least 28 days after transplantation (Table 4). This survival duration was similar for both allogeneic and semiallogeneic trophoblast transplants.
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DISCUSSION
These results emphasize the autonomous role that trophoblast cells play in their own differentiation and in both stimulating and evading maternal immune responses during pregnancy. Equine invasive trophoblast cells were found to differentiate and to function after ectopic transplantation. Transplanted chorionic girdle cells developed into binucleate, eCG-secreting endometrial cup cells in the vulvar mucosa or subdermal skin in a time frame that paralleled this developmental process in the uterus during pregnancy.
Unexpectedly, the monoclonal antibody specific for horse trophoblast (i.e., 102.1) labeled molecules expressed by the epidermal cells of the skin (Fig. 4, A and B, arrowheads). This antibody was originally described as a trophoblast-specific molecule in the horse, one of only a few such molecules identified in any species [26]. With the exception of the trophoblast, this antibody had failed to label 18 adult and 6 fetal horse tissues. Although skin from fetuses at Days 35–60 of pregnancy were tested, skin from adult horses was never examined. Antibody 102.1 may identify an epitope that is common to the epithelium of adult skin and fetal trophoblast, or it could identify a molecule shared between the trophoblast and skin.
Studies in mice have also reported autonomous differentiation and function of trophoblast in ectopic sites. When ectoplacental cone cells from Day 8 of pregnancy were transplanted to the subcutaneous tissues of host mice, these early trophoblastic cells were able to morphologically differentiate into placental-like cells [32]. Murine blastocysts transplanted to the kidney capsule of pseudopregnant mice have also been found to be functionally active by prolonging the luteal phase induced by copulation [33].
One aspect of trophoblast differentiation not studied was invasion across an epithelial barrier, as occurs in the uterus at Days 36–38 of equine pregnancy [34]. This property of chorionic girdle cell invasion was not addressed in this study because the invasive trophoblast tissue strips were placed directly within the vulvar mucosa and subdermal skin via an incision. Despite the method of trophoblast transplantation used here, the phenotypic markers of invasion were identical to those observed for endometrial cup cells in vivo.
Equine invasive trophoblast cells evoked robust cellular and humoral immune responses after ectopic transplantation, and these responses were similar to those that occur during normal pregnancy. Specifically, the ratio of CD4+ to CD8+ T cells surrounding transplanted trophoblast was similar to that described for endometrial cups during normal horse pregnancy [21]. As with MHC class I compatible and incompatible pregnancies in horses [21, 35], no obvious differences were observed in the leukocyte responses to allogeneic, semiallogeneic, and MHC class I compatible trophoblast transplants. This suggests that in MHC compatible trophoblast transplants the cellular immune response might be directed against minor histocompatibility antigens [35]. Alternatively, the lymphocyte accumulation might have an inflammatory origin, not an antigen-specific immune basis. Equine invasive trophoblast might secrete lymphocyte-specific chemokines that traffic T lymphocytes to the site of trophoblast invasion. Secondary lymphoid-tissue chemokine (SLC) is one of several lymphocyte-specific chemokines that induce the migration of naïve T cells from the peripheral circulation to secondary lymphoid organs [36]. Other chemokines that elicit transendothelial chemotaxis of unstimulated peripheral blood T lymphocytes include monocyte chemotactic protein (MCP)-1, MCP-2, MCP-3, RANTES (regulated upon activation, normal T-expressed, and presumably secreted), macrophage inflammatory protein (MIP)-1
, and MIP-1ß [37].
Human and rodent pregnancies are marked by a striking accumulation of the so-called uterine NK cells in the decidua [38, 39]. During naturally occurring ectopic pregnancy in humans, NK cell accumulations appear to be found only when decidualization has occurred [40]. During experimental ectopic pregnancy in mice, no NK cells were found after transplantation of trophoblast to the kidney capsule [41], but decidualization was not observed in these sites. Equine pregnancy does not involve the development of decidual tissues. Therefore, the accumulation of T lymphocytes around transplanted equine trophoblast may be expected to occur, as it does in the uterus during pregnancy.
During early equine pregnancy, cytotoxic antibody to the MHC class I antigens of the sire appears shortly after development of the endometrial cups in virtually all mares carrying MHC class I incompatible fetuses [19]. These antibodies are usually detectable by Day 60 of pregnancy in primiparous mares, or approximately 20 days after the chorionic girdle cells invade the endometrium. In mares carrying MHC class I compatible fetuses, no cytotoxic antibody titers are detectable in serum samples collected at any time during pregnancy [19].
To our knowledge, host antibody responses to ectopic trophoblast transplants have not been reported previously in any other species. After equine trophoblast transplantation, the onset of cytotoxic antibody to the graft antigens was similar to that seen during a primary antibody response to the conceptus during pregnancy. There appeared to be stronger antibody responses to the lymphocytes of the sire than to the lymphocytes of the dam in fully allogeneic trophoblast transplants. This finding was surprising, because both paternal and maternal MHC class I antigens are codominantly expressed by equine trophoblast [42] and, in the few cases studied, both paternal and maternal MHC class I antigens have been found to be immunogenic [42, 43]. The antibody responses to allogeneic trophoblast transplants were stronger than the responses to semiallogeneic trophoblast transplants. This finding may relate to the physiological state of the recipient, because recipients of the semiallogeneic transplants were also donors of the conceptus tissues and, therefore, were pregnant until the day of the transplant.
The pattern of MHC class I antigen down-regulation in these experiments was similar to that described during early equine pregnancy as chorionic girdle cells differentiate into endometrial cup cells [18]. This finding was consistent for allogeneic, semiallogeneic, and MHC class I compatible trophoblast transplants. Previous studies have shown that the tempo of MHC class I antigen down-regulation does not appear to be influenced by the maternal antibody response, because the loss of expression of MHC class I antigens was not different in MHC compatible and incompatible pregnancies [18]. Compared to endometrial cup cells, transplanted chorionic girdle cells appeared to be slower in down-regulating MHC class I molecules. This suggests that the site of implantation and/or the physiological state of the pregnant mother may partially influence expression of these molecules.
Equine invasive trophoblast cells were identified in tissue samples for at least 28 days after transplantation to sites other than the uterus. Despite ongoing humoral and cellular immune responses, transplanted trophoblasts were able to survive at least twice as long as a conventional skin allograft (2 wk). The exact mechanisms by which horse trophoblast cells defend themselves against immune attack remain unknown. The findings in this study suggest that down-regulation of MHC class I antigens may be an important defense mechanism during pregnancy. However, expression of MHC class I molecules on trophoblast between Days 14 and 28 after transplantation did not appear to compromise the survival of these cells in the presence of host CD8+ T cells. These findings support previous evidence that suggests additional mechanisms may allow successful pregnancy even when MHC class I antigens are highly expressed on trophoblast [44, 45].
In summary, the findings presented here suggest that the trophoblast cells at the fetal-maternal interface play a key role in protection of the fetus from maternal immune attack during pregnancy, and that the trophoblast transplant model has the potential to provide a means to study the mechanisms underlying trophoblast differentiation, maternal sensitization, and immune responses to the fetus during pregnancy. The ability of equine trophoblast transplants to sensitize the recipient's immune system and to evoke a vigorous immune response provides strong evidence that the cells of the fetal-placental unit are primarily responsible for the immune responses observed during early pregnancy.
ACKNOWLEDGMENTS
The authors wish to thank Jane Miller and Laurie Lantagne for technical help and James Hardy for assistance with horse breeding. Slides of the immunohistochemical labeling of Day 47 endometrial cups (Fig. 2, C, E, and G) were generously provided by Dr. Julio Oriol.
FOOTNOTES
First decision: 11 August 2000.
1 Supported by National Institutes of Health grants NICHD-15799, NICHD-34086, and the Dorothy Russell Havemeyer Foundation, Inc. A.P.A. was supported by an Institutional NRSA Training Grant (T32 RR07059), and subsequently, by an Individual NRSA Grant (F32 HD08575). 
2 Correspondence: A. Paige Adams, J.A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Hungerford Hill Road, Ithaca, NY 14853. FAX: 607 256 5608; apa7{at}cornell.edu
Received: October 9, 2000.
REFERENCES
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