INTRODUCTION

Although the birth of Dolly, the first offspring produced by somatic cell nuclear transfer (SCNT), happened more than 15 yr ago [1], SCNT is still a very inefficient process, with less than 10% of transferred embryos resulting in a live offspring [123–4]. In cattle, Day 28–32 pregnancy rates in SCNT and in vitro fertilization (IVF)-produced embryos are comparable (approximately 40%) [56–7]. While loss of pregnancies produced by IVF is approximately 13%–16% between Days 30 and 90 of gestation, and, in in vivo-produced embryos, pregnancy loss during this time period is about 5%, pregnancy losses range from 50% to 100% in SCNT pregnancies [8].

Abnormal placentation appears to be the main cause of these embryonic losses [9]. During the first trimester of SCNT pregnancies, hypoplastic, partially developed placentae with rudimentary cotyledons have been described [9]. In the later stages of gestation, placentae are grossly abnormal with membranes that are thickened and edematous, and placentomes that are reduced in number and hypertrophic [8].

One of the mechanisms cattle and other species use to escape from maternal immune attack is to downregulate the expression of trophoblast major histocompatibility complex class I (MHC-I) proteins during the first trimester of pregnancy. This is a mechanism to protect the semiallogeneic conceptus from attack by the maternal immune system. Unlike other mammals, in cattle, placental MHC-I expression is temporally and regionally regulated [10]. In the interplacentomal region, a significant number of trophoblast cells are positive for classical MHC-I (MHC-Ia) and nonclassical MHC-I (MHC-Ib) proteins from the sixth month of pregnancy on, while the placentomal villous trophoblast cells, the area of intimate contact between fetal cells and the maternal epithelium, are negative throughout gestation [10, 11].

Immunological recognition of allogeneic MHC-I proteins facilitates successful detachment of the bovine placenta at parturition. An increased incidence of placental retention in normal gestations has been associated with MHC-I compatibility between the mother and the fetus [1213–14]. Davies et al. [13] found that MHC-I-compatible pregnancies had decreased numbers of trophoblast and epithelial cells undergoing apoptosis, decreased expression of IL2 at the fetal-maternal interface, and incomplete degranulation of binucleate cells.

While MHC-I incompatibility seems to be beneficial for placental detachment at term, the expression of MHC-I proteins by SCNT embryos during the first trimester of pregnancy appears to be detrimental to embryonic survival [13, 15]. In SCNT pregnancies from 34 to 63 days, trophoblast cells were found to express MHC-I proteins, while age-matched control pregnancies were negative for MHC-I [15]. This abnormal MHC-I expression likely results from incomplete reprogramming of somatic cell nuclei during the SCNT process. In the same study, SCNT pregnancies had an increased number of endometrial cluster of differentiation (CD)3⁺ T lymphocytes that was likely a response to the abnormal MHC-I expression [15]. A maternal immune response against trophoblast MHC-I proteins is likely one of the causes of the high first-trimester embryonic mortality rate observed in SCNT pregnancies.

The present study investigated the expression of MHC-I proteins by trophoblast cells, endometrial lymphocyte populations, and endometrial cytokines at Day 35 of gestation in MHC-I-compatible and -incompatible pregnancies. Our hypothesis is that inappropriate placental MHC-I expression by SCNT conceptuses induces a uterine immune response against the placenta in MHC-I-incompatible pregnancies, while conceptuses that are MHC-I compatible or established by artificial insemination (AI) do not trigger such a response.

MATERIALS AND METHODS

Materials

PBS was purchased from Fisher Scientific (Fair Lawn, NJ), as were hydrogen peroxide and bovine serum albumin (BSA). Normal goat serum, aminoethyl carbazole (AEC) substrate kits, and streptavidin peroxidase were obtained from Invitrogen (Camarillo, CA). Detailed information about the monoclonal antibodies is shown in Table 1. Monoclonal antibodies against bovine MHC-I, MHC-II, CD3, CD4, CD8, CR2 (previously known as CD21), γ/δTCR, CSF2 (previously known as GM-CSF), and their respective isotype-matched negative controls were purchased from the Washington State University Monoclonal Antibody Center (Pullman, WA). The FOXP3 monoclonal antibody (clone 157B/F4) was a generous donation from Dr. Alison Banham at the University of Oxford [16]. The mouse anti-human CD68 antibody (clone EBM11, ascites, 2.3 μg/ml) was purchased from Dako (Carpinteria, CA). Monoclonal antibodies against NCR1 (previously known as NKp46), IL12, and TNF (previously known as TNFα) were purchased from AbD Serotec (Kidlington, U.K.). The biotinylated anti-mouse IgG secondary antibody was purchased from Vector Laboratories (Burlingame, CA). Fluoromount-G mounting medium was purchased from Southern Biotech (Birmingham, AL).

TABLE 1

Monoclonal antibodies used for immunohistochemistry.

a 

Washington State University Monoclonal Antibody Center (Pullman, WA).

b 

Donated by Dr. Alison Banham (University of Oxford, Oxford, U.K.).

c 

Dako (Carpinteria, CA).

d 

AbD Serotec (Kidlington, U.K.).

RESULTS

Gross Morphology and Embryonic Survival

Embryonic mortality rates between 28 and 35 days were significantly increased in the MHC-I heterozygous-incompatible pregnancies compared with the MHC-I homozygous-compatible and -incompatible, and control pregnancies (100%, 11.1%, 12.5%, and 12.5%, respectively; P < 0.001; Table 2). There was no significant difference between the MHC-I homozygous-compatible, homozygous-incompatible, and control pregnancies for embryonic mortality rates. The presence of grossly visible placental vasculature, readily seen as blood vessels filled with bright red blood, was completely correlated with embryonic viability (Table 2 and Supplemental Fig. S1). Embryonic crown-to-rump length and weight of SCNT embryos did not significantly differ between groups (P = 0.32; Fig. 1).

FIG. 1

Crown-to-rump length (cm) and weight of embryos at Day 35 ± 1 of gestation. SCNT pregnancies were either MHC-I homozygous-compatible (n = 9), homozygous-incompatible (n = 8), or heterozygous-incompatible (n = 5). Age-matched control pregnancies (n = 8) were established by AI. There was no significant difference between any of the groups.

TABLE 2

Comparison of embryonic mortality rates and placental vascularization.

a 

Embryonic mortality was assessed between Pregnancy Days 28 and 35 ± 1.

b 

Placental vascularization was assessed grossly at Day 35 ± 1 of gestation.

c 

The heterozygous-incompatible SCNT group was significantly different from the other groups (P < 0.001).

MHC-I Expression by Trophoblast Cells

In the control animals, the trophoblast expression of MHC-I proteins was nearly undetectable, including in the placenta of the one dead embryo. Trophoblast cells from SCNT pregnancies expressed MHC-I proteins at different levels. The heterozygous-incompatible pregnancies expressed significantly higher levels of MHC-I proteins compared to the control, homozygous-compatible, and homozygous-incompatible pregnancies (P < 0.001, P = 0.006, and P = 0.01, respectively; Figs. 2 and 3). Although both the homozygous-compatible and -incompatible pregnancies expressed higher levels of MHC-I proteins than the control pregnancies, statistical significance was only achieved for the homozygous-compatible group (P = 0.035), not for the homozygous-incompatible group (P = 0.13; Fig. 2).

FIG. 2

Percent area of MHC-I⁺ trophoblast cells in the placenta, and dispersed CD3⁺ and CD4⁺ T cells in the endometrium in control (n = 8), MHC-I homozygous-compatible SCNT (n = 9), MHC-I homozygous-incompatible SCNT (n = 8), and MHC-I heterozygous-incompatible SCNT (n = 5) pregnancies at Day 35 ± 1 of pregnancy. Age-matched control pregnancies were established by AI. Groups with different superscripts were significantly different (P < 0.05).

FIG. 3

Immunohistochemical labeling of trophoblast for MHC-I (A–D), and endometrial tissue for CD3 (E–H) and CD4 (I–L) of cows at Day 35 ± 1 of pregnancy. A, E, and I) Pregnancies established by AI (control). B, F, and J) MHC-I homozygous-compatible SCNT pregnancies. C, G, and K) Homozygous-incompatible SCNT pregnancies. D, H, and L) Heterozygous-incompatible SCNT pregnancies. Bar = 50 μm.

Endometrial CD3⁺, CD4⁺, CD8⁺, and FOXP3⁺ Cells

Endometrial lymphocyte infiltration and proliferation was assessed by the percent area of the superficial and deep endometrial stroma occupied by these cells. In the interplacentomal endometrium the presence of dispersed CD3⁺, CD4⁺, CD8⁺, and FOXP3⁺ cells in the superficial stroma followed the same trend as the trophoblast MHC-I expression (Figs. 2FIG. 3–4). All SCNT pregnancies had increased numbers of dispersed CD3⁺ cells compared to the control pregnancies (P < 0.001, P < 0.001, and P < 0.001 for MHC-I homozygous compatible, homozygous incompatible, and heterozygous incompatible, respectively). There were no significant differences between the SCNT pregnancies for the infiltration of CD3⁺ lymphocytes.

FIG. 4

Percent area of dispersed CD8⁺, FOXP3⁺, and γ/δTCR⁺ lymphocytes in the endometrium of Day 35 ± 1 pregnancies established by SCNT, which were MHC-I homozygous compatible (n = 9), MHC-I homozygous incompatible (n = 8), or MHC-I heterozygous incompatible (n = 5). Age-matched control pregnancies were established by AI (n = 8). Groups with different superscripts were significantly different (P < 0.05).

Endometrial infiltration of CD4⁺ lymphocytes was significantly affected by embryonic viability (P = 0.002) as well as treatment (P < 0.001). This lymphocyte population was increased in live compared to dead fetuses in all treatment groups, which is the opposite of what would be predicted if the CD4⁺ cells were accumulating in response to the presence of a dead embryo. These cells were increased in SCNT homozygous- and heterozygous-incompatible pregnancies compared to the homozygous-compatible (P = 0.027 and P = 0.02, respectively) and control groups (P < 0.001 and P < 0.001, respectively; Figs. 2 and 3). The CD8⁺ and FOXP3⁺ cells were significantly increased in the MHC-I heterozygous-incompatible group compared to the control group (P = 0.01 for CD8⁺ cells and P < 0.001 for FOXP3⁺ cells; Fig. 4). Among the SCNT pregnancies, the MHC-I heterozygous-incompatible group was significantly increased compared with the homozygous-compatible and -incompatible pregnancies for CD8⁺ cells (P = 0.018 and P = 0.020, respectively) and FOXP3⁺ cells (P < 0.001 and P = 0.041, respectively; Fig. 4). Interestingly, while the CD3⁺, CD4⁺, CD8⁺, and FOXP3⁺ cells were disseminated throughout the superficial and glandular endometrial stroma in control pregnancies, in the SCNT pregnancies, the majority of these cells were located in the superficial stroma under the uterine epithelium. Lymphocytes were also found organized in large lymphoid nodules in the superficial endometrial stroma in the heterozygous-incompatible and, to a lesser extent, in the homozygous-compatible and -incompatible pregnancies (Fig. 3). These nodules were composed mainly of CD4⁺ cells, with the remaining cells consisting of CD8⁺, CR2⁺, and a very small percentage of γ/δTCR⁺ cells.

Endometrial CR2⁺, MHC-II⁺, and NCR1⁺ Cells

Figure 5 illustrates the area percent of dispersed CR2-, MHC-II-, and NCR1-positive cells in the different treatment groups. Very few CR2⁺ cells were found in the endometrium of pregnant cows at 35 days of pregnancy, and these cells were scattered throughout the glandular stroma. No significant difference was seen among treatment groups (P = 0.92). The MHC-II⁺ cells were significantly more abundant in the endometrium of cows carrying SCNT conceptuses compared with the control group (homozygous compatible, P = 0.002; homozygous incompatible, P = 0.011; heterozygous incompatible P = 0.003). These cells were mainly located immediately adjacent to the basement membrane of the uterine epithelium. The number of NCR1⁺ cells was dramatically increased in the heterozygous-incompatible pregnancies compared to the control, homozygous-compatible, and homozygous-incompatible groups (P = 0.009, P = 0.011, and P = 0.010, respectively).

FIG. 5

Percent area of dispersed cells positive for CR2, MHC-II, and NCR1 in the Day 35 ± 1 pregnant endometrium. Pregnancies were established by either AI (control, n = 8) or SCNT. SCNT pregnancies were MHC-I homozygous compatible (n = 9), MHC-I homozygous incompatible (n = 8), or MHC-I heterozygous incompatible (n = 5). Groups with different superscripts were significantly different (P < 0.05).

Endometrial and Placental CD68⁺ Cells

CD68 is a pan-macrophage marker. In all treatment groups, CD68⁺ cells were abundant in the endometrial stroma. The endometrial CD68⁺ cells were numerically increased in the MHC-I-incompatible treatment groups, but the increase was not statistically significant (P = 0.331; Fig. 6). As shown in Figure 6, increased numbers of macrophages were found in the placental tissue of MHC-I heterozygous-incompatible pregnancies compared with the control, homozygous-compatible, and homozygous-incompatible pregnancies (P = 0.02, P < 0.01, and P < 0.01, respectively). Two of the dead MHC-I heterozygous-incompatible conceptuses, the two with the smallest embryos, had particularly large numbers of CD68⁺ macrophages that comprised 3.9% and 11% of the area of the placenta, respectively. It is likely that these embryos died earlier than the other ones, and that more maternal macrophages infiltrated the placenta to clean up the cellular debris.

FIG. 6

Percent area of CD68⁺ cells in the endometrium and placenta of pregnancies at Day 35 ± 1 of gestation. SCNT pregnancies were MHC-I homozygous compatible (n = 9), MHC-I homozygous incompatible (n = 8), or MHC-I heterozygous incompatible (n = 5). Age-matched control pregnancies were established by AI (n = 8). Groups with different superscripts were significantly different (P < 0.05).

Cytokine Immunostaining

We were only able to conduct immunohistochemistry for TNF, IL12, and CSF2. Because cytokines are secreted proteins and are often dispersed in the tissue section, it was not possible to do a precise measurement of the area of positive cells. Therefore, cytokine protein expression was evaluated visually and scored on a scale from 0 to 5.

Immunostaining for TNF, IL12, and CSF2 is depicted in Figure 7. Immunostaining for TNF revealed that the MHC-I heterozygous- and homozygous-incompatible treatment groups had increased production of this cytokine compared to the homozygous-compatible and control groups. The mean TNF scores for the control, MHC-I homozygous-compatible, MHC-I homozygous-incompatible, and MHC-I heterozygous-incompatible pregnancies were 1.2, 2.1, 2.8, and 3.8, respectively. IL12 was only increased in the heterozygous-incompatible group compared to the other groups. Mean scores for IL12 staining in the control, homozygous-compatible, homozygous-incompatible, and heterozygous-incompatible pregnancies were 1.2, 1.0, 2.1, and 3.6, respectively. Interestingly, CSF2 was slightly increased in control compared to the SCNT pregnancies, with mean scores of 1.6 in the control, 0.9 in the homozygous-compatible, 1.2 in the homozygous-incompatible, and 1.3 in the heterozygous-incompatible pregnancies.

FIG. 7

Immunohistochemical labeling of cells for TNF (A–D), IL12 (E–H), and CSF2 (I–L) in the endometrium of cows at Day 35 ± 1 of pregnancy. A, E, and I) Pregnancies established by AI (control). B, F, and J) MHC-I homozygous-compatible SCNT pregnancies. C, G, and K) Homozygous-incompatible SCNT pregnancies. D, H, and L) Heterozygous-incompatible SCNT pregnancies. Bar = 50 μm.

DISCUSSION

In cattle, trophoblast MHC-I expression is shut down during the first trimester of the pregnancy to avoid maternal immune recognition. As shown here and by others [15], trophoblast cells from SCNT conceptuses express significant amounts of MHC-I proteins during the first trimester of pregnancy compared with control pregnancies established by AI.

As a consequence, a pronounced endometrial lymphocyte infiltration composed mainly of CD3⁺ T lymphocytes was observed. The vast majority of both the dispersed lymphocytes and the lymphocytes in lymphoid nodules were CD4⁺ T helper (Th) cells (Figs. 3 and 4). Furthermore, because the lymphoid nodules were excluded when the leukocyte populations were quantified, the relative number of CD3 and CD4 lymphocytes shown in Figure 2 for the MHC-I-incompatible pregnancies, which tended to have more lymphoid nodules, are low estimates. The accumulation of CD4⁺ lymphocytes in the endometrium of MHC-I-incompatible SCNT pregnancies suggests that MHC-I proteins are processed and presented to the maternal immune system indirectly by MHC-II⁺ endometrial antigen-presenting cells. The indirect recognition of MHC-I proteins is also supported by the presence of significant numbers of MHC-II⁺ cells in the superficial stroma of the endometrium. It is likely that delivery of MHC-I proteins to the maternal immune system happens through the migration and fusion of MHC-I⁺ binucleate trophoblast cells with the maternal endometrial epithelium and eventual death of hybrid trinucleate cells [181920–21].

Placental abnormalities have been suggested to be the main cause for the increased fetal demise of SCNT-produced calves [4, 9, 22, 23]. SCNT embryos and fetuses themselves present few macroscopic and histological abnormalities [23]. In the present study, there was complete correlation between the presence of grossly visible placental vasculature and embryonic mortality (Table 2 and Supplemental Fig. S1). It is likely that lack of visible blood vessels was due to embryonic circulatory failure and an absence of red blood cells in the placental vasculature. Lack of grossly visible placental blood vessels is not evidence for failure of placental vasculogenesis. Placental vascular development can only be assessed histologically, which was not done. Embryonic mortality between Days 28 and 35 of gestation was dramatically increased in the MHC-I heterozygous-incompatible group compared with other pregnancies. The fact that all of the embryos in this group were dead at the time of tissue collection is a potential confounding factor. Nevertheless, our data suggest that lymphocyte infiltration was the cause and not the consequence of fetal demise. The evidence for this conclusion is that the number of endometrial lymphocytes was not increased in the control pregnancy that had a dead fetus. This is also in accord with a previous study in which the only dead fetus observed in the control group did not trigger an increased endometrial lymphocytic infiltration [15]. Furthermore, the lack of a difference in fetal weight and crown-to-rump length suggests that SCNT and AI embryos were developmentally comparable and that embryonic death occurred immediately prior to sample collection.

Recent evidence indicates that T regulatory cells play an essential role in tolerance of the fetal allograft in mice and humans. In mice, the absence of T regulatory cells leads to implantation failure, due to a maternal immune response against fetal alloantigens [24, 25]. Expression of the transcription factor, FOXP3, is critical for the development and function of T regulatory cells. Deficient expression of FOXP3 is characterized by a lethal lymphoproliferative autoimmune disease in mice [26] and humans [27]. In the present study, we observed an increase in cells expressing FOXP3 in the heterozygous-incompatible group, which followed the same trend as the number of CD3⁺, CD4⁺, and CD8⁺ T cells present in the endometrium. This suggests that T regulatory cells respond to the same chemotactic signals as other T lymphocytes in the predominantly proinflammatory endometrial environment of MHC-I heterozygous-incompatible pregnancies. An alternative explanation is that FOXP3⁺ cells were specifically recruited to the endometrium as a way to control T cell responses in an attempt to prevent immune-mediated rejection of the conceptus.

As with the other T lymphocyte populations, γ/δTCR⁺ T cell infiltration was minimal in control pregnancies. Interestingly, the pattern of endometrial infiltration of these cells in the SCNT pregnancies was opposite to that of the other immune cell populations. Although the difference was not statistically significant, γ/δTCR⁺ T cells were more abundant in the endometrium of cows carrying MHC-I homozygous-compatible pregnancies than in the uterus of cows carrying MHC-I heterozygous- and homozygous-incompatible conceptuses. The role of γ/δTCR⁺ cells during pregnancy is still unclear. However, Hoek et al. [28] demonstrated that circulating bovine γ/δTCR⁺ cells could function as regulatory cells in culture systems. While in humans and rodents only 1%–10% of T lymphocytes are γ/δTCR⁺, in ruminants the percent of lymphocytes that are γ/δTCR⁺ can be quite large (20%–40%) [29]. A recent study showed that γ/δTCR⁺ cells are significantly increased both in peripheral blood and in the decidua of pregnant compared with nonpregnant women [30], and that these cells are highly biased toward IL10 and TGFB1 (previously known as TGF-β) production [30, 31]. Fan et al. [30] also found that these cells promoted trophoblast proliferation and invasion, and decreased trophoblast apoptosis through IL10 secretion. Gamma/deltaTCR⁺ cells accumulate in the endometrial epithelium of pregnant sheep as a result of unknown systemic signals [32, 33]. These cells may contribute to the growth of the conceptus and immune tolerance to fetal antigens. The increased mortality rate of the MHC-I heterozygous-incompatible embryos between 28 and 35 days of gestation may be compounded by the lower number of regulatory γ/δTCR⁺ cells in the endometrium providing a less-favorable immunological environment for embryonic survival.

Macrophages seem to play an essential role during early pregnancy, and the population of macrophages is expanded in the endometrium during pregnancy as early as Day 13 of gestation [34]. During attachment, apoptosis of endometrial epithelial cells and syncytial cells, hybrid cells resulting from fusion of endometrial epithelial cells and binucleate trophoblast cells, is critical for tissue remodeling and trophoblast invasion. The effective removal of apoptotic cells and cell debris by endometrial macrophages is a fundamental process during attachment because it promotes adequate tissue remodeling and prevents the release of paternal antigens. There was considerable variation in the number of CD68⁺ endometrial macrophages in all of the experimental groups, so the difference between the groups was not statistically significant (Fig. 6). Nevertheless, the number of endometrial macrophages in the MHC-I-incompatible pregnancies was increased in comparison to the control group. One explanation for the increased number of macrophages in the MHC-I heterozygous pregnancies is that these macrophages were attracted to the endometrium in response to the death of the conceptus. However, this explanation seems unlikely, since there were just as many macrophages in the MHC-I homozygous-incompatible pregnancies that had viable embryos. Another explanation for the increased numbers of endometrial macrophages in the MHC-I homozygous- and heterozygous-incompatible pregnancies is that endometrial macrophages were attracted or proliferated in conjunction with the increase in the number of CD4⁺ T lymphocytes (Fig. 2). This makes sense, since the primary role of CD4⁺ Th1 cells is to activate macrophages.

The signals that initiate the migration and differentiation of monocytes into macrophages in the pregnant endometrium still remain to be elucidated. Macrophages have a very high degree of plasticity, and differentiate according to their microenvironment. Macrophages are classified into two phenotypes: M1 and M2. The M1 phenotype promotes inflammation and cytotoxicity, while M2 macrophages are involved in immunosuppression and angiogenesis [35, 36]. In cattle pregnancies, at least a portion of the endometrial macrophages are M2 macrophages, which are involved in angiogenesis, immunoregulation, tissue remodeling, and apoptosis [37]. Trophoblast cells from healthy pregnancies seem to play a major role in stimulating endometrial macrophages to acquire an immunosuppressive, M2 phenotype. Exposure of macrophages to in vitro-cultured trophoblast debris induces the expression of immunosuppressive factors, such as IL10, IL6, IL1 receptor antagonist (IL1RN), and indoleamine 2,3-dioxygenase 1, and decreases the secretion of IL1B (previously known as IL1β), IL12 (previously known as IL12p70), and CXCL8 (previously known as IL8) [38]. Additionally, endometrial macrophages have been shown to secrete high levels of vascular endothelial growth factor A (previously known as VEGF), which suggests that they are involved with vascular remodeling [39].

We found that placental macrophages were increased in the MHC-I heterozygous-incompatible pregnancies compared to the control and other SCNT pregnancies. Since all of the MHC-I heterozygous embryos were dead at the time of tissue collection, the macrophages present in these placentae were probably maternal macrophages that were responding to the death of the embryo by invading the conceptus to phagocytize cellular debris.

The number of MHC-II⁺ cells was increased in the SCNT pregnancies compared with the control pregnancies. It would be reasonable to conclude that the majority of these MHC-II⁺ cells are macrophages; however, Oliveira and Hansen [40] have demonstrated that, in the interplacentomal endometrium of normal pregnancies, the vast majority of the MHC-II⁺ cells adjacent to the luminal epithelium do not express markers specific for macrophages, such as CD68 or CD14. Therefore, it is likely that a significant portion of the MHC-II⁺ cells that we observed in the superficial stroma were dendritic cells rather than macrophages. The CD68⁺ cells that were present could be mainly of the M2 phenotype, responsible for angiogenesis and immunoregulation, but not antigen presentation. While it is unclear if endometrial M2 macrophages are MHC-II negative, tumor-associated M2 macrophages have been shown to have significantly decreased MHC-II expression compared to M1 macrophages [41]. The MHC-II⁺ cell population adjacent to the endometrial glands was also mostly negative for CD68 and CD14, but was CR2⁺. Other data collected by our group suggest that these cells are likely dendritic cells (C.R. Thacker, H.M. Rutigliano, and C.J. Davies, unpublished results).

Natural killer (NK) cells recognize self-MHC-I proteins using receptors that deliver inhibitory signals that prevent destruction of the target cell. When there is absence or down-regulation of MHC-I, or the expression of nonself-MHC-I proteins, these inhibitory signals are not delivered and the target cell is killed either by the release of cytotoxic granzymes [42] or by induction of apoptosis [43]. In this study, the presence of increased numbers of NK cells in the MHC-I heterozygous-incompatible group suggests that these cells were recruited as a result of the proinflammatory environment in the endometrium. It is possible that NK cells interact with trophoblast cells that express allogeneic MHC-I proteins and induce their apoptosis.

Although at a lower magnitude, the endometrial lymphocyte infiltration in the contralateral uterine horn to the pregnancy followed the same trend as in the ipsilateral horn (Supplemental Fig. S2). This suggests the involvement of both local and systemic signals in lymphocyte accumulation. Majewski et al. [33], using a model where ovine pregnancies were confined to only one uterine horn, demonstrated that factors of maternal or conceptus origin act systemically, rather than locally, to regulate endometrial lymphocyte populations during pregnancy.

Unfortunately, we were not able to conduct immunohistochemistry for many of the cytokines in which we were interested, but the IL12 and TNF cytokine expression data presented here are in agreement with our lymphocyte population data, which suggests that there is a pronounced T cell and macrophage response mainly in the MHC-I heterozygous-incompatible pregnancies. IL12 is produced mainly by macrophages and dendritic cells. Its primary function is to stimulate the differentiation of naïve T cells into Th1 cells, which produce IFNG (previously known as IFNγ) and TNF. TNF is primarily secreted by M1 macrophages, but is also produced by CD4⁺ lymphocytes and NK cells; it is responsible for a variety of functions, including local inflammation, endothelial activation, apoptosis, and T and B cell proliferation. Our gene expression data, which will be presented in a subsequent aricle (H.M. Rutigliano et al., unpublished data), are consistent with what we have shown here, and demonstrates that, in the endometrium of MHC-I heterozygous-incompatible pregnancies, Th1 proinflammatory cytokines, such as IFNG, TNF, and IL12, are upregulated.

We have observed that MHC-I heterozygous SCNT conceptuses trigger an exacerbated endometrial immune response, which probably leads to fetal death. Davies et al. [13] showed that MHC-I homozygous conceptuses produced by SCNT had considerably higher survival rates from Pregnancy Days 28 to 90, 28 to 200, and 28 to term compared with MHC-I heterozygous conceptuses. The exacerbated immune response and decreased embryonic survival in the MHC-I heterozygous-incompatible pregnancies can be explained by the fact that trophoblast cells from these pregnancies express two MHC-I haplotypes that are antigenically different from the maternal haplotypes, while the homozygous-incompatible pregnancies express only one unmatched haplotype. The increased antigenicity of the MHC-I heterozygous conceptuses triggers an augmented immune response against fetal antigens in the endometrium, which is accompanied by increased levels of IFNG. It is known that IFNG increases MHC-I expression in somatic cells [44]. Therefore, a feedback loop may be formed with antigenic MHC-I proteins expressed by trophoblast cells of MHC-I-incompatible SCNT conceptuses promoting release of IFNG, which, in turn, promotes further expression of MHC-I by trophoblast cells.

To our knowledge, this is the first study to determine embryonic survival and uterine immune responses in MHC-I-compatible and -incompatible SCNT pregnancies. We have demonstrated that MHC-I compatibility between the fetus and the dam influences the maternal immune response to the conceptus. MHC-I-matched or -compatible SCNT pregnancies have lower endometrial lymphocytic infiltration and better embryonic survival rates than MHC-I-incompatible pregnancies.