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.

The invasive and fully antigenic trophoblast of the chorionic girdle portion of the equine fetal membranes has the capacity to survive and differentiate after transplantation to ectopic sites. The objectives of this study were to determine: (i) the survival time of ectopically transplanted allogeneic trophoblast cells in non-pregnant recipient mares, (ii) whether equine Chorionic Gonadotrophin (eCG) can be delivered systemically by transplanted chorionic girdle cells, and (iii) if eCG delivered by the transplanted cells is biologically active and can suppress behavioral signs associated with estrus. Ectopically transplanted chorionic girdle survived for up to 105 days with a mean lifespan of 75 days (95% CI 55–94), and secreted sufficient eCG for the hormone to be measurable in the recipients’ circulation. Immunohistochemical labeling of serial biopsies of the transplant sites and measurement of eCG profiles demonstrated that graft survival was similar to the lifespan of equine endometrial cups in normal horse pregnancy. The eCG secreted by the transplanted cells induced corpora lutea formation and sustained systemic progesterone levels in the recipient mares, effects that are also observed during pregnancy. This in turn caused suppression of estrus behavior in the recipients for up to three months. Thus, ectopically transplanted equine trophoblast provides an unusual example of sustained viability and function of an immunogenic transplant in a recipient with an intact immune system. This model highlights the importance of innate immunoregulatory capabilities of invasive trophoblast cells and describes a new method to deliver sustained circulating concentrations of eCG in non-pregnant mares.

Human CRTh2+ Th2 cells express IL‐25 receptor (IL‐25R) and IL‐25 has been shown to potentiate production of Th2 cytokines. However, regulation of IL‐25R and whether it participates in Th2 differentiation of human cells have not been examined. We sought to characterize IL‐25R expression on CD4+ T cells and determine whether IL‐25 plays a role in Th2 differentiation. Naïve human CD4+ T cells were activated in the presence of IL‐25, IL‐4 (Th2 conditions) or both cytokines to assess their relative influence on Th2 differentiation. For experiments with differentiated Th2 cells, CRTh2‐expressing cells were isolated from differentiating cultures. IL‐25R, GATA3, CRTh2 and Th2 cytokine expression were assessed by flow cytometry, qRT‐PCR and ELISA. Expression of surface IL‐25R was induced early during Th2 differentiation (2 days). Addition of IL‐25 to naïve CD4+ T cells revealed that it induces expression of its own receptor, more strongly than IL‐4. IL‐25 also increased the proportions of IL‐4‐, GATA3‐ and CRTh2‐expressing cells and expression of IL‐5 and IL‐13. Activation of differentiated CRTh2+ Th2 cells through the TCR or by CRTh2 agonist increased surface expression of IL‐25R, though re‐expression of CRTh2 following TCR downregulation was impeded by IL‐25. These data suggest that IL‐25 may play various roles in Th2 mediated immunity. We establish here it regulates expression of its own receptor and can initiate Th2 differentiation, though not as strongly as IL‐4.

This research tested the hypothesis that immunological sensitization of mares by skin allografting, followed by the establishment of pregnancy using semen from the skin-graft donor, would give rise to secondary immune responses to the developing horse conceptus, resulting in an earlier demise of the fetally derived endometrial cups. Maiden mares received skin allografts from a stallion homozygous for Major Histocompatibility Complex (MHC) antigens and/or equivalent autografts and were subsequently mated to the skin-graft donor stallion during the next two breeding seasons. Mares that had been immunologically primed to the foreign MHC class I antigens of the skin-graft donor stallion developed strong secondary antibody responses early in their first pregnancies, whereas autografted mares made weak primary antibody responses in their first pregnancies and strong secondary responses in their second pregnancies. In contrast, histological examination of the endometrial cups after surgical pregnancy termination at Day 60 of gestation revealed no discernible differences between allografted and autografted mares, and there were no significant differences in the concentrations and/or duration of secretion of the endometrial cup-specific hormone, equine chorionic gonadotrophin (eCG), between allografted and autografted mares, nor in either group between first and second pregnancies. The vigorous antibody response observed in the pregnant allografted mares supported the first part of our hypothesis, providing evidence of systemic immunological priming. However, there was a lack of an equivalent heightened cellular response to the endometrial cups. These findings provided strong evidence for an asymmetric immune response to the conceptus, characterized by strong humoral immunity and a dampened cellular response.