MATERNAL IMMUNOTOLERANCE: DEFINING THE DEVELOPMENT OF T CELLS DURING EARLY PREGNANCY IN THE HORSE

Abstract

Pregnancy acceptance requires immune tolerance of the embryo, which is initiated upon maternal exposure to the paternal antigen into the uterus. Stimulation is believed to be initiated by paternal antigen presentation within the seminal plasma (SP) fraction to developing regulatory T cell (Treg) populations within the maternal immune system. Tregs then suppress effector T cells, such as Th1. The mechanisms in which this is modulated have yet to be fully elucidated, but the equine model may offer unique insights, as SP is frequently reduced during assisted reproductive techniques (ART) such as insemination with cryopreserved semen. Therefore, we aimed to assess developing Treg and Th1 populations following insemination, in addition to evaluating the impact of SP exposure on these populations. To do so, peripheral blood mononuclear cells (PBMCs) were collected from mares prior to insemination in addition to weekly following ovulation for 28 days. Following insemination, mares were categorized into groups based on breeding protocol; mares were bred with fresh semen (n=10), cooled semen (n=8), and frozen semen (n=6). RNA was isolated from PBMC for qRT-PCR of the transcription factors associated with Treg (FoxP3, TGF-β, CD25) and Th1 (TBX21, IFN-γ) development. Statistics were performed utilizing SAS 9.4, where the effect of time, pregnancy outcome, and SP exposure was assessed. FoxP3 expression was found to increase in expression over time (p=0.04) in pregnant animals, while no changes were noted for TGF-β (p=0.38), CD25 (p=0.67), TBX21 (p=0.39) or IFN-γ (p=0.14). While a significant effect of pregnancy status was noted for IFN-γ (p=0.05), noted as increased IFN-γ expression in the nonpregnant animals, no effect of pregnancy status was noted when assessing FoxP3 (p=0.61), TGF-β (p=0.66), CD25 (p=0.29), or TBX21 (p=0.93). A significant effect of SP exposure at time of breeding was noted for CD25 (p=0.04) with a trend noted for expression of TGF-β (p=0.06). In both Treg-related transcripts, expression levels were found to be highest in mares bred with fresh semen (full SP exposure) in comparison to cooled or frozen semen, where SP was reduced. In conclusion, these findings suggest that exposure to seminal plasma at the time of breeding may influence the development of maternal immune tolerance by promoting Treg-associated signaling pathways. Mares bred with fresh semen, and therefore exposed to greater concentrations of seminal plasma, demonstrated increased expression of Treg-associated transcripts compared to mares bred with cooled or frozen semen. Collectively, these data support a role for seminal plasma in shaping early maternal immune responses following insemination and highlight a potential immunological consequence of reduced seminal plasma exposure during assisted reproductive techniques in the horse.