Molecular evidence for natural killer-like cells in equine endometrial cups

ObjectivesTo identify equine orthologs of major NK cell marker genes and utilize them to determine whether NK cells are present among the dense infiltration of lymphocytes that surround the endometrial cup structures of the horse placenta during early pregnancy.Study designPCR primers were developed to detect the equine orthologs of NKP46, CD16, CD56, and CD94; gene expression was detected in RNA isolated from lymphocytes using standard 2-step reverse transcriptase (RT) PCR and products were cloned and sequenced. Absolute real-time RT-PCR was used to quantitate gene expression in total, CD3+, and CD3- peripheral lymphocytes, and invasive trophoblast. Lymphocytes surrounding the endometrial cups (ECL) of five mares in early pregnancy were isolated and NK marker gene expression levels were assayed by quantitative RT-PCR.Main outcome measuresAbsolute mRNA transcript numbers were determined by performing quantitative RT-PCR and comparing values to plasmid standards of known quantities.ResultsNKP46 gene expression in peripheral CD3- lymphocytes was higher than in CD3+ lymphocytes, CD16 levels were higher in the CD3+ population, and no significant differences were detected for CD56 and CD94 between the two groups. Expression of all four NK cell markers was significantly higher in lymphocytes isolated from the endometrial cups of pregnant mares compared to PBMC isolated from the same animal on the same day (NKP46, 14-fold higher; CD94, 8-fold higher; CD16, 20-fold higher; CD56, 44-fold higher).ConclusionsThese data provide the first evidence for the expression of major NK cell markers by horse cells and an enrichment of NK-like cells in the equine endometrium during pregnancy.