PD-L1 expression in trophoblasts contributes to maternal-fetal immune tolerance in the canine placenta

To elucidate the mechanisms of maternal-fetal immune tolerance in the canine endotheliochorial placenta, we investigated the localization and functional significance of programmed death-ligand 1 (PD-L1). Immunohistochemical analysis revealed region-specific PD-L1 expression, with negative staining in syncytiotrophoblasts and cytotrophoblasts within the labyrinth zone, and positive staining in monolayered columnar trophoblasts within the junctional zone. As the junctional zone is the primary interface for maternal tissue invasion, this site-specific expression suggests that PD-L1 functions as an immunological barrier. To verify this mechanism at the cellular level, we established primary canine trophoblast cells (CD45^-/CD90^-/Pan-cytokeratin⁺) purified from placental tissues. These cells were PD-L1 negative under unstimulated conditions; however, PD-L1 expression was markedly increased upon stimulation by the addition of with culture supernatants containing T-cell-derived cytokines, including interferon-gamma (IFN-γ). Furthermore, functional analysis by co-culture with canine peripheral blood mononuclear cells revealed that trophoblasts significantly inhibited Concanavalin A-stimulated proliferation of CD4⁺ and CD8⁺ T cells and IFN-γ production in the culture supernatant. This immunosuppressive effect was significantly reversed by administration of a neutralizing anti-canine PD-L1 antibody. Therefore, trophoblasts actively suppress T-cell responses via the PD-L1 pathway. These findings highlight the importance of local immune regulation in canine pregnancy maintenance and provide a novel perspective for understanding the pathophysiology of reproductive disorders such as abortion.