Dynamic switching of chemokine production and stable im-munosuppressive activity of murine placental erythroid cells during mid and late pregnancy

Nucleated erythroid cells (NECs) are emerging as important immune regulators at the maternal-fetal interface, expanding in the spleen and placenta during pregnancy to support fetomaternal tolerance. However, their chemokine profiles, migratory capacity, and suppressive functions across gestation remain incompletely understood. Allogeneic pregnancy was induced by crossing CBA females with C57Bl males. Placentas and spleens were collected at mid (E12.5) and late (E19.5) gestation. TER-119⁺ NECs were isolated by positive magnetic sorting. Chemokine production was measured by multiplex analysis; chemokine receptor mRNA expression by real-time PCR. Immunosuppressive molecules (PD-L1, TGF-β, ROS) were analyzed by flow cytometry. T-cell proliferation was assessed by CFSE dilution, and immune cell migration toward placental NECs by Transwell assay. CD45⁺ placental NECs were major PD-L1, TGF-β, and ROS producers, with maximal expression at E19.5, indicating multimodal suppressive protection in the third trimester. CCL17 and CXCL9 production switched from spleen to placenta as pregnancy progressed (E12.5 → E19.5). Conversely, splenic NECs constitutively produced high CCL22, whereas placental NECs produced minimal CCL22 and persistently low CXCL5. Splenic NECs predominantly expressed CCR3 and CXCR4 compared to non‑erythroid splenocytes. CCL2/CCL4 blockade enhanced immune cell migration toward E19.5 placental NECs, suggesting atypical chemokine decoy receptor (e.g., ACKR2) involvement. Placental NECs potently suppressed T‑cell proliferation from E12.5 onward, with stable suppressive capacity maintained despite pronounced chemokine profile remodeling. Placental NECs undergo dynamic chemokine reprogramming (CCL17/CXCL9 switching from spleen to placenta) while maintaining stable T‑cell suppression via PD-L1, TGF-β, and ROS. Enhanced migration after CCL2/CCL4 blockade reveals a complex chemokine network. These findings advance understanding of pregnancy immunobiology and may inform complications such as preterm birth and preeclampsia.