Spatiotemporal Dynamics of NK Cell Immune Checkpoints Tim-3 and Lag-3 in Human Pregnancy: An Integrated Peripheral...

Pregnancy represents a unique state of immune adaptation requiring precise regulation to maintain tolerance toward the semi-allogeneic fetus. Although natural killer (NK) cells and T cells play central roles in maternal-fetal immunity, gestational stage-specific expression patterns of immune checkpoint molecules Tim-3 and Lag-3, especially regarding their soluble forms, remain poorly characterized. This study comprehensively investigates T-cell immunoglobulin and mucin domain-3 (Tim-3) and Lymphocyte-activation gene 3 (Lag-3) in maternal immune regulation by integrating peripheral blood and decidual tissue analyses. In decidual NK (dNK) cells, single-cell RNA sequencing identified four distinct subsets (dNK1, dNK2, dNK3, and proliferating NKp cells) and revealed subset-specific immune checkpoint profiles: Tim-3 was highly expressed in dNK1 and dNK2, whereas Lag-3 showed consistently low expression. Patients with recurrent pregnancy loss (RPL) exhibited significantly reduced Tim-3 expression in key dNK subsets. Gene set enrichment analysis (GSEA) of Tim-3+ dNK cells identified significant enrichment in immune-related pathways, including chemokine signaling, cytokine-cytokine receptor interaction, and apoptosis. Peripheral analyses revealed dynamic gestational changes: Tim-3+ NK cells peaked early and declined, while Tim-3+ T cells gradually increased. Soluble Tim-3 (sTim-3) levels negatively correlated with membrane-bound Tim-3 on NK cells, suggesting shedding-mediated regulation. Collectively, these findings underscore the spatial and temporal specificity of immune checkpoint regulation during pregnancy and indicate that dysregulated Tim-3 expression in both decidual and peripheral NK cells may contribute to impaired maternal-fetal immune tolerance in RPL.