Serial intravascular labeling resolves dynamic decidual NK residency and gestational remodeling toward T-bet-Low states

Decidual natural killer (dNK) cells are the predominant immune population at the maternal-fetal interface during early pregnancy. However, how vascular trafficking, tissue residency, proliferation, and apoptosis-associated activity are coordinated to establish and maintain the dNK population throughout pregnancy remains unclear. We hypothesized that gestational remodeling of the dNK compartment reflects coordinated changes in these cellular processes in association with progressive T-bet downregulation. We combined serial intravascular labeling (SIVL), flow cytometry, BrdU incorporation, active caspase-3 detection, spatial imaging, and Tbx21 fate mapping to examine NK cells in virgin mice and across gestation. Pregnancy altered CD49a/CD49b-defined dNK-cell composition and redistributed dNK cells among SIVL-defined vascular-accessibility states, supporting a model of dynamic dNK-cell tissue residency. Trafficking-associated marker scores displayed continuous gradients of tissue retention, circulatory/egress potential, and chemokine responsiveness rather than defining a single resident state. Spatial imaging demonstrated greater dNK-cell density in the mesometrial decidua and preferential CD31⁺ vessel contact by recently intravascular dNK cells during early and mid-gestation. BrdU incorporation and active caspase-3 positivity peaked during early pregnancy and varied across SIVL-defined states. Tbx21 fate mapping identified a progressive and selective shift from current T-bet expression toward ex-T-bet and non-fate-mapped Eomes⁺ dNK states. T-bet-low states preferentially maintained proliferation at mid-gestation, whereas current T-bet expression was associated with greater circulatory/egress potential. However, active caspase-3 was not selectively increased in current T-bet-expressing cells. Collectively, these findings identify pregnancy-induced dNK-cell remodeling as a coordinated process integrating dynamic tissue residency, vascular accessibility, local proliferation, and apoptosis-associated activity, alongside a progressive shift toward T-bet-low dNK states.