PD-1+ NK cell subsets in high grade serous ovarian cancer: an indicator of disease severity and a target for combined immunecheckpoint blockade

Abstract
Background Ovarian cancer (OC) is the fifth leading cause of cancer-related death among women, with High-Grade
Serous Ovarian Carcinoma (HGSC) representing the most aggressive and prevalent subtype. Despite promising results
in other malignancies, immune checkpoint blockade has shown limited efficacy in HGSC, highlighting the need for
alternative immunotherapeutic targets.
Methods We conducted an integrated analysis combining multiparametric flow cytometry, RNA sequencing,
multiplex immunohistochemistry, and functional assays to characterize NK cells isolated from peripheral blood,
peritoneal fluid, primary tumor tissue, and metastases in 60 HGSC patients.
Results We identified a distinct population of PD-1⁺ NK cells enriched in HGSC tumors and metastatic sites but
absent in healthy donors. These cells, characterized by a CD56dimNKG2A⁺KIR⁺/⁻NKp46⁺CD57low phenotype, displayed
impaired cytotoxicity against autologous HGSC targets, correlating with poorer prognosis. Crucially, this dysfunction
was reversible upon combined blockade of PD-1/PD-L1, NKG2A, and KIRs. Spatial and molecular profiling revealed
that these cells localize within PD-L1⁺/HLA-E⁺ tumor niches, suggesting that immune suppression is spatially andmolecularly coordinated. Transcriptomic analysis confirmed their altered functional state and highlighted actionable
checkpoint targets.
Conclusions Our findings uncover a previously underappreciated population of dysfunctional PD-1⁺ NK cells in
HGSC and demonstrate that their suppression is reversible through combinatorial checkpoint inhibition. These
insights support the development of spatially-informed, NK-targeted immunotherapies for HGSC patients, particularly
those resistant to T cell-based strategies.