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Abstract

The lung is a major site of immune interaction, where epithelial and immune cells cooperate to maintain barrier integrity during infection and inflammation. Alveolar Type II (AT2) cells are specialized epithelial cells in the lung alveoli. In lung infection, they recruit and activate immune cells thus driving inflammation. Among these, natural killer (NK) cells act as rapid responders, yet how local factors such as epithelial stress and oxygen deprivation influence their effector functions is not fully understood. The transcription factor hypoxia-inducible factor (HIF)-1α regulates cellular adaptation to tissue hypoxia and survival by upregulating glycolysis and angiogenesis, but also by limiting NK responses. The aim of this study was to establish an in vitro model combining primary human NK cells with the AT2-derived epithelial-like A549 cell line to mimic viral and bacterial infection- and hypoxia-associated conditions in the lung and to analyze NK cells effector functions, immune receptor expression, hypoxia-induced signaling, and ligand-dependent immune modulation. NK cells co-cultured with A549 cells exhibited increased HIF-1α expression and elevated Tumor necrosis factor (TNF)-α, Interferon (IFN)-γ, and Interleukin (IL)-8 levels, suggesting that epithelial cell-derived soluble factors and oxygen deprivation jointly contribute to NK cell metabolic activation. Furthermore, the study identified significant modulation of immune receptors, including DNAX accessory molecule (DNAM-1) particularly within the CD56dim NK cell subset. DNAM-1 was consistently downregulated across NK cell subsets, likely due to ligand-induced internalization and degradation following persistent engagement with CD155 ligand on A549 cells. Silencing CD155 in A549 cells partially restored DNAM-1 expression without affecting HIF-1α levels, indicating a direct, contact-dependent mechanism independent of HIF-1α. Building upon these insights from the co-culture model, the study next examined NK cell receptor expression in human lung tissue to determine whether similar phenotypic adaptations occur in lung tissue derived NK cells ex vivo. Profiling of 24 immune receptors on NK cells from lung tissue and peripheral blood of lung surgery patients revealed seven distinct NK cell clusters based on their receptor expression patterns. Clusters 1 and 2 were more frequent in peripheral blood, clusters 3 and 5 were enriched in lung tissue, and cluster 6 was exclusively expressed in lung tissue also showing high expression of tissue-resident markers CD69, CD103 and CD49a. Clusters 4 and 7 were similarly represented in both tissues. Together, these results highlight that epithelial stress, ligand engagement, and hypoxia collectively reshape the NK cell phenotype by modulating receptor expression and adjusting activation thresholds within the lung microenvironment.