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Background: In contrast to the extensive knowledge about human natural killer (NK) cells in peripheral blood, relatively little is known about NK cells in the human lung. Knowledge about the composition, differentiation, and function of human lung NK cells is critical to better understand their role in diseases affecting the lung, including asthma, chronic obstructive pulmonary disease, infections, and cancer., Objective: We sought to analyze and compare the phenotypic and functional characteristics of NK cells in the human lung and peripheral blood at the single-cell level., Methods: NK cells in human lung tissue and matched peripheral blood from 132 subjects were analyzed by using 16-color flow cytometry and confocal microscopy., Results: CD56 dim CD16 + NK cells made up the vast majority of NK cells in human lungs, had a more differentiated phenotype, and more frequently expressed educating killer cell immunoglobulin-like receptors compared with NK cells in peripheral blood. Despite this, human lung NK cells were hyporesponsive toward target cell stimulation, even after priming with IFN-α. Furthermore, we detected a small subset of NK cells expressing CD69, a marker of tissue residency. These CD69 + NK cells in the lung consisted predominantly of immature CD56 bright CD16 - NK cells and less differentiated CD56 dim CD16 + NK cells., Conclusion: Here, we characterize the major NK cell populations in the human lung. Our data suggest a model in which the majority of NK cells in the human lung dynamically move between blood and the lung rather than residing in the lung as bona fide tissue-resident CD69 + NK cells., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)