Differential secretion of TNF-α and IFN-γ by human peripheral blood-derived NK subsets and association with functional maturation

Abstract

Natural killer cells can be separated into three major subsets (free, binder, and killer) based on their ability to bind and kill sensitive target cells. The nonbinder, nonkiller free cells are the most immature and can be activated to become binders and killers. Natural killer (NK) cells synthesize and secrete several cytokines that are intimately involved in NK activation. This study investigated the secretion of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) by purified NK cells and NK subsets following activation by various stimuli. K562 target cells stimulated secretion of both TNF-α and IFN-γ by both the binder and the killer subsets but not by the free subset. IFN-α activated the secretion of IFN-γ only, whereas IL-2 activated the secretion of both TNF-α and IFN-γ by the binder and killer subsets and secretion was augmented by the addition of K562 to the cultures. Phorbol myristate acetate (PMA) and ionophore stimulated TNF-α and IFN-γ secretion in both the binder and the killer subsets, though IFN-γ secretion was more pronounced in the binder subset. Activation of TNF-α and IFN-γ secretion was dependent on de novo protein synthesis. Analysis at the single-cell level demonstrated that the binder subset had the highest frequency of cells secreting IFN-γ. These results demonstrate that both the binder and the killer subsets can be activated to secrete TNF-α and IFN-γ, whereas the free NK subset secretes little or no TNF-α and IFN-γ following activation. These data suggest that the ability of NK cells to secrete TNF-α and IFN-γ following activation correlates with the functional stage of maturation of NK cells.