SRP325939 PRJNA742047 RNA contents of clonal normal human NK3.3-derived extracellular vesicles Natural killer (NK) cells, critical for proper immune function, destroy tumor cells primarily through the release of granules containing potent cytolytic molecules. NK cells also release these molecules within membrane-bound exosomes and microvesicles collectively known as extracellular vesicles (EVs). Our study investigates the features and anti-tumor function of EVs isolated from our well described NK cell line - NK3.3 - the only published clonal, normal human cell line to date. We show that NK3.3 EVs contain the cytolytic molecules perforin, granzymes A and B, and granulysin, and an array of common EV proteins. We previously showed that the E3 ubiquitin ligase identified by our laboratory, natural killer lytic-associated molecule (NKLAM/RNF19b), is localized to NK granules and is essential for maximal NK killing; our current study shows NKLAM situated within the NK3.3 EV membrane. Analysis of NK3.3 EV contents also identified multiple RNA species including miRNAs associated with anti-tumor activity. We demonstrate that treatment of an array of hematopoietic and non-hematopoietic tumor cell lines with NK3.3 EVs inhibits proliferation and induces apoptosis and cell death while leaving normal cells unaffected. This suggests that NK3.3 EVs have the potential to be effective immunotherapeutic agents.