Natural killer (NK) cells play a crucial role in innate immunity as effectors against tumor and pathogen-infected cells. NK-mediated host defense against tumor cells is strongly impaired in patients with head and neck squamous cell carcinoma (HNSCC). Tumor secretion of various immune suppressive mediators contributes to massively compromised immune functions. Herein we demonstrate that NK cell cytotoxicity against tumor cells of HNSCC can be efficiently stimulated by single-stranded immunostimulatory RNA (ss-isRNA). Stimulation with ss-isRNA results in an increased production of interferon-γ and effector proteins perforin and granzyme B. Our investigations revealed that supernatants of permanent HNSCC cell lines negatively affect the ss-isRNA-triggered stimulation of cytolytic NK cell functions. Stimulation of cytotoxicity requires Toll-like receptor 7 (TLR7). Increased expression of NK cell TLR7 was shown in response to ss-isRNA. These results suggest ss-isRNA as a potential immunostimulatory tool of human NK cells against HNSCC.

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