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Natural killer (NK) cells play a vital role in eliminating tumorigenic cells. Efficient locating and killing of target cells in complex three‐dimensional (3D) environments are critical for their functions under physiological conditions. However, the role of mechanosensing in regulating NK‐cell killing efficiency in physiologically relevant scenarios is poorly understood. Here, we report that the responsiveness of NK cells is regulated by tumor cell stiffness. NK‐cell killing efficiency in 3D is impaired against softened tumor cells, whereas it is enhanced against stiffened tumor cells. Notably, the durations required for NK‐cell killing and detachment are significantly shortened for stiffened tumor cells. Furthermore, we have identified PIEZO1 as the predominantly expressed mechanosensitive ion channel among the examined candidates in NK cells. Perturbation of PIEZO1 abolishes stiffness‐dependent NK‐cell responsiveness, significantly impairs the killing efficiency of NK cells in 3D, and substantially reduces NK‐cell infiltration into 3D collagen matrices. Conversely, PIEZO1 activation enhances NK killing efficiency as well as infiltration. In conclusion, our findings demonstrate that PIEZO1‐mediated mechanosensing is crucial for NK killing functions, highlighting the role of mechanosensing in NK‐cell killing efficiency under 3D physiological conditions and the influence of environmental physical cues on NK‐cell functions. [ABSTRACT FROM AUTHOR]

CTLs kill target cells via fusion of lytic granules ( LGs) at the immunological synapse ( IS). Soluble N-ethylmaleimide-sensitive factor attachment protein receptors ( SNAREs) function as executors of exocytosis. The importance of SNAREs in CTL function is evident in the form of familial hemophagocytic lymphohistiocytosis type 4 that is caused by mutations in Syntaxin11 ( Stx11), a Qa- SNARE protein. Here, we investigate the molecular mechanism of Stx11 function in primary human effector CTLs with high temporal and spatial resolution. Downregulation of endogenous Stx11 resulted in a complete inhibition of LG fusion that was paralleled by a reduction in LG dwell time at the IS. Dual color evanescent wave imaging suggested a sequential process, in which first Stx11 is transported to the IS through a subpopulation of recycling endosomes. The resulting Stx11 clusters at the IS then serve as a platform to mediate fusion of arriving LGs. We conclude that Stx11 functions as a t- SNARE for the final fusion of LG at the IS, explaining the severe phenotype of familial hemophagocytic lymphohistiocytosis type 4 on a molecular level. [ABSTRACT FROM AUTHOR]