LFA-1 mechanical dose controls DNAM-1 signalling and perforin polarization in NK cells

Natural killer (NK) cells are essential effectors of the innate immune system, playing a vital role in controlling viral infections and tumour progression by limiting their spread and reducing tissue damage. NK cell activity is tightly regulated by a balance of activating and inhibitory receptors on their surface, with extensive research focusing on how receptor expression levels affect NK cell function. However, the mechanical properties of lymphocyte function-associated antigen-1 (LFA-1) are still not well understood. Specifically, how LFA-1 mediates mechanical forces in NK cells has not been fully elucidated. To investigate this, we quantified and modulated LFA-1 tension in NK cells using DNA-based tension gauge tethers (TGTs) functionalized with intercellular adhesion molecule-1 (ICAM-1). By using multiplexed TGTs that combine unzip and shear geometries while maintaining a constant molecular density, we enabled precise control over the mechanical dosage delivered via LFA-1-mediated adhesion by modulating the proportion of ligand-receptor bonds above a defined force threshold. This method allowed fine-tuning of LFA-1-mediated mechanotransduction and its downstream effects, including LFA-1/DNAM-1 colocalization and perforin polarization toward the cell-substrate interface. Our findings demonstrate that high-tension LFA-1/ICAM-1 interactions are associated with enhanced perforin polarization and receptor organization at the immune synapse, consistent with increased NK cell activation.