Summary Immune cells communicate through dynamic cell-cell junctions known as immune synapses. Although the biochemical properties of these synapses have been studied extensively, we know little about their mechanical activities and how these activities contribute to immune function. We use cytotoxic lymphocytes as a model system to investigate the origins and purposes of synaptic force. Cytotoxic lymphocytes fight pathogens and cancer by forming an immune synapse with an infected or transformed target cell and then secreting toxic granzymes and the pore forming protein perforin into the intercellular space. Work from our lab and others suggests critical roles for mechanical forces both in triggering lymphocyte activation and in enhancing the efficiency of killing responses. Our proposed studies, which are divided into two Specific Aims, will address the functional relevance and molecular bases of synaptic force in these contexts. Aim 1 builds on preliminary data indicating that cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells detect the physical stiffening of target cells and use this mechanosensing capacity to identify and destroy cancer cells invading the metastatic niche. To determine if and how this mechanical form of immunosurveillance, which we call mechanosurveillance, shapes anti-tumor immunity in vivo, we will apply multiple murine metastasis models, atomic force microscopy, and analysis of clinical immunotherapy trials. Aim 2 is premised on prior work indicating that CTLs use mechanical force to potentiate the pore forming activity of secreted perforin. This sort of physicochemical synergy demands a high degree of coordination between mechanical and secretory output within the synapse, but how lymphocytes achieve this coupling remains unknown. We have developed an imaging-based biophysical approach to address this problem, which will enable us to establish the mechanochemical choreography of cytotoxicity with unprecedented precision. Our proposed studies will employ technically innovative methods, including super-resolution imaging of lymphocyte force exertion against micron-scale biophysical probes. They will also introduce a number of innovative concepts, including the idea that cellular rigidity can trigger immunosurveillance and the idea that lymphocyte subsets employ distinct mechanical signatures to specify their effector responses. Our work will also address a simple but technically vexing issue that has constrained the field for some time, namely whether mechanobiological principles actually influence immunity in vivo. Understanding the biophysical dimensions of immune synapse function could potentially reveal new strategies for the modulation and assessment of lymphocyte activity in the clinic. As such, the studies described herein are highly relevant to the NIH mission in that they will contribute to the advancement of knowledge that could improve human health.