On arrested neutrophils a focal adhesive cluster of ~200 high affinity (HA) β₂-integrin bonds under tension is sufficient to trigger Ca²⁺ flux that signals an increase in activation in direct proportion to increments in shear stress. We reasoned that a threshold tension acting on individual β₂-integrin bonds provides a mechanical means of transducing the magnitude of fluid drag force into signals that enhance the efficiency of neutrophil recruitment and effector function. Tension gauge tethers (TGT) are a duplex of DNA nucleotides that rupture at a precise shear force, which increases with the extent of nucleotide overlap, ranging from a tolerance of 54pN to 12pN. TGT annealed to a substrate captures neutrophils via allosteric antibodies that stabilize LFA-1 in a high- or low-affinity conformation. Neutrophils sheared on TGT substrates were recorded in real time to form HA β₂-integrin bonds and flux cytosolic Ca²⁺, which elicited shape change and downstream production of reactive oxygen species. A threshold force of 33pN triggered consolidation of HA β₂-integrin bonds and triggered membrane influx of Ca²⁺, whereas an optimum tension of 54pN efficiently transduced activation at a level equivalent to chemotactic stimulation on ICAM-1. We conclude that neutrophils sense the level of fluid drag transduced through individual β₂-integrin bonds, providing an intrinsic means to modulate inflammatory response in the microcirculation.

In this study, we investigated the biophysical interaction between cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and CD80. CTLA-4 is a key molecule in immunosuppression, and CD80 is a costimulatory receptor promoting T cell activation. We observed that after cell-cell contact was established between breast cancer cells and antigen presenting cells (APCs), CTLA-4 expressed on the breast cancer cells bind to CD80 expressed on the APCs, and underwent trans-endocytosis to deplete CD80. Force measurement and live cell imaging revealed that upon binding to CD80, forces generated by breast cancer cells and transmitted via CTLA-4 were sufficiently strong to displace CD80 from the surface of APCs to be internalized by breast cancer cells. We further demonstrated that because of the force-dependent trans-endocytosis of CD80, the capacity of APCs to activate T cells was significantly attenuated. Furthermore, inhibiting force generation in cancer cells would increase the T cell activating capacity of APCs. Our results provide a possible mechanism behind the immunosuppression commonly seen in breast cancer patients, and may lead to a new strategy to restore anti-tumor immunity by inhibiting pathways of force-generation.