Deepali Balasubramani

Immunoreceptors experience forces that modulate their activities; however, demonstrating this in vivo has been limited by technical challenges. As a first step toward meeting this challenge, we adapted a synthetic Notch (SynNotch) receptor system to report force transmission through immunoreceptors in vivo by replacing the native ligand‐binding domain with a receptor‐specific antibody and rewiring Notch signaling to drive enhanced green fluorescent protein or luciferase expression. We expressed SynNotch on Jurkat cells targeting CD40 or T cell receptor (TCR) and characterized their activation in coculture with B or T cells, defining the requirements, optimal conditions, and kinetics of activation. Using complementary mechanobiology approaches, we quantified the exogenous force required for reporter activation and verified that activation depends on forces generated by receptor‐expressing sender cells rather than SynNotch‐expressing receiver cells. By implanting sensors and targets into immunocompromised mice, we visualized mechanically activated reporter expression on CD40 and TCR‐targeting SynNotch cells in vivo. Furthermore, CD40 and TCR signaling was amplified when the receptor bore force against mechanical support from immobilized ligand, indicating that force functions as a biologically relevant co‐stimulus. Together, our results establish mechanically activated SynNotch reporters as a useful strategy for detecting receptor‐associated mechanical signaling across two‐dimensional coculture, three‐dimensional organoid, and in vivo systems.