Li, M.; Lyu, J.; Li, K.; Dravid, A.; Balasubramani, D.; Kazemipour Ashkezari, A. H.; Choi, H.-K.; Kwong, G. A.; Singh, A.; Zhu, C.

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 forces on immunoreceptors in vivo by replacing the native ligand-binding domain with a receptor-specific antibody and rewiring Notch signaling to drive expression of EGFP or luciferase. 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 reporter 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 gated reporter activation on CD40 and TCR-targeting SynNotch cells in vivo. Furthermore, signaling through CD40 and TCR was amplified by allowing the receptor to bear force against mechanical support from immobilized ligand, indicating that force functions as biologically relevant co-stimulus that immune cells detect and interpret to guide immune signaling. Together, our results establish mechanically gated SynNotch reporters as a useful strategy for detecting receptor-associated mechanical signaling across 2D coculture, 3D organoid, and in vivo systems.