(A) Conventional methods of mutating the pMHC to alter the binding half-life also change the binding interface, which changes several parameters at once. By contrast, optogenetic control allows light intensity to control ligand binding half-life while keeping the binding interface constant. Therefore, no other aspects of the receptor-ligand interaction change. Red and blue lines highlight the binding interfaces. (B) Schematic of experimental setup. Jurkat cells expressing a live cell DAG reporter and a Zdk-CAR are exposed to an SLB functionalized with purified, dye-labeled LOV2. In the dark, LOV2 binds to and accumulates under the receptor, and stimulates DAG production, recruiting the reporter to the plasma membrane. Blue light excites LOV2, inducing its dissociation from the receptor to terminate signaling. (C) Montage from a time course in which cells were alternately stimulated in the presence or absence of blue-light. White arrows highlight cells with low to undetectable receptor occupancy, and red arrows highlight cells with high receptor occupancy. (D and E) Cells with very different levels of receptor occupancy (D), can have similar DAG levels (E), suggesting that receptor occupancy is not a good predictor of DAG levels. Top blue bars indicate the presence of blue light.