Using optogenetic tools to test the kinetic proofreading model of T cell receptor ligand discrimination
The kinetics and intensity of stimulation are thought to affect T cell signaling at multiple levels. For example, at the level of the receptor, ligand binding kinetics have been suggested to discriminate agonistic from non-agonist pMHCs. Downstream of the receptor, small changes in the intensity of stimulation can sharpen into a large all-or-nothing activation of Erk. However, precisely tuning the kinetics and intensity of stimulation and measuring the effects has remained challenging, limited in part by a lack of appropriate tools. Here we report the use of a LOV2-based optogenetic system to control the kinetics and intensity stimulation through a chimeric antigen receptor. Signaling through the receptor is initiated by the binding of LOV2 to the extracellular binding domain. Varying the intensity of blue light tunes the ligand binding kinetics from sub-second to tens of seconds. Other biophysical aspects of the interaction are not changed, allowing us to cleanly measure the effects ligand binding kinetics have on receptor signaling. Downstream of the receptor, titrating the intensity of blue light titrates the strength of the signal. By using several fluorescent live cell readouts of proximal signaling, we can measure single cell “dose response curves,” seeing if different portions of the signaling pathway respond in a linear, switch-like, adaptive or integrated manner to upstream signals. Optogenetic control of receptor signaling allows us to study T cell signaling with greater control than ever before, allowing us to directly ask how cells respond to changes in the kinetics and intensity of stimulation.