Abstract
Linker for activation of T cells (LAT) is a membrane-surface scaffold protein that assembles key signaling molecules upon T cell receptor (TCR) activation. Recent observations have revealed LAT assembly occurs in discretized condensates traceable to single molecular TCR activation events. Moreover, the condensation process occurs abruptly, but after a distinct delay, indicative of a type of phase transition. Here we examine the effects of costimulatory (CD80, CD86) and inhibitory (PD-L1) ligands on the nucleation propensity of LAT condensation. We utilize single molecule mobility tracking to resolve the moment a pMHC molecule binds to TCR combined with intracellular imaging to measure the time delay between pMHC:TCR binding and nucleation of the corresponding LAT condensate. These delay time distributions reflect the propensity function for stochastic nucleation of the LAT condensate and provide a measure of the effective signal strength from a single, activated TCR. The results reveal that CD28 stimulation by CD80 or CD86 causes a distinct reduction in the mean delay time to LAT condensation while PD-L1 is mildly antagonistic to the process. Collectively, these observations strengthen evidence that probability and timing of the LAT nucleation process itself is tightly associated with signal propagation downstream from individual TCR activation events.