Signaling through the T cell receptor (TCR) underlies the adaptive immune response. This signaling has classically been described as a cascade: a series of events that happen sequentially after TCR engagement. In the cascade, TCR engagement triggers condensation of the linker for activated T cells (LAT), which enables the Tec kinase ITK to activate Plcγ1, unleashing the cell-wide response. This cascade has largely been investigated using methods that activate many TCRs asynchronously, however, leaving open questions concerning the timing and ordering of events. We examined the kinetics of recruitment of ITK to LAT condensates by activating only single TCRs using a supported-lipid bilayer system and primary mouse T cells, imaging them with TIRF microscopy. We find that although LAT condensation is strictly dependent on TCR engagement, ITK recruitment does not depend on TCR engagement. Upon initial cellular adhesion through LFA-ICAM interactions, ITK is recruited to the plasma membrane and clusters. The lipid-binding PH-TH module of ITK is required for its recruitment and the SH2 domain of ITK is required for its clustering with the scaffold SLP76. Strikingly, ITK is already present by the time TCR engagement triggers LAT condensation. Our data suggest that the full TCR signaling unit (also referred to as the signalosome) forms piecewise, with some components not dependent on TCR activation. This represents a form of signal priming in which cell adhesion sensitizes the system for single-molecule detection by TCR.
Eisen et al. (Sun,) studied this question.