Kinetic regulation of TCR activation as a potential mechanism for CD28 co-stimulation

Abstract T cell receptor (TCR) engagement initiates a chain of proximal TCR signaling events including phosphorylation of TCRζ, recruitment of upstream kinases, followed by recruitment of signaling and adapter proteins. These events occur within seconds of TCR engagement and are important determinants of the T cell response. Yet, we lack a precise understanding of the spatiotemporal relationships between these molecular events due to the limitations of conventional light microscopy. We used stimulatory antibodies and ligands against the TCR to trigger formation of TCR microclusters (MC), which are submicron-sized basic signaling units formed during T cell activation. Formation of these signaling subunits was then studied using TIRF and TIRF-SIM to delineate how the CD28 co-stimulatory signal alters the kinetics and molecular stoichiometry of TCR proximal signaling events and subsequent effects on the immunoresponse. Our results show that CD28 co-stimulation specifically accelerated recruitment of ZAP70 to the TCRζ chain in the MCs, as revealed by a decreased kinetic lag between detection of TCRζ and the recruitment of ZAP70. We also observed that CD28 co-stimulation markedly changed Lck dynamics in T cells, resulting in Lck enrichment within MCs and a concomitant increase in co-localization of Lck with ZAP70 and TCRζ. CD28 co-stimulation led to spatial segregation between activated and inhibited species of Lck, raising the possibility of formation of localized Lck activation sites within MCs. These results suggest that CD28 co-stimulation may lower the TCR activation threshold by enhancing the activated form of Lck in the TCR MCs, thereby decreasing the energy barrier for a rate-limiting kinetic step in TCR proximal signaling.