Raychaudhuri, K.; Rangu, R.; Ma, A.; Alvinez, N.; Tran, A. D.; Pallikkuth, S.; McIntire, K. M.; Garvey, J. A.; Yi, J.; Samelson, L. E.

T cell activation requires T cell receptor (TCR) engagement, which initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCR{zeta} chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response. Currently we lack a full understanding of the molecular mechanism of CD28 activation. TCR microclusters (MC) are submicron-sized molecular condensates and basic signaling units that form immediately after TCR ligation. Our results show that CD28 co-stimulation specifically accelerated recruitment of ZAP70 to the TCR{zeta} chain in MCs and increased ZAP70 activation. This CD28-mediated acceleration of ZAP70 recruitment was driven by enhanced Lck recruitment to the MCs. A greater spatial separation between active and inactive species of Lck was also observed in the MCs as a consequence of CD28 co-stimulation. These results suggest that CD28 co-stimulation may lower the TCR activation threshold by enhancing the activated form of Lck in the TCR MCs.