Antigen affinity modulates ERK pulsing frequency during T cell activation

T cells achieve precise antigen discrimination by relying on the temporal stability of T cell receptor (TCR) interactions with antigens. Given that time is central to antigen discrimination, we used real-time, single-cell imaging in a controlled TCR-antigen system to characterize extracellular signal-regulated kinase (ERK) signaling dynamics as a function of antigen affinity to better understand the temporal patterns of signaling downstream of the TCR. We found that intermediate-affinity antigens elicited pulsatile ERK activity at different frequencies and that T cell activation correlated with the cumulative amount of ERK activity. Mechanistically, we found that the ERK pulsing frequency depended on the rate of activity of the Src family kinase Lck at the plasma membrane, whereas mitogen-activated protein kinase (MAPK) kinase (MEK) modulated the amplitude of ERK signaling. Moreover, we showed that ERK activity dynamics in T cells depended on members of two upstream MAP3K groups: mixed lineage kinases (MLKs) and RAFs, which played distinct roles promoting or sustaining the formation of upstream signaling condensates containing the transmembrane adaptor molecule LAT. Together, our findings reveal insights into the spatiotemporal organization of TCR signaling activities and their roles in T cell activation.