Beyond the TCR, Antigen Discrimination in T Cells Continues in the LAT:GRB2:SOS Protein Condensate

Abstract

T cells activate in response to a stochastic series of single molecule binding events between T cell receptors (TCRs) and peptide ligands presented on major histocompatibility complexes (pMHCs) accumulated at a cell-cell interface. Discrimination between ligands with subtly different affinities ensures selective and robust immunological responses upon TCR triggering. Formation of protein condensates based on the scaffolding protein Linker for Activation of T cells (LAT) is a key intermediate step in TCR signaling. LAT is multi-phosphorylated on tyrosine residues by the kinase, ZAP70, which is activated by triggered TCRs. The phosphotyrosines recruit Grb2, which, via interactions with the multivalent GEF, SOS, forms an extended LAT:Grb2:SOS assembly that resembles a phase transition. We mapped single pMHC:TCR binding events in T cells expressing fluorescent protein-tagged LAT and show that LAT assemblies are discrete and have characteristic features that are independent of the originating binding event. Furthermore, LAT assemblies can become spatially uncoupled from the pMHC:TCR complex. These individual assemblies are capable of deforming the plasma membrane and creating local domains that may participate in protein segregation or control reaction kinetics. The probability of LAT assembly is a function of the pMHC affinity, suggesting a second layer of kinetic proofreading that exists beyond the triggered TCR. We recently introduced a molecular impulse-response assay which maps pMHC:TCR binding events to early T cell activation. Results here argue that LAT assemblies may confer a molecular memory that connects a series of spatiotemporally correlated binding events to cellular decision-making. By extending the impulse-response assay, we show that activating cells experience a characteristic, fast rate of LAT assembly. Taken together, we present new molecular dimensions to T cell activation that could provide actionable therapeutic insights.