Proofreading and single-molecule sensitivity in T-cellreceptor signaling by condensate nucleation

T-cells display the remarkable ability to detect single foreign peptides displayed on target cells, while ignoring highly abundant self peptides. This selectivity has been explained by kinetic proofreading in the T-cell receptor (TCR) signaling pathway, which prevents responses to short-lived binding events regardless of their abundance. However, the biochemical mechanisms that drive kinetic proofreading have remained unclear. Here, using computational modeling, we show that these key signaling properties of the TCR pathway can emerge from the dynamics of LAT phosphorylation, diffusion, and condensation following TCR-pMHC binding. In this model, time delays in LAT condensate nucleation underlie kinetic proofreading, enabling selective signaling responses to high-affinity pMHC ligands. The cooperativity in the nucleation and growth of LAT condensates also provides a mechanism to amplify weak signals from single foreign peptides and for condensates to grow with increasing antigen numbers. In contrast to other models, condensate-nucleation proofreading predicts a dependence of signal strength on pMHC spacing at fixed number, a prediction we validated experimentally using a protein scaffold to present pMHCs at defined intervals. Our results suggest that nucleation-condensation proofreading underlies the remarkable antigen detection capabilities of the TCR signaling pathway. SignificanceTo fight infections and cancer, T-cells must selectively recognize low levels of foreign peptides from pathogens or cancer cells, but the mechanisms that enable these properties have remained unclear.Using mathematical modeling and experiments, we find that T-cells can selectively detect single foreign peptides through a clustering process where a key protein downstream of the T-cell receptor forms condensates containing hundreds of signaling proteins. This condensate nucleation process can explain experimentally observed features of T-cell signaling, including our finding that the size of signaling clusters depends on the distance between foreign peptides. Our work reveals a key role for the condensation of signaling molecules in setting the spatial and temporal thresholds that control the sensitivity and selectivity of T-cells.