UCSF

T cells measure the lifetime of antigen binding events to discriminate self from foreign antigens. The full mechanism behind this kinetic proofreading behavior remains elusive. Previously, we developed a light-gated immune receptor to directly test the kinetic proofreading model in T cell antigen signaling. We found evidence of a kinetic proofreading mechanism upstream of the generation of the signaling lipid diacylglycerol (DAG). Here, we modify our assay to be compatible with more live-cell biosensors, and measure the magnitude of kinetic proofreading behavior at multiple upstream events in T cell antigen signaling. We found that kinetic proofreading behavior exists in receptor activation, with greater proofreading at the formation of LAT clusters, and even greater proofreading at the generation of DAG. These data suggest that the mechanism of proofreading is distributed among receptor activation, LAT cluster formation, and DAG generation from fully mature LAT clusters. Having kinetic proofreading steps beyond the individual receptor creates the possibility of cooperatively between multiple active receptors for antigen discrimination.