LAT assembly into a two-dimensional protein condensate is a prominent feature of antigen discrimination by T cells. Here, we use single-molecule imaging techniques to resolve the spatial position and temporal duration of each pMHC:TCR molecular binding event while simultaneously monitoring LAT condensation at the membrane. An individual binding event is sufficient to trigger a LAT condensate, which is self-limiting, and neither its size nor lifetime is correlated with the duration of the originating pMHC:TCR binding event. Only the probability of the LAT condensate forming is related to the pMHC:TCR binding dwell time. LAT condenses abruptly, but after an extended delay from the originating binding event. A LAT mutation that facilitates phosphorylation at the PLC-γ1 recruitment site shortens the delay time to LAT condensation and alters T cell antigen specificity. These results identify a function for the LAT protein condensation phase transition in setting antigen discrimination thresholds in T cells.

Under physiological conditions, peptide-major histocompatibility complex (pMHC) molecules can trigger T cell receptors (TCRs) as monovalent ligands that are sparsely distributed on the plasma membrane of an antigen-presenting cell. TCRs can also be triggered by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit nonphysiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCRb Fab 0 antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab 0 -DNA ligand efficiently triggers TCR as a monomer when membrane associated and exhibits a potency and activation profile resembling agonist pMHC. In this report, we explore the geometric requirements for efficient TCR triggering and cellular activation by Fab 0 -DNA ligands. We find that T cells are insensitive to the ligand binding epitope on the TCR complex but that length of the DNA tether is important. Increasing, the intermembrane distance spanned by Fab 0 -DNA:TCR complexes decreases TCR triggering efficiency and T cell activation potency, consistent with the kinetic-segregation model of TCR triggering. These results establish design parameters for constructing synthetic TCR agonists that are able to activate polyclonal T cell populations, such as T cells from a human patient, in a similar manner as the native pMHC ligand.

SUMMARYLAT assembly into a two-dimensional protein condensate is a prominent feature of antigen discrimination by T cells. Here, we use single-molecule imaging techniques to resolve the spatial position and temporal duration of each pMHC:TCR molecular binding event while simultaneously monitoring LAT condensation at the membrane. An individual binding event is sufficient to trigger a LAT condensate, which is self-limiting, and neither its size nor lifetime is correlated with the duration of the originating pMHC:TCR binding event. Only the probability of the LAT condensate forming is related to the pMHC:TCR binding dwell time. LAT condenses abruptly, but after an extended delay from the originating binding event. A LAT mutation that facilitates phosphorylation at the PLC-γ1 recruitment site shortens the delay time to LAT condensation and alters T cell antigen specificity. These results identify a role for the LAT protein condensation phase transition in setting antigen discrimination thresholds in T cells.

Under physiological conditions, peptide-MHC (pMHC) molecules can trigger T-cell receptors (TCRs) as monovalent ligands, sparsely distributed on the plasma membrane of an antigen-presenting cell. TCR can also be activated by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit non-physiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCRβ Fab′ antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab′-DNA ligand efficiently activates TCR as a monomer when membrane-associated and exhibits a potency and activation profile resembling agonist pMHC. In this report, we explore the geometric requirements for effective TCR triggering and cellular activation by Fab′-DNA ligands. We find that T cells are insensitive to the ligand binding epitope on the TCR complex, but that length of the DNA tether is important. Increasing the intermembrane distance spanned by Fab′-DNA:TCR complexes decreases TCR triggering efficiency and T cell activation potency. These results establish design parameters for construction of synthetic TCR agonists that are able to activate polyclonal T cell populations, such as T cells from a human patient, in a similar manner as the native pMHC ligand.

kinetics relative to each TCR binding event in living primary T cells with high spatiotemporal, multi-color resolution. We observe that after a pMHC binds to a TCR that a discrete LAT condensation occurs after an extended delay. While this timing is likely set, in part, by a competition of kinase-phosphatase pressure, the phosphorylation of LAT has a similarly extended delay. This suggests that phospho-LAT requires an additional rate-limiting step to trigger aggregation. We report the discovery of the molecular events that control the delay time to LAT condensation. Additionally, we find that this timing is tunable and critical for antigen discrimination. These data demonstrate, and are the first to visualize, decisive kinetic bottlenecks leading to LAT condensation and thus antigen discrimination.