Discrete LAT condensates encode antigen information from single pMHC:TCR binding events.

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.

Height, but not binding epitope, affects the potency of synthetic TCR agonists.

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-TCRß Fab' antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab'-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'-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, 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.

How the T cell signaling network processes information to discriminate between self and agonist ligands.

T cells exhibit remarkable sensitivity and selectivity in detecting and responding to agonist peptides (p) bound to MHC molecules in a sea of self pMHC molecules. Despite much work, understanding of the underlying mechanisms of distinguishing such ligands remains incomplete. Here, we quantify T cell discriminatory capacity using channel capacity, a direct measure of the signaling network's ability to discriminate between antigen-presenting cells (APCs) displaying either self ligands or a mixture of self and agonist ligands. This metric shows how differences in information content between these two types of peptidomes are decoded by the topology and rates of kinetic proofreading signaling steps inside T cells. Using channel capacity, we constructed numerically substantiated hypotheses to explain the discriminatory role of a recently identified slow LAT Y132 phosphorylation step. Our results revealed that in addition to the number and kinetics of sequential signaling steps, a key determinant of discriminatory capability is spatial localization of a minimum number of these steps to the engaged TCR. Biochemical and imaging experiments support these findings. Our results also reveal the discriminatory role of early negative feedback and necessary amplification conferred by late positive feedback.

Breakage of the oligomeric CaMKII hub by the regulatory segment of the kinase.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is an oligomeric enzyme with crucial roles in neuronal signaling and cardiac function. Previously, we showed that activation of CaMKII triggers the exchange of subunits between holoenzymes, potentially increasing the spread of the active state (Stratton et al., 2014; Bhattacharyya et al., 2016). Using mass spectrometry, we show now that unphosphorylated and phosphorylated peptides derived from the CaMKII-α regulatory segment bind to the CaMKII-α hub and break it into smaller oligomers. Molecular dynamics simulations show that the regulatory segments dock spontaneously at the interface between hub subunits, trapping large fluctuations in hub structure. Single-molecule fluorescence intensity analysis of CaMKII-α expressed in mammalian cells shows that activation of CaMKII-α results in the destabilization of the holoenzyme. Our results suggest that release of the regulatory segment by activation and phosphorylation allows it to destabilize the hub, producing smaller assemblies that might reassemble to form new holoenzymes.

Mapping the stochastic sequence of individual ligand-receptor binding events to cellular activation: T cells act on the rare events.

T cell receptor (TCR) binding to agonist peptide major histocompatibility complex (pMHC) triggers signaling events that initiate T cell responses. This system is remarkably sensitive, requiring only a few binding events to successfully activate a cellular response. On average, activating pMHC ligands exhibit mean dwell times of at least a few seconds when bound to the TCR. However, a T cell accumulates pMHC-TCR interactions as a stochastic series of discrete, single-molecule binding events whose individual dwell times are broadly distributed. With activation occurring in response to only a handful of such binding events, individual cells are unlikely to experience the average binding time. Here, we mapped the ensemble of pMHC-TCR binding events in space and time while simultaneously monitoring cellular activation. Our findings revealed that T cell activation hinges on rare, long-dwell time binding events that are an order of magnitude longer than the average agonist pMHC-TCR dwell time. Furthermore, we observed that short pMHC-TCR binding events that were spatially correlated and temporally sequential led to cellular activation. These observations indicate that T cell antigen discrimination likely occurs by sensing the tail end of the pMHC-TCR binding dwell time distribution rather than its average properties.

Isolation of a Structural Mechanism for Uncoupling T Cell Receptor Signaling from Peptide-MHC Binding.

TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human T cell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds within the TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy.