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

Abstract

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.

Structural principles of peptide-centric chimeric antigen receptor recognition guide therapeutic expansion

“…In an analogous manner to TCRs, both antigen density and pHLA dissociation rate from the 10LH CAR can contribute to our observation that HLA-A*23:01, which has approximately two orders of magnitude reduced binding to 10LH relative to HLA-A*24:02 (2.6 μM versus 11 nM K D ), shows potent CAR T cell killing of tumor cells ( 14 ), because both interactions show similar k d values by SPR (0.0085 s −1 versus 0.0022 s −1 ). These results allow us to establish a relationship between pHLA dissociation rate from the scFv—expressed as CAR dwell time—and CAR T cell activation, cytokine release, and cytotoxicity, established by previous studies focusing on both TCRs and CARs ( 28 – 30 ).…”

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

“…Finally, in single-cell molecule experiments, pMHC-bound TCRs give rise to pLAT clusters only in a fraction of cases (25-50%), even when they were bound for very long times 24 . This cap on TCR-induced clustering likely arises from feedback mechanisms which deactivate pMHC-bound TRCs over time.…”

“…Having shown that pLAT alone can undergo clustering in our model, we proceeded to ask whether pLAT can form clusters in response to TCR pMHC engagement and LAT phosphorylation. Single-molecule studies had previously shown that single TCRs can induce LAT clusters with a variable time delay after pMHC binding 24 . In these experiments TCRs are only tracked after they bind to a pMHC, and the peptides used are very high affinity.…”

“…This network of polyvalent interactions causes these molecules to form condensates tethered to the membrane by LAT 20,21,22 , and recruits a variety of other molecules which act together with PLCγ and SOS to transmit the activation signal into the T-cell 9,23 . These clustering events have been overlooked by existing kinetic proofreading models, which generally assume that the reaction system is well-mixed, even though it has been determined experimentally that this is not the case 20,21,22,24 . More recent work has explored the condensation of LAT with Grb2, SOS, and PLCγ using coarse grained molecular dynamics simulations 21,25 .…”

Barrier and signal transduction functions could explain the lipid asymmetry of the plasma membrane

“…Another important and emerging feature of signal transduction at the plasma membrane is the formation of membrane proximal, liquid‐liquid separated cytoplasmic condensates at the signaling sites. [ 7 ] In these condensates, proteins are highly concentrated and how this interface affects the diffusion of plasma membrane associated signaling molecules would be an interesting avenue to explore.…”