UC Berkeley

An adaptive immune response begins with a T cell accurately identifying a pathogen-derived peptide loaded into MHC protein on the surface of an antigen presenting cell (APC). T cells engage pMHC with their T cell receptor (TCR) and must discriminate between agonist pMHC molecules, present on APC surfaces at extremely low copy numbers (e.g., tens of molecules), and hundreds to thousands of self pMHC. To be both highly sensitive and precise, T cells have evolved elaborate signaling systems to minimize error. Antigen discrimination is largely based on the binding duration kinetics (dwell time) of pMHC:TCR complexes with agonist pMHC exhibiting long dwell times. LAT is an integral membrane scaffold protein that is intrinsically disordered, and upon its phosphorylation by the kinase Zap70 at multiple tyrosine sites, LAT undergoes a condensation phase transition, being crosslinked by various adaptor and signaling proteins. LAT condensation is a critical feature of productive TCR signaling.

Using single molecule imaging techniques, we observe that an individual LAT condensate is induced by a single pMHC:TCR complex and constitutes a unit of productive TCR signaling. Accumulation of a sufficient number of these condensates is correlated with whole cell activation. LAT condensates form abruptly after an extended delay from the onset of pMHC:TCR binding. Condensate lifetime and size carry no antigen information as they are completely uncorrelated with the dwell time of the originating pMHC:TCR complex. Instead, dwell time correlates with the probability of LAT condensate formation. Condensate delay time is shortened by accelerating the phosphorylation of a unique tyrosine site on LAT, Y136, that exclusively binds the PIP2 lipase PLCγ1. Perturbing delay time kinetics subsequently alters antigen specificity.

We discover that PLCγ1 plays a novel structural role in nucleating LAT condensates while locked in its autoinhibited conformation by facilitating early crosslinking of LAT. PLCγ1 recruits to LAT coincident with condensate formation, and altering the cytosolic concentration of PLCγ1 modulates condensation delay time and probability, indicating that condensate nucleation is a major kinetic bottleneck step. Nucleation occurs when PLCγ1 binds LAT at Y136 and an unknown binding partner, through likely SLP-76, with its SH3 domain, suggesting that LAT must be tetravalent for in vivo condensates to form. Thus, PLCγ1 works cooperatively with LAT Y136 phosphorylation to control LAT condensation and antigen discrimination in T cells for accurate immune response.