TCR-pMHC bond length controls TCR ligand discrimination

Abstract

12T-cell receptors (TCRs) detect specifically and sensitively a small number of agonist peptide-1 major histocompatibility complexes (pMHCs) from an ocean of structurally similar self-2 pMHCs to trigger antigen-specific adaptive immune responses [1][2][3][4] . Despite intense efforts, the 3 mechanism underlying TCR ligand discrimination remains a major unanswered question in 4 immunology. Here we show that a TCR discriminates between closely related peptides by 5 forming TCR-pMHC bonds with different lengths, which precisely control the accessibility 6 of CD3z immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation. 7Using in situ fluorescence resonance energy transfer (FRET) 3,5 , we measured the 8 intermolecular length of single TCR-pMHC bonds and the intramolecular distance of 9 individual TCR-CD3z complexes at the membrane of live primary T cells. We found that an 10 agonist forms a short TCR-pMHC bond to pull the otherwise sequestered CD3z off the inner 11 leaflet of the plasma membrane, leading to full exposure of its ITAMs for strong 12 phosphorylation. By contrast, a structurally similar weaker peptide forms a longer bond 13 with the TCR, resulting in partial dissociation of CD3z from the membrane and weak 14 phosphorylation. Furthermore, we found that TCR-pMHC bond length determines 2D TCR 15 binding kinetics and affinity, T-cell calcium signaling and T-cell proliferation, governing the 16 entire process of signal reception, transduction and regulation. Thus, our data reveal the 17 fundamental mechanism by which a TCR deciphers the structural differences between 18 foreign antigens and self-peptides via TCR-pMHC bond length to initiate different TCR 19 signaling for ligand discrimination. 20It remains elusive how a TCR deciphers the structural differences among peptides and 21properly propagates surface recognition signals across the cell plasma membrane to CD3 22 intracellular ITAM domains to induce distinct T-cell signaling. The TCR conformational change and accessary molecules ICAM-1 and B7-1, respectively ( Fig. 1b-e, Extended Data Fig. 2a and 3-1 4, Supplimentary Movie 1-3). For cell surface FRET1, we readily detected FRET signals for three 2 agonist pMHCs but not for a null pMHC on lipid bilayer and glass surface, and the FRET 3 efficiencies (EFRET) were positively correlated with the pMHC potencies in activating T cells 9 . The 4 average synaptic EFRET1 was 0.79, 0.54 and 0.29 for the super agonist K5, agonist MCC and weak 5 agonist 102S, respectively (Fig. 1d). However, no synaptic FRET was found for the null pMHC 6 ( Fig. 1b and d). These data validated the specificity of our cell surface TCR-pMHC FRET and 7 were consistent with a previous report 3 . In contrast, the transmembrane TCR-CD3z FRET 8 efficiencies were inversely correlated with the pMHC potencies with the highest FRET observed 9 for the null ligand ( Fig. 1c and e). In the presence of agonist pMHCs K5, MCC and 102S, the 10 transmembrane FRET was only detected at the TCR-CD3z co-localized microclusters but not 11 o...