Cooperative ectodomain interaction among TCRαβ, CD3γε, and CD3δε enhances TCR mechanotransduction

Abstract

The T-cell receptor (TCR) complex comprises TCRαβ, CD3γε, CD3δε, and CD3ζζ. TCRαβ engagement with peptide-bound major histocompatibility complex (pMHC) triggers CD3 phosphorylation, which is regulated by mechanical force. However, the inner workings of the TCR mechanotransduction machinery remains unclear. TCR ectodomain (ECD) interactions have been inferred from structural and mutagenesis studies. Due to their extreme weakness, however, direct measurements of affinity had failed and of force regulation have never been attempted. Here we measured two-dimensional affinities and force-dependent lifetimes of interactions among TCRαβ, CD3γε, and CD3δε ECDs, showing a cooperative CD3δε–TCRαβ–CD3γε catch bond with longer-lasting lifetime that exceeds the TCR–pMHC bond lifetime. Molecular dynamics stimulations revealed a central interacting region surrounded by TCR ECDs and identified critical interacting residues at their interfaces. Interfering TCR ECD interactions by antibodies impaired TCR–pMHC interaction and T cell function. Mutating residues that mediate TCR ECD cis-interactions with CD3s altered the catch bond of TCR–pMHC trans-interaction, which correlates with changed T cell cytokine production. Thus, TCR mechanotransduction is supported by cooperative TCR ECD interactions, which regulates T cell function. Our results provide a missing link between pMHC ligation and CD3 signaling and may guide future TCR engineering design for immunotherapies.

Catch bond models may explain how force amplifies TCR signaling and antigen discrimination

“…1e-g), indicating the main conformational change responsible for TCR–pMHC-II catch-slip bond is unfolding rather than tilting. The validity of this model is supported by its excellent fitting to our published data of mouse WT or MT TCRs on naïve T cells (3.L2) or hybridomas (2B4) interacting with their respective p:I-E k ligands 5,32 (Fig. 4d, Supplementary Table 4 and 5).…”

“…The effects of changing θ and are shown in the upper and lower panels, respectively, for the indicated parameter values. d, Fitting of class II model-predicted 1/ k ( F ) curves to experimental bond lifetime vs force data ( points , mean ± sem) of 3.L2 TCR on CD4 − CD8 + naive T cells interacting with indicated p:I-E k’ s 5 ( upper ) or WT and indicated mutant 2B4 TCRs on hybridomas interacting with K5:I-E k 32 . e, Dimensional metrics, Δ t ( left ), scaled relative length of bond lifetime L ( middle ), and intensity of catch bond I ( right ) vs reciprocal % change (relative to WT) in the peptide dose required to achieve half-maximum hybridoma IL-2 production (1/EC 50 ) 33 (3.L2, red ) or in the area under the dose response curve (AUC) 34 (2B4, blue ) plots.…”

“…7). Since mutations on the 2B4 TCR were designed to perturb its Cαβ interaction with the CD3 subunits, their effect on the bond profile and T cell function might be weaker than the effects of the altered peptide ligands for the 3.L2 TCR 5,32 . Nevertheless, the correlations between ligand potency and the three model parameters θ , n *, and for the 2B4 system are comparable to those for the 3.L2 system (Fig.…”

“…With the exception of a recent paper that observed T cell pulling on TCR by ∼2 pN forces using spider silk peptide-based force probe 41 , five publications from two laboratories demonstrated that TCR experienced endogenous forces of 12-19 pN using DNA-based force probes 13,14,42-44 .Also, except for a single paper that failed to observe catch bonds in the 1G4 TCRαβ ectodomain using a flow chamber 45 , extensive data in 10 papers from four laboratories have demonstrated catch bonds in 11 membrane TCRs (including 1G4) using biomembrane force probe and optical tweezers 4-8,13,15,26-28 . These experiments prompted us to develop two mathematical models for TCR catch bonds, one with class I and the other with class II pMHC, based on Kramer’s kinetic theory and accounted for the 3D coarse-grained structures, molecular elasticity, and conformational changes of the TCR–pMHC-I/II complexes.…”

“…All data are included in the article and Supplementary Materials. Previously published bond lifetime data were re-analyzed for models fitting 41–6,8,13,15,32,39 .…”