Structural Basis of γδ T-Cell Receptor Crosslinking and Activation by a Spontaneously Dimerized Fab

Authors

Li, Z., Fan, Y., Li, J., Zhao, Y., Chen, L., Chen, Y., Wang, Z., Zheng, Y., Zhang, Y., Wang, J., Wu, H., Wu, D., Wang, L.

Abstract

The {gamma}{delta} T-cell receptor ({gamma}{delta} TCR) mediates immune responses in {gamma}{delta}T-cells, playing crucial roles in immune defense, tissue surveillance, and homeostasis. While recent studies suggest that {gamma}{delta} TCRs engage ligands multivalently (such as butyrophilins), the activation mechanism of the {gamma}{delta} TCR remains elusive. Here, we present the structures of the V{gamma}9V{delta}2 (clone G115) TCR in both the inactive and the Fab-crosslinked active states. The {gamma}{delta} TCR is a lipoprotein complex consisting of a TCR-{gamma}{delta} heterodimer surrounded by three CD3 dimers, CD3{delta}{epsilon}, CD3{gamma}{epsilon}, CD3{zeta}{zeta}. Lipids form a 'barrier' that isolates CD3{zeta} from the other {gamma}{delta} TCR subunits. Unexpectedly, we discover that the Fab fragment of the UCHT1 antibody dimerizes upon binding the V{gamma}9V{delta}2 TCR. This dimerized Fab acts as an agonist by crosslinking the receptor. Our cryo-EM structures of the Fab-crosslinked V{gamma}9V{delta}2 TCR reveal rigid 2:2 Fab:TCR tetramers in three conformations. Structural analysis identifies three Fab dimer interfaces, one between the light chains and two symmetrical ones between light chains and heavy chains. In addition to the expected Fab-CD3{epsilon} interface, glycan-Fab interactions likely contribute to the Fab:TCR tetramer assembly. We show that the dimerized V{gamma}9V{delta}2 TCR exhibits conformational plasticity. Furthermore, lipidomic mass spectrometry demonstrates that cholesterol and phosphatidylethanolamine are enriched within the inactive {gamma}{delta} TCR complex but are significantly depleted upon activation by the dimerized Fab. Our findings reveal a Fab-dimerization-driven activation mechanism, provide a basis for engineering rigid TCR-crosslinking agonists, and suggest that multivalency-induced clustering may represent a general paradigm for {gamma}V{delta} TCR activation.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 16 Oct 2026.

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