Cryo-EM structural insights into Vγ9Vδ2 TCR activation via multiple butyrophilins

Human Vγ9Vδ2 T cells detect cancerous and pathogen-infected cells through an "inside-out" mechanism wherein intracellular domains of butyrophilin (BTN) proteins recognize phosphoantigens (pAgs) to prime BTN ectodomains to engage with the T cell receptor (TCR) to activate Vγ9Vδ2 T cells. Here, we present cryo-electron microscopy (cryo-EM) structures, including the full-length BTN3A1-BTN3A2-BTN2A1 complex stabilized by the pAg HMBPP and an HMBPP-primed BTN multimer complex engaged with the Vγ9Vδ2 TCR. The HMBPP-primed BTN3A1-BTN3A2-BTN2A1 complex confirms that this pAg bridges the intracellular B30.2 domains of BTN3A1 and BTN2A1, while also revealing an association of the ectodomains of BTN3A2 and BTN2A1. Upon TCR engagement, the TCR undergoes a substantial conformational change and BTN3A2-BTN2A1 ectodomain interaction dissociates, allowing BTN2A1 to bind to the lateral surface of the Vγ9 chain, while BTN3A2 binds to the apical surface of the Vδ2 chain. Beyond demonstrating that BTN3A2 as a bona fide TCR ligand, this dissociation/binding sequence suggests a "pliers-like gripping" mechanism for TCR activation. We also obtained cryo-EM structures of BTN proteins in complex with functional antibodies, further elucidating the overall Vγ9Vδ2 T cell activation process. Thus, our structural insights into how BTN proteins sense antigens and drive TCR activation lay the groundwork for developing targeted γδ T cell immunotherapies.