Molecular glue binding behavior of phosphoantigens to alpaca butyrophilins

Vγ9Vδ2 T cells that respond to phosphoantigen (pAg) function as crucial sentinels of the immune system to eradicate pathogen-infected cells and tumor cells. Alpaca (Vicugna pacos) is the first nonprimate species identified to possess the pAg-reactive Vγ9Vδ2 T cell subset. However, the molecular mechanism accounting for the pAg recognition of alpaca Vγ9Vδ2 T cells remains unclear. Here, we report the crystal structures of alpaca butyrophilin 3 (VpBTN3) B30.2 domain in complex with the exogenous pAg analog, HMBPP-08, which is a valuable tool for studying the mechanism of butyrophilin-dependent Vγ9Vδ2 T cell activation, and the endogenous pAg analogue, dimethylallyl (S)-thiolodiphosphate (DMASPP). We elucidated that the function of pAgs is governed by their structural differences. Notably, DMASPP acts as a molecular glue in the interaction between the intracellular B30.2 domains of heterologous butyrophilins in alpaca and human. Interestingly, although HMBPP-08 has stronger affinity than DMASPP to VpBTN3 B30.2 domain, HMBPP-08 did not promote heterologous VpBTNs interaction. These findings establish a novel theoretical framework elucidating the mechanisms of Vγ9Vδ2 T cell activation and demonstrate the conserved evolutionary mechanisms underlying cross-species immune adaptation.