BTN3A1 Discriminates γδ T Cell Phosphoantigens from Nonantigenic Small Molecules via a Conformational Sensor in Its B30.2 Domain

Human Vγ9/Vδ2 T-cells detect tumor cells and microbial infections by recognizing small phosphorylated prenyl metabolites termed phosphoantigens (P-Ag). The type-1 transmembrane protein Butyrophilin 3A1 (BTN3A1) is critical to the P-Ag-mediated activation of Vγ9/Vδ2 T-cells; however, the molecular mechanisms involved in BTN3A1-mediated metabolite sensing are unclear, including how P-Ag's are discriminated from nonantigenic small molecules. Here, we utilized NMR and X-ray crystallography to probe P-Ag sensing by BTN3A1. Whereas the BTN3A1 immunoglobulin variable domain failed to bind P-Ag, the intracellular B30.2 domain bound a range of negatively charged small molecules, including P-Ag, in a positively charged surface pocket. However, NMR chemical shift perturbations indicated BTN3A1 discriminated P-Ag from nonantigenic small molecules by their ability to induce a specific conformational change in the B30.2 domain that propagated from the P-Ag binding site to distal parts of the domain. These results suggest BTN3A1 selectively detects P-Ag intracellularly via a conformational antigenic sensor in its B30.2 domain and have implications for rational design of antigens for Vγ9/Vδ2-based T-cell immunotherapies.