NanoBRET Saturation Analysis with a Novel HaloTag Ligand Reveals Butyrophilin Heteromeric Interactions Controlling...

Human γ9δ2 T cells detect microbial phosphoantigens (pAg) through butyrophilin (BTN) family proteins; however, how multiple BTN paralogs assemble into functional signaling complexes remains unresolved. Because γ9δ2 T cell activation requires coordinated action of several BTN members rather than a single receptor pair, defining their organization in living cells is essential. Here, we apply a live-cell NanoBRET saturation analysis using a novel HaloTag ligand to quantitatively resolve BTN oligomerization dynamics. We found that BTN3A1 exists as higher-order basal oligomers. Coexpression of BTN3A2 or BTN3A3 redistributes these assemblies into defined BTN3 dimers. The BTN3 dimers have a baseline interaction with BTN2A1 dimers that is altered by IgV domain interactions and by classical pAgs such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate. Mutation of a BTN3A1 IgV epitope disrupts a restraint on BTN2A1 and triggers spontaneous γ9δ2 T cell activation, which is independent of pAg. In contrast, BTN3A3 forms a strong, constitutive tetramer with BTN2A1 in the absence of BTN3A1/BTN3A2. This complex potentiates detection of nonclassical pAgs such as mevalonate diphosphate, and disruption of its juxtamembrane/B30.2 intersection abolishes both complex formation and signaling. BTN3A2 suppresses both interaction and function of BTN3A3 complexes, in contrast to its stimulatory effect on BTN3A1 complexes. Together, these results reveal that γ9δ2 T cell activation is controlled by ligand-dependent activation of competing BTN complexes. More broadly, this work establishes quantitative NanoBRET analysis as a general framework for dissecting multiprotein membrane receptor organization in living cells.