Report Phosphoantigen sensing combines TCR-dependent recognition of the BTN3A IgV domain and germline interaction with BTN2A1

Vy9V82 T cells play critical roles in microbial immunity by detecting target cells exposed to pathogen-derived phosphoantigens (P-Ags). Target cell expression of BTN3A1, the "P-Ag sensor,"and BTN2A1, a direct ligand for T cell receptor (TCR) Vy9, is essential for this process; however, the molecular mechanisms involved are unclear. Here, we characterize BTN2A1 interactions with Vy9V82 TCR and BTN3A1. Nuclear magnetic reso-nance (NMR), modeling, and mutagenesis establish a BTN2A1-immunoglobulin V (IgV)/BTN3A1-IgV struc-tural model compatible with their cell-surface association in cis. However, TCR and BTN3A1-IgV binding to BTN2A1-IgV is mutually exclusive, owing to binding site proximity and overlap. Moreover, mutagenesis in-dicates that the BTN2A1-IgV/BTN3A1-IgV interaction is non-essential for recognition but instead identifies a molecular surface on BTN3A1-IgV essential to P-Ag sensing. These results establish a critical role for BTN3A-IgV in P-Ag sensing, in mediating direct or indirect interactions with the y8-TCR. They support a composite-ligand model whereby intracellular P-Ag detection coordinates weak extracellular germline TCR/BTN2A1 and clonotypically influenced TCR/BTN3A-mediated interactions to initiate Vy9V82 TCR triggering.