Butyrophilin 3A1 engages the CDR2δ loop of the Vγ9Vδ2+ T cell receptor

Phosphoantigen (pAg) recognition by Vγ9Vδ2 + T cells plays a critical role in immunity to pathogens and cancer. The butyrophilin (BTN) family of molecules have emerged as key regulators of γδ T cells; however, the underlying mechanisms remain unclear. Here, we demonstrate an interaction between BTN3A1 and Vγ9Vδ2 + T cell receptor (TCR) in a cell-free assay, confirming that BTN3A1 is a direct ligand for the Vγ9Vδ2 + TCR. Furthermore, immobilized recombinant BTN2A1 plus BTN3A1 extracellular domains are sufficient to activate Vδ2 + T cells. Finally, we show that intracellular pAg accumulation can modulate binding of Vγ9Vδ2 + TCR in a BTN3A1-dependent manner, indicating the important role of TCR binding to BTN3A1 in pAg recognition. These data confirm that BTN3A1, in conjunction with BTN2A1, is a direct ligand for Vγ9Vδ2 + TCR, providing the key interactions required for pAg recognition.