Whilst αβ T cell recognise MHC-Ag complexes for immunity, very little is known about the Ag-presenting elements of γδ T cells. Recently, butyrophilin (BTN) molecules have emerged as key regulators of γδ T cell immunity, however, the mechanism by which they activate the γδTCR is unknown. In humans, most circulating γδ T cells are phosphoantigen (pAg)-reactive Vγ9Vδ2+ T cells, which play a critical role in immunity to most bacterial and apicomplexan parasites infections as well as cancer. γδ T cells are also emerging as important mediators of anti-viral immunity to a broad range of viral infections, ranging from hepatitis B and C, cytomegalovirus, EBV and human immunodeficiency virus infections and respiratory infections, such influenza and SARS-CoV-2. Recent studies have shown BTN molecules are up-regulated by sensors of cellular stress and viral infection, AMP kinase and NLRC5 respectively, potentially co-ordinating Vγ9Vδ2+ T cell responses to virally infected cells.
To explore how BTNs co-ordinate γδ T cell immunity, we solved the crystal structure of Vγ9Vδ2+ TCR in complex with BTN member 2A1 (BTN2A1), revealing that BTN2A1 engages the side of the γδTCR. Intriguingly, we also found that a second ligand, namely BTN3A1, can bind the exposed apical surface of Vγ9Vδ2+ TCR alongside BTN2A1. However, BTN3A1 binding only occurred following cross-linking with an agonist anti-BTN3A1 mAb, or alternatively, following mutation of a negative regulatory residue, Lys53 of the TCR δ-chain. Indeed, we demonstrate that mutation of the Lys53 residue confers spontaneous reactivity of the γδ T cell to BTN2A1–BTN3A1 expressing cells.
Our findings reveal a new paradigm in immune activation, whereby a single γδTCR recognises two BTN molecules, the so-called ‘two-ligand, one-receptor’ model of immune activation, and offer a potential new avenue for treatment of viral infection.