Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing

V gamma 9V delta 2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1' s P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to V gamma 9V delta 2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for V gamma 9V delta 2 TCR-mediated responses. Surface plasmon resonance experiments established V gamma 9V delta 2 TCRs used germline-encoded V gamma 9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to V gamma 9V delta 2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the V gamma 9V delta 2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.