The Outer Face of the IgV Domain of Butyrophilin 3A1 is Critical for the Sensing of Prenyl Pyrophosphates by Human Vγ2Vδ2 T cells: A New Paradigm for γδ T Cell Recognition

Abstract γδ T cells expressing Vγ2Vδ2 TCR play critical roles in human immunity to microbes and tumors. These cells are uniquely able to use their TCRs for pattern recognition to monitor phosphorylated metabolites in isoprenoid biosynthesis in a process requiring butyrophilin (BTN) 3A1. Metabolites are sensed by binding to the intracellular B30.2 domain and requires the juxtamembrane coiled coil region. The role played by the extracellular domains (ECD) is unclear. To assess the ECD, surface IgV and IgC residues were mutagenized to alanine. Mutant BTN3A1 cDNAs were expressed in 293 T embryonic kidney cells lacking BTN3A1, -A2, and -A3, and the transfected cells pulsed with zoledronic acid and used to stimulate Vγ2Vδ2 T cells. Mutation of 13 residues scattered on the IgC domain had no effect on stimulation. Mutation of 22 residues scattered on the inner, side, and outer faces of the IgV domain also had no effect. However, 4 residues localized to a region on the outer face of the IgV domain either abrogated or diminished stimulation. Others have identified 2 additional residues in this region and mapped recognition to the Vγ2 HV4 loop paralleling BTNL3 and Btnl6 recognition by other γδ TCR. BTN3A2 and BTN3A3 can complement mutant BTN3A1 proteins suggesting that they form heterodimers or can alter their conformation. Modeling of the potential binding interface identifies BTN3 residues that can form ionic or hydrogen bonds to HV4. Given that our previous studies have shown the requirement for all CDRs of Vγ2Vδ2 TCR, a putative second ligand for the conventional binding side is also required. Taken together, we propose that Vγ2Vδ2 T cell stimulation is dependent on the simultaneous binding of BTN3A1 to HV4 and a second ligand to the conventional Vγ2Vδ2 binding site.