Butyrophilin-like 3 Directly Binds a Human Vγ4+ T Cell Receptor Using a Modality Distinct from Clonally-Restricted Antigen

Butyrophilin (BTN) and butyrophilin-like (BTNL/Btnl) heteromers are major regulators of human and mouse gamma delta T cell subsets, but considerable contention surrounds whether they represent direct gamma delta T cell receptor (TCR) ligands. We demonstrate that the BTNL3 IgV domain binds directly and specifically to a human V gamma 4(+) TCR, LES'' with an affinity (similar to 15-25 mu M) comparable to many alpha beta TCR-peptide major histocompatibility complex interactions. Mutations in germline-encoded V gamma 4 CDR2 and HV4 loops, but not in somatically recombined CDR3 loops, drastically diminished binding and T cell responsiveness to BTNL3-BTNL8-expressing cells. Conversely, CDR3 gamma and CDR3 delta loops mediated LES TCR binding to endothelial protein C receptor, a clonally restricted autoantigen, with minimal CDR1, CDR2, or HV4 contributions. Thus, the gamma delta TCR can employ two discrete binding modalities: a non-clonotypic, superantigen-like interaction mediating subset-specific regulation by BTNL/BTN molecules and CDR3-dependent, anti-body-like interactions mediating adaptive gamma delta T cell biology. How these findings might broadly apply to gamma delta T cell regulation is also examined.