Butyrophilin-like 3 and 8 tetramers clamp Vγ4Vδ1 T cell receptors for antigen-independent activation.

BTNL3 and BTNL8, butyrophilin-like (BTNL) family receptors, are predominantly expressed on intestinal epithelial cells. The BTNL3-BTNL8 complex selectively activates human Vγ4⁺ T cells, which play critical roles in intestinal immune homeostasis and tissue damage repair. Here, we report the structure of the BTNL3-BTNL8-Vγ4Vδ1 T cell receptor (TCR) complex. The structure reveals that BTNL3-BTNL8 forms an antigen-independent tetramer, in contrast to the phosphoantigen-driven association of BTN2A1-BTN3A1. Two BTNL3-BTNL8 heterodimers clamp a head-to-head TCR homodimer to form a 2:2:2 BTNL3:BTNL8:Vγ4Vδ1 TCR complex, with the Vγ4 HV4 and CDR2 loops jointly engaging BTNL3. Structural alignment indicates that the CD1d binding site on TCR overlaps spatially with one monomer within a TCR dimer, suggesting that TCR dimerization sterically precludes simultaneous engagement of CD1d molecules. Together, our results elucidate the structural mechanism of BTNL3-BTNL8-mediated engagement and activation of Vγ4Vδ1 TCR, offering insights into γδ TCR signaling initiation.