Extended Data Fig. 6: Residues in the CFG face of BTN3A1 IgV domain are required for interacting with BTN2A1 and Vγ9Vδ2+ TCR.

Summary of the effect of single-residue mutations within the (a) IgV domain or (b) IgC domain of BTN3A1 on anti-BTN3A reactivity (mAb clones 103.2 and 20.1) as well as binding in cis to BTN2A1 as measured by FRET, and binding to G115 γδTCR tetramer. +++ high expression; + intermediate expression; - low expression;–no expression; ND – no binding data due to lack of BTN3A1 expression or lack of 20.1 mAb binding. (c) Förster resonance energy transfer (FRET, depicted on x-axis) between anti-BTN2A1 (clone 259, not depicted) and anti-BTN3A (clone 103.2, depicted on y-axis) mAb staining on gated BTN2A1⁺BTN3A1⁺ NIH-3T3 cells, 48 h after co-transfection with WT BTN2A1 plus the indicated BTN3A1 mutant, or as irrelevant controls, BTN2A1 plus PD-L2 or BTN3A1 plus CD80. Mutants in red were excluded from analysis due to diminished BTN3A1 staining. Mutants in green are those which reduced FRET levels. Representative one of six independent experiments, except for CD80 and PD-L2 controls which were included in three experiments. (d) Surface representation of BTN3A1 V-dimer depicting residue side chains that upon mutation led to an abrogation of BTN3A1 association with BTN2A1 (green), or those which did not impact the interaction with BTN2A1 (black), as determined by the FRET assay (left). The BTN3A1 surface on the right depicts atoms that contacted BTN2A1 based on the crystal structure (green, reproduced from Extended Data Fig. 5b). (e) G115 tetramer-PE staining of BTN3A1 WT or mutant-transfected NIH-3T3 cells following pre-incubation with anti-BTN3A-AF647 (mAb clone 20.1). Mutants in red were excluded from analysis due to diminished BTN3A1 mAb 20.1 staining. Mutants in green are those which impaired G115 tetramer staining. Representative of one of three independent experiments. (f) CD25-PE expression on purified pre-expanded Vδ2⁺ γδ T cells following co-culture with NIH-3T3 cells that were co-transfected with BTN2A1 plus the indicated BTN3A1 mutant, or alternatively control BTNL3 plus BTNL8, ± zoledronate (5 µM) for 24 h. Data are from one of three independent experiments, each with two donors. (g) Surface of BTN2A1 V-dimer depicting residues that contact BTN3A1 based on the BTN2A1–BTN3A1 crystal structure in yellow, residues that contact Vγ9Vδ2⁺ TCR based on the G115 TCR–BTN2A1 crystal structure in blue, and residues that overlap and contact both in green. (h) Superimposition of BTN2A1 component within BTN2A1–G115 Vγ9Vδ2⁺ TCR and BTN2A1–BTN3A1 structures, showing BTN2A1 (green ribbon)–BTN3A1 (blue mesh) and BTN2A1 (not depicted)–G115 Vγ9Vδ2⁺ TCR (surface render).