Extended Data Fig. 2: Soluble Vγ9Vδ2⁺ TCR tetramers interact with BTN3A1 expressing cells.

(a) BTN2A1 tetramer, BTN3A1 tetramer, control mouse CD1d tetramer, or SAv-PE staining of human HEK293T cells transfected with plasmids co-encoding GFP and either G115 Vγ9Vδ2⁺ or control 9C2 Vγ5Vδ1⁺ γδTCRs. Plots gated on GFP⁺ cells. Data from one of 10 independent experiments. Inset – median fluorescence intensity (MFI) of PE parameter. (b) Vγ9Vδ2⁺ TCR tetramer-PE (clones TCR3, TCR6, TCR7 and G115) or streptavidin (SAv.) control staining of mouse NIH-3T3 cells transfected with BTN2A1, BTN3A1 or no DNA following pre-incubation with anti-BTN3A mAb clones 20.1 (red) or 103.2 (blue), or isotype control (IgG1,κ, black). Representative of N = 4 (TCR3, TCR6 and TCR7) or 2 (G115) experiments. (c) γδTCR tetramer or SAv control staining of HEK293T BTN2A.BTN3A^KO cells transfected with plasmids co-encoding GFP and either BTN2A1, BTN3A1 or control BTNL3, which were pre-incubated with anti-BTN3A mAb 20.1 or isotype control (mouse IgG1,κ) antibody. Representative of one of two independent experiments. (d) SDS-PAGE separation of wild-type and Vδ1–chimeric γδ TCRs alone (LHS) or mixed with excess SAv (RHS). (e) Staining of BTN2A1, BTN3A1 or control BTNL3-transfected NIH-3T3 cells with chimeric γδTCR tetramers comprised of the TCR6, TCR7 or G115 pAg-reactive γ-chains, plus either the pAg-reactive Vδ2⁺ or the 9C2 Vδ1⁺ δ-chains ± anti-BTN3A mAb 20.1 (red) or isotype control (IgG1,κ, black). Inset of panels (c) and (e), MFI of PE for mAb 20.1-treated cells (red numbers) or isotype control (IgG1,κ)-treated BTN3A1⁺ cells (black numbers) on GFP⁺ gated events, as depicted on the SAv controls and applied to all samples. Representative of N = 3 (TCR6 and G115) or 5 (TCR7 or streptavidin (SAv)) experiments.