Fig 2: BTN2A1 and BTN3A1 Synergize to Potentiate P-Ag Sensing in Rodent Cells(A) Expression of BTN2A1, BTN3A1, or both genes in transduced BW cells.(B) Production of IL-2 by TCR-MOP transductants in response to HMBPP-treated CD80+ BW cells transduced to express BTN2A1, BTN3A1, both, or untransduced controls. Percentage activation is normalized against the maximum response obtained from CD80+ CHO cells expressing both BTN2A1 and BTN3A1 in the presence of 10 µM HMBPP.(C) Production of IL-2 from TCR-MOP transductants in response to HMBPP-treated CD80+ CHO cells transduced with either BTN2A1, BTN3A1, both genes, or untransduced controls, with responses normalized as in (B).Error bars in (B) and (C) represent standard deviation for three independent experiments. Differences between untransduced and BTN2-transduced cells were significant (p < 0.05), as were those between the BTN2A1-transductant and BTN2A1+BTN3A1-transductant in the presence of HMBPP.(D) MOP-TCR tetramer staining of transduced BW cells.(E) Staining of transduced 293T cells with V?9Vd2 TCRs.(F) MOP-TCR tetramer staining or anti-BTN2A1 mAb staining of BTN2A1 and BTN3A1-transduced CD80+ BW cells versus untransduced controls in the presence and absence of Zol.See also Figure S2.

Fig 3: BTN2A1 Forms Disulphide-Linked Homodimers at the Cell Surface(A) Homology model of BTN2A1 homodimer.(B) C-terminal region of BTN2A1 homology model indicating close proximity of Cys residues.(C) Non-reducing (NR) or reducing (R) SDS-PAGE analysis of CHO-cell expressed BTN2A1 and BTN2A2 protein.(D) Effects of BTN2A1C247W mutation on IL-2 production by TCR-MOP in response to HMBPP-treated CD80+ CHO cells expressing BTN2A1 and BTN3A1. Error bars indicate standard deviation for three independent stimulation experiments. Percentage activation is normalized against the maximum response obtained from CHO cells expressing both BTN2A1 and BTN3A1 in the presence of 1 µM HMBPP.(E) Model of V?9Vd2-BTN2A1 interaction incorporating BTN2A1 homodimer formation, and bilateral V?9Vd2 interaction with BTN2A1 IgV domain.See also Figure S4.

Fig 4: Direct BTN2A1 Binding to Germline-Encoded Regions of V?9 Is Essential for P-Ag Sensing(A) (Top panel) Injection of BTN2A1 IgV (25 µM) over surfaces with immobilized V?9Vd2 TCR (2,457 resonance units (RU)) and control surfaces comprising V?4Vd5 TCR (2,351 RU), V?2Vd1 TCR (1,800 RU), or streptavidin alone. Notably, signals over streptavidin alone and control TCR surfaces are equivalent. (Bottom panel) Injection of BTN2A1 IgV (24 µM) over surfaces with immobilized G115 (V?9Vd2; 3,109 RU), MOP (V?9Vd2; 3,108 RU), and V?9Vd1 (2,774 RU) TCRs and LES TCR control (V?4Vd5; 2,885 RU).(B) Equilibrium affinity measurements and Scatchard analysis (inset) of BTN2A1 IgV binding to the G115 (Kd = 39.5 µM) and MOP (Kd = 48.4 µM) V?9Vd2 TCRs and V?9Vd1TCR (Kd = 47.9 µM).Data in (A) and (B) are representative of eight to nine independent experiments.(C) Model of the BTN2A1-V?9 interaction mode based on the proposed BTNL3-V?4 interaction, with expanded panel showing potential contacts at the V?9-BTN2A1 IgV interface.(D) Effects of seven alanine substitutions in proposed BTN2A1 interface residues on V?9Vd2 TCR interaction, indicating affinity of mutant BTN2A1 relative to WT BTN2A1 calculated in the same experiment. Data shown are representative of two independent experiments.(E) Effects of BTN2A1 R124A and R124E mutations on IL-2 production by TCR-MOP in response to HMBPP-treated BTN3A1 and BTN2A1 expressing CD80+ CHO cells.(F) Predicted involvement of V?9 HV4 and CDR2 residues in BTN2A1 interaction.(G) Effects of V?9-E70 mutation (HV4) on IL-2 production by TCR-MOP in response to HMBPP-treated BTN3A1 and BTN2A1 expressing CD80+ CHO cells.(H) Effects of mutations in Vd2 CDR2 (R51A) or a deletion in CDR3 (?CDR3) on IL-2 production by TCR-MOP in response to HMBPP-treated BTN3A1 and BTN2A1 expressing CD80+ CHO cells.In (E), (G) and (H), error bars indicate standard deviation for three independent stimulation experiments. Percentage activation is normalized against the maximum response obtained from CHO cells expressing both BTN2A1 and BTN3A1 in the presence of 1 µM HMBPP. Differences between WT and mutants in (E), (G), and (H) were significant, except for TCR-MOP E70A at 1 µM. See also Figure S3.