Figure 3.

Direct BTN2A1 Binding to Germline-Encoded Regions of Vγ9 Is Essential for P-Ag Sensing

(A) (Top panel) Injection of BTN2A1 IgV (25 μM) over surfaces with immobilized Vγ9Vδ2 TCR (2,457 resonance units (RU)) and control surfaces comprising Vγ4Vδ5 TCR (2,351 RU), Vγ2Vδ1 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 (Vγ9Vδ2; 3,109 RU), MOP (Vγ9Vδ2; 3,108 RU), and Vγ9Vδ1 (2,774 RU) TCRs and LES TCR control (Vγ4Vδ5; 2,885 RU).

(B) Equilibrium affinity measurements and Scatchard analysis (inset) of BTN2A1 IgV binding to the G115 (K_d = 39.5 μM) and MOP (K_d = 48.4 μM) Vγ9Vδ2 TCRs and Vγ9Vδ1TCR (K_d = 47.9 μM).

Data in (A) and (B) are representative of eight to nine independent experiments.

(C) Model of the BTN2A1-Vγ9 interaction mode based on the proposed BTNL3-Vγ4 interaction, with expanded panel showing potential contacts at the Vγ9-BTN2A1 IgV interface.

(D) Effects of seven alanine substitutions in proposed BTN2A1 interface residues on Vγ9Vδ2 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 Vγ9 HV4 and CDR2 residues in BTN2A1 interaction.

(G) Effects of Vγ9-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 Vδ2 CDR2 (R51A) or a deletion in CDR3 (ΔCDR3) 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.