Key role played by BTN3A1 in PAg-induced activation of Vγ9Vδ2 T cells. (A) Surface expression of CD277 molecules on shRNA 284 transduced-HEK293FT cells, knocked down for the expression of endogenous CD277 molecules and selectively re-expressing, after transfection, silently mutated CD277 isoforms (BTN3A1^m, BTN3A2^m, or BTN3A3^m). shControl indicates clone 3, random shRNA sequence; sh#284, shRNA-specific for CD277, clone 30; and empty vector, transfection of an empty plasmid. The values for the percentage of CD277⁺ cells are indicated. Data are representative of more than 3 independent experiments. (B) Expression of CD107a on Vγ9Vδ2 T cells (line GUI) after coculture with HEK293FT cells re-expressing BTN3A1^m, BTN3A2^m, or BTN3A3^m isoforms and pretreated for 2 hours with NBP (pamidronate, 250μM) or anti-CD277 mAb (20.1, 10 μg/mL). (C) TNF-α release from Vγ9Vδ2 T cells (line GUI) induced by grading doses of soluble PAg (BrHPP) in coculture with HEK293FT cells re-expressing BTN3A1, BTN3A2, or BTN3A3 isoforms. Data are mean ± SD and are representative of 3 independent experiments. *P < .05 (paired Student t test). (D) Expression of CD107a on Vγ9Vδ2 T cells (line AL) after coculture with HEK293FT cells expressing full-length BTN3A1, BTN3A3, truncated BTN3A1 proteins lacking the intracellular B30.2 domain (BTN3A1^mΔB30.2), or chimeric BTN3A3 proteins assembled with the BTN3A1 intracellular B30.2 domain (BTN3A3^mB30.2A1). Target cells were pretreated for 2 hours with anti-CD277 mAb (20.1, 10 μg/mL) or NBP (pamidronate, 250μM). Data are mean ± SD of the percentage of CD107a⁺ γδ T cells (n = 3). *P < .05 (Student t test).