Involvement of both TCR and NKG2D during ULBP4-induced γδT-cell activation. (A) MTT assay evaluating inhibition of cytotoxicity by PBMC-Vδ2T cells against EL4-ULBP4 cells. Anti-NKG2D (20 μg/mL), anti-TCRγδ (10 μg/mL), a combination of both, and control mAb. Data represent means ± SD of 3 independent experiments (*P < .05, **P < .01). (B-C) Effect of TCR and NKG2D signal on IFN-γ secretion and granule release. Immobilized rULBP4, anti-NKG2D mAb, anti–pan-TCRγδ mAb, and Ig control were precoated on microtiter plates. PBMC-Vγ9/δ2T cells were incubated. In blocking assay, CsA (100ng/mL), Ly294002 (50 μM), or wortmannin (1 μM) was added to some cultures. Supernatants collected after 48 hours (B) or after 5 hours (C) were measured for IFN-γ secretion (B) or BLT esterase release (C) by ELISA. Each error bar represents means ± SD of triplicate samples.