Broad activation of human Vγ9Vδ2 T cells by anti-CD277 mAbs. (A) Frequency of human γδ T cells in IL-2–supplemented ex vivo PBMCs after a 2 week-incubation in the presence of anti-CD277 mAb (#20.1; 10 μg/mL). The values for the percentage of γδ T cells within PBLs are indicated. No stim. indicates no stimulation. (B) Intracellular stainings of IFN-γ and TNF-α in ex vivo human PBL-γδ T cells after incubation for 5 hours with anti-CD277 mAb (#20.1; 10 μg/mL). Numbers inside or adjacent to outlined areas indicate the values for the percentage of IFN-γ⁺ (top row) or TNF-α⁺ (bottom row) cells within γδ⁻ and γδ⁺ PBL subsets. (C) Expression of CD69 on ex vivo human PBL subsets after incubation for 7 hours with either anti-CD277 mAb (#20.1; 10 μg/mL) or soluble PAg (BrHPP; 3μM). MFI indicates geometric mean of fluorescence intensity. Data in graph are mean ± SD (n = 3 healthy donors). *P < .05 (Student t test). **P < .005 (Student t test). (D) Stainings of CD107a/b (left), IFN-γ (middle), and TNF-α (right) of Vγ9Vδ2 (clone GR4), Vγ8Vδ3 (clone 73R9), CD8⁺ αβ (clone 13), and invariant NK (iNKT, line MAD11) human T cells after treatment with anti-CD277 mAb (#20.1; 10 μg/mL, 5 hours). PMA/Iono. indicates nonspecific activation induced by PMA and ionomycin. (A-B,D) Data are representative of at least 3 experiments. (E) Intracellular Ca²⁺ levels were measured by videomicroscopy within clusters of Fura-2 AM-loaded polyclonal human Vγ9Vδ2 (γδ) T-cell lines after addition (t = 0 minutes) of anti-CD277 mAb (#20.1; 10 μg/mL) and compared with CD8⁺ αβ (αβ) or isolated γδ T cells. IgG1 indicates isotype control. Values correspond to the mean of emissions (340/380 nm ratio) measured among all T cells present in the field. γδ, n = 70; αβ, n = 30; isolated γδ, n = 30; and IgG1, n = 30.