Figure 4
Figure 4. Abrogation of Vγ9Vδ2 T-cell responses by a blocking anti-CD277 mAb. (A) Surface stainings of Raji cells with either 20.1 and 103.2 anti-CD277 mAbs. Filled histogram represents IgG isotype control. (B) Stainings of CD107a/b (top), IFN-γ (middle), and TNF-α (bottom) of Vγ9Vδ2 T cells (clone GR4) after coculture with Raji cells pretreated for 2 hours with either 20.1 or 103.2 anti-CD277 mAbs (10 μg/mL). Numbers adjacent to outlined areas indicate the percentage of CD107a/b+, IFN-γ+, and TNF-α+ γδ T cells. Positive control indicates PAg (BrHPP, 3μM); and (−), no activation. (C) Staining of CD107a/b on Vγ9Vδ2 T cells (line GUI) after coculture with 721.221 B cells, pretreated, or not, for 2 hours with NBP (pamidronate; 250μM). Cocultures were performed in the presence of either control IgG or 103.2 anti-CD277 Abs (10 μg/mL). Numbers adjacent to outlined areas indicate the percentage of CD107a/b+ γδ T cells. (D) TNF-α production by Vγ9Vδ2 T cells (clone GR4) after activation induced by grading doses of soluble PAg (BrHPP) or anti-CD3 mAb (UCHT1, inset) in the presence of control IgG or anti-CD277 (103.2) Abs (10 μg/mL). (E) Top: Effects of grading doses of 103.2 anti-CD277 mAb on IL-2 release by γδTCR53/4-CD28+ hybridoma cells coexpressing both an MBP-specific αβ TCR (RT1Bl/MHC II) and a human Vγ9Vδ2 TCR. Human Raji cells transduced for RT1Bl expression were cocultured with hybridoma T cells in the presence of Guinea pig myelin basic protein peptide (0.1 μg/mL) or sec-butylamine (1mM). Bottom: Activation induced by soluble PAg (BrHPP, 3μM). IgG indicates isotype control. IL-2 production (ELISA) is presented relative to those of cells activated in the absence of antibody. *P < .05 (paired Student t test). **P < .005 (paired Student t test). (F) Expression of CD107a on Vγ9Vδ2 T cells (polyclonal line AL) after coculture with Mycobacterium bovis BCG-infected THP-1 cells, in the presence or in the absence of 103.2 anti-CD277 mAb (10 μg/mL). The values for the percentage of CD107a+ γδ T cells are indicated on the y-axis. MOI indicates multiplicity of infection; and (−), no antibody. Data are mean ± SD (n = 3 experiments). *P < .05 (Student t test). ***P < .0005 (Student t test).

Abrogation of Vγ9Vδ2 T-cell responses by a blocking anti-CD277 mAb. (A) Surface stainings of Raji cells with either 20.1 and 103.2 anti-CD277 mAbs. Filled histogram represents IgG isotype control. (B) Stainings of CD107a/b (top), IFN-γ (middle), and TNF-α (bottom) of Vγ9Vδ2 T cells (clone GR4) after coculture with Raji cells pretreated for 2 hours with either 20.1 or 103.2 anti-CD277 mAbs (10 μg/mL). Numbers adjacent to outlined areas indicate the percentage of CD107a/b+, IFN-γ+, and TNF-α+ γδ T cells. Positive control indicates PAg (BrHPP, 3μM); and (−), no activation. (C) Staining of CD107a/b on Vγ9Vδ2 T cells (line GUI) after coculture with 721.221 B cells, pretreated, or not, for 2 hours with NBP (pamidronate; 250μM). Cocultures were performed in the presence of either control IgG or 103.2 anti-CD277 Abs (10 μg/mL). Numbers adjacent to outlined areas indicate the percentage of CD107a/b+ γδ T cells. (D) TNF-α production by Vγ9Vδ2 T cells (clone GR4) after activation induced by grading doses of soluble PAg (BrHPP) or anti-CD3 mAb (UCHT1, inset) in the presence of control IgG or anti-CD277 (103.2) Abs (10 μg/mL). (E) Top: Effects of grading doses of 103.2 anti-CD277 mAb on IL-2 release by γδTCR53/4-CD28+ hybridoma cells coexpressing both an MBP-specific αβ TCR (RT1Bl/MHC II) and a human Vγ9Vδ2 TCR. Human Raji cells transduced for RT1Bl expression were cocultured with hybridoma T cells in the presence of Guinea pig myelin basic protein peptide (0.1 μg/mL) or sec-butylamine (1mM). Bottom: Activation induced by soluble PAg (BrHPP, 3μM). IgG indicates isotype control. IL-2 production (ELISA) is presented relative to those of cells activated in the absence of antibody. *P < .05 (paired Student t test). **P < .005 (paired Student t test). (F) Expression of CD107a on Vγ9Vδ2 T cells (polyclonal line AL) after coculture with Mycobacterium bovis BCG-infected THP-1 cells, in the presence or in the absence of 103.2 anti-CD277 mAb (10 μg/mL). The values for the percentage of CD107a+ γδ T cells are indicated on the y-axis. MOI indicates multiplicity of infection; and (−), no antibody. Data are mean ± SD (n = 3 experiments). *P < .05 (Student t test). ***P < .0005 (Student t test).

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