Figure 3
Figure 3. Vγ9Vδ2 TCR implication in anti-CD277 mAb-induced activation. (A) Confocal microscopy. CD3ε (red) and CD277 (green) distribution in conjugates of Vγ9Vδ2 T cells (line AL) and HEK293 cells expressing EmGFP tagged-CD277 molecules. HEK293 cells were pretreated for 2 hours with anti-CD277 mAb (20.1; 10 μg/mL) and washed before coculture with Vγ9Vδ2 T cells. Composite pictures showing overlays of both colors and bright-field pictures. Confocal images of representative γδ T-cell–HEK293 conjugates are shown. Bars represent 10 μm. (−) indicates untreated cells. One experiment representative of 3 is shown. (B) Surface expression of CD69 on human Jurkat T-cell γδ TCR transductants (human Vγ8Vδ3 or Vγ9Vδ2 TCRs) after incubation for 4 hours with either anti-CD3 (OKT3; 10 μg/mL) or anti-CD277 (20.1; 10 μg/mL) mAbs. WT indicates wild-type Jurkat cells with no TCR expression. The values for the MFI of stained cells are represented in the graph. **P < .005 (Student t test). ***P < .0005 (Student t test). Data are representative of 3 independent experiments. (C) IFN-γ response in PBL-derived human αβ T cells expressing a human Vγ9Vδ2 TCR after incubation with NBP (pamidronate)- or anti-CD277 (20.1; 10 μg/mL) mAb-treated Daudi cells. Production of IFN-γ was determined via ELIspot analysis. Data are mean ± SEM. **P < .005 (2-way ANOVA). ***P < .0005 (2-way ANOVA). Comparable results were obtained in 3 independent experiments. (D-E) IL-2 release by murine T-cell hybridomas, transduced for the expression of a human Vγ9Vδ2 TCR, after coculture with human Raji cells in the presence of anti-CD277 mAb (20.1). (D) IL-2 release by mouse γδTCR58C-CD28+ hydridoma cells. IgG indicates isotype control. Data are representative of 4 independent experiments. *P < .05 (paired Student t test and Mann-Whitney test). (E) IL-2 release by γδTCR53/4-CD28+ (αβ TCR− variant) rat/mouse T-cell hybridoma in the presence of Raji cells pretreated with grading doses of anti-CD277 mAb (20.1). Data are mean ± SEM and are representative of 3 independent experiments. *P < .05 (paired Student t test). **P < .005 (paired Student t test). IL-2 production in both panels is presented relative to those of cells cocultured with Raji cells in the absence of antibody with (100%) or without 3mM sec-butylamine (0%).

Vγ9Vδ2 TCR implication in anti-CD277 mAb-induced activation. (A) Confocal microscopy. CD3ε (red) and CD277 (green) distribution in conjugates of Vγ9Vδ2 T cells (line AL) and HEK293 cells expressing EmGFP tagged-CD277 molecules. HEK293 cells were pretreated for 2 hours with anti-CD277 mAb (20.1; 10 μg/mL) and washed before coculture with Vγ9Vδ2 T cells. Composite pictures showing overlays of both colors and bright-field pictures. Confocal images of representative γδ T-cell–HEK293 conjugates are shown. Bars represent 10 μm. (−) indicates untreated cells. One experiment representative of 3 is shown. (B) Surface expression of CD69 on human Jurkat T-cell γδ TCR transductants (human Vγ8Vδ3 or Vγ9Vδ2 TCRs) after incubation for 4 hours with either anti-CD3 (OKT3; 10 μg/mL) or anti-CD277 (20.1; 10 μg/mL) mAbs. WT indicates wild-type Jurkat cells with no TCR expression. The values for the MFI of stained cells are represented in the graph. **P < .005 (Student t test). ***P < .0005 (Student t test). Data are representative of 3 independent experiments. (C) IFN-γ response in PBL-derived human αβ T cells expressing a human Vγ9Vδ2 TCR after incubation with NBP (pamidronate)- or anti-CD277 (20.1; 10 μg/mL) mAb-treated Daudi cells. Production of IFN-γ was determined via ELIspot analysis. Data are mean ± SEM. **P < .005 (2-way ANOVA). ***P < .0005 (2-way ANOVA). Comparable results were obtained in 3 independent experiments. (D-E) IL-2 release by murine T-cell hybridomas, transduced for the expression of a human Vγ9Vδ2 TCR, after coculture with human Raji cells in the presence of anti-CD277 mAb (20.1). (D) IL-2 release by mouse γδTCR58C-CD28+ hydridoma cells. IgG indicates isotype control. Data are representative of 4 independent experiments. *P < .05 (paired Student t test and Mann-Whitney test). (E) IL-2 release by γδTCR53/4-CD28+ (αβ TCR− variant) rat/mouse T-cell hybridoma in the presence of Raji cells pretreated with grading doses of anti-CD277 mAb (20.1). Data are mean ± SEM and are representative of 3 independent experiments. *P < .05 (paired Student t test). **P < .005 (paired Student t test). IL-2 production in both panels is presented relative to those of cells cocultured with Raji cells in the absence of antibody with (100%) or without 3mM sec-butylamine (0%).

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