Fig. 4.

Pharmacological inhibition of CFTR significantly increased IFN-γ release and cytotoxic activity in γδ T cells and triggered V_m-induced Ca²⁺ influx. a Flow cytometry analysis of splenic γδ T cells treated with PMA (50 ng/ml) and ionomycin (1 μg/ml) stimulation for 6 h in the presence of the CFTR inhibitor CFTR_inh172 (5 μM) or vehicle DMSO was performed to detect IFN-γ and IL-17 production (n = 4, *p < 0.05). b In vitro-expanded γδ T cells were pretreated with the CFTR inhibitor CFTR_inh172 (5 μM) or vehicle DMSO for 2 h and then cocultured with CFSE-labeled B16 cells at the indicated ratio. Six hours later, dead B16 cells were identified by PI staining (n = 3, *p < 0.05). c A representative immunoblot analysis (from three independent experiments) of the indicated proteins in the whole-cell lysate of in vitro-expanded γδ T cells stimulated in the presence of the CFTR inhibitor CFTR_inh172 (5 μM) or vehicle DMSO with anti-CD3 (10 μg/ml) and anti-CD28 (1 μg/ml) antibodies for 2, 5, 10, 15, or 30 min is shown; band densities were quantified with ImageJ and ratios of indicated intensities were shown. act: actin. d The cytoplasmic V_m of γδ T cells treated with the CFTR inhibitor CFTR_inh172 (10 μM) or vehicle DMSO (1 × 10⁶ cells for each group) followed by gramicidin (10 μM) was measured, and the relative V_m in WT or CFTR_inh172-treated γδ T cells was quantified (n = 3, **p < 0.01). e In vitro-expanded γδ T cells were pretreated with CFTR_inh172 (10 μM) or the vehicle DMSO (1 × 10⁶ cells for each group) for 2 h in Ca²⁺-free medium. Changes in the [Ca²⁺]_i were monitored by flow cytometry, and the overlay of the Ca²⁺ influx profiles after the addition of CaCl₂ (5 mM) to the extracellular medium in the presence of thapsigargin (1 μM) is shown. The relative peak [Ca²⁺]_i in WT or CFTR_inh172-treated γδ T cells was quantified (n = 3, *p < 0.05). f In vitro-expanded γδ T cells were treated with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 6 h in the presence of the CFTR inhibitor CFTR_inh172 (5 μM) or vehicle DMSO with the addition of FK506 and then evaluated to detect IFN-γ and IL-17 production (*p < 0.05, ns not significant, n = 3). g Flow cytometry analysis of the phosphorylated NFATc1 level in in vitro-expanded WT or CFTR_inh172-treated γδ T cells stimulated with soluble anti-CD3 (10 μg/ml) and anti-CD28 (1 μg/ml) antibodies for 30 min was performed (n = 3, **p < 0.01). Dotted line: isotype control