Fig. 3: Qualification of γδT-cell apoptotic resistance after cytokine withdrawal.

When γδT cells were cultured with IL-2, IL-15, and IL-2 plus IL-15 for 14 days, cytokines were removed and apoptosis was analyzed by measuring the level of Annexin V and 7-AAD by flow cytometry. A Flowchart of the experimental setup to study effects of IL-15 stimulation and withdrawal in γδT cells. B Annexin V⁺ γδT cells were analyzed at day 14 (HC, n = 7; NB, n = 7). Data were mean ± SEM from four independent experiments. Statistical analysis was performed using one-way ANOVA and Dunnett’s multiple comparisons. C Representative gating strategy for the flow-cytometric analysis. γδT cells fostered with different conditions were examined after cytokine withdrawal 0 h, 24 h, 48 h, 72 h, 96 h. D, E the proportion of Annexin V⁺ γδT cells were analyzed at each time point after cytokine withdrawal (HC, n = 7; NB, n = 7). Data were mean ± SEM from four independent experiments. Statistical analysis was performed using one-way ANOVA and Dunnett’s multiple comparisons. *, p < 0.05; **, p < 0.01; ***, p < 0.001. F Representative gating strategy for the flow-cytometric analysis. Mitochondrial membrane potential was analyzed by flow cytometry following staining with JC-1 when cytokines were withdrawn for 72 h. JC-1 aggregates are representative of high mitochondrial membrane potential and JC-1 monomers are representative of low mitochondrial membrane potential (apoptosis). G JC-1 monomer percentage was analyzed (HC, n = 11; NB, n = 7). Data were mean ± SEM from six independent experiments. Statistical analysis was performed using one-way ANOVA and Dunnett’s multiple comparisons. **, p < 0.01; ***, p < 0.001. H Annexin V^- 7-AAD^- γδT cells represented viable cells were assessed (HC, n = 7; NB, n = 7). Data were mean ± SEM from four independent experiments. Statistical analysis was performed using one-way ANOVA and Dunnett’s multiple comparisons.