FIG. 5.

Effects of VacA on ATP levels and mitochondrial membrane polarization. Primary human CD4⁺ T cells were activated with CD3/CD28 and cultured in the presence of IL-2 for 7 days as described in Materials and Methods. Cells were then incubated for 16 h without IL-2 and then treated with the indicated additives for 8 h without IL-2. (A) After treatment, cells were stimulated with IL-2 for 24 or 48 h and then the total ATP levels were analyzed using Cell Glow reagent as described in Materials and Methods. (B) Cell viability analysis for panel A. Cells were treated as stated for panel A, and then at 24 and 48 h cells were analyzed for viability based on forward and side scatter analysis with flow cytometry. (C) For analysis of mitochondrial membrane potential and cell viability, cells were stimulated with IL-2 for 24 h and stained using Mito Flow reagent as described in Materials and Methods. Cell viability was determined based on forward and side scatter analysis and PI staining (data not shown), and mitochondrial membrane potential was determined as described in Materials and Methods. Asterisks indicate P < 0.05 compared to PBS-treated cells at the same time point. Statistical significance between groups was determined by a two-tailed t test. Results are representative of three experiments performed in triplicate with T cells isolated from different donors. DNP, dinitrophenol; NaN₃, sodium azide; Rap, rapamycin.