Cell viability assessed by flow cytometry by annexin V–PE and 7-AAD staining. (A) Among PHA-stimulated T lymphocytes (> 70% CD25⁺ events in all cases), cell viability significantly decreases by increasing the drug concentration. Mean percentage (SD) of annexin V–PE–negative and 7-AAD–negative CD3⁺ cells (viable T lymphocytes) for the different doses of bortezomib in PHA-stimulated samples: 42.13 (7.03) at 0 nM; 46.14 (9.47) at 1 nM; 35.85 (12.24) at 10 nM; 21.2 (11.9) at 100 nM; and 12.87 (7.72) at 1000 nM. (B) Among resting T lymphocytes, only a minority of cells express CD25 (< 3% CD25⁺ events in all cases). Cell viability assessed as annexin V–PE–negative and 7-AAD–negative events is not significantly affected by the increments in the drug's concentration. Mean percentage (SD) of annexin V–PE–negative and 7-AAD–negative CD3⁺ cells (viable T lymphocytes) for the different doses of bortezomib in unstimulated samples: 60.64 (6.03) at 0 nM; 65.6 (3.04) at 1 nM; 64.6 (3.37) at 10 nM; 63.21 (4.96) at 100 nM; and 53.08 (4.32) at 1000 nM. (C) Comparison of the percentage (±95% confidence interval) of annexin V–PE–negative and 7-AAD–negative CD3⁺ cells between PHA-stimulated and unstimulated (control) T lymphocytes at the different doses of bortezomib. Median, 50% CI, and 95% CI are shown.