Expression kinetics of intracellular perforin and degranulation following anti-CD3 and IL-21/IL-15 stimulation. IL-15, but not IL-21, induces significant degranulation and cytotoxicity in anti-CD3–stimulated CD8 T cells. (A) PBMCs were stimulated with anti-CD3 (30 ng/mL), IL-21 (50 ng/mL), and a combination for 1, 2, or 5 hours. Intracellular perforin (dashed line) and surface CD107a (solid line) expression were analyzed in CD3⁺CD8⁺ T cells by flow cytometry at baseline and after 1, 2, and 5 hours of culture in healthy individuals (top row) and patients with HIV (bottom row). Values depicted represent mean ± SEM. (B) PBMCs from patients with HIV were stimulated with anti-CD3 (30 ng/mL), IL-21 (50 ng/mL), and IL-15 (50 ng/mL) for 5 to 6 hours and analyzed for surface CD107a expression. Bars represent mean values and one standard error above mean for percent CD107⁺ CD8⁺ T cells. *P < .05. (C) A representative dotplot showing perforin and CD107a expression after 5 hours of stimulation in PBMCs from a patient with HIV. Numbers in quadrants indicate percentages of CD8⁺ T cells that are positive or negative for perforin, CD107a, or both. Boxed quadrants show differential effects of IL-21 and IL-15 on anti-CD3–induced degranulation and show that IL-21 induces de novo synthesis of perforin and does not promote degranulation. (D) Cytotoxicity of purified CD3 T lymphocytes from a healthy volunteer and a patient with HIV in a redirected cytotoxicity assay as described in “Patients, materials, and methods.” Purified CD3 T lymphocytes were cultured in medium or stimulated with IL-21 or IL-15 for 24 hours, and cytotoxicity was measured against P815 cell line. Data are shown as the percentage lysis that was determined as [(% 7-AAD staining sample − % 7-AAD staining of negative control) / (100% 7-AAD staining of negative control)] × 100.