Granulysin is essential for Vγ9Vδ2 antiparasitic activity. The S1 T-cell line was infected with the lentiviral vector containing the granulysin-specific shRNA sequences GNLYsh1, GNLYsh2, or a NS shRNA sequence, and their antiparasitic activity has been assessed in a standard parasite inhibition assay. (A) RT-PCR assessment of granulysin mRNA levels in the noninfected S1 cell line (Ctrl) or S1 cell line infected with NS, GNLYsh1 (Gnly1), or GNLYsh2 (Gnly2) shRNA constructs. β2-Microglobulin mRNA levels were measured as a control. (B) Granulysin and perforin protein levels were detected in S1 (Ctrl), NS, Gnly1, and Gnly2 cell lines by Western blotting. Actin expression was measured as a loading control. (C) Intracellular granulysin expression was measured using flow cytometry in the NS, Gnly1, and Gnly2 cell lines. The percentage of granulysin-positive cells and the MFI of granulysin intracellular staining are indicated in the top right corner of each panel. Dotted lines represent isotype control antibody. (D) Antiparasitic activity of the NS, Gnly1, and Gnly2 cell lines. Antiparasitic activity (percentage) was calculated as 100 − [(average % parasitemia of duplicate with cells in the presence of IL-2 + IL-15/average % parasitemia of duplicate with cells in the presence of IL-2) × 100]. Data represent the mean ± SD of 2 independent experiments (n = 2; *P ≤ .05 by Mann-Whitney rank sum test comparing NS with Gnly1 and Gnly2 T-cell lines).