rVV-induced effector cells from vaccinated mice kill CD4⁺ T cells from HBZ-Tg mice. (A) Schema of the cytotoxicity assay using flow cytometry. Ly5.2 target cells were cocultured with Ly5.1 effector cells. Annexin V– and Ly5.2-positive cells were measured to evaluate cytotoxicity. (B) Cytotoxicity of effector cells from rVV-Tax M22–vaccinated mice. (C) HBZ-PA/HBZ-PB–pulsed EL-4 cells or (D) primary Ly5.2⁺CD4⁺ T cells from HBZ-Tg mice were cocultured with Ly5.1⁺ HBZ effector cells, and cytotoxicity against target cells was measured. Ly5.1 and Ly5.2 were used as markers to discriminate effector T cells and target T cells. (E-F) Cytotoxicity of HBZ-specific effector cells against HBZ-overexpressing CD4⁺ T cells. (E) Primary Ly5.1⁺CD4⁺ T cells were transduced with HBZ and used as target cells in coculture with Ly5.2⁺ HBZ effector cells. The GFP⁺ target cells among Ly5.1⁺ and CD4⁺ cells were measured. (F) Relative index = E:T_(x:1)/E:T_(0:1) in GFP-positive cells (%). (G-I) In vivo cytotoxicity assay. (G) Naïve mice were vaccinated 6 times and inoculated with Tracer dye–labeled HBZ-Tg CD4⁺ T cells; 24 hours later, the percentage of labeled cells was measured in the CD4⁺ population. (H-I) Transferred HBZ-Tg CD4⁺ T cells in spleen and mesenteric, submandibular, and inguinal lymph nodes of recipient mice. The percentage of tracer dye–positive cells is shown in the dot plots. The data from WT VV-vaccinated mice was used as a reference. The bars represent the mean ± SD of all mice. At least 2 independent experiments were performed. *P < .05 by Student t test. i.v., intravenous. SSC, side scatter; FSC, forward scatter; E:T, effector:target; GFP, green fluorescent protein.