Enhanced antileukemia cytotoxicity of γδ PBL cultures activated with pan–T-cell mitogen. (A) γδ peripheral blood lymphocytes (γδ PBLs) were MACS-sorted from the peripheral blood of healthy volunteers (left panel), and stimulated with either HMB-PP and IL-2 or PHA and IL-2 for 4 to 19 days. Activation was evaluated by flow cytometry for CD69 up-regulation (middle panels; levels in freshly isolated control cells are shaded), and total cell numbers are shown on the right panel. (B-C) Preactivated (for 14 days, as in panel A) γδ PBLs were coincubated with DDAOse-labeled leukemia cells for 3 hours. Tumor cell lysis was evaluated by annexin-V staining using flow cytometry. (B) Representative results of 6 different donors for the Bv173 leukemia cell line. Percentages refer to annexin-V+ tumor cells. Basal tumor cell apoptosis (in the absence of γδ PBL) was < 5%. (C) Summary of the results of 6 different donors with 4 leukemia target cell lines. Error bars represent SD (n = 6, *P < .05; **P < .01). (D) Real-time PCR quantification of GzmB mRNA levels in freshly isolated, HMB-PP and IL-2–activated and PHA and IL-2–activated γδ PBL. Data in this figure are representative of 2 to 3 independent experiments with similar results.