Rejection of live IL12-AML cells is CD8⁺T-cell dependent and generates long-lasting leukemia-specific immunity. (A) Groups of SJL mice were injected IP on 3 consecutive days with 150 μg of purified MoAb of either 53.672 hybridoma (▴) or GK1.5 hybridoma (•). One day after the last injection, 1 mouse from each group was killed and the depletion was verified in spleen cells by flow cytometry. The remaining mice were injected IV with 10⁵live IL12-AML cells, and the antibody injections continued twice weekly for 3 weeks. Control mice were treated with isotype control rat IgG (□). All mice depleted of CD8⁺ T cells developed lethal leukemia, whereas only 20% of CD4⁺ T-cell–depleted mice developed leukemia and 80% rejected the IL12-AML cells. All of the control mice rejected the IL12-AML cells. (B) Groups of mice were injected IV with 10⁵ live IL12-AML (•) or control (□) cells. All IL12-AML–injected mice rejected the leukemic cells, whereas all control mice developed lethal leukemia. Mice that rejected IL12-AML cells were challenged 4 months later with 10⁵ live wt AML cells. All of the challenged mice were immune against AML cells and did not develop leukemia. Three months after the challenge, 3 mice were killed and their splenocytes were used for CTL assays. (C) Splenocytes from vaccinated mice were stimulated in vitro with irradiated AML cells as described in Materials and Methods. Six days later, cells were harvested and used as effector cells in the indicated E:T ratios. Target cells (autologous H-2^s AML or control H-2^b EL-4 cells) were incubated with ⁵¹Cr for 90 minutes. The standard 4-hour CTL assays were set up in a total volume of 0.2 mL/well in 96-well microtiter plates. All conditions were set up in quadruplicate. As the control in the experiment, normal SJL splenocytes were tested for CTL activity on AML (naive/AML) and EL-4 cells (naive/EL-4). Splenocytes from vaccinated mice lysed autologous AML (vac/AML) but not EL-4 cells (vac/EL-4).