Serial vaccination with 32Dp210-derived whole cell vaccines in non-tumor-bearing mice stimulates robust antileukemic cytolytic activity. Naïve C3H mice were treated with 4 vaccinations with 2 × 10⁶ irradiated 32Dp210-Luc, 32Dp210-mIL-15-IL-15Rα, 32Dp210-CD80, or 32Dp210-IL-15/IL-15Rα/CD80. In the first experimental group of mice, splenocytes were harvested and restimulated in vitro with irradiated (100 Gy) 32Dp210 parent cells for 5 days. Thereafter, cells were harvested and purified by Ficoll density gradient for ex vivo assays (A-F). In a parallel experimental group, vaccinated mice were challenged with IV inoculation of 32Dp210 leukemia and monitored for survival (G). (A) Cytolytic activity of splenocytes in ex vivo assays is greatest after stimulation by 32Dp210-IL-15/IL-15Rα/CD80 vaccine. Splenic effectors cells and 1 × 10⁵ CellTrace DDAO-SE labeled 32Dp210 target cells were cocultured at different ratios (red bars, 1:1; blue bars, 5:1; green bars, 10:1) for 48 hours. X-axis: naïve: indicates splenocytes from uninjected C3H mice; 32Dp210: splenocytes from mice vaccinated with the parent 32Dp210 cell line; vaccine expressing IL-15/IL-15Rα, CD80, or IL-15/IL-15Rα/CD80 (below graph): depict assays with splenocytes from C3H mice treated with lentivirally transduced 32Dp210 vaccines expressing the indicated transgenes. The mean percentage of apoptotic cells, defined by detection of activated caspase-3 by antibody staining, is depicted on the y-axis (± SEM). (B) ELIspot assay of IFN-γ expression in splenocytes from vaccinated mice shows increased frequencies of cytotoxic cells after treatment with all transduced 32Dp210 vaccines. Splenocytes from unvaccinated mice (naïve), or from mice vaccinated with irradiated parent cells 32Dp210 cells, or with engineered 32Dp210 cells expressing each of the transgene cassettes, indicated below the bar graphs, were cocultured with irradiated 32Dp210 cells, and the frequency of IFN-γ positive cells was quantified. The mean number of spots per well per containing 3 × 10⁵ cells in triplicate samples is depicted on the y-axis ± SEM. (C and D) Highest levels of intracellular IFN-γ expression are observed in CD3⁺CD8⁺ and CD3⁺CD4⁺ T cells from 32Dp210-IL-15/IL-15Rα/CD80-vaccinated mice after secondary stimulation, whereas stimulation with BCR-ABL-loaded splenocytes produces IFN-γ levels comparable to unstimulated controls. In panels C and D, the y-axis depicts the percent IFN-γ positive CD3⁺CD8⁺ T cells (C) and CD3⁺CD4⁺ T cells (D) in splenocytes from vaccinated non-tumor-bearing mice. X-axis: naïve = unvaccinated mice; 32Dp210 = vaccinated weekly, 4 times with unmodified irradiated 32Dp210 parent cells; -IL-15/IL-15Rα = vaccinated with 32Dp210-IL-15/IL-15Rα; -CD80 = vaccinated with 32Dp210-CD80; -IL-15/IL-15Rα/CD80 = vaccinated with 32Dp210-IL-15/IL-15Rα/CD80. Splenocytes were stimulated for 20 hours with either media alone, C3H splenocytes loaded with an irrelevant control peptide, or BCR-ABL peptide, or with unmodified 32Dp210 cells as indicated. (E) Comparative ELIspot assays of IFN-γ expression in splenocytes from vaccinated mice confirm increased frequencies of cytotoxic cells after treatment with all transduced 32Dp210 vaccines but no significant stimulation by BCR-ABL-loaded cells. ELIspot assays were performed as in panel A. C3H splenocytes were loaded with either BCR-ABL peptide or control peptide and cocultured with splenocytes from vaccinated animals as described in panel A, depicted on the x-axis. The mean number of spots per well per containing 3 × 10⁵ cells in triplicate samples is depicted on the y-axis (± SEM). (F) Splenocytes from non-tumor-bearing mice treated with the 32Dp210-derived vaccines show high levels of lytic activity to 32Dp210 targets, but not to human BCR-ABL peptide loaded syngeneic C3H cells. Splenocytes from vaccinated non-tumor-bearing C3H mice, as described in panel A were stimulated ex vivo for 5 days with irradiated 32Dp210 cells and used as effectors. Unmodified 32Dp210 cells, BCR-ABL, or control peptide-loaded splenocytes from naïve C3H mice were used as targets. Ex vivo lytic activity was assayed by intracellular staining of active caspase-3 after coculture of effectors and targets at an effector-to-target ratio = 10:1 for 24 hours. X-axis depicts cells from different experimental vaccine groups as in panel A. The mean percentage of apoptotic cells, defined by detection of activated caspase-3 by antibody staining, is depicted on the y-axis (± SEM). (G) Treatment with transduced 32Dp210-derived vaccines confers greater survival after leukemia challenge than does administration of untransduced irradiated 32Dp210 cell vaccines. Mice were treated weekly 3 times with 32Dp210-derived vaccines as described previously. Thereafter, they were inoculated IV with 1 × 10⁴ 32Dp210-luc cells and longitudinally monitored by in vivo bioluminescence imaging for tumor progression and survival. Percent survival is depicted on the y-axis, and duration of survival on the x-axis. Animals surviving the initial leukemia challenge were rechallenged with a second IV inoculation of 32Dp210 leukemia (indicated by downward arrow), 150 days after the initial tumor challenge.