Primary coculture of splenocytes from normal naïve mice with transduced, irradiated 32Dp210-derived vaccines increases proliferation of CD3⁺CD8⁺and CD3⁺CD4⁺T cells. Whole splenocyte populations from normal naïve mice were labeled with CFSE and cultured alone (no stimulation), or in the presence of irradiated untransduced 32Dp210 cells, or with 32Dp210 cells transduced with lentiviral vectors carrying CD80, IL-15-IL-15Rα, or IL-15-IL-15Rα-CD80 (depicted above each graph) for 12 days. The percent dividing cells is indicated in each plot. After coculture, cells were stained with anti-CD3, anti-CD8, and anti-CD4 antibodies, and quantified by flow cytometric analyses. (A) Proliferation of CD3⁺CD8⁺ T cells. (B) Proliferation of CD3⁺CD4⁺ T cells. Bar graphs to the right of the flow plots depict the mean percentage of dividing cells from triplicate wells ± SEM. 32Dp210-luc indicates luciferase-positive parental leukemia cell line. The gene or genes expressed by each of the transduced 32Dp210 lines are indicated above each of the plots. (C and D) IL-15/IL-15Rα and CD80 expressed by the 32Dp210-IL-15/IL-15Rα/CD80 vaccine are highly synergistic in stimulating the proliferation of purified T cells from naïve normal mice. Splenocytes were harvested, and T cells were purified, labeled with CFSE, and cocultured with irradiated 32Dp210-derived vaccines in primary stimulation assays for 12 days. Thereafter, proliferation was quantified by flow cytometric analyses. (C) Proliferation of CD3⁺CD8⁺ T cells; (D) proliferation of CD3⁺CD4⁺T cells. The genes expressed by the 32Dp210-derived vaccines in each stimulation assay are indicated above each plot. The percent dividing cells is indicated in each plot. Controls include culture with media alone (no stimulation) and coculture with the untransduced parental 32Dp210 cells. (E-F) Secondary stimulation assays of splenocytes harvested from non-tumor-bearing mice treated with 32Dp210 vaccines and restimulated ex vivo with parental 32Dp210 cells exhibit enhanced proliferative capacity. Splenocytes were harvested from C3H mice previously vaccinated weekly 4 times with irradiated 32Dp210, 32Dp210-IL15-IL15Rα, 32Dp210-CD80, or 32Dp210-IL-15/IL15-Rα/CD80 cell vaccines. Cells were labeled with CFSE and cocultured for 12 days with untransduced 32Dp210 parent cells treated with 100 Gy γ-irradiation. Thereafter, cocultured splenocytes were stained with anti-CD3, CD4, and CD8 antibodies, and proliferation was quantified by flow cytometry. The percent dividing cells is indicated in each plot. (E) Proliferation of CD3⁺CD8⁺ T cells. (F) Proliferation of CD3⁺CD4⁺ T cells. Bar graphs to the right depict the mean percentage of dividing cells in triplicate wells ± SEM. Transgenes expressed by each vaccine cell line are indicated above each of the plots and below the corresponding bar graphs.