Double treatments restore capacity of lymphocytes within the tumor to produce IL-2 and proliferate. Tumors were harvested on day 7 and single cell suspensions were prepared. Pools of cells from 3–5 mice were combined and subsequently stained with cell trace. Cells were cultured with or without plate-bound anti-CD3 for 48 h then treated with anti-CD3/anti-CD28-stimulation in the presence of Brefeldin A for 6 h. Cells were then stained for production of IL-2, IFN-γ and TNF-α by intracellular flow cytometry. A. A representative FACS plot showing proliferation via cell trace dilution on the x-axis and intracellular IL-2 staining on the y-axis. A pool of five mice was analyzed. B. Statistical analysis of the amount of proliferating CD3+CD8+ cells in the non-stimulated (left) and stimulated (right) group. Only double treatments show a significant increase in proliferation compared to non-stimulated and single treatments when tested with a one-way Anova. Bars represent mean +/− SEM of a total of 4 pools collected out of 2 experiments. C. The percentages of IFN-γ+ (open bar), IFN-γ+ and IL-2+ (gray bar), and proliferating IFN-γ+IL-2+ cells (filled bar) were calculated within the CD3+CD8+ cell population. D. The percentages of polyfunctional (TNF-α+IFN-γ+IL-2+ proliferated) CD8+ T cells (black) compared to other less functional subsets is shown. Dark gray represents no proliferation, light gray represents no IL-2 production, white represents no IFN-γ production for each treatment group. C and D were calculated based on the results shown in A and B.