ESC vaccination induces tumor cell-specific, Th1-mediated cytokine response in CD8⁺ T cells.

<p>C57BL/6 mice (6/group) were immunized twice (days 0 and 14) with HBSS (control) or irradiated 1×10⁶ ESC alone, or irradiated 1×10⁶ ESC+irradiated 1×10⁶ STO-GM, or irradiated 1×10⁶ STO-GM cells alone, s.c. in the right flank. Ten days after the boost, mice were euthanized and spleens were removed. Splenocytes from vaccinated and control mice were co-cultured with LLC lysate (50 µg/ml) for an additional 4 days. Effectors were harvested and stimulated for 4 hours with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of Brefeldin A (1 µl/ml). After restimulation, effectors were harvested, Fc receptors were blocked, and stained for surface expression of CD4, CD8 and intracellular expression of cytokines and analyzed by flow cytometry. (<b>A, B</b>) Dot plots showing TNF-α and IFN-γ expression in CD8⁺ cells in splenocyte cultures obtained from control and ESC/STO-GM vaccinated mice. Numbers in quadrants represent the percentages of each subpopulation. (<b>C, D</b>) Bar graphs showing percentages of CD8⁺TNF-α⁺, CD8⁺IFN-γ⁺, and CD8⁺IL2⁺ cells in splenocyte cultures derived from control, ESC alone, STO-GM alone and ESC/STO-GM vaccinated mice. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042289#s2" target="_blank">Results</a> are expressed as percentages of total cells. *, p<0.05; relative to control group; ANOVA. (<b>E, F</b>) Unstimulated spleen cells from vaccinated and control mice were directly treated with PMA/ionomycin/Brefeldin A and stained for intracellular cytokine expression. Two independent cell culture assays were performed with cells isolated from 6 mice per group; data from one representative assay is shown. Error bars represent mean ± SD.