ESC vaccination-induced CD8⁺ T-effector responses are maintained in long-term surviving animals.

<p>Long-term surviving mice from the ESC/STO-GM group were re-challenged s.c with 1×10⁵ LLC cells 60 days after initial tumor inoculation. (<b>A</b>) Tumor growth was monitored daily in all animals until sacrifice due to tumors exceeding 5% of body weight. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042289#s2" target="_blank">Results</a> represent a summation of two independent experiments with 10 mice/group. **, p<0.001; relative to naïve control; log-rank test. (<b>B–D</b>) Spleens were isolated from vaccinated survivors and control naïve mice 10 days after the tumor injection. (<b>B</b>) Splenocytes 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, cells were harvested, Fc receptors were blocked, and stained for intracellular expression of cytokines, and analyzed by flow cytometry. Bar graphs showing percentage of intracellular IFN-γ expression in CD8⁺ cells (*, <i>p</i><0.05; relative to naïve control; <i>t</i> test). <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. (<b>C</b>) Dot plots showing the percentages of CD44⁺ and CD8⁺ cells in splenocytes derived from long-term surviving ESC vaccinated and control naïve mice. Numbers in the quadrants represent percentages of each subpopulation. (<b>D</b>) Bar graph showing the percentages of CD44⁺CD8⁺ cells (*, <i>p</i><0.05; relative to naïve control; <i>t</i> test). <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. Data for B–D are representative of two independent experiments with 4 mice/group. Error bars represent mean ± SD.