Luate the immunologic response to tumor ablation with thermal ablation, such as radiofrequency and cryoablation [16?8]. In the present study, we found that the percentages of CD3+ T lymphocytes and CD4+ T lymphocytes, as well as the CD4+/CD8+ ratio, of tumor-bearing rats was lower than that in the non-tumor-bearing group before operation. Both ITI-007 manufacturer surgical tumor resection and IRE treatment reduced the percentage of CD8+ T lymphocytes in tumor-bearing rats, but there was no statistically significant difference between the twoFigure 3. Changes in T lymphocyte subset percentage (A, B, C) and CD4+/CD8+ ratio (D). *p,0.05; #p.0.05. doi:10.1371/journal.pone.0048749.gImmunologic Response to IREFigure 4. Changes in cytokine IFN-c-positive (A) and IL-4-positive (B) splenocytes. doi:10.1371/journal.pone.0048749.ggroups. The percentages of CD3+ T lymphocytes and CD4+ T lymphocytes as well as the CD4+/CD8+ ratio of the surgical resection group and IRE group increased after operation (P,0.05), and those in IRE group increased more rapidly. Such changes were even more prominent at 14 and 21 days after operation. However, the indexes were similar in the IRE group and surgical resection group 7 days after operation (P.0.05). At 21 days after IRE treatment, the rats in the IRE group had similar percentages of CD3+ and CD4+ cells and a similar CD4+/CD8+ ratio compared with the non-tumor-bearing rats. This result demonstrated that surgical resection could remove the tumor tissue but not evoke a great immune response, while the increased percentage of CD4+ T helper cells and relatively stable percentage of CD8+ suppressor T lymphocytes following IRE treatment could give rise to the increased CD4+/CD8+ cell ratio, suggesting an enhancement in host immunity after IRE treatment. In most malignant diseases, elevated levels of serum sIL-2R are observed [19,20]. Serum sIL-2R is 
a useful parameter for evaluating disease stage and monitoring the disease progression during posttreatment follow-up [21,22]. In this study, we also found that the serum sIL-2R (soluble interleukin-2 receptor) level in the peripheral blood exhibited the same change as the T lymphocytes. This result indicated that the immune response was strengthened after tumor ablation with irreversible electroporation. IL-10 is a multifunctional cytokine with both immunosuppressive and antiangiogenic functions, and it may have both tumor-promoting and –Benzocaine inhibiting properties [23,24]. It was found to be a more powerfuloutright inhibitor of T-helper 1 T cells (Th1) functions than IL-4 [25?7]. In our study, the IL-10 level decreased with time in the surgical resection group and the IRE group, and it was significantly different from those in the sham operation group and the control group. However, there was no significant difference in the serum IL-10 levels of the IRE group and the surgical resection group. This indicated that IRE treatment, like tumor resection, could release the immunosuppression caused by high IL-10. Furthermore, it is known that T-cells exert their effector functions partly by producing and releasing cytokines. Th1 and Th2 cells are characterized by their distinct cytokine expression patterns. Th1 cells secrete IFN-c and IL-2, whereas Th2 cells produce IL-4, IL-5 and IL-10 [28]. A cytokine profile analysis of the percentage of IFN-c and IL-4-positive splenocytes showed that there was no statistically significant difference 22948146 between the five groups before operation. The percentage of IFN-c-p.Luate the immunologic response to tumor ablation with thermal ablation, such as radiofrequency and cryoablation [16?8]. In the present study, we found that the percentages of CD3+ T lymphocytes and CD4+ T lymphocytes, as well as the CD4+/CD8+ ratio, of tumor-bearing rats was lower than that in the non-tumor-bearing group before operation. Both surgical 
tumor resection and IRE treatment reduced the percentage of CD8+ T lymphocytes in tumor-bearing rats, but there was no statistically significant difference between the twoFigure 3. Changes in T lymphocyte subset percentage (A, B, C) and CD4+/CD8+ ratio (D). *p,0.05; #p.0.05. doi:10.1371/journal.pone.0048749.gImmunologic Response to IREFigure 4. Changes in cytokine IFN-c-positive (A) and IL-4-positive (B) splenocytes. doi:10.1371/journal.pone.0048749.ggroups. The percentages of CD3+ T lymphocytes and CD4+ T lymphocytes as well as the CD4+/CD8+ ratio of the surgical resection group and IRE group increased after operation (P,0.05), and those in IRE group increased more rapidly. Such changes were even more prominent at 14 and 21 days after operation. However, the indexes were similar in the IRE group and surgical resection group 7 days after operation (P.0.05). At 21 days after IRE treatment, the rats in the IRE group had similar percentages of CD3+ and CD4+ cells and a similar CD4+/CD8+ ratio compared with the non-tumor-bearing rats. This result demonstrated that surgical resection could remove the tumor tissue but not evoke a great immune response, while the increased percentage of CD4+ T helper cells and relatively stable percentage of CD8+ suppressor T lymphocytes following IRE treatment could give rise to the increased CD4+/CD8+ cell ratio, suggesting an enhancement in host immunity after IRE treatment. In most malignant diseases, elevated levels of serum sIL-2R are observed [19,20]. Serum sIL-2R is a useful parameter for evaluating disease stage and monitoring the disease progression during posttreatment follow-up [21,22]. In this study, we also found that the serum sIL-2R (soluble interleukin-2 receptor) level in the peripheral blood exhibited the same change as the T lymphocytes. This result indicated that the immune response was strengthened after tumor ablation with irreversible electroporation. IL-10 is a multifunctional cytokine with both immunosuppressive and antiangiogenic functions, and it may have both tumor-promoting and -inhibiting properties [23,24]. It was found to be a more powerfuloutright inhibitor of T-helper 1 T cells (Th1) functions than IL-4 [25?7]. In our study, the IL-10 level decreased with time in the surgical resection group and the IRE group, and it was significantly different from those in the sham operation group and the control group. However, there was no significant difference in the serum IL-10 levels of the IRE group and the surgical resection group. This indicated that IRE treatment, like tumor resection, could release the immunosuppression caused by high IL-10. Furthermore, it is known that T-cells exert their effector functions partly by producing and releasing cytokines. Th1 and Th2 cells are characterized by their distinct cytokine expression patterns. Th1 cells secrete IFN-c and IL-2, whereas Th2 cells produce IL-4, IL-5 and IL-10 [28]. A cytokine profile analysis of the percentage of IFN-c and IL-4-positive splenocytes showed that there was no statistically significant difference 22948146 between the five groups before operation. The percentage of IFN-c-p.