CD8+ cell accumulation into the tumor. (a) Multiplex immunohistochemical staining of the tumors 7 days after the therapy. Top: cytokeratin staining to mark tumor tissue. Middle: lymphocyte marker staining; CD8 (magenta), CD4 (cyan) and Foxp3 (orange). The inset in the picture of CD25-PIT shows examples of CD8+ cell (gray filled arrowhead), CD4+Foxp3− cell (white filled arrowhead) and CD4+Foxp3+ cell (open arrowhead). bottom: merged image. Scale bar, 100 μm. (b) intratumoral CD8+ cell density was significantly higher in the CD25-PIT group and the combined PIT group than the control group (n = 5; one-way ANOVA followed with Tukey's test; *, p < 0.05, **, p < 0.01). (c) intratumoral CD8+/CD4+Foxp3+ (Treg) ratio was also increased in these two groups compared with the control group (n = 5; one-way ANOVA followed with Tukey's test; *, p < 0.01; **, p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)