Fig. 4: Tumors treated by the combinations of E7 vaccines before or after radiation showed bigger difference in the ratios of CD8⁺/Treg cells.

Local established tumor treatments were followed by the experimental procedures described as in Fig. 3a. On days 7 and 14 after radiation, tumors were collected for flow cytometry, immunofluorescent staining, and RT-PCR analyses. a Percentage of Tregs in CD4⁺ cells analyzed using flow cytometry. b The ratio of CD8⁺/Tregs in tumors analyzed using flow cytometry. c The ratio of CD8⁺/CD45⁻ cells in tumors analyzed using flow cytometry. d From each group 3 tumors were used for the immunofluorescent staining of CD8. Slides were incubated with anti-CD8 primary antibodies overnight in 4 °C and then with corresponding secondary antibodies (RED), then stained the nucleus with DAPI (BLUE). 10 to 12 fields of view in each treatment group were imaged and analyzed (Upper). The pictures were merged by image software (Lower). Percentage of CD8⁺ positive staining cells was analyzed using Image-Pro Plus software. e, f Whole tumor RNAs were isolated from the tumors of treated mice. Real-time PCR measurement of the expression of CXCL9, CCL5, CXCL10, CXCR3 in tumors after radiation for 7 days (e) and 14 days (f). Data were presented as mean ± SD; n = 3 to 4 mice/group. Experiments were repeated three times and two-tailed t-test was used for comparisons of biological replicates. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.