Fig. 7.
Ad-TD-nsIL-12 efficacy is dependent on hamster CD3+/CD4− immune cell subsets. a, b Syrian hamsters were inoculated subcutaneously with 2 × 106 HPD1NR cells. The established tumors were injected i.t. with 1 × 109 PFU Ad-TD-nsIL-12 or PBS (n = 7/group) on day 0, 2, 4, 6, 8, and 10. Control IgG and either mouse anti-hamster CD3 mAb (4F11) a or CD4 mAb b were injected i.p. at doses of 500 μg/injection every fourth day from the day before the viral therapy to the end of the experiment and FACS analysis used to confirm the depletion. Mean tumor volumes and SEM are shown for each group. Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. *p < 0.05, ***p < 0.001. c–g 2 × 106 HPD1NR cells were seeded into the right flank of Syrian hamsters. When tumor volumes reached 300 mm3, nine hamsters per group were each injected i.t with PBS, 1 × 109 PFU Ad-TD-LUC or Ad-TD- IL-12/nsIL-12 on day 0. On days 3, 7, and 21 tumors were collected and processed for IHC. c Representative images of immunohistochemical staining for CD3 and CD4 at day 7 (×200). d−f Quantitative scores of lymphocyte infiltration within tumors. Lymphocytes were counted in 5 HPFs randomly selected from each tumor section (×200). The scoring was conducted within the tumor and stroma; necrotic areas were avoided. The extent of lymphocyte infiltration was categorized into the following four grades: 1, <25 cells/HPF; 2, 25–49 cells/HPF; 3, 50–75 cells/HPF; 4, >75 cells/HPF. Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. *p < 0.05, **p < 0.01, ***p < 0.001. g Spleens and lymph nodes were collected and analyzed by FACS for CD3 and CD4 expression at the time points shown. Mean expression and SEM is plotted (n = 3/group). Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. *p < 0.05, **p < 0.01, ***p < 0.001