Fig. 8: Therapeutic effects of neoadjuvant combination therapy are elicited by cellular and humoral immunity.
a, Treatment schedule of neoadjuvant agonist therapies for s.c. KPC tumors and blood plasma collection or serum transfer to naive recipients. b, Blood plasma was collected 2 weeks after tumor resection, heparinized and diluted at a 1:50 ratio with PBS, and added to cultured KPC cells. The binding of plasma IgG to KPC cells was visualized by fluorescently labeled sheep anti-mouse IgG (red). Scale bar, 1 mm. c, Similarly, blood plasma collected from different mouse groups was incubated with KPC cells in single-cell suspension, and the binding of plasma IgG to these cells was detected by the right shift of the cell population in FACS analysis (indicated by red gating). IgM binding was undetectable compared with the sham and intact mouse (non-immune plasma) controls in this assay. d, Percentage fraction of IgG-bound KPC cells in total KPC cells. Plasma samples from 10–14 different mice from each group were examined for KPC cell binding. A representative result of three experiments. **P < 0.01. e, Bone marrow was collected from the left and right femurs and tibias of each agonist-treated or untreated mouse 2 weeks after tumor resection, and bone marrow leukocytes were analyzed by flow cytometry using Blimp1 and CD44 staining as markers for long-lived plasma cells. One experiment. N = 5 per group. *P < 0.05, **P < 0.01, ****P < 0.0001. f, Blood serum was collected from different donor mouse groups (sham surgery, agonist untreated, combination therapy treated) and transferred to naive recipient mice several times after tumor inoculation. The tumor growth in these animals was monitored for 20 days. One experiment. N = 20 tumors were analyzed for each serum transferred group. N = 10 for controls receiving no serum transfer. ***P < 0.001. g, Neoadjuvant treatment and tumor resection followed by tumor reinoculation were carried out in wild-type or B cell-deficient CD79a knockout mice as described in Fig. 3e. Tumor size was compared between different mouse groups on day 9 after tumor reinoculation. The results of two independent experiments were combined. N = 20 tumors were analyzed for each group. ****P < 0.0001. h, Neoadjuvant treatment and tumor resection followed by tumor reinoculation were carried out in wild-type mice as described in Fig. 3e. Starting from one day before the reinoculation of KPC tumors, anti-NK1.1, anti-CD8 or anti-Ly6G was administered (i.p.) to deplete NK cells, CD8+ T cells or neutrophils, respectively. Tumor size was compared between different mouse groups on day 6 after tumor reinoculation. One experiment. N = 20 tumors were analyzed for each group. *P < 0.05, ****P < 0.0001. Data are presented as the mean ± s.e.m. and analyzed using one-way (d, e, g and h) or two-way (f) ANOVA with Tukey’s test for statistical significance. All replicates represent biological replicates.
