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. 2019 Nov 5;10:2472. doi: 10.3389/fimmu.2019.02472

Figure 2.

Figure 2

DTT-neoAg induces neoantigen-specific antibodies and antitumor cellular immune responses in mouse. The statistical significances were determined by Student's T test. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. (A) A schematic diagram of the experimental design. (B–D) Female C57BL/6 mice (n = 5) were immunized subcutaneously on day 0, 12, 24 with DTT-neoAg vaccine. One week after the third immunization, mouse sera were collected by orbital blood sampling, and the antibody responses were analyzed by ELISA. (B) ELISA with sera in 1:100 dilution and with indicated coating antigens. (C) The antibody titers against the neoantigen. (D) The percentage of CD19+ B cells in the lymphocytes of immunized mice. Seven days after the third immunization, the splenocytes of the vaccinated mice were stained with anti-CD19-FITC, and the percentage of CD19+ B cells in the lymphocytes was analyzed by flow cytometry. (E) The antibody subclass analyses. The sera were 1:100 diluted. (F–H) DTT-neoAg vaccine induced cellular immune response. Six to eight weeks old female C57BL/6 mice (n = 3) were immunized with DTT-neoAg or PBS formulated with Alum + CpG, respectively, at 2-week intervals. Seven days after the third immunization, the splenocytes were stimulated with CTB-neoAg or PBS for 72 h. Cell proliferation (F) and stimulation indices (G) were measured by CCK8 kit. (H) The splenocytes from DTT-neoAg-treated mice or PBS-treated mice were stimulated with CTB-neoAg (30 μg/mL) for 72 h, and were used as effector cells, then co-cultured with target cells B16F10 at indicated ratios for 4 h at 37°C. The percentage of cell lysis was determined by LDH assay.