Figure 5.
Figure 5. Enhanced tumor-specific immunogenicity and protective effect(s) of TNP-470–treated DC vaccine in prophylactic B16 melanoma vaccination model. (A) Schematic diagram of prophylactic setting of vaccination model. Mice were immunized with PBS (solvent control), DC without tumor lysate pulsing (DC control), tumor lysate-pulsed TNP-470/vehicle-treated DC vaccine. (B) Lymphocytes from immunized mice in different groups were collected and stimulated with tumor lysate (50 µg/mL) for 24, 48 and 72 hours ex vivo. Culture supernatant was detected for secretion of IL-2 (left) and IFN-γ (right). **P < .01 and *** P < .001 TNP-DC vaccine vs vehicle DC vaccine. (C) To quantify amount of tumor-specific CTLs from mice in different groups, lymphocytes were stimulated with tumor lysate (50 µg/mL) for 72 hours ex vivo, followed by re-stimulation of PMA/Ionomycin for 6 hours. Cells were then stained with CD3, CD8 and IFN-γ. Tumor-specific CTLs were determined by flow cytometric analysis of CD3+CD8+IFN-γ+ cells. *P < .05 vehicle DC vaccine vs PBS solvent control. **P < .01 TNP-DC vaccine vs DC control. ***P < .001 TNP-DC vaccine vs PBS solvent control. ##P < .01 TNP-DC vaccine vs vehicle DC vaccine. Data presented as mean ± SD (n = 6) from 2 independent studies. (D) Tumor-specific cytolytic activity. Lymphocytes from mice in different groups were collected and stimulated with tumor lysate (50 µg/mL) for 72 hours ex vivo, then harvested as effector cells (E), and B16-F10 cells were used as target cells (T). Specific lysis was detected by LDH assay at E:T ratio = 100:1. **P < .01 TNP-DC vaccine vs vehicle DC vaccine; ***P < .001 TNP-DC vaccine vs PBS solvent control and DC control. Data presented as mean ± SD (n = 6) from 2 independent studies. (E) Percentages of tumor-free mice in different vaccination groups (n = 6). *P < .05 TNP-DC vaccine vs PBS solvent control. #P < .05 TNP-DC vaccine vs DC control. (n = 6, Modified Kaplan-Meier Survival Analysis). (F) Weight of solid tumor developed on mice on day 26 (end point). *P < .05 TNP-DC vaccine vs PBS solvent control. **P < .01 TNP-DC vaccine vs DC control. Data presented as mean ± SD (n = 6) from 2 independent studies.

Enhanced tumor-specific immunogenicity and protective effect(s) of TNP-470–treated DC vaccine in prophylactic B16 melanoma vaccination model. (A) Schematic diagram of prophylactic setting of vaccination model. Mice were immunized with PBS (solvent control), DC without tumor lysate pulsing (DC control), tumor lysate-pulsed TNP-470/vehicle-treated DC vaccine. (B) Lymphocytes from immunized mice in different groups were collected and stimulated with tumor lysate (50 µg/mL) for 24, 48 and 72 hours ex vivo. Culture supernatant was detected for secretion of IL-2 (left) and IFN-γ (right). **P < .01 and *** P < .001 TNP-DC vaccine vs vehicle DC vaccine. (C) To quantify amount of tumor-specific CTLs from mice in different groups, lymphocytes were stimulated with tumor lysate (50 µg/mL) for 72 hours ex vivo, followed by re-stimulation of PMA/Ionomycin for 6 hours. Cells were then stained with CD3, CD8 and IFN-γ. Tumor-specific CTLs were determined by flow cytometric analysis of CD3+CD8+IFN-γ+ cells. *P < .05 vehicle DC vaccine vs PBS solvent control. **P < .01 TNP-DC vaccine vs DC control. ***P < .001 TNP-DC vaccine vs PBS solvent control. ##P < .01 TNP-DC vaccine vs vehicle DC vaccine. Data presented as mean ± SD (n = 6) from 2 independent studies. (D) Tumor-specific cytolytic activity. Lymphocytes from mice in different groups were collected and stimulated with tumor lysate (50 µg/mL) for 72 hours ex vivo, then harvested as effector cells (E), and B16-F10 cells were used as target cells (T). Specific lysis was detected by LDH assay at E:T ratio = 100:1. **P < .01 TNP-DC vaccine vs vehicle DC vaccine; ***P < .001 TNP-DC vaccine vs PBS solvent control and DC control. Data presented as mean ± SD (n = 6) from 2 independent studies. (E) Percentages of tumor-free mice in different vaccination groups (n = 6). *P < .05 TNP-DC vaccine vs PBS solvent control. #P < .05 TNP-DC vaccine vs DC control. (n = 6, Modified Kaplan-Meier Survival Analysis). (F) Weight of solid tumor developed on mice on day 26 (end point). *P < .05 TNP-DC vaccine vs PBS solvent control. **P < .01 TNP-DC vaccine vs DC control. Data presented as mean ± SD (n = 6) from 2 independent studies.

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