NF-κB activation during intradermal DNA vaccination is essential for eliciting tumor protective antigen-specific CTL responses

Figures & data

Figure 1. Type 1 interferons are not required for generating CTL responses. Wild-type (WT) and IFNαR^−/− mice on 129sv background were vaccinated twice with the OVA-encoding plasmid pOVA. The percentages of IFN-γ producing CD8⁺ T lymphocytes were detected after in vitro stimulation with OVA and control peptides (A). In vivo cytotoxicity was measured by quantifying OVA pulsed CFSE^High labeled target cells and compared with control pulsed CFSE^Low labeled cells in WT and IFNαR^−/− mice vaccinated with pOVA. The mean percentage ± SEM of specific killing for each case is indicated (B). Tyrosinase-related protein 2 (TRP2) encoding DNA was used to vaccinate WT and IFNαR^−/− 129sv mice. IFN-γ producing CD8 T lymphocytes were measured after in vitro stimulation with TRP2 and control peptides (C). WT and IRF3^−/− mice on C57BL/6 background were vaccinated with TRP2-encoding plasmid and the percentages of IFN-γ-producing CD8 T cells were measured by flow cytometry after in vitro stimulation with TRP2 and control peptides (D). Bars indicate the mean ± SEM.

Figure 2. Toll-like receptor 9 (TLR9) and myeloid differentiation primary response gene 88 (MyD88) are not essential for CTL induction. Wild-type (WT), TLR9^−/− and MyD88^−/− mice on C57BL/6 background were vaccinated twice. Levels of IFN-γ producing CD8 T cells induced by DNA vaccination against OVA and TRP2 were measured two weeks after the second DNA vaccination by flow cytometry. Bars indicate the mean ± SEM (A and C). In vivo cytotoxicity was measured by quantifying OVA pulsed CFSE^High labeled target cells and compared with control pulsed CFSE^Low labeled cells in WT, TLR9^−/− and MyD88^−/− mice vaccinated with the OVA encoding plasmid pOVA. The mean percentage ± SEM of specific killing for each case is indicated (B). WT and IL-1β^−/− IL-18^−/− mice on C57BL/6 background were vaccinated with OVA or TRP2 encoded plasmid as described previously. The percentages of IFN-γ-producing CD8 T cells were measured by flow cytometry after in vitro stimulation with TRP2 and control peptides (D). Functionality of OVA-specific CTLs was measured by in vivo cytotoxicity. Lymph nodes were harvested one day after mice were injected i.v. with OVA peptide pulsed CFSE^High labeled splenocytes and control peptide pulsed CFSE^Low splenocytes. Bars indicate the mean percentage ± SEM of OVA-specific killing (E).

Figure 3. NF-κB activation during intradermal DNA vaccination mediates the generation of CTLs against the tumor-associated antigen TRP2. C57BL/6 mice were electroporated with NFκB luciferase reporter plasmid and IκBα-SR encoding plasmid. One day after DNA electroporation, in vivo luminescence was measured after intraperitoneal (i.p.) injection of luciferin. Representative images are shown (A). C57BL/6 mice were vaccinated twice against TRP2. One group was co-electroporated with IκBα-SR encoding plasmid and the control group with the empty vector. Percentage of TRP2-specific IFN-γ-producing CD8 T cells were measured two weeks after the second DNA vaccination by flow cytometry after in vitro stimulation with TRP2 and control peptides. Bars indicate the mean ± SEM (B). C57BL/6 mice vaccinated with pVAX, pTRP2 or co-electroporated with pTRP2 and pIκBα-SR were challenged by i.v. injection of 10⁵ B16F10 melanoma cells. Two weeks after injection lungs were excised and melanoma foci were enumerated. The mean percentage ± SEM of melanoma foci is shown for each group (C).