PMC

In vivo tumor protection experiment examining the antitumor effects generated by pcDNA3-CRT/LT against LT-expressing tumor. (A) Schematic diagram of experiment schedule. (B) Survival curve of vaccinated mice subjected to tumor challenge. Note that vaccination with pcDNA3-CRT/LT greatly prolonged mouse survival after tumor challenge.

In vivo tumor treatment experiment with DNA vaccines. (A) Schematic diagram of experiment schedule. (B) Survival curve of tumor-bearing mice treated with various DNA vaccines. Note that pcDNA3-CRT/LT had the greatest therapeutic effect and extended the survival of B16/LT tumor-bearing mice.

pcDNA3-CRT/LT generates the most LT-specific (aa 19–27) CD8⁺ T cells. C57BL/6 mice (5 mice/group) were immunized with DNA vaccines by gene gun in the same schedule as Figure A. Pooled splenocytes from mice vaccinated with pcDNA3 vector (control), pcDNA3-LT, and pcDNA3-CRT/LT were collected and cultured in vitro with either no peptide or amino acid 19–27 then stained for intracellular IFN-gamma and CD8⁺ T cell surface marker. (A) Representative flow cytometry dot plot of LT-specific CD8⁺ T cell activation after stimulation with amino acid sequence 19–27. (B) Representative bar graph of flow cytometric data.

MHC class I H-2^b binding restriction of LT aa 19–27 epitope identified by intracellular cytokine staining and flow cytometry. MHC class I binding restriction was determined by C1R transfectants, C1R/D^b and C1R/K^b , that were pulsed with immunodominant LT peptide (aa 19–27). (A) Representative flow cytometric data showing the amounts of LT-specific IFN-γ⁺ CD8⁺ T-cells after splenocytes from pcDNA3-CRT/LT vaccinated mice are stimulated by LT peptide-pulsed C1R, C1R/D^b, or C1R/K^b cells in vitro. (B) Representative bar graph of flow cytometric data showing the proportions of LT-specific IFN-γ + CD8+ T cells per 3 × 10⁵ splenocytes following in vitro stimulation as described above. Note that LT epitope (aa 19–27) is H-2k^b-restricted.

The effect of CD8 T cells on tumor protection of the pCDNA3-CRT/LT vaccine. (A) Schematic diagram of the vaccination regimen for in vivo antibody depletion experiments. C57BL/6 mice (5 per group) were vaccinated by gene gun with pcDNA3-CRT/LT DNA vaccine on D0. Vaccinated mice were boosted two times at the same dose and regimen at one week intervals. Beginning 1 day after last vaccination, vaccinated mice were intraperitoneally injected with anti-CD8 monoclonal antibody other day. Antibody-depleted mice were then challenged with B16/LT tumor (1 × 10⁵ cells/mouse) subcutaneously in the right flank on D22. Mice were monitored for evidence of tumor growth by inspection, palpation and tumor size was measured twice a week. (B) Survival analysis of B16/LT tumor-bearing mice treated with pcDNA3-CRT/LT DNA vaccine.

Identification of MHC class I-restricted immunodominant LT epitope using overlapping peptides and splenocytes from mice vaccinated with pcDNA3-CRT/LT. (A) Schematic diagram of vaccination schedule in vivo. C57BL/6 mice (5 mice/group) intradermally received pcDNA, pcDNA3-LT, or pcDNA3-CRT/LT 3 times by gene gun at a 7-day interval. Vaccination with pcDNA3-CRT/LT produced the most LT-specific CD8+ T cells therefore pooled splenocytes from pcDNA3-CRT/LT vaccinated mice were cultured in vitro with various overlapping LT peptides to find the immunodominant LT epitope. (B) Representative bar graph of flow cytometric data indicating the pool containing peptides #1-10 activated the most LT-specific CD8+ T cells. (C) Representative bar graph of flow cytometric data indicating that out of peptides #1 through 10, LT peptide #4 was able to activate the most LT-specific CD8+ T cells. (D) Representative bar graph of flow cytometric data suggesting amino acid 19–27 (IAPNCYGNI) of the LT antigen may be the immunodominant epitope determining the specificity of the vast majority of LT-specific CD8+ T cells. (E) Representative bar graph of flow cytometric data confirming that the suggested amino acid 19–27 (IAPNCYGNI) of the LT antigen may be the immunodominant epitope determining the specificity of the vast majority of LT-specific CD8+ T cells, and not the peptide occupying positions 20–27 (APNCYGNI). Ova (SIINFEKL) was used a negative control.