Figure 1

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.