Intracellular cytokine staining with flow cytometry analysis reveals immunodominant CD8+ T cell epitope in MusPV1 E6 and its MHC class I restriction.

<p>Schematic of immunization schedule of C57BL/6 mice with DNA vaccines expressing CRT (calrecticulin) fused to different MusPV1 proteins and subsequent harvest of splenocytes (A). Bar graph of flow cytometry data after intracellular cytokine staining of splenocytes for interferon-γ and CD8 after harvest from CRT/mE6-vaccinated mice and stimulation with mE6 peptide library pools (B). Bar graph of flow cytometric analysis of intracellular cytokine staining of splenocytes for interferon-γ and CD8 after harvest from CRT/mE6E7L2-vaccinated mice and stimulation with both mE6 and mE7 peptide library pools (C). Bar graph of flow cytometry data after intracellular cytokine staining of splenocytes for interferon-γ and CD8 after harvest from CRT/mE6 and stimulated with candidate 9mer peptides to map the MHC class I epitopes of MusPV1 E6 (D). Bar graph of flow cytometry analysis showing percentages of interferon-γ expressing mE7-specific CD8+ T cells after co-incubation with 293-K^b or 293-K^d cells that were transfected with either CRT/mE6 or CRT-alone plasmid (E). All data was repeated and representative images provided.