Binding affinity and in vivo immunogenicity of DKK1 peptides. Peptide binding assay showing (A) affinity and (B) stability (fluorescence index) of P20 and P66 and their heteroclitic peptides for HLA-A*0201 molecules. HIV-pol and Flu-matrix peptides were used as positive controls. (A) T cells were incubated with 100 μg/mL peptides overnight. (B) T2 cells were incubated with 100 μg/mL peptides for different time points, and analyzed for surface HLA-A*0201 expression. Details are provided in “Materials and methods.” (C) Intracellular staining for CD8⁺ IFN-γ–expressing T cells in the splenocytes of mice immunized with Flu-matrix peptide (Flu), P20, or P66v peptides. Splenocytes were restimulated with or without the immunizing peptides for 5 days before analysis. (D) HLA-A*0201-peptide-tetramer staining showing DKK1 peptide-specific CD8⁺ T cells in the splenocytes. (E) Cytotoxicity of the splenocytes against mouse DCs pulsed with the immunizing peptides (Flu+, P20+, or P66v+) or unpulsed DCs (Flu⁻, P20⁻, or P66v⁻). Representative results of 3 independent experiments are shown. The error bars in panels A, B, and E indicate SD of the 3 experiments. The numbers in the quadrants in panels C and D show the percentages of HLA-A*0201-DKK1 peptide (P20 or P66v)–tetramer positive staining CD8⁺ cells.