Figure 4.

Mutated p53 R248W and Kras G12D/V peptides are more immunogenic than corresponding wt peptides when used for vaccination of A2.DR1 dtg mice and comprise HLA class I and II restricted epitopes. CD8⁺ (A) and CD4⁺ (B) T cell recall responses against mutated and wt peptides tested in A2.DR1 dtg mice immunized with group A and C long peptide cocktails (vaccination regimen: peptides in PBS-based formulations including 50 μg CpG ODN 1668 as an adjuvant, twice on a bi-weekly basis) or untreated mice are shown. In vitro recall responses were obtained from combined IFN-γ secretion assays and intracellular cytokine stainings performed with splenic CD90⁺ purified T cells from immunized mice. IFN-γ secretion of CD8⁺ T cells upon in vitro recall against respective single peptides presented by CD11c⁺ DCs are displayed. Each peptide and control sample was tested in triplicates. Results are plotted as means ± SEM. Differences were tested for by unpaired, two-tailed t-test. (C) Putative class I and II epitopes (highlighted in gray) within the long p53 R248W and Kras G12V peptide sequences. (D) – (G) T cell recall responses against mutated and wt Kras G12(V) and p53 R248(W) peptides tested in A2.DR1 dtg mice immunized with the long peptides Kras G12V or p53 R248W. In vitro recall responses were obtained from two-color cytokine secretion assays performed with splenic pan-T cells from immunized mice. Percentages of IFN-γ/IL-2 (D, E) double-positive CD8⁺ (C, D) and IFN-γ (F), IL-2 (G) positive CD4⁺ T cells upon in vitro recall against short and long Kras or p53 peptides presented by CD11c⁺ DCs are displayed. Each peptide and control sample was tested in triplicates. Results are plotted as means of triplicate assays ± SEM. Differences were tested for by unpaired, two-tailed t-test. ns: not significant.