Summary of pre-selection experiments.

<p>Summary of pre-selection experiments.</p

Predictive value of modifications.

<p>IFN-γ ELISpot on spleen cells of mice vaccinated with 75 nmol of either WT peptide or CPLs and stimulated for 16 hours with 0.1 nmol WT peptide or CPL per well. The three different modifications are based on final selected peptides for each epitope: <b>(A)</b> am-phg on P₁ based on G1, <b>(B)</b> 4-FPHE on P₁ and 2-AOC on P₉ based on F5 and <b>(C)</b> NLE on P₂ based on N53. X-axis depicts peptide used for vaccination. White boxes represents restimulation with WT peptide and grey boxes restimulation with CPL. Bars are min to max, with line at mean. Although it appears difficult to predict whether a modification will work in a certain epitope, an effective modification in one epitope is in some cases also effective in other epitopes. Bars represent a minimum of three mice (GILGFVFTL and FMYSDFHFI) and a maximum of eight (NMLSTVLGV). Data were statistically analyzed using a Mann-Whitney test. * p<0.05; ** p<0.01 compared to the WT equivalent.</p

FP binding scores HLA-A*0301 peptides.

<p>FP binding scores HLA-A*0301 peptides.</p

FP binding scores of selected HLA-A*0201 peptides.

<p>FP binding scores of selected HLA-A*0201 peptides.</p

Vaccination with CPLs shows enhanced IFN-γ responses in vivo compared to vaccination with WT peptide.

<p>Mice were vaccinated with different doses of WT peptide or CPLs on day 0 and day 21 and two weeks later spleen cells were isolated and restimulated with homologous peptides or WT peptide. Responses were measured by IFN-γ ELISpot after 16 hours stimulation with 0.1 nmol peptide/well. Mice were vaccinated with mock (not shown), 10, 25, 50 or 100 nmol of WT GILGFVFTL or with the indicated CPLs. Spleen cells were restimulated with homologous <b>(A)</b> or WT <b>(B)</b> peptide. Overall, responses were highest after stimulation with CPL G1. For FMYSDFHFI mice were vaccinated with mock (not shown), 25, 50 or 75 nmol of WT peptide or the indicated CPLs. Cells were restimulated for 16 hours with homologous <b>(C)</b> or WT <b>(D)</b> peptide. Three out of four CPLs (F5, F100 and F111) induced higher responses compared to WT-peptide vaccination. For NMLSTVLGV mice were mock vaccinated or vaccinated with a dose of 75 nmol of WT peptide or respective CPLs. Spleen cells were restimulated with homologous <b>(E)</b> or WT <b>(F)</b> peptide. CPL N172 induced most T cells that responded to homologous stimulation, whereas N53 induced most T cells responding to WT peptide. Mock-vaccinated mice in experiments shown in Fig 2A-2D showed comparable responses to mock-vaccinated mice in Fig 2E-2F. Fig 2A-2D depict three mice per dose. Data in Fig 2E and 2F are derived from 7–8 mice per group, with the exception of the mock, for which three mice were included. Bars are min to max, with line at mean. Data were statistically analyzed using a Mann-Whitney test. * p<0.05; *** p<0.001 compared to the WT equivalent.</p

Binding affinity dose-response curves of CPLs and WT peptides.

<p>The IC₅₀ curves of the selected CPLs show increased HLA binding affinity compared to IC₅₀ curves of the corresponding WT-peptides. To generate IC₅₀ curves the FP-based competition assay was performed using threefold peptide dilutions in the presence of a standard amount of tracer peptide. Shown are averages and their standard deviation of three independent experiments. Curves of CPLs are shifted to the left compared to WT peptides, indicating that a lower dose of CPLs is needed to inhibit tracer binding.</p

Flow cytometry analysis on CD8⁺ T cell responses of CPL- and WT-vaccinated mice.

<p>Dot plots showing IFN-γ production by CD8⁺ T cells of mice vaccinated with 75 nmol of either WT peptide or CPL (G1, F5, N53 and N172). In the upper panel, the respective WT-peptide control of that particular experiment is shown. In the lower panel, the CPL-induced IFN-γ responses are shown. Spleen cells (2*10⁶/well) were stimulated O/N with 1 nmol/well WT peptide. Highest responders of each group are shown. Vaccination with F5 and N53 induced the largest improvement in IFN-γ production compared to WT peptide-vaccinated mice. Negative control (mock stimulated splenocytes) had an average of 0.07% with a SD of 0.1%.</p