TILs preferentially respond to v-peptides. A, Percentages of peptides among all predicted class I-peptides, nv-class I-peptides, and v-class I-peptides that stimulated the TILs. B, Proliferations of TILs after stimulation with 5
v-class I-peptides used in the first to fourth vaccinations for 5 days. Five replicate wells were tested for proliferation. C, Percentages of peptides among all predicted class II-peptides, nv-class II-peptides, and v-class II-peptides that stimulated the TILs. D, Proliferations of TILs were stimulated with 5
v-class II-peptides used in the first to fourth vaccinations for 5 days. Five replicate wells were tested for proliferation. E, Comparison of the proliferation levels of TILs after stimulation with positive nv- and v-D-class I/II-neopeptides. TILs were stimulated with v-class II-peptides in the presence of a blocking anti-HLA-DR/DP/DQ (TÜ39) antibody or isotype control. Proliferations of TILs (F) and concentrations of IFNγ in the supernatant (G) were detected 5 days after stimulation. Five replicate wells were set up for each condition, and cells were collected and pooled from replicate wells for proliferation test. H, Expression levels of activation markers CD69 and CD25 as well as cytotoxicity marker granzyme B on/in CD4+ TILs after stimulation with v-class II-peptides for 5 days. Five replicate wells were set up for each condition, and cells were collected and pooled from replicate wells for testing. (#) indicates the peptide that was used in the first to fourth vaccinations. Data are expressed as mean ± SEM, and P values were determined using an unpaired t test.