Fig. 5.

Immunogenicity and antitumour effect of ppCT vaccine. a In vivo immunogenicity in HHD-DR3 mice. Mice were immunized four times at 1-week intervals with the ppCT vaccine or vehicle plus adjuvant control. One week after the final immunization, splenocytes were recovered and cultured in medium or restimulated with each peptide or the peptide cocktail; then IFN-γ secretion was measured. Three-to-five mice per group were included. Results are means (±SEM) of three independent experiments (n = 12). b In vivo cytotoxic activity. Splenocytes were loaded with ppCT peptides and injected i.v. 2 days after the last immunization. Surviving target cell frequencies were detected in blood 6, 24 and 48 h later. Five mice per group were included. Results are means (±SEM) of two independent experiments (n = 10). c Tumour progression. 10⁶ D122-HHD-ppCT cells were injected s.c. At days 1, 7 and 21 (arrows), mice were vaccinated with ppCT vaccine or vehicle plus adjuvant and tumour volume was measured every third day. d Tumour weight. Tumours were recovered at day 27 after engraftment and weighed. e Absolute TIL counts. Numbers of TILs per milligram of tumour. Three-to-five mice per group were included. Results in c–e are given as means (±SEM) of four independent experiments (n = 16). f IGR-Heu tumour growth in NSG mice. NSG mice were engrafted with IGR-Heu tumour pieces and, at day 10, adoptively transferred with healthy donor PBMCs. At days 11 and 17, mice were vaccinated i.v. with the ppCT vaccine or with vehicle plus adjuvant, and tumour growth was recorded every 2 days. g Tumour weight. Tumours were recovered at day 34 after engraftment and weighed. Bars are mean ± SEM. Results are from one experiment out of six. h Absolute number of human IFN-γ-producing CD8⁺ T cells. Tumours were recovered, dissociated and human CD45⁺ leukocytes were isolated and restimulated with the ppCT peptide cocktail. Total number of IFN-γ-producing CD3⁺CD8⁺ cells was determined. Five mice per group were included. Results in f–h are means (±SEM) of one experiment out three (n = 16). *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed Student’s unpaired t test)