δ-Catenin peptide vaccines repress hepatocellular carcinoma growth via CD8⁺ T cell activation

Figures & data

Figure 1. δ-Catenin might be a potential target for designing cancer peptide vaccines. (A) H22 cells were treated by 2% O₂ or 8 Gy ionization irradiation for 6 h. The mRNA levels of Ctnnd2 were examined by qRT-PCR. (B) The amino acid sequences of δ-Catenin were aligned with sequences of δ-Catenin peptide vaccines by Invitrogen AlignX.

Figure 2. δ-Catenin peptide vaccines could inhibit the growth of subcutaneous tumors formed by liver cancer cells. (A-B) Experimental design of the therapeutic model for examining the functions of δ-Catenin peptide vaccines in vivo. (C) 5×10⁵ H22 cells were injected subcutaneously into ICR mice. δ-Catenin peptide vaccine 1, peptide vaccine 2 or combined vaccines were immunized as shown in (A). Tumor sizes were measured and tumor volumes were calculated at the indicated time to draw the curves (*P < 0.05, **P < 0.01). (D) 5×10⁵ Hepa1-6 cells were injected subcutaneously into C57 BL/6 mice. δ-Catenin peptide vaccine 1 and peptide vaccine 2 were combined to be immunized as shown in (B). Tumor sizes were measured and tumor volumes were calculated at the indicated time to draw the curves (*P < 0.05, **P < 0.01). (E) ICR mice were sacrificed at Day 24 as shown in (A). Tumor were harvested and tumor weights were measured (*P < 0.05, **P < 0.01). (F) C57 BL/6 mice were sacrificed at Day 24 as shown in (B). Tumor were harvested and tumor weights were measured (*P < 0.05, **P < 0.01). (G) ICR mice were sacrificed at Day 24 as shown in (A). Tumors harvested were shown. (H) C57 BL/6 mice were sacrificed at Day 24 as shown in (A). Tumors harvested were shown.

Figure 3. δ-Catenin peptide vaccines might stimulate the activation of CTLs in PB and the infiltration of CTLs in tumors to have the anti-tumor effect. (A-B) ICR mice were sacrificed at Day 24 as shown in Fig. 2A. Peripheral blood was harvested. The percentages of CTLs were examined by flow cytometry. (C-D) C57 BL/6 mice were sacrificed at Day 24 as shown in Fig. 2B. Peripheral blood was harvested. The percentages of CTLs were examined by flow cytometry. (E) ICR mice were sacrificed at Day 24 as shown in Fig. 2A. Tumors were homogenized in PBS to be the lysates with a concentration of 10 mg/mL proteins. The concentration of IFN-γ in tumor lysates was measured by Elisa. (F) C57 BL/6 mice were sacrificed at Day 24 as shown in Fig. 2B. Tumors were homogenized in PBS to be the lysates with a concentration of 10 mg/mL proteins. The concentration of IFN-γ in tumor lysates was measured by Elisa. (G-H) ICR mice were sacrificed at Day 24 as shown in Fig. 2A. Tumors were harvested and the infiltration of CD8⁺ T cells was measured by immunofluorescence.

Figure 4. δ-Catenin peptide vaccines could stimulate the activation of CTLs in vitro. (A-B) Lymphocytes from ICR mice were cultured in vitro with different treatment for 48 h. The percentages of CTLs were examined by flow cytometry. (C-D) CD3⁺ T cells and CD3⁻ APCs from ICR mice were isolated by FACS. T cells and APCs were co-cultured with different treatment in vitro for 48 h. The percentages of CTLs were examined by flow cytometry. (E-F) Lymphocytes from C57 BL/6 mice were cultured in vitro with different treatment for 48 h. The percentages of CTLs were examined by flow cytometry. (G-H) CD3⁺ T cells and CD3⁻ APCs from C57 BL/6 mice were isolated by FACS. T cells and APCs were co-cultured with different treatment in vitro for 48 h. The percentages of CTLs were examined by flow cytometry.

Figure 5. δ-Catenin peptide vaccines could enhance the killing activity of T cells in vitro. (A) Lymphocytes from ICR mice were cultured in vitro with different treatment for 48 h. The concentration of IFN-γ in supernatant was measured by Elisa. (B) CD3+ T cells and CD3- APCs from ICR mice were isolated by FACS for 48 h. T cells and APCs were co-cultured with different treatment in vitro. The concentration of IFN-γ in supernatant was measured by Elisa. (C) Lymphocytes from ICR mice were cultured in vitro with different treatment for 48 h. Then lymphocytes were co-cultured with H22 cells as the ratio 1:1, 5:1, 10:1, 25:1 and 50:1 for 4 h. The cell killing efficiency of lymphocytes was measured by examining the relative LDH released levels in the supernatant. (D) Lymphocytes from C57 BL/6 mice were cultured in vitro with different treatment for 48 h. The concentration of IFN-γ in supernatant was measured by Elisa. (E) CD3+ T cells and CD3- APCs from C57 BL/6 mice were isolated by FACS for 48 h. T cells and APCs were co-cultured with different treatment in vitro. The concentration of IFN-γ in supernatant was measured by Elisa. (F) Lymphocytes from C57 BL/6 mice were cultured in vitro with different treatment for 48 h. Then lymphocytes were co-cultured with Hepa1-6 cells as the ratio 1:1, 5:1, 10:1, 25:1 and 50:1 for 4 h. The cell killing efficiency of lymphocytes was measured by examining the relative LDH released levels in the supernatant.

Figure 6. δ-Catenin peptide vaccines enhanced the activation of CD8+ T cells via the activation of ERK signaling(A) Lymphocytes from ICR mice were cultured in vitro with different treatment for 1 h. Then the CD8⁺ T cells were isolated by FACS. (B) Cell lysates of CD8⁺ T cells from Fig. 6A were indicated to immunoblotting. (C) Lymphocytes from ICR mice were cultured in vitro with different treatment for 4 h. Then the CD8⁺ T cells were isolated by FACS. The mRNA levels of T-bet were examined by qRT-PCR. (D) Lymphocytes from ICR mice were cultured in vitro with different treatment for 4 h. Then the CD8⁺ T cells were isolated by FACS. The mRNA levels of Eomes were examined by qRT-PCR. (E) Lymphocytes from C57 BL/6 mice were cultured in vitro with different treatment for 1 h. Then the CD8⁺ T cells were isolated by FACS. (F) Cell lysates of CD8⁺ T cells from Fig. 6E were indicated to immunoblotting. (G) Lymphocytes from C57 BL/6 mice were cultured in vitro with different treatment for 4 h. Then the CD8⁺ T cells were isolated by FACS. The mRNA levels of T-bet were examined by qRT-PCR. (H) Lymphocytes from C57 BL/6 mice were cultured in vitro with different treatment for 4 h. Then the CD8⁺ T cells were isolated by FACS. The mRNA levels of Eomes were examined by qRT-PCR.

Figure 7. δ-Catenin peptide vaccines prolonged the survival time of mice after tumors were resected (A) Experimental design of the therapeutic model for examining the functions of δ-Catenin peptide vaccines in vivo. (B) Death of mice in Fig. 7A were recorded and survival curves were drawn.