Co-immunization with L-Myc enhances CD8+ or CD103+ DCs mediated tumor-specific multi-functional CD8+ T cell responses

Abstract

Renal carcinoma shows a high risk of invasion and metastasis without effective treatment. Herein, we developed a chitosan (CS) nanoparticle-mediated DNA vaccine containing an activated factor L-Myc and a tumor-specific antigen CAIX for renal carcinoma treatment. The subcutaneous tumor models were intramuscularly immunized with CS-pL-Myc/pCAIX or control vaccine, respectively. Compared with single immunization group, the tumor growth was significantly suppressed in CS-pL-Myc/pCAIX co-immunization group. The increased proportion and mature of CD11c⁺ DCs, CD8⁺ CD11c⁺ DCs and CD103⁺ CD11c⁺ DCs were observed in the splenocytes from CS-pL-Myc/pCAIX co-immunized mice. Furthermore, the enhanced antigen-specific CD8⁺ T lymphocyte proliferation, cytotoxic T lymphocyte (CTL) responses, and multi-functional CD8⁺ T cell induction were detected in CS-pL-Myc/pCAIX co-immunization group compared with CS-pCAIX immunization group. Of note, the depletion of CD8 T cells resulted in the reduction of CD8⁺ T cells or CD8⁺ CD11c⁺ DCs and the loss of anti-tumor efficacy induced by CS-pL-Myc/pCAIX vaccine, suggesting the therapeutic efficacy of the vaccine was required for CD8⁺ DCs and CD103⁺ DCs mediated CD8⁺ T cells responses. Likewise, CS-pL-Myc/pCAIX co-immunization also significantly inhibited the lung metastasis of renal carcinoma models accompanied with the increased induction of multi-functional CD8⁺ T cell responses. Therefore, these results indicated that CS-pL-Myc/pCAIX vaccine could effectively induce CD8⁺ DCs and CD103⁺ DCs mediated tumor-specific multi-functional CD8⁺ T cell responses and exert the anti-tumor efficacy. This vaccine strategy offers a potential and promising approach for solid or metastatic tumor treatment.

Keywords: CAIX; CS nanoparticles; L-Myc; multi-functional CD8+ T cells; renal carcinoma; tumor vaccine.

FIGURE 1

Characterization of CS‐DNA nanoparticle vaccine. (A) Size intensity curves. (B) Surface zeta potential. (C) Gel blocking analysis of CS‐DNA. (D) The micrographs of CS‐DNA nanoparticles were acquired by transmission electron. Scale bars, 200 nm. (E and F) The expression of L‐Myc or CAIX was detected by western blot in muscular tissues from mice immunized with CS‐pL‐Myc or CS‐pCAIX. CS‐Vector was used as the corresponding control. (G and H) Quantification of L‐Myc or CAIX expression by densitometry in (E) and (F). Data are from one representative experiment of three performed and presented as the mean ±SD. The different significance was set at ***P <.001

FIGURE 2

Therapeutic effects of CS‐pL‐Myc/pCAIX vaccine in subcutaneous hCAIX‐Renca tumor model. (A) Schema of the subcutaneous hCAIX‐Renca‐tumor model intramuscularly inoculated with various vaccines on day 7 after tumor inoculation. (B) Tumor volume. (C) Tumor volumes. (D) Tumor weight. (E) Survival curve. (F) The frequencies of T, CD4⁺ T, CD8⁺ T, NK, DCs, macrophages, or MDSCs were analyzed in TIL of mice treated with vaccines. (G) Total numbers of various immune cells in (F). Data are from one representative experiment of three performed and presented as the mean ±SD. The different significance was set at *P <.05, **P <.01, and ***P <.001; ns, not significant

FIGURE 3

Co‐immunization with L‐Myc enhances the induction and mature of DC subsets. Mice immunized with vaccines were sacrificed on the 42 days post tumor inoculation, the proportions of DCs subsets were analyzed by flow cytometry in spleen. (A) The representative flow cytometry of CD11c⁺, CD103⁺CD11c⁺ or CD8⁺CD11c⁺ DCs subpopulation. (B‐D) Statistical analysis of the percentages of DCs subsets in (A). (E) The expression of CD80, CD86, or MHC‐II on CD11c⁺ cells. (F‐H) Statistical analysis of the frequencies of CD11c⁺CD80⁺, CD11c⁺CD86⁺ or CD11c⁺MHC II⁺ cells in (E). The data shown are the representative of three experiments. Data are means ±SD. The different significance was set at**P <.01, ***P <.001

FIGURE 4

CS‐pL‐Myc/pCAIX co‐immunization promotes the tumor‐specific CD8 T cell responses. (A) The lymphocytes isolated from spleen of immunized mice were stimulated with CAIX protein (10 μg/ml) in vitro; the percentages of EdU⁺ cell was assessed in gated CD8 T cells by flow cytometry. (B) RTCA assay were used to measure the CTL activity. (C and D) IFN‐γ‐secreting T lymphocytes were detected by ELISPOT assay. (E and F) Flow cytometry were performed on splenocytes to assess the proportion of TNF‐α⁺CD8⁺, IL‐2⁺CD8⁺, and IFN‐γ⁺CD8⁺ T cells. The data shown are the representative of three experiments. Data are means ±SD. The different significance was set at**P <.01, ***P <.001

FIGURE 5

CS‐pL‐Myc/pCAIX co‐immunization increased the induction of multi‐functional CD8⁺ T cells. 42 days after the tumor inoculation, splenocytes or TILs from vaccine immunized mice were stimulated in vitro. (A and B) The proportions of t TNF‐α⁺IL‐2⁺CD8⁺ T cells in spleen or TILs were detected by flow cytometry. (C and D) The percentages of TNF‐α⁺IFN‐γ⁺CD8⁺ T cells. (E and F) The percentages of IL‐2⁺IFN‐γ⁺CD8⁺ T cells. (G and H) The proportions of TNF‐α⁺IL‐2⁺IFN‐γ⁺CD8⁺ T cells. The data shown are the representative of three experiments. Data are means ±SD. The different significance was set at **P <.01, ***P <.001

FIGURE 6

Anti‐tumor effect induced by CS‐pL‐Myc/pCAIX vaccine is dependent on CD8 T cell immune responses. In the depleted group of mice, 0.5 mg of anti‐mouse CD8 mAb was injected intraperitoneally at day −2, 5 and 12 after vaccination. (A) Tumor progression. (B) Tumor volumes. (C) Tumor weights. (D) Survival rate. (E and F) The proportions of CD8⁺ T cells and CD8⁺CD11c⁺ DCs in spleen or TILs. (G) Immunochemistry staining for CD8⁺ T cells of tumor tissues (×200 magnification). (H) Staining scores were analyzed in (G). The experiments were conducted with five mice per group. The data shown are the representative of three experiments. Data are means ±SD.**P <.01, ***P <.001

FIGURE 7

CS‐pL‐Myc/pCAIX co‐immunization suppressed the lung metastasis in renal carcinoma model by enhancing multi‐functional CD8⁺ T cell responses. (A) Schema of lung metastasis model establishment and experiment. (B) The present images of lung metastasis tumors excised from mice. (C) The numbers of metastatic nodules. (D) Lung tissues performed by H&E staining. (E) CD8⁺ T cells of lung tissues were detected by immunochemistry staining (×200 magnification). (F) The frequencies of CD8⁺ T cells were analyzed by flow cytometry in the lung tumor tissues. (G) Intracellular staining of TNF‐α, IFN‐γ, and IL‐2 of multi‐functional CD8⁺ T cells in stimulated splenocytes. (H) CAIX‐specific IFN‐γ‐secreting T lymphocyte cells were quantified by ELISPOT assay. (I) The CTL was detected by RTCA assay. (J) Cell index by RTCA assay were analyzed in (I). The experiments were performed with five mice per group. Data are from one representative experiment of three performed and presented as the mean ±SD. The different significance was set at *P <.05, **P <.01, ***P <.001