Effective antitumor immunity against murine gliomas using dendritic cells transduced with hTERTC27 recombinant adenovirus

Abstract

hTERTC27, a 27-kDa hTERT C-terminal polypeptide has been demonstrated to cause hTERT-positive HeLa cell apoptosis and inhibits the growth of mouse melanoma. hTERTC27 has been associated with telomere dysfunction, regulation of gene-regulated apoptosis, the cell cycle and activation of natural killer (NK) cells, but its mechanism of action is not fully understood. Here, we report that dendritic cells (DCs) transduced with hTERTC27 can increase T-cell proliferation, and augment the concentration of interleukin-2 (IL-2) and interferon-γ (IFN-γ) in the supernatants of T cells. It can also induce antigen-specific cytotoxic T lymphocytes (CTL) against glioma cells in vitro. Moreover, hTERTC27 gene-transduced DCs exhibit a very potent cytotoxicity to glioma cells in vivo. It could prolong the survival time and inhibit the growth of glioma-bearing mice. These data suggest that hTERTC27 gene-transduced DCs can efficiently enhance immunity against gliomas in vitro and in vivo.

Figure 1

Cells derived from mouse bone marrow exhibited typical morphological and phenotypic characteristics of mature DCs. (A) Photomicrograph of DCs (original magnification, ×200). After 7 days of culture, the majority of the cells displayed the pricking and dendritic eminences on the cell surface. (B) The phenotype of the cells was analyzed by flow cytometry for the expression of the surface markers. These cells expressed high levels of CD80, CD86 and MHC-II.

Figure 2

Adenovirus-mediated gene transfer was highly efficient. Strong EGFP expression in DCs transfected with the adenovirus vector were observed under phase contrast microscope and fluorescence microscope (×100). (A) DCs transfected with rAd-C27. (B) DCs transfected with rAd-EGFP.

Figure 3

rAd-C27 DCs expressed hTERTC27 protein. The results show that only the DCs infected with rAd-C27 could express hTERTC27 protein. Lane 1, DCs transduced with rAd-C27; lane 2, DCs transduced with rAd-EGFP; lane 3, DCs.

Figure 4

DC stimulatory ability was detected in allogeneic mixed lymphocyte reactions (MLR) at varied ratios of DCs to T cells. Allogeneic T cells were mixed with 1×10⁵ rAd-C27 DCs, rAd-EGFP DCs and normal control DCs, stimulator/responder (S/R) ratios varying from 1:5 to 1:40, and incubated for 3 days. ^*P<0.05.

Figure 5

rAd-C27-DCs promote CTL generation in vitro. The DCs were infected with rAd-C27 at a multiplicity of infection (MOI) of 200. The percentage lysis of target cells was determined by using the CCK8 assay. The cytotoxic activity of rAd-C27 DCs was greater than that of rAd-EGFP DCs and DCs at the effector/target ratios of 20:1 and 40:1 (^*P<0.01).

Figure 6

The concentration of IL-2 and IFN-γ in the supernatants of T cells were assayed with ELISA. The T cells were co-cultured with the rAd-C27 DCs, rAd-EGFP DCs and normal control DCs at a stimulator/responder ratio of 1:10. The concentration of both IL-2 and IFN-γ in T cells co-cultured with Advc27-EGFP DCs group were higher than in the other two groups. ^*P=0.001.

Figure 7

In vivo experiments using the intracranial tumor model. (A) The experimental schema shows tumor implantation, DCs injection, CTL, TUNEL, and survival observation time. (B) Effect of DCs on the experimental glioma in C57BL/6 mice. Kaplan-Meier survival curve of intracranial glioma-bearing C57BL/6 mice that were injected twice with rAd-C27 DCs, rAd-EGFP DCs or DCs.

Figure 8

rAd-C27 DCs induced cytotoxicity against murine glioma in vivo. Splenocytes harvested on Day 7 after the second intracerebra injection were restimulated by GL261 and their cytotoxicity was detected by the CCK8 assay. The cytotoxicity elicited by rAd-C27 DCs was higher than the other groups at effector/target (E/T) ratios of 5:1, 10:1 and 40:1.

Figure 9

Inhibition of tumor growth in GL261 mice. On Day 21 after tumor implantation, four mice from each group were euthanized to obtain brain tissues and compare the tumor volume. (A) The brain tissues stained with hematoxylin and eosin (H&E) demarcated the tumor outline. (B) The average tumor size in different groups after GL261 glioma cells implantation.