Immunotherapy for Lewis lung carcinoma utilizing dendritic cells infected with CK19 gene recombinant adenoviral vectors

Abstract

Dendritic cells (DCs) as 'professional' antigen-presenting cells (APCs) initiate and regulate immune responses to various antigens. DC-based vaccines have become a promising modality in cancer immunotherapy. Cytokeratin 19 (CK19) protein is expressed at high levels in lung cancer and many other tumor cells, suggesting CK19 as a potential tumor‑specific target for cancer immune therapy. We constructed a recombinant adenoviral vector containing the CK19 gene (rAd-CK19). DCs transfected with rAd-CK19 were used to vaccinate C57BL/6 mice bearing xenografts derived from Lewis lung carcinoma (LLC) cells. The transfected DCs gave rise to potent CK19-specific cytotoxic T lymphocytes (CTLs) capable of lysing LLC cells. Mice immunized with the rAd‑CK19-DCs exhibited significantly attenuated tumor growth (including tumor volume and weight) when compared to the tumor growth of mice immunized with rAd-c DCs or DCs during the 24-day observation period (P<0.05). The results revealed that the mice vaccinated with the rAd-CK19-DCs exhibited a potent protective and therapeutic antitumor immunity to LLC cells in the subcutaneous model along with an inhibitive effect on tumor growth compared to the mice vaccinated with the rAd-c DCs or DCs alone. The present study proposes a meaningful mode of action utilizing rAd-CK19 DCs in lung cancer immunotherapy.

Figure 1

Construction of rAd-CK19 and transduction of DCs with rAd-CK19 and cell viability. (A) Demonstrative figure of the construction of the recombinant adenovirus and CK19 gene, using AdMax system. The shuttle plasmid pDC316-EGFP-cmv-CK19 was transfected into HEK-293 cells with the plasmid pBH-Glox E1, 3 Cre containing 5 type adenovirus genome. Then the recombinant, replication-deficient adenovirus rAd-CK19 gene was generated. (B) Transduction of DCs with rAd-CK19 and cell viability. After infection for 48 h, GFP expression was evaluated and the percentage of viable cells was evaluated using trypan blue staining; Q1, % of GFP-positive cells by flow cytometry. (C) Assay of CK19 mRnA expression in LLC cells, rAd-CK19-DCs and DCs by RT-PCR.

Figure 2

(A) Identification of CK19 expression in LLC cells by immunofluorescence (magnification, ×100). (B) Expression of CK19 protein in LLC cells and DC2.4s by western blotting. (C) CK19 expression in DC2.4 and rAd-CK19-DCs. DC+vector was used as a negative control.

Figure 3

Variation of surface markers on CK19-DCs. The expression levels of MHC class II, CD86 or CD80 on CK19-DCs, DCs or vector-DCs were analyzed by FCM, and CK19-DCs exhibited markedly increased expression of MHC class II, CD80 and CD86.

Figure 4

Induction of specific T cells by mice vaccinated with rAd-c DCs. Mice were subcutaneously vaccinated 3 times during a 3-day interval with DCs (○), rAd-CK19-DCs (■) or rAd-c DCs (△). After the last vaccination, spleen cells were isolated and stimulated with mitomycin C-treated LLC cells for 5 days. The stimulated cells were then assayed for cytolytic function using LLC cells as target cells. Results are shown as means ± SD, ^*P<0.01 compared with rAd-c DCs or DCs; ^**P<0.001 compared with rAd-c DCs or DCs.

Figure 5

Effect of rAd-CK19-DCs on tumor growth in mice. LLC cells were subcutaneously injected into mice into the right flanks on day 1. When tumors grew to 4–6 mm, the mice were divided into groups with similar tumor size (6 mice/group). Then the mice were injected with rAd-c DCs (△), rAd-CK19 DCs (■) or DCs (○). Tumor volume was measured every 2 days. Mean ± SD of tumor volume is shown. The CK19-DC vaccine significantly attenuated the tumor growth as compared to that in the control groups.

Figure 6

Capacity of CK19-DCs to sitimulate T cell proliferation. Proliferation of T cells was evaluated by MTT assay. The SI yielded 10.30±0.93 and 15.78±1.95 in the CK19-DCs compared with 5.67±0.58 and 6.99±1.51 in the rAd-c DCs (P<0.01), and 7.12±0.66 and 9.63±0.89 in the DCs at 24 and 48 h. The results are presented as the mean ± SD (n=5), ^*P<0.01.