In Vitro Induction of Anti-Leukemia CTL Effect by Dendritic Cells Infected with Recombinant Adenovirus Vectors Containing Human Survivin Genes.

Abstract

Relapse after chemotherapy could lead to poor prognosis in patients with leukemia. By using tumor-specific immunotherapy to eradicate minimal residual diseases could prevent disease relapse. Our study aims at exploring whether peripheral blood derived dendritic cells (DC) expressing human survivin can enhance their capability to induce human T lymphocytes into tumor-specific cytotoxic T lymphocytes (CTL) for immunotherapy. In our experiments the full length survivin cDNA was obtained from recombinant plasmid pcDNA3.0-survivin by PCR. The PCR products were double-digested with restriction endonucleases KpnIand HindIII, and inserted orientationally into pshuttle-CMV plasmid. The plasmid of pshuttle-CMV-survivin was lined with PmeI, and the fragments containing survivin genes were reconfirmed by DNA sequencing. The plasmid of pshuttle-CMV-survivin was transfected into E.coli BJ5183. After homologous recombination in bacteria, the extracted plasmids of positive clones were lined with PacI, transfected into HEK293 cells with liposome Lipofectaminei 2000 and then identified by PCR method. The high-titer adenovirus supernatants were harvested. Mononuclear cells were obtained from healthy donors by density centrifuge. The washed mononuclear cells were cultured in complete medium (CM) supplemented with 10% fetal calf serum (FCS) at a final concentration of 2×106 cells/ml. After incubated for 120minutes, the adherent cells were used for inducing CTL, and cultured in CM with 1000 U/ml GM-CSF, 1000 U/ml IL- 4 and 50 u/ml TNF-α. The morphology of DC was dynamically analyzed with an inverted microscope and an electron-microscope, and their surface immunophenotyping were determined by flow cytometry. The function of dendritic cells was assayed by mixed leukocyte reaction (MLR) tests. DCs were collected on day 7 and divided into two groups, ad-survivin group and the control group. Two days later, 5×105 cells of each group were placed to a 24-well plate as stimulation cells, and then 6×105 of T lymphocytes (responding cell) were added to each well. On day 14, cells were collected as effector cells for cytotoxicity test. LDH release test was used to evaluate cytotoxicity of CTL. The results showed that the recombined adenovirus-survivin was constructed successfully and its titer was about 2.65×109 pfu/ml. Under inverted microscope and electron-microscope the morphology of these cells is consistent with typical DCs. They are non-adherent and have dendrictic projections. The immunophenotypic analysis of these cells showed positive for CD80, CD 86, CD83 and HLA-DR. The levels of IL-12 and IFN-γ in the culture supernatants, assayed by ELISA were 50.5pg/ml and 82.5pg/ml, respectively. The expression of survivin in transfected dentritic cells was confirmed by Western blot analysis. A 16.5×103 KD band was detected by Western blot. In MLR assay, DCs infected with Ad-survivin can induce high allogeneic lymphocytes reaction at the ratios of 1 to 5, 1 to10, 1 to 50 and 1 to 100. DCs infected with Ad-survivin have much higher activity of CTL to HL-60 cells than control DC. Our conclusion is that DCs infected with Ad-survivin can induce ant-leukemic cells CTL response in vitro. Therefore, adenovirus vectors containing survivin might be the promising candidates for leukemic vaccine development.