The Antitumor Effect Of Human Cord Blood-derived Dendritic Cells Modified By The Livin α Gene In Lung Cancer Cell Lines

Part oneConstruction of a replication-defective recombinant adenovirus vector encoding the livin a gene to infect UCB-derived DCs[Background]The inhibitors of apoptosis proteins (IAPs) inhibit apoptosis by interacting with the pro-apoptotic caspases via their baculoviral IAP repeat (BIR). Livin is a novel member of the IAP family. It has two isoforms that are termed livin a (298amino acids residues) and livin β (280amino acids residues). Livin is specifically expressed by the majority of tumor cells, but it is not expressed in normal adult tissues. Since anti-livin autoantibodies have been identified in the serum of the majority of cancer patients, immunologic tolerance to livin is thought to be very weak, making livin a promising candidate target for immunotherapy. Transduction through replication-defective recombinant adenovirus (rAd) vector is one of efficient method. The gene encoded by the rAd could continually expressed for at least2weeks in the body, at the same time without integration into the genome of the host, ensuring the safety of receptor. Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells and is resistant to replicative senescence and immunosenescence. HLA-mismatched cord blood-derived transplants are associated with a lower incidence and severity of acute or chronic graft-versus-host disease (GVHD). Thus, UCB cells are an ideal source of cells for allogeneic antitumor transplants. Due to the biological activity of livin, we used a rAd vector encoding the livin a gene to infect UCB-derived DCs to generate an antigen-specific vaccine that can be applied to cancer immunotherapy.[Objective]To aimed to construction of recombinant adenoviral vector encoding the livin a gene. Isolation and induction of UCB-derived DCs in vitro. Useing a rAd-livin a to infect UCB-derived DCs to generate an antigen-specific vaccine.[Methods]The shuttle plasmid pDC316-EGFP-cmv-livin a and the helper plasmid pBHGlox_E1,3Cre were co-transfected with HEK-293cells to produce the recombinant adenovirus. PCR was used to evaluate whether the rAd-livin a vector had been generated successfully. The tissue culture infectious dose50(TCID50) method was used to determined the titer of the virus. UCB samples were isolated following cesarean sections. The precursor DCs were isolated from mononuclear cells in the Umbilical cord blood through anti-CD34microbeads. GM-CSF, IL-4and SCF were added to induce the maturation of UCB-derived DCs. The flow cytometry was used to detected EGFP-positive cells to evaluation of the infection efficiency of restructured adenoviruses in DCs, and the trypan blue staining were used to evaluation of cell viability.[Results]The results showed that the target gene detected by PCR was consistent with the expected gene livin a (897bp). The titer of virus was2.7×1010TCID50/ml. DCs infected with either rAd-c or rAd-livin a fluoresced green when viewed using a fluorescent microscope. Optimal MOI for recombinant adenovirus infection of DC cells is200, at which88%of the cells expressed EGFP and69%of the cells were viable.[Conclusions]Our study had constructed successfully the recombinant adenovirus vector encoding human livin a gene. Being cultured with combination cytokines protocols promote the induction and maturation of UCB-derived DC precursors which was isolated using anti-CD34microbeads. UCB-derived rAd-livin a-infected DC vaccines was generated, paving the way for the next step of research. Part twoThe antitumor effect and related mechanism of human cord blood-derived dendritic cells modified by the livin a gene in lung cancer cell lines[Background]A variety of immune cells and immune molecules participate in antitumor immune responses. CD8+CTLs and CD4+Thl cells play key roles in antitumor activity. T lymphocytes specifically recognize antigen peptides presented by APCs in the context of MHC I molecules and become activated and secrete cytokines. We propose using DCs loaded with tumor antigen as a DC vaccine to induce or enhance antitumor immune responses. As livin a contains virtually all the antigenic epitopes of livin β, We hypothesize that rAd-livin a-infected DCs expressing the complete livin gene present a wide variety of molecular epitopes in the context of MHC I, so the rAd-livin α-infected DCs may also present other HLA-restricted livin antigens in either HLA-A2or other HLA subtypes. Thus, rAd-livin α-infected DCs could be widely applied to the treatment of cancer, regardless of HLA subtype. we infected DCs with rAd-livin a. Due to these immune mechanisms and the in vivo immune environment, there are multiple cell types in the human immune system that can mediate non-specific antitumor effects, such as macrophages, NK and LAK cells. So In this study, we chose non-adherent UCB-derived MNCs cultured with IL-2as effector cells instead of CD8+T lymphocytes sorted from MNCs.[Objective]In this study, we expanded on previous work to show that livin a gene-modified DC vaccines derived from UCB can activate multiple innate effector cell types in the human immune system in addition to CTLs to exert antitumor effects. Besides, we also examined the T cell differentiation and the specific anti-livin T lymphocytes to further explore the possible antitumor mechanism of rAd-livin a DC vaccines.[Methods]We used western blots to analyze livin expression in H460, HBE and A549cells, in rAd-c-infected and rAd-livin α-infected DCs and in MNCs. The expression of DC cell surface molecules (CDla, CD80, CD86and HLA-DR) and the T cell subsets was analyzed by flow cytometry. Cytotoxicity assay. Livin-specific effector T cells was detected by An IFN-y ELISPOT assay and flow cytometry. An annexin V/PI assay was performed to detected the cytotoxic activity of the effector cells.[Results]The livin protein is expressed in A549and H460cells and rAd-livin α-infected DCs, but not in HBE cells, rAd-c-infected DCs or MNCs. Expression of DC surface molecules is increased when DCs infected with adenovirus. Effector cells that had been stimulated by HLA-24+rAd-livin a-infected DCs and were then exposed to HLA-24presenting livin polypeptide generated a statistically greater number of IFN-y spots than which stimulated by rAd-c-infected DCs. Effector cells stimulated with rAd-livin α-infected DCs or with proteins that had undergone multiple freeze-thaw cycles mediated similar levels of cytotoxicity but effector cells stimulated with rAd-livin a-infected DCs mediated less non-specific cytotoxicity than those stimulated with proteins from A549or H460cells that had undergone multiple freeze-thaw cycles.[Conclusions]The present study demonstrated that infection with rAd can promote the maturation of UCB-derived DCs. And rAd-livin a infected-DC can sensitize autologous T lymphocytes to effectively kill livin-expressing tumor cells without inducing autoimmunity against normal tissue.