The Antitumor Effects And Immunological Mechanism Of SLC And BMDC Vaccines Co-deliveryed By SLC Peptide Gene And Tumor RNA

Chemokines belong to a family of small proteins that control the migration of certain leukocyte populations. SLC(secondary lymphoid tissue chemokine) is a specifical chemokine that originally was shown to be strongly expressed in secondary lymphoid organs, particularly in the high endothelial venules(HEV) and in the T cell zone of the lympho nodes(LN). SLC has been described as a chemotactic factor for dendritic cells, T cells and NK cells mediated through the chemokine receptor CCR7.The chemokine receptor CCR7 is a specific 7- transmembrane, G-protein coupled receptor expressed in T, NK, and dendritic cells in a time-ordered and stimulus-dependent manner. High affinity receptors binding initiates signal transduction cascades relevant to the re-organization of actin filaments within the cell, including calcium mobilization, MAPK, p21Ras-family and PI-3K activation as a result of cell migration. Amplification of signaling from the CCR7 is regulated primarily by the pertussis toxin (PTX)-sensitive heterotrimeric GTP-binding protein Gai. NIK(nuclear factor κB-inducing kinase) and nuclear factor kB were reported also involved in signal transduction of SLC/CCR7 axis. So we hyperthesied that there is a cross talking pathway relevant to signal transduction decades between the cell migration and proliferation.Development of effective antitumor immune response depends on timely interactions of effector cells, including APCs and different subsets of T cells. Dendritic cells are most powerful APCs that possess the unique ability to prime naive T cells in vivo. Efficient retrieval and transport of DCs into T-cell areas of the LN mediated by SLC/CCR7 axis are central to the development of an immune response. In addition to CCR7, mouse SLC interacts with the chemokine receptor CXCR3, as do several chemokines with antiangiogenic properties such as IP-10 and Mig. The potential broad spectrum of actions of SLC, in the recruitment of dendritic cells and effector cells as well as in theinhibition of angiogenesis, prompted us to analyze its role in antitumor activity.To further study the bio-activity and the cross talking pathway between its signal transduction mediated by SLC/CCR7 axis, and to explore the antitumor effects and immunological mechanism of SLC and DC vaccine, we inserted into Hepal-6 cells and DCs a cDNA coding for the SLC using rAAV(recombinant adeno-associated virus) as delivery vector because of its broad host range, low host immunity, high-efficiency transduction, long-term expression and native tumor suppressor properties in vivo. To enhance its capability of inducing primary antigen-specific CTLs, we also transfected DC with tumor RNA.The main results are as follows.1. SLC induced proliferation of T lymphocytes in addition to its migration mediated by SLC/CCR7 axis. The effect could be significantly reduced by PTX. Total RNA were prepared from T cells and BMDCs with or without SLC stimulation for G Protein-coupled Receptors Signaling PathwayFinder (MM-025) Gene Array analysis. CyclinDl, El, E2 localized on the protein tyrosine kinase pathway were considered increased upon SLC stimulation in both T cells and BMDCs through. VCAM-1 (Vascular cell adhesion molecule 1) and ICAM-l(Intercellular adhesion molecule 1) localized on the NFkB pathway were also increased. Besides, SLC could upregulate the expression of some cytokines including Interleukin 2 and 6 in T cells.2. The new recombinant AAV encoding SLC (named as rAAV-SLC) was constructed successfully using a pAAV Helper-Free System. The favorite gene was detected by fluorescent microscope and polymerase chain reaction. Flow cytometry for titration of infectious rAAV-SLC was performed using a 530/15nm (FITC) band pass filter. CMV promoter specific qPCR(quantitative real-time PCR) was performed to quantify the copy number of viral genomes <> The infectious titer of rAAV-SLC was 5xlO8IU/ml, the genome titer of rAAV-SLC was 1.2xl0nparticles/ml.3. The rAAV-SLC cells could transfect a mouse hepatocellular carcinoma (HCC) cell line Hepal-6 at high efficiency, showed reduced tumorigenicity...