Study Of DC Transfected With Tumor Cell Total RNA Inducing Anti-tumor Immunity Of Lung Cancer In Vitro

Objective:To investigate whether blood-derived DC transfecting with unfractionated RNA (total RNA) extracted from tumor cells of lung cancer can induce the effective generation of Ag-specific CTL and antitumor immunity in vitro.Methods:DCs were generated from lung cancer patients concentrated leukocyte fraction of blood. Peripheral blood mononuclear cells (PBMC) were further separated by density-gradient centrifugation and cells were resuspended and allowed plastic adherence. The adherent cell fraction was used for DC culture in CM supplemented with rhGM-CSF , rhIL-4 and rhTNF- a . After 5 days of culture, DCs were harvested. DC preparations fulfilling immature DC phenotypic criteria were used for subsequent RNA transfection. Total RNA was isolated from tumor cell line Calu-6 of lung cancer. Pulsing of autologous DC with Calu-6 RNA encapsulated by liposome. FACS analyses the phenotype of transfected DC and untransfrcted DC. MLR assay was done for comparing the function oftransfected DCs and untransfected DCs to stimulate allogeneic T lymphocyte proliferation. Measure the quantity of IL-12 secreted by transfected DC. RNA pulsed DCs were not only used as stimulators for CTL, but also serve as cellular targets in cytotoxicity assays. Measure the induction of CTL response in LDH assay. What's more, IFN-y secreted by sensitive CD8+T lymphocytes were also identified by EL1SA.Results:Wse can obtain fully mature and functional DCs by culture PBMC exposed to GM-CSF and IL-4 for 5 days. FACScan analysis indicate that immature DCs transfected with total RNA exhibited increased expression of membrane molecular such as CDla, CD83, CD86, HLA-ABC and HLA-DR, and were more potent in stimulating allogeneic T cell proliferation than untransfected DC. CTL primed by DC/Calu-6 RNA was higher in lysis of cognate target cells (DC/Calu-6 RNA) than CTL primed by DC/823RNA or DC (p<0.05). CTL against cognate targets induce more effective lysis than LAK against the same targets (p<0.05). In addition, sensitive CD8+T lymphocytes stimulated by DC/Calu-6RNA secreted more IFN-y when restimulated by cognate Ag than restimulated by other Ags such as DC/823RNA or DC (p<0.05).Conclusion:In this study we show that DCs transfected with total RNA isolated from lung cancer cells are remarkably effective in stimulating tumor specific T-cell response in vitro while obviating the need to identify the antigens involved. The tumor-specific CTLs can only effectively lysed theantigen-specific targets (DC/Calu-6). Such specific lysis is more potent than nonspecific lysis by LAK. What's more, this vaccine can induce the sensitive CD8+T lymphocytes to secrete IFN-y, Thl type cytokine to assistant CTL, NK and macrophage function. This study provides a scientific foundation for further investigation in vivo of this approach.