| Objective: In order to further improve the immunogenicity of exosomes, we prepared exosomes vaccine (Ex/GPI-IL-2) derived from Glycosyl-phosphatidylinositol (GPI) anchored IL-2 (GPI-IL-2) gene modified human transitional cell carcinoma of bladder cells(T24 cells), analyze the morphology and immunoprotein compositionin, also investigate the antitumor efficiency of the special cytotoxic T lymphocyte induced by Ex/GPI-IL-2. Offer a theoretical basis for immunotherapy of bladder transitional cell carcinoma.Methods: 1. A fusion gene of a DNA oligo encoding GPI-anchor signal sequence attaching to human IL-2 cDNA was generated by annealing two synthesized, resulting in plasmid pEGFP-N1-IL2gpi. Then the transfected human transitional cell carcinoma of bladder cells were selected by G418, we respectively constructed T24/GPI-IL-2 and T24/p cell lines stably expressing GPI-IL-2 and GFP proteins, the GPI-anchored IL-2 proteins expressed on the cell surface were detected by immunofluorescence and ELISA analysis. 2. exosomes (Ex/GPI-IL-2, Ex/p and Ex) were respectively isolated and purified by ultrafiltration and sucrose gradient centrifugation from supernatants of T24/GPI-IL-2, T24/p and T24 cells, and characterized by transmission electron microscopy and western blot analyses. 3. Isolated mononuclear cells by Ficoll density gradient centrifugation from peripheral blood, adherent cells were cultured in RPMI1640 containing rhGM-CSF and IL-4. Inverted microscope and scanning electron microscope were used to observe the morphology of cultured DCs. DCs were pulsed with Ex/GPI-IL-2, Ex/p and Ex, the mixed lymphocyte reaction study were used to determine the proliferation effect of T lymphocytes cells induced by Ex/GPI-IL-2, and the cytotoxic sduty were used to determine the cytotoxicity target to T24, BIU87 and RC2 cells of allogeneic T lymphocytes induced by Ex/GPI-IL-2.Rusult: 1. Successfully construction of the pEGFP-N1-IL2gpi plasmid and confirmed by restriction enzyme digestion and DNA sequencing analysis, then established of two stable cell lines expressing GPI-IL-2 fusion proteins and green fluorescent proteins(GFP). 2. Ex/GPI-IL-2, Ex/p and Ex were isolated and purified by serial centrifugation and sucrose gradients ultracentrifugation respectively from supernatants of T24/GPI-IL-2, T24/p and T24 cells, they were small vesicular of 30-90 nm saucer-shaped in diameter. Western blotting confirmed that the exosomes contained heat shock protein 70(HSP70), intercellular adhesion molecule-1(ICAM-1) and melanoma antigen family A member-1 (MAGE-1), there was no obvious difference in quantity of those proteins. GPI-IL-2 gene modified tumor cells can make the exosomes contain anchored IL-2 by ELISA anlysis. 3. These typical DCs had a variety of branching forms on days of 7 culture. The stimulate index of T cells induced by Ex/GPI-IL-2, Ex/p and Ex pulsed DCs respective was 2.78±0.25, 1.94±0.32 and 1.92±0.28, the cytotoxicity of CTL induced by Ex/GPI-IL-2, Ex/p and Ex pulsed DCs target to T24 cells respective was 53.01±2.25%, 37.22±2.49% and 37.85±1.57%, Correspondingly, the release of IFN-γby activated T cells respective was 1054.82±35.20pg/ml, 678.02±17.97pg/ml and 683.23±13.99pg/ml. The cytotoxicity of CTL induced by Ex/GPI-IL-2 target to T24, BIU-87 and RC-2 respective was 53.01±2.25%, 46.61±2.60% and 31.38±2.33%.Conclusions: Transitional cell carcinoma of bladder derived exosomes contained HSP70, ICAM-1 and MAGE-1, exosomes derived from GPI-IL-2 gene modified tumor cells contain IL-2 with the increased anti-tumor effects. Our study provided a feasible approach for exosomes-based tumor immunotherapy. |
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