ABSTRACT
<p><b>OBJECTIVE</b>To prepare IL-2-anchored and tumor-derived exosomes vaccine, and investigate the antitumor efficiency of the special cytotoxic T-lymphocytes induced by Ex/GPI-IL-2.</p><p><b>METHODS</b>To construct pEGFP-N1-IL2gpi plasmid coding a fusion gene of a DNA oligo encoding GPI-anchor signal sequence attaching to human IL-2 cDNA. Then T24 cell lines stably expressing GPI-IL-2 proteins (T24/GPI-IL-2) were established. Ex/GPI-IL-2 were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and the morphology and molecule markers were analyzed. The mixed lymphocyte reaction study and cytotoxic study were performed to determine the proliferative effect of T lymphocytes and the cytotoxicity induced by Ex/GPI-IL-2.</p><p><b>RESULTS</b>The pEGFP-N1-IL2gpi plasmid was successfully constructed, and cell lines stably expressing GPI-IL-2 fusion proteins were established. Ex/GPI-IL-2 were small vesicular and saucer-shaped in diameter of 30-90 nm, containing heat shock protein 70, intercellular adhesion molecule-1, MAGE-1 and GPI-IL-2. Ex/GPI-IL-2-pulsed could dendritic cells induce proliferation of T cells and cytotoxic immune response more efficiently (P<0.05).</p><p><b>CONCLUSIONS</b>GPI-IL-2 gene-modified tumor cells can make the exosomes containing GPI-IL-2 with an increased anti-tumor effect. Our study provides a feasible approach for exosome-based tumor immunotherapy of bladder transitional cell tumors.</p>
Subject(s)
Humans , Cancer Vaccines , Allergy and Immunology , Carcinoma, Transitional Cell , Allergy and Immunology , Metabolism , Pathology , Cell Line, Tumor , Cell Proliferation , Exosomes , Genetics , Metabolism , Glycosylphosphatidylinositols , Metabolism , HSP70 Heat-Shock Proteins , Metabolism , Intercellular Adhesion Molecule-1 , Metabolism , Interleukin-2 , Metabolism , Melanoma-Specific Antigens , Metabolism , Plasmids , Recombinant Fusion Proteins , Metabolism , T-Lymphocytes, Cytotoxic , Allergy and Immunology , Transfection , Urinary Bladder Neoplasms , Allergy and Immunology , Metabolism , PathologyABSTRACT
<p><b>OBJECTIVE</b>To prepare a vaccine of IL-12-anchored exosomes derived from renal cancer cells and to evaluate its antitumor effect in vitro.</p><p><b>METHODS</b>A mammalian co-expression plasmid of glycolipid-anchor-IL-12 (GPI-IL-12) was constructed by subcloning IL-12A chain gene (P35 subunit) and a fusion gene containing GPI-anchor signal sequence and IL-12B chain gene (P40 subunit) in pBudCE4.1. Confocal laser scanning microscopy and flow cytometry were used to analyze the expression of the fusion proteins. Transmission electron microscopy and Western blot were used to identify the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of IL-12-anchored exosomes was determined by IFN-gamma release assay.</p><p><b>RESULTS</b>Mammalian co-expression plasmids were successfully constructed. Confocal laser scanning microscopy and flow cytometric analysis of the RC-2-GPI-IL-12 transfectants showed the expression of IL-12 on the cell surface. Exosomes were purified by ultrafiltration and sucrose gradient centrifugation, which were 30-80 nm in diameter, typically saucer-shaped, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. (80.0 +/- 9.6) pg/ml of IL-12 was detected in 10 microg/ml exosomes and it significantly induced the release of IFN-gamma. Stimulation with EXO-IL-12 could efficiently induce antigen-specific cytotoxic T lymphocytes (CTL), resulting in more significant cytotoxic effects in vitro.</p><p><b>CONCLUSION</b>A vaccine of exosomes-GPI-IL-12 can be obtained from the culture supernatant of renal cancer cells modified to express anchored IL-12. This vaccine expressing IL-12 and tumor associated antigen G250 may become a new strategy for the treatment of renal cancer.</p>
Subject(s)
Humans , Antigens, Neoplasm , Metabolism , Cancer Vaccines , Allergy and Immunology , Carbonic Anhydrase IX , Carbonic Anhydrases , Metabolism , Cell Line, Tumor , Cytotoxicity, Immunologic , Exosomes , Genetics , Metabolism , Glycosylphosphatidylinositols , Genetics , Metabolism , HSP70 Heat-Shock Proteins , Metabolism , Intercellular Adhesion Molecule-1 , Metabolism , Interferon-gamma , Bodily Secretions , Interleukin-12 , Genetics , Metabolism , Kidney Neoplasms , Metabolism , Pathology , Plasmids , T-Lymphocytes, Cytotoxic , Cell Biology , Allergy and Immunology , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To isolate and purify exosomes derived from human bladder transitional cell carcinoma T24 cells, analyze the morphology and protein composition, and investigate the antitumor effect of specific cytotoxic T lymphocytes induced by exosomes.</p><p><b>METHODS</b>Exosomes were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and characterized by electron microscopy and Western blot. Dendritic cells were amplified and purified from peripheral blood and pulsed with exosomes. Then they were co-cultured with T cells, and divided into 3 groups: exosome-pulsed DC group, unplused DC group and control group. Alamar-Blue assay was used to evaluate the specific cytolytic activity.</p><p><b>RESULTS</b>The exosomes were in size about 30 approximately 90 nm saucer-shaped membranous vesicles. HSP70, ICAM-1 and CK20 were detected by Western blot. The CTL induced by DC pulsed with exosomes had significant cytolytic activity (P < 0.01).</p><p><b>CONCLUSION</b>The exosomes derived from T24 cells are loaded with immunoprotein HSP70 and ICAM-1, and DC pulsed with exosomes can promote the anti-tumor effect of CTLs in vitro.</p>