Objective: To investigate the recombinant calreticulin (rCRT) mediated antitumor immune response. Methods: Cell proliferation was determined by MTT method, apoptosis was evaluated by DNA fragmentation and CRT expression and cell localization were assayed by western blotting, QT-RT-PCR and immunofluorescence assays. The mouse melanoma cell line B16-F1 was treated with polyamine analogue BENS to induce apoptosis and incubated with rCRT to get rCRT coated on the membrane, and then the cells were used to immune BALB/c mice as a cell-antigen. Immunized animals were rechallenged by live B16-F1 cells and then tumor generation ratio and the lactate dehydrogenase release assay were used to evaluate antitumor effects of rCRT-mediated immunity. Results: BENS induced apoptosis of B16-F1 cells without the redistribution of CRT within the cells. When B16-F1 cells coated with rCRT were used as cell-antigen to inoculate the animals, the mice obtained the ability in inhibiting proliferation of homologous tumor cells in vivo. Comparing with the positive control group, the splenocytes from those inoculated mice have an obvious enhancement on their cytolytic effects specifically against B16-F1 cells. Conclusion: rCRT coated on the cell surface can enhance immunogenicity of apoptotic tumor cells and mediated effective anti-tumor immunoresponse in mice.
In the process of cell apoptosis induced by specific reagents,calreticulin(CRT)in endoplasmic reticulum is transferred and coated onto the cell membrane.As a sort of specific ligand,the CRT on the surface of apoptotic cells could mediate recognition and clearance of apoptotic cells by phagocytes.In this research we discovered that mitoxantrone could induce apoptosis of mouse melonoma B16-F1 tumor cells,accompanied by the membrane translocation and coating of CRT.When mitoxantrone-treated B16-F1 cells were used as antigen to inoculate mice,the mice acquired an ability to suppress proliferation of homologous tumor cells.Splenocytes from these mice showed an increased cytolytic effect on homologous B16-F1 cells but no such effect on non-homologous H22 tumor cells.All these results suggested that mitoxantrone-treated apoptotic B16-F1 cells could be used as a sort of cell vaccine to initiate effective anti-tumor immunoresponse in mice.
