Dendritic Cells By Epstein-barr Virus Vaccine Stimulation For Epstein-barr Virus Infection-mediated Tumor Therapy Study

Epstein-Barr virus (EBV) is a human herpesvirus, which is endemic in all human populations. More than 95% of the human population worldwide becomes infected with the virus during childhood and then carry the virus for life. If the initial infection is delayed until adolescence, infectious mononucleosis frequently results. The virus is also linked with several important human cancers including Burkitt's lymphoma (BL) nasopharyngeal carcinoma (NPC) and Hodgkin's disease and so on. In these tumor cells there were mainly four kinds of virus-encoded proteins (EA MM VGA and EBNA). EB virus envelope glycoprotein gp340, one of these viral antigens, can elicit virus-neutralizing antibodies.. ADCC and the antiviral GTL responses. Thus, a number of vaccines against the infection have been developed based on the EBV membrane antigen gp340. Dendritic cells (DG) are the most potent antigen-presenting cells (APC), which are called custocyte system together with monocyte and macrophage. and play a critical role in the induction of naive and memory CD4 and CD8 T cells in the recent research. Using DG pulsed with antigen or peptide can be successfully applied in tumor immunotherapy. Against this background, we sought to isolate GD34+ hematopoietic stem cells from normal bone marrow and cultured in GM-CSF.. TNF- ci IL-4 and JL-3 for two weeks to obtain large amount of DCs with the antigen capturing and processing capacity in vitro. Then DGs following EBV membrane antigen gp340 exposure we investigate the use of DCs to generate specific CTL responses to EBV-associated tumor. The main results are described as follows: 1. By Mini-MACS CD34+ hematopoietic stem cells, present at a frequency of about 0.5% in normal bone marrow, can be rapidly and efficiently enriched to a purity of more than 90%. This method may facilitate further studies on the generation of high purity of DCs. These suggest the bone- marrow-derived dendritic cells could be used in tumor biotherapy. 2. CD34+ hematopoietic stem cells of bone marrow can differentiate into large amount of DCs when cultured in presence of GM-CSF.. TNF- a and IL- 4 in vitro for two weeks. More DGs could be obtained when IL-3 was also added. The number of DCs is 5 to 10 times as much as the number of CD34+ hematopoietic stem cells. 3. The DCs expanded from human normal bone marrow have typical d~ndritic morphology and express high levels of major CD1 on cell surface and could stimulate proliferation of allogeneic-T lymphocytes. Therefore, cultured DCs that maintain the antigen capturing and processing capacity and asepsis may facilitate further studies on DGs and their clinic applications. 4. We designed an alternative approach that DGs were pulsed with the EBV envelope glycoprotein gp340. The result indicated that DGs could efficiently present EBV membrane antigen glycoprotein gp340. Moreover, gp340-loaded DCs were higher stimulatory in mixed leukocyte reaction than DCs. 5. In cytotoxicity assays we designed these experiment groups including (1) coculture of DCs and T cell, (2) coculture of gp340-loaded DCs and T cell, (3) coculture of gp340 and T cell. Only gp340-loaded DGs can induce special CTLs against tumor cell concerned with the infection of EBV. Conclusion and prospect: 1. Antigen-presenting cells (APC) play a critical role in the course of EBV membrane antigen including humoral and cellular immune responses...