The Transfection Of DCs With Recombinant Vaccinia Carrying EBV-LMP2A Gene And LMP2A-specific CTL Induction In Vitro

The program aims at inducing the Epstein-Barr virus (EBV)-LMP2A specific cytotoxic T lymphocyte (CTL) by co-culturing dendritic cells (DCs) and T lymphocyte in vitro. EBV is a ubiquitous human y -herpes virus that has been associated with several malignant diseases. These diseases include nasopharyngeal carcinoma (NPC), Burkitt lymphoma, approximately 50% of Hodgkin's disease, and lymphoproliferative disorders in the immunocompromised patients. Radiotherapy and chemotherapy are the primary treatments for those patients, but none of such treatments can eliminate tumor cells thoroughly, for example, in NPC patients. Therefore, it is necessary to explore any other alternative treatments for such EBV-associated diseases. Priming of CTL response requires the presentation of the relevant antigen by professional antigen-presenting cells (APCs) capable of providing ostimulation? DCs are efficient APCs for CTL induction and may be an attractive adjuvant for anti-tumor immunity. Since DCs own the most potent function of antigen-presentation and can stimulate the proliferation of naive T cells. First, monocytes were isolated from normal human peripheral blood mononuclear cells (PBMCs) and cultured with cytokines including GM-C SF, IL-4 and TNF- a , to induce and expand mature DCs. Next, DCs were infected with recombinant vaccinia carrying 4 EBV latent membrane protein (LMP)-2A gene and then were co-cultured with autologous PMBCs, with the stimulation from IL-2. CTLs were finally gained and assayed. Results are showed briefly as following: 1 .PBMCs were cultured in GM-C SF, IL-4 and TNF- a . Ten days later, large amount of mature and functional DCs were obtained. Surface marker assay by FACS showed that DCs expressed CD83 (94.2%), which is the relative specific marker of DCs, and HLA-ABC (98.3%), HLA-DR (96%), CD4O (97.7%) and CD8O (98.2%). Mixed lymphocyte reaction (MLR) suggested that DCs induced through such pathway were strongly potent to stimulate the proliferation of allo-T cells. 2.EBV-LMP2A gene can be successfully transfected into DCs through recombinant vaccinia carrying such gene. Suitable conditions include M0110, incubation for 12 hours, radioaction by ultraviolet in 35cm distance for 30 minutes. 3.DCs transfected with LMP-2A gene were cultured with autologous PBMCs at the ratio 1: 20, added IL-2 and half-renewed cell media every 4 days. At the 15th day, CTL cytotoxicity assays were performed by LDH kits. Our results suggested that EBV-specific CTL can be induced by DCs infected with recombinant vaccinia carrying EBV-LMP2A gene in vitro, although LMP2A itself is weakly immunogenic. Such results can provide experimental basis for clinical treatments of EBV-associated diseases.