Experimental study of transfecting adenovirus carrying G250 antigen gene to dendritic cells and to activate immune effector cells in kidney cancer treatment

Abstract

Objective To investigate T cell-mediated effects of dendritic cells(DC)transfected with kidney associated antigen G250 gene using adenovirus(Ad)as vector in the treatment of kidney cancer. Methods Peripheral blood mononuclear cells(PBMC)were isolated by standard FicollPaque density gradient centrifugation of heparinized blood obtained from healthy donors.After removal of the nonadherent ceils,the adherent cells were divided into gene transfer group,protein pulsed group and control group.The gene transfer group was infected with Ad/G250 virus;the protein pulsed group was added with G250 protein; the control group was added with PBS.The DCs were cultured and proliferated in vitro using recombinant human granulocyte macrophage colony stimulating factor and inlerleukin-4.The autologous T cells were added into the 3 DC groups(responders:dendritics,20:1).After 5 d co-culture,3 eytotoxic T lymphocyte(CTL)groups were collected.TheG250 mRNA expression was detected in transduced DCs using RT-PCR.The G250 protein translation products and cell surface marker of DCs were analyzed by flow cytometry.The specific cytolytic activities of CTL to different target cells(renal carcinoma 786-0 cell line and lung cancer A549 cell line)were assessed by MTT method. Results Ad/G250 transfected DC successfully and the G250 expression was confirmed by RT-PCR. The expression levels of CD_(80), CD_(83). HLA-DR and CD_(86) in the Ad/G250 group were higher than those in the other 2 groups. The cytotoxicity to 786-0 of T lymphocytes activated by Ad/G250 transfecting DCs(83. 4±2. 8)% was greater than those of T lymphocytes in the protein pulsed group(79. 6±2. 4)% and control group(77. 3±2. 1)%. There were no significant differences of the cytotyxicities to A549 among the 3 cytotoxic T lymphocyte groups(F=0. 373.P=0.693). Conclusions Ad-loading DCs may be useful for immunotherapeutic protocols against self-antigens for the kidney cancer. Key words: Dendritic cells; Carcinoma, renal cell; Adenoviridae; T-lymphocytes, cytotoxic