E Kilger1, G Pecher, A Schwenk, W Hammerschmidt. 1. GSF-National Research Center for Environment and Health, Institut für Klinische Molekularbiologie und Tumorgenetik, Munich, Germany.
Abstract
BACKGROUND: EBV immortalized B-cells can be used as antigen presenting cells (APC) to stimulate specific T-cell responses. Mini-Epstein-Barr virus (mini-EBV) plasmids contain all functional elements of Epstein-Barr virus (EBV) necessary to immortalize B-cells in vitro. These immortalized B-cells are incapable of releasing infectious virus in contrast to cells immortalized by wildtype EBV. In addition, mini-EBVs can be modified in E. coli to alter their genetic composition or adopt new genes. METHODS: We constructed a mini-EBV plasmid carrying an expression cassette for the human tumor antigen mucin encoded by the gene MUC-1. Primary human B-cells were infected with the MUC-1 carrying mini-EBV plasmid packaged into an EBV coat and immortalized B-cell clones were expanded in vitro. These B-cells were analyzed by FACS analyses for the expression of mucin and co-stimulatory molecules and were subsequently used as antigen presenting cells (APC) to stimulate peripheral blood mononuclear cells from healthy donors. RESULTS: Several B-cell lines were established that were shown to be free of helper virus or wildtype EBV. These B-cells expressed the relevant tumor-specific epitopes of mucin and the co-stimulatory ligands B7.1 and B7.2 necessary for efficient T-cell activation. Using the mucin expressing B-cells as antigen presenting cells (APC) mucin-epitope specific cytotoxic T-cells were established. CONCLUSIONS: Virus-free B-cell lines expressing tumor-associated epitopes such as mucin or other antigens of interest provide an unlimited and safe source of APC to generate antigen specific T-cells which could be used for clinical trials in adoptive immune therapy or cancer vaccines.
BACKGROUND:EBV immortalized B-cells can be used as antigen presenting cells (APC) to stimulate specific T-cell responses. Mini-Epstein-Barr virus (mini-EBV) plasmids contain all functional elements of Epstein-Barr virus (EBV) necessary to immortalize B-cells in vitro. These immortalized B-cells are incapable of releasing infectious virus in contrast to cells immortalized by wildtype EBV. In addition, mini-EBVs can be modified in E. coli to alter their genetic composition or adopt new genes. METHODS: We constructed a mini-EBV plasmid carrying an expression cassette for the humantumor antigen mucin encoded by the gene MUC-1. Primary human B-cells were infected with the MUC-1 carrying mini-EBV plasmid packaged into an EBV coat and immortalized B-cell clones were expanded in vitro. These B-cells were analyzed by FACS analyses for the expression of mucin and co-stimulatory molecules and were subsequently used as antigen presenting cells (APC) to stimulate peripheral blood mononuclear cells from healthy donors. RESULTS: Several B-cell lines were established that were shown to be free of helper virus or wildtype EBV. These B-cells expressed the relevant tumor-specific epitopes of mucin and the co-stimulatory ligands B7.1 and B7.2 necessary for efficient T-cell activation. Using the mucin expressing B-cells as antigen presenting cells (APC) mucin-epitope specific cytotoxic T-cells were established. CONCLUSIONS: Virus-free B-cell lines expressing tumor-associated epitopes such as mucin or other antigens of interest provide an unlimited and safe source of APC to generate antigen specific T-cells which could be used for clinical trials in adoptive immune therapy or cancer vaccines.
Authors: A Schepers; M Ritzi; K Bousset; E Kremmer; J L Yates; J Harwood; J F Diffley; W Hammerschmidt Journal: EMBO J Date: 2001-08-15 Impact factor: 11.598