Expression of mucin (MUC-1) from a Mini-Epstein-Barr virus in immortalized B-cells to generate tumor antigen specific cytotoxic T cells
✍ Scribed by Ellen Kilger; Gabriele Pecher; Arvid Schwenk; Wolfgang Hammerschmidt
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 254 KB
- Volume
- 1
- Category
- Article
- ISSN
- 1099-498X
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✦ Synopsis
Background EBV immortalized B-cells can be used as antigen presenting cells (APC) to stimulate speci®c 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 modi®ed 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-speci®c epitopes of mucin and the co-stimulatory ligands B7.1 and B7.2 necessary for ef®cient T-cell activation. Using the mucin expressing B-cells as antigen presenting cells (APC) mucin-epitope speci®c 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 speci®c T-cells which could be used for clinical trials in adoptive immune therapy or cancer vaccines.