Expression of mucin (MUC-1) from a mini-Epstein-Barr virus in immortalized B-cells to generate tumor antigen specific cytotoxic T cells.
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.
Kilger
E
E
GSF-National Research Center for Environment and Health, Institut für Klinische Molekularbiologie und Tumorgenetik, Munich, Germany.
Pecher
G
G
Schwenk
A
A
Hammerschmidt
W
W
D017951:Antigen Presentation; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D015870:Gene Expression; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D018396:Mucin-1 / Q000235:genetics* / Q000276:immunology*; D010446:Peptide Fragments / Q000235:genetics* / Q000276:immunology*; D010957:Plasmids / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D017931:DNA Primers; D000939:Epitopes; C116055:MUC1 tandem repeat peptide; D018396:Mucin-1; D010446:Peptide Fragments