Blood donor derived dendritic cells and cytotoxic T cells for specific fusion-gene adoptive immunotherapy.
OBJECTIVE
Therapeutic immunological reagents tailored to individual patients have been shown to be a viable treatment strategy for some forms of leukaemia. This work investigates the possibility of using blood donations as a source of leukaemia-specific immune therapeutics.


METHODS
The acute promyelocytic cell line NB4 carrying the PML-RAR alpha fusion was used as a target for cytotoxic T lymphocytes (CTL) stimulated to recognize the fusion. Stimulation of CTL was by production of dendritic cells pulsed with plasmid vectors containing polymerase chain reaction (PCR)-generated sequences of PML-RAR alpha derived from NB4 cells. PCR primer design included a Kozak consensus sequence to allow correct translation of the nucleic acid into protein. Identification of specific cytotoxicity was by both Granzyme B ELISPOT and by (51)Cr-release assays.


RESULTS
Specific CTL activity targeting NB4 cells can be generated from donor-derived peripheral blood mononuclear cells. However, individual donors appear to respond differently to the length of stimulatory sequence encoded in the vector. Use of an internal methionine in the PML gene, which also satisfies the Kozak rules, allows translation in vitro and, thus, might provide a suitable start site for stimulation using acute promyelocytic leukaemia-specific sequence.


CONCLUSIONS
The work presented here suggests that blood donor derived dendritic cells can be used to stimulate leukaemia-specific CTL from the same donation ex vivo. This would enable the generation of patient-specific therapeutics from major histocompatibility (MHC)-matched allogeneic donors. However, different MHC-matched donors might vary in their response depending on the length of the antigenic sequence.
Gallagher
R C J
RC
SNBTS Cell Therapy Group, Centre for Regenerative Medicine, Edinburgh, UK. ronnie.gallagher@ed.ac.uk
Waterfall
M
M
Samuel
K
K
Turner
M L
ML
D000595:Amino Acid Sequence; D001782:Blood Donors; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D003602:Cytotoxicity, Immunologic; D003713:Dendritic Cells / Q000276:immunology*; D050939:Gene Fusion; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics / Q000276:immunology / Q000628:therapy; D008969:Molecular Sequence Data; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014162:Transfection
D015514:Oncogene Proteins, Fusion; C093476:promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein