Adenovirus-Mediated MUC1 gene transduction into human blood-derived dendritic cells.
MUC1 protein is widely expressed on various human cancer cells and has a specific highly glycosylated core structure with multiple tandem repeats, which may include an immunogenic peptide sequence. The potency of MUC1 protein to induce human histocompatibility leukocyte antigen-class I-restricted cytotoxic T-lymphocyte (CTL) induction remains to be fully clarified in human beings. In the current study, we made MUC1-expressing human dendritic cells (DCs) using recombinant adenovirus vector. Adenovirus vector plasmid containing human MUC1 cDNA, pAdHM4-MUC1 was constructed using in vitro ligation with a shuttle vector, pHMCMV5. Adenovirus vector expressing MUC1 was generated by the transfection of PacI-digested recombinant vector plasmid into 293 cells. Human blood DCs were obtained from 7-day culture of monocytes with recombinant human (rh) granulocyte-macrophage (GM) colony-stimulating factor (CSF) and (rh)interleukin (IL)-4. Then, 1 x 10(6) DCs were incubated with viral supernatant at a multiplicity of infection of 200 for 24 h in the presence of rhGM-CSF and rhIL-4. Flow cytometric analysis showed that 30% to 40% of the transduced DCs expressed MUC I protein; by contrast, nontransduced or transduced DCs with mock virus expressed only small amounts of MUC1 protein. Adenovirus-mediated MUC1 gene transduction into DCs had no significant effect on DC surface marker expressions or functions such as mixed leukocyte reaction. Furthermore, MUCI-specific CD8+ CTLs could be induced from healthy donor blood lymphocytes using MUC1-expressing DCs as stimulators. These results suggested that MUC1 gene-transduced DCs are a functional and potent tool for triggering a CTL response against MUC1 cancer cells.
Maruyama
K
K
Growth Factor Division, National Cancer Center Research Institute, Tokyo, Japan.
Akiyama
Y
Y
Nara-Ashizawa
N
N
Hojo
T
T
Cheng
J Y
JY
Mizuguchi
H
H
Hayakawa
T
T
Yamaguchi
K
K
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D019556:COS Cells; D003601:Cytotoxicity Tests, Immunologic; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007959:Lymphocyte Culture Test, Mixed; D018396:Mucin-1 / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
C116055:MUC1 tandem repeat peptide; D018396:Mucin-1; D010446:Peptide Fragments