PURPOSE: The human carcinoembryonic antigen (CEA) is expressed in several tumor types, including colorectal cancer, and is a tumor-associated antigen used as a target for antigen-specific immunotheraphy. CEA is a self-antigen associated with development, expressed in fetal cells and rarely expressed in normal colorectal epithelial cells. The induction of immune response to CEA is very difficult. In this study, we attempted to increase the tumor immunity specific to CEA by using dendritic cells pulsed with fusion proteins of CEA and Tat (transactivator of transcription), which transduces extracellular proteins into cytoplasm and causes antigens to be presented with MHC class I pathway. METHODS: The Tat gene was amplified in the PNL4-3 HIV plasmid and then inserted into PCEP4 plasmid vector. The CEA gene was cloned from cDNA from LoVo human colorectal cell line and then amplified through polymerase chain reaction method. After cloning of PCEP4 plasmid vector, the dendritic cell was sensitized and internalized with CEA and Tat-CEA protein. Then the Western blot analysis of the expression of CEA in the gene-modified dendritic cell and the immunofluorescent staining of the expression of CEA in CEA or Tat-CEA-pulsed dendritic cell were performed. A detection of IFN-gamma-releasing CD8 cell and a cytotoxicity of T-cell were was assesed using ELISPOT assay. The Immunoglobulin (Ig) G isotypes were analyzed with enzyme-linked immunosorbent assay. The statistical significance was assessed using Students t-test. RESULTS: CEA pulsed in dendritic cells was distributed over the cell surface and TatCEA was observed in the cytoplasm. The cellular immune responses by immunization with dendritic cells pulsed with TatCEA (322/10(4) lymphocytes) were significantly increased compared with those with CEA (244/10(4) lymphocytes) by IFN-gamma ELISPOT assay (P<0.05). The cytotoxic T lymphocyte (CTL) activity using mouse T-cell, EL-4 pulsed peptide (EAQNTTYL) as target cells was 23.3+/-2.75% (E:T=1:100) in the CEA group and 22.9+/-2.23% (E:T=1:100) in the TatCEA group. In ELISA analysis of the IgG isotype, the titer of IgG2a and IgG3, representing Th1 immune response, was lower than that of IgG1, representing Th2 immune response, in both the CEA group and the TatCEA group. CONCLUSIONS: These results suggest that TatCEA could be used for the development of a tumor vaccine and cellular immunotherapy using CTLs induced in vitro.
Animals ; Blotting, Western ; Carcinoembryonic Antigen ; Cell Line ; Clone Cells ; Cloning, Organism ; Colorectal Neoplasms ; Cytoplasm ; Dendritic Cells ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Enzyme-Linked Immunospot Assay ; Epithelial Cells ; Equidae ; Genes, tat ; HIV ; Humans ; Immunity, Cellular ; Immunization ; Immunoglobulin G ; Immunoglobulins ; Immunotherapy ; Lymphocytes ; Mice ; Plasmids ; Polymerase Chain Reaction ; T-Lymphocytes