Purification of antigenic peptide from murine hepatoma cells recognized by Class-I major histocompatibility complex molecule-restricted cytotoxic T-lymphocytes induced with B7-1-gene-transfected hepatoma cells.
OBJECTIVE
It has been reported that expression of costimulatory molecules, such as B7, on tumors is essential for priming tumor-specific cytotoxic T-lymphocytes (CTLs). Here, we have attempted to induce murine hepatoma-specific CTLs by immunizing with the B7-1-gene-expressing hepatoma cells, and to identify the epitope(s) presented on the hepatoma cells.


METHODS
The B7-1-gene encoding plasmid was transferred into the murine hepatoma cell line, Hepa1-6. Syngeneic C57BL/6 mice were immunized with the B7-1-transfected cells via various routes to prime CTLs. The mild acid elution method was used to isolate antigenic fractions from the class-I major histocompatibility complex (MHC) molecules on the Hepa1-6 cells. Cytotoxicity was measured by standard 51Cr-releasing assay. The effect of the CTLs on hepatoma growth was evaluated in hepatoma-bearing SCID mice to which the cells were preadministered.


RESULTS
A clone, termed L1, highly expressing the B7-1-gene, has been established. Killer cells generated from mice immunized intraperitoneally with L1 cells eliminated both L1 and Hepa1-6 cells, and also another syngeneic hepatoma cell line, Hepa1-clc7. The killer cells were CD8+ and the class-I MHC molecule-restricted CTLs which might recognize hepatoma-specific antigenic peptide(s) in association with the D(b)class-I MHC molecules. A functional peptide fraction was obtained from eluted fluid of the Hepa1-6 cells. In addition, intravenous preadministration of the CTLs inhibited the hepatoma growth in SCID mice.


CONCLUSIONS
The hepatoma epitope-specific CTLs which suppressed hepatoma growth in vivo could be generated with the B7-1-gene-transfected hepatoma cells. These results will be useful in establishing immunotherapy against hepatocellular carcinoma.
Department of Microbiology and Immunology, Nippon Medical School, Tokyo, Japan.
K
K
Nakatsuka
H
H
Sugiyama
Y
Y
Nakagawa
H
H
Takahashi
D000818:Animals; D000951:Antigens, Neoplasm / Q000302:isolation & purification*; D018122:B7-1 Antigen / Q000235:genetics / Q000276:immunology*; D016827:CD8 Antigens / Q000032:analysis; D000939:Epitopes / Q000302:isolation & purification; D005260:Female; D005805:Genes, MHC Class I; D007694:Killer Cells, Natural / Q000276:immunology; D008114:Liver Neoplasms, Experimental / Q000276:immunology* / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D010455:Peptides / Q000276:immunology*; D010957:Plasmids; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D018122:B7-1 Antigen; D016827:CD8 Antigens; D000939:Epitopes; D010455:Peptides