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

Abstract

Background/Aim: 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 moleculerestricted CTLs which might recognize hepatoma-specific antigenic peptide(s) in association with the D bclass-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.