Identification of a new HLA-A*0201-restricted CD8+ T cell epitope from hepatocellular carcinoma-associated antigen HCA587

Abstract

For the development of peptide-based cancer immunotherapies, we aimed to identify specific HLA-A*0201-restricted CTL epitopes in hepatocellular carcinoma (HCC) associated antigen HCA587, which has been identified as a member of the cancer/testis (CT) antigens highly expressed in HCC. We first combined the use of an HLA-A*0201/peptide binding algorithm and T2 binding assays with the induction of specific CD8(+) T cell lines from normal donors by in vitro priming with high-affinity peptides, then IFN-gamma release and cytotoxicity assays were employed to identify the specific HLA-A*0201 CD8(+) T cell epitope using peptide-loaded T2 cells or the HCA587 protein(+) HCC cell line HepG2. In the six candidate synthesized peptides, two peptides showed higher binding ability in T2 binding assays. No. 2 peptide, encompassing amino acid residues FLAKLNNTV (HCA587(317-325)), was able to activate a HCA587-specific CD8(+) T-cell response in human lymphocyte cultures from two normal donors and two HCC patients, and these HCA587-specific CD8(+) T cells recognized peptide-pulsed T2 cells as well as the HCA587 protein(+) HCC cell line HepG2 in IFN-gamma release and cytotoxicity assays. The results indicate that no. 2 peptide is a new HLA-A*0201-restricted CTL epitope capable of inducing HCA587-specific CTLs. Our data suggest that identification of this new HCA587/HLA-A*0201 peptide FLAKLNNTV may facilitate the design of peptide-based immunotherapies for the treatment of HCA587-bearing HCC patients.

Fig. 1

Facs analysis of T2 binding assay. The binding affinity of each HCA587 peptide and control (flu) peptide (50 µg/ml) was determined by incubation with T2 cells overnight in serum-free medium containing human β₂-M (5 µg/ml).

Fig. 2

Effector activity of in vitro peptide-primed CD8⁺ T cells in interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assays. CD8⁺ T cells from two different normal donors (each donor shown in separate column) were, respectively, stimulated four times with HCA587 no. 2 and no. 3 peptide (each shown in separate rows). The CD8⁺ T cells (2·5 × 10⁴/well) were then restimulated with T2 cells (5 × 10⁴/well) loaded with various peptides (10 µg/ml) in IFN-γ release ELISPOT assays. Number of specific responsive spots per well is shown as mean ± standard deviation from three parallel wells. The peptide-stimulated specific CD8⁺ T cells response was dramatically blocked by anti-HLA class I monoclonal antibody (mAb) (W6/32), but not by anti-HLA class II mAb (L243).

Fig. 3

Effector activity of in vitro recombinant HCA587 protein-primed CD8⁺ T cells in interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assays. Peripheral blood lymphocytes (PBLs) from two different normal donors (donor 1 and donor 2) were stimulated four times with HCA587 recombinant protein, then the effector CD8⁺ T cells were positively purified with Dynabeads. The effector CD8⁺ T cells (2·5 × 10⁴/well) were restimulated with T2 cells (5 × 10⁴/well) loaded with various peptides(10 µg/ml) in IFN-γ release ELISPOT assays. Number of specific responsive spots per well is shown as mean ± standard deviation from three parallel wells.

Fig. 4

Analysis of HCA587 mRNA expression in HCC cell lines. HCA587 mRNA expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR) from the cDNA samples prepared from HCC cell lines and testis: lane 1, HepG2; lane 2, HLE; lane 3, SMMC-7721; lane 4, Bel-7402; lane 5, Bel-7405; lane 6, testis as positive control; lane 7, H₂O as negative control.

Fig. 5

Analysis of HCA587 protein expression in hepatocellular carcinoma (HCC) cell lines. HCA587 protein expression was assessed by immunochemical staining in HCA587 mRNA⁺ HepG2 cells (a), HCA587 mRNA^– HLE cells (b) and SMMC-7721 (c) with anti-HCA587 polyclonal antibody. The slides were then stained with the peroxidase-labelled goat-anti-rabbit IgG. The colour was developed with diaminobenzidine (DAB) chromogen and the slides were counterstained with haematoxylin.

Fig. 6

In vitro HCA587 no. 2 peptide-primed CD8⁺ T cells from the two donors recognized the hepatocellular carcinoma (HCC) cell lines loaded with no. 2 peptide (10 µg/ml) or not. The interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assays were performed using the peptide-reactive CD8⁺ T cells (2·5 × 10⁴/well) from donor 1 and donor 2 to estimate its activity against HCA587 protein⁺ HepG2 cells (5 × 10⁴/well), HCA587 protein^– HLE cells (5 × 10⁴/well) and SMMC-7721 (5 × 10⁴/well). Number of specific responsive spots per well is shown as mean ± standard deviation from three parallel wells.

Fig. 7

Granzyme B cytotoxic activity of no. 2 peptide-specific bulk CTLs from two HLA-A*0201 hepatocellular carcinoma (HCC) patients on HCC cell lines. Effector cells(2·5 × 10⁴/well) were run against HepG2, HLE, SMMC-7721 targets (5 × 10⁴/well) in a 4-h assay at 37°C. Data is presented as spots per well ± standard deviation and is representative of three experiments with similar results.