Identification of human leukemia antigen A*0201-restricted epitopes derived from epidermal growth factor pathway substrate number 8

Abstract

T-cell-mediated immunotherapy of hematological malignancies requires selection of targeted tumor-associated antigens and T-cell epitopes contained in these tumor proteins. Epidermal growth factor receptor pathway substrate 8 (EPS8), whose function is pivotal for tumor proliferation, progression and metastasis, has been found to be overexpressed in most human tumor types, while its expression in normal tissue is low. The aim of the present study was to identify human leukemia antigen (HLA)-A*0201-restricted epitopes of EPS8 by using a reverse immunology approach. To achieve this, computer algorithms were used to predict HLA-A*0201 molecular binding, proteasome cleavage patterns as well as translocation of transporters associated with antigen processing. Candidate peptides were experimentally validated by T2 binding affinity assay and brefeldin-A decay assay. The functional avidity of peptide-specific cytotoxic T lymphocytes (CTLs) induced from peripheral blood mononuclear cells of healthy volunteers were evaluated by using an enzyme-linked immunosorbent spot assay and a cytotoxicity assay. Four peptides, designated as P455, P92, P276 and P360, had high affinity and stability of binding towards the HLA-A*0201 molecule, and specific CTLs induced by them significantly responded to the corresponding peptides and secreted IFN-γ. At the same time, the CTLs were able to specifically lyse EPS8-expressing cell lines in an HLA-A*0201-restricted manner. The present study demonstrated that P455, P92, P276 and P360 were CTL epitopes of EPS8, and were able to be used for epitope-defined adoptive T-cell transfer and multi-epitope-based vaccine design.

Figure 1

Affinity and stability of peptide binding to HLA-A*0201. (A) Affinity of candidate epitopes to the HLA-A*0201 molecule. ^*P<0.01, P455 vs. P276, P360, P92, negative control and T2. ^#P<0.01, P360 vs. P276, P92, negative control, positive control and T2. ^**P<0.01, P276 vs. negative control, positive control and T2. ^##P<0.01, P92 vs. negative control, positive control and T2. Values are expressed as the mean ± standard deviation (n=3). (B) Binding stability of candidate epitopes to HLA-A*0201 molecule. HLA, human leukemia antigen.

Figure 2

EPS8 expression of cell lines. (A) mRNA expression of EPS8, detected by reverse transcription quantitative polymerase chain reaction. Relative gene expression levels of EPS8 were compared using the 2^−ΔΔCt method, with GAPDH used as the internal control. Values are expressed as the mean ± standard deviation (n=3). (B) Protein expression of EPS8, detected by western blot analysis. GAPDH was used as the control. EPS8, epidermal growth factor receptor pathway substrate 8.

Figure 3

The HLA-A phenotype of target cells. (A) MCF-7, (B) T2, (C) THP-1 and (D) K562 cells. Cells were stained with HLA-A2-FITC monoclonal antibody and detected by flow cytometry. T2 cells were used as a positive control. The blue background lines represent T2 cells. MCF-7 cells were HLA-A*0201-positive, and THP-1 and K562 were HLA-A*0201-negative. FITC, fluorescein isothiocyanate; HLA, human leukemia antigen.

Figure 4

Interferon-γ secretion of specific CTLs in response to corresponding peptides. The number of SFCs was evaluated using an ELISPOT assay against four candidate epitopes of EPS8, with each experiment performed in triplicate with 2×10⁵ cells/well, and the average number of spots was calculated. PBMCs only served as a negative control. PHA stimulation served as a positive control. Bar graphs indicate the mean ± standard deviation. (A) Images of SFCs for volunteer 1. (B) Images of SFCs for volunteer 2. (C) Comparison of the number of SFCs of each candidate’s epitopes. Values are expressed as the mean ± standard deviation (n=3). ^✩P<0.05 compared to negative control, X-VIVO medium. PBMC, peripheral blood mononuclear cell; PHA, phytohaemagglutinin-M; CTL, cytotoxic T lymphocyte; EPS8, epidermal growth factor receptor pathway substrate 8; SFC, spot forming cell.

Figure 5

Functional characterization of candidate epitope-specific CTLs. LDH release assay using a ratio of effectors to targets of 100:1. (A) Results for volunteer 1; (B) Results for volunteer 2. Values are expressed as the mean ± standard deviation of LDH release. ^*P<0.05 compared to negative control. CP, cognate peptides, NP, irrelevant peptide; CTL, cytotoxic T lymphocyte.