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Comparative Study
. 2008 Aug;99(8):1656-62.
doi: 10.1111/j.1349-7006.2008.00866.x.

Erythropoietin-producing hepatocyte B6 variant-derived peptides with the ability to induce glioma-reactive cytotoxic T lymphocytes in human leukocyte antigen-A2+ glioma patients

Mingyue Jin  1 , Yoshihiro Komohara, Shigeki Shichijo, Ryuya Yamanaka, Junichi Nikawa, Kyogo Itoh, Akira Yamada
Affiliations
Comparative Study

Erythropoietin-producing hepatocyte B6 variant-derived peptides with the ability to induce glioma-reactive cytotoxic T lymphocytes in human leukocyte antigen-A2+ glioma patients

Mingyue Jin et al. Cancer Sci. 2008 Aug.

Abstract

We recently cloned a variant form of erythropoietin-producing hepatocyte (Eph)B6, a member of the Eph receptor tyrosine kinase family. In the present study, we examined the expression of the EphB6 variant (EphB6v) in a panel of brain tumor cell lines and glioblastoma tissues and we found that EphB6v was preferentially expressed in malignant brain tumors, such as glioblastomas and anaplastic astrocytomas. The EphB6v has a unique 54 amino acid sequence at the C-terminal that is not found in normal EphB6. Therefore, we attempted to identify antigenic peptides unique to EphB6v for immunotherapy. The two EphB6v-derived peptides exhibited the ability to bind to human leukocyte antigen (HLA)-A0201 molecules, and each of them was able to induce cytotoxic T lymphocytes in vitro in the peripheral blood mononuclear cells of HLA-A2(+) glioma patients. The cytotoxicity was mediated by peptide-specific CD8(+) T cells in an HLA-A2-restricted manner. The expression of EphB6v was also observed in different types of cancer (e.g. lung, colon, stomach, liver and pancreatic) cells. Taken together, the two peptides derived from EphB6v might be appropriate targets for peptide-based specific immunotherapy for HLA-A2(+) patients with various cancers.

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Figures

Figure 1
Figure 1
Comparison of erythropoietin‐producing hepatocyte (Eph)B6 expression in various cell lines and malignant glioma tissues. The mRNA levels of EphB6 and the EphB6 variant (EphB6v) in (a) brain tumor cell lines, (b) glioblastoma tissues and (c) non‐tumor cells were detected by reverse transcription polymerase chain reaction as described in Materials and Methods. Tubulin was used as an internal control. (d) Semiquantitative amplification of template cDNA in this system was confirmed by using serial dilution of cDNA samples from KNS‐60 cells. (e) The protein expression of EphB6v in brain tumor cell lines was confirmed by Western blot analysis using rabbit antiserum to EphB6v 472–486 peptide. PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin.
Figure 2
Figure 2
Binding capacity of erythropoietin‐producing hepatocyte B6 variants (EphB6v)‐3 and ‐4 peptides to human leukocyte antigen (HLA)‐A0201 molecules was examined by an HLA stabilizing assay using RMA‐S‐A0201 cells. Each point indicates the mean fluorescence intensity (MFI) of HLA expression from duplicate assays. DMSO, dimethylsulfoxide solvent alone; EBV, Epstein–Barr virus.
Figure 3
Figure 3
The cytotoxicity of erythropoietin‐producing hepatocyte B6 variants (EphB6v)‐3 (upper panels) and ‐4 (lower panels) peptide‐induced cytotoxic T lymphocytes from human leukocyte antigen (HLA)‐A2+ patients (right panels) and healthy donors (left panels) against KNS‐81 (EphB6v+HLA‐A2−), KNS‐81‐A0201 (EphB6v+HLA‐A2+), U251MG (R596) (EphB6v−HLA‐A2+), and phytohemagglutinin (PHA) blasts (EphB6v+HLA‐A2+) was examined by standard 6‐h 51Cr‐release assay. Representative results are shown. E/T, effector/target.
Figure 4
Figure 4
Cold‐target inhibition and antibody blocking of the cytotoxicity of erythropoietin‐producing hepatocyte B6 variant (EphB6v) peptide‐induced cytotoxic T lymphocytes (CTL) from malignant glioma patients and healthy donors. The purified CD8+ T‐cell fraction of peptide‐induced CTL from (a,c) human leukocyte antigen (HLA)‐A2+ malignant glioma patients or (b) healthy donors were tested for cytotoxicity against KNS‐81‐A0201 cells in the presence of cold T2 cells preloaded with either the corresponding peptide or the HIV peptide (a, b) or anti‐class I HLA (W6/32) mAb (c). Anti‐CD14 monoclonal antibody (mAb) was also used as an isotype‐matched control mAb. Cytotoxicity was significantly inhibited by the addition of either cold‐target cells or W6/32. *Values are statistically significant from that of corresponding controls as determined by Student's t‐test, P < 0.05. E/T, effector/target.
Figure 5
Figure 5
The expression of erythropoietin‐producing hepatocyte B6 (EphB6) was examined in pancreatic cancer, stomach cancer, colon cancer, liver cancer and lung cancer cell lines, as described in Materials and Methods. EphB6 expression was detected in all tested cancer cell lines; in particular, the EphB6 variant was preferentially expressed in hepatocellular carcinoma and in lung squamous and adenocarcinoma cell lines. Tubulin was used as an internal control. SCC, squamous cell carcinoma.
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