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Heat shock protein DNAJB8 is a novel target for immunotherapy of colon cancer-initiating cells.

Morita R, Nishizawa S, Torigoe T, Takahashi A, Tamura Y, Tsukahara T, Kanaseki T, Sokolovskaya A, Kochin V, Kondo T, Hashino S, Asaka M, Hara I, Hirohashi Y, Sato N - Cancer Sci. (2014)

Bottom Line: In the present study, we found that a heat shock protein (HSP) 40 family member, DnaJ (Hsp40) homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in CSC/CIC derived from colorectal cancer (CRC) cells rather than in non-CSC/CIC.Overexpression of DNAJB8 enhanced the expression of stem cell markers and tumorigenicity, indicating that DNAJB8 has a role in CRC CSC/CIC.A CTL clone specific for DNAJB8 peptide showed higher killing activity to CRC CSC/CIC compared with non-CSC/CIC, and CTL adoptive transfer into CRC CSC/CIC showed an antitumor effect in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan.

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Antitumor effect of DNAJB8-specific cytotoxic T lymphocyte (CTL) clone. (a) Interferon-γ enzyme-linked immunospot assay. (b) 51Cr release assay. We evaluated specific cytotoxic activity against peptide-pulsed T2-A24 cells in healthy donors. (c, d) 51Cr release assay using the DNAJB8_143(9)-specific CTL clone. We established CTL clones recognizing DNAJB8_143(9) and evaluated cytotoxic activity against side population (SP) cells and main population (MP) cells derived from HT29 cells. (e) Tumor growth of HT29 cells in a therapeutic adoptive transfer model. HT29 cells were inoculated subcutaneously into the back of five NOD/SCID mice and CTL clone cells or PBS was injected intravenously 3 weeks later. Tumor growth was measured weekly. Data represent means ± SD. Differences between groups were examined for statistical significance using the Student's t-test.
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fig04: Antitumor effect of DNAJB8-specific cytotoxic T lymphocyte (CTL) clone. (a) Interferon-γ enzyme-linked immunospot assay. (b) 51Cr release assay. We evaluated specific cytotoxic activity against peptide-pulsed T2-A24 cells in healthy donors. (c, d) 51Cr release assay using the DNAJB8_143(9)-specific CTL clone. We established CTL clones recognizing DNAJB8_143(9) and evaluated cytotoxic activity against side population (SP) cells and main population (MP) cells derived from HT29 cells. (e) Tumor growth of HT29 cells in a therapeutic adoptive transfer model. HT29 cells were inoculated subcutaneously into the back of five NOD/SCID mice and CTL clone cells or PBS was injected intravenously 3 weeks later. Tumor growth was measured weekly. Data represent means ± SD. Differences between groups were examined for statistical significance using the Student's t-test.

Mentions: The PBMC of two healthy volunteer donors (A and B) were stimulated using a mixture of DNAJB8_22(9), DNAJB8_99(9) and DNAJB8_143(9) and then the reactivity for each peptide was evaluated using a IFN-γ ELISpot assay and 51Cr release assay. Interferon-γ secretion was observed for DNAJB8_22(8)-pulsed and DNAJB8_143(9)-pulsed target cells from both donors using a IFN-γ ELISpot assay (Fig. 4a), whereas cytotoxic activity was detectable for only DNAJB8_143(9)-pulsed target cells using a 51Cr release assay (Fig. 4b). Therefore, DNAJB8_143(9) peptide is a candidate for DNAJB8-targeting immunotherapy. We generated four CTL clones specific for DNAJB8_143(9) from donor A (CTL clone #21, 67 and 84) and one clone from donor B (CTL clone #70) and performed further analysis using CTL clone #84. The DNAJB8_143(9)-specific CTL clone showed cytotoxic activity for T2-A24 cells pulsed with DNAJB8_143(9) peptide but not for T2-A24 cells without the peptide or for K562 cells (Fig. 4c). To verify whether this CTL clone can recognize endogenously presented DNAJB8_143(9) peptide of DNAJB8-positive CRC CSC/CIC, we performed a 51Cr release assay using SP cells derived from HT29 cells. The DNAJB8_143(9)-specific CTL clone showed greater cytotoxic activity for HLA-A*2402+ HT29-SP cells than for HLA-A*2402+ HT29-MP cells or HLA-A*2402-DNAJB8- K562 cells (Fig. 4d). These results indicate that DNAJB8_143(9) peptide is an immunogenic epitope and that the endogenously processed peptide is presented on the surface of SP cells.


Heat shock protein DNAJB8 is a novel target for immunotherapy of colon cancer-initiating cells.

Morita R, Nishizawa S, Torigoe T, Takahashi A, Tamura Y, Tsukahara T, Kanaseki T, Sokolovskaya A, Kochin V, Kondo T, Hashino S, Asaka M, Hara I, Hirohashi Y, Sato N - Cancer Sci. (2014)

Antitumor effect of DNAJB8-specific cytotoxic T lymphocyte (CTL) clone. (a) Interferon-γ enzyme-linked immunospot assay. (b) 51Cr release assay. We evaluated specific cytotoxic activity against peptide-pulsed T2-A24 cells in healthy donors. (c, d) 51Cr release assay using the DNAJB8_143(9)-specific CTL clone. We established CTL clones recognizing DNAJB8_143(9) and evaluated cytotoxic activity against side population (SP) cells and main population (MP) cells derived from HT29 cells. (e) Tumor growth of HT29 cells in a therapeutic adoptive transfer model. HT29 cells were inoculated subcutaneously into the back of five NOD/SCID mice and CTL clone cells or PBS was injected intravenously 3 weeks later. Tumor growth was measured weekly. Data represent means ± SD. Differences between groups were examined for statistical significance using the Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig04: Antitumor effect of DNAJB8-specific cytotoxic T lymphocyte (CTL) clone. (a) Interferon-γ enzyme-linked immunospot assay. (b) 51Cr release assay. We evaluated specific cytotoxic activity against peptide-pulsed T2-A24 cells in healthy donors. (c, d) 51Cr release assay using the DNAJB8_143(9)-specific CTL clone. We established CTL clones recognizing DNAJB8_143(9) and evaluated cytotoxic activity against side population (SP) cells and main population (MP) cells derived from HT29 cells. (e) Tumor growth of HT29 cells in a therapeutic adoptive transfer model. HT29 cells were inoculated subcutaneously into the back of five NOD/SCID mice and CTL clone cells or PBS was injected intravenously 3 weeks later. Tumor growth was measured weekly. Data represent means ± SD. Differences between groups were examined for statistical significance using the Student's t-test.
Mentions: The PBMC of two healthy volunteer donors (A and B) were stimulated using a mixture of DNAJB8_22(9), DNAJB8_99(9) and DNAJB8_143(9) and then the reactivity for each peptide was evaluated using a IFN-γ ELISpot assay and 51Cr release assay. Interferon-γ secretion was observed for DNAJB8_22(8)-pulsed and DNAJB8_143(9)-pulsed target cells from both donors using a IFN-γ ELISpot assay (Fig. 4a), whereas cytotoxic activity was detectable for only DNAJB8_143(9)-pulsed target cells using a 51Cr release assay (Fig. 4b). Therefore, DNAJB8_143(9) peptide is a candidate for DNAJB8-targeting immunotherapy. We generated four CTL clones specific for DNAJB8_143(9) from donor A (CTL clone #21, 67 and 84) and one clone from donor B (CTL clone #70) and performed further analysis using CTL clone #84. The DNAJB8_143(9)-specific CTL clone showed cytotoxic activity for T2-A24 cells pulsed with DNAJB8_143(9) peptide but not for T2-A24 cells without the peptide or for K562 cells (Fig. 4c). To verify whether this CTL clone can recognize endogenously presented DNAJB8_143(9) peptide of DNAJB8-positive CRC CSC/CIC, we performed a 51Cr release assay using SP cells derived from HT29 cells. The DNAJB8_143(9)-specific CTL clone showed greater cytotoxic activity for HLA-A*2402+ HT29-SP cells than for HLA-A*2402+ HT29-MP cells or HLA-A*2402-DNAJB8- K562 cells (Fig. 4d). These results indicate that DNAJB8_143(9) peptide is an immunogenic epitope and that the endogenously processed peptide is presented on the surface of SP cells.

Bottom Line: In the present study, we found that a heat shock protein (HSP) 40 family member, DnaJ (Hsp40) homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in CSC/CIC derived from colorectal cancer (CRC) cells rather than in non-CSC/CIC.Overexpression of DNAJB8 enhanced the expression of stem cell markers and tumorigenicity, indicating that DNAJB8 has a role in CRC CSC/CIC.A CTL clone specific for DNAJB8 peptide showed higher killing activity to CRC CSC/CIC compared with non-CSC/CIC, and CTL adoptive transfer into CRC CSC/CIC showed an antitumor effect in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan.

Show MeSH
Related in: MedlinePlus