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Membrane-bound HSP70-engineered myeloma cell-derived exosomes stimulate more efficient CD8(+) CTL- and NK-mediated antitumour immunity than exosomes released from heat-shocked tumour cells expressing cytoplasmic HSP70.

Xie Y, Bai O, Zhang H, Yuan J, Zong S, Chibbar R, Slattery K, Qureshi M, Wei Y, Deng Y, Xiang J - J. Cell. Mol. Med. (2010)

Bottom Line: We found that EXO(HSP) were able to more efficiently stimulate maturation of DCs with up-regulation of Ia(b) , CD40, CD80 and inflammatory cytokines than EXO(HS) after overnight incubation of immature bone-marrow-derived DCs (5 × 10⁶ cells) with EXO (100 μg), respectively.We demonstrate that EXO(HSP) are able to stimulate type 1 CD4(+) helper T (Th1) cell responses, and more efficient P1A-specific CD8(+) cytotoxic T lymphocyte (CTL) responses and antitumour immunity than EXO(HS) .In addition, we further elucidate that EXO(HSP) -stimulated antitumour immunity is mediated by both P1A-specific CD8(+) CTL and non-P1A-specific natural killer (NK) responses.

View Article: PubMed Central - PubMed

Affiliation: Research Unit, Division of Health Research, Saskatchewan Cancer Agency, Department of Oncology, University of Saskatchewan, Saskatoon, Canada.

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Phenotypic analysis of J558HSP-released EXO. (A) J558HSP, J558HS and J558 tumour cells were stained with a panel of Abs (solid lines) or isotype-matched irrelevant Abs (dotted lines), and analysed by flow cytometry. (B) J558HSP, J558HS and J558 tumour cells as well as irradiated J558 tumour cells were stained with FITC-annexin V (solid lines) or FITC anti-rat IgG (dotted lines) and analysed by flow cytometry. (C) Electron micrograph of EXO. The image shows small cup-shaped vesicles of 60–100 nm in diameter, which are representative of EXO released by tumour cells. Bar, 100 nm. (D) Western blot analysis of tumour cell-released EXO and tumour cell lysates was performed with a panel of antibodies. (E) Flow cytometric analysis of EXO. EXO were grouped for analysis of expression of surface molecules. (F) EXOneo, EXOHS and EXOHSP released from J558neo, J558HS and J558HSP tumour cells were stained with a panel of FITC antibodies (solid lines) or isotype-matched irrelevant FITC antibodies (dotted lines), and analysed by flow cytometry. One representative experiment of two is displayed.
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fig01: Phenotypic analysis of J558HSP-released EXO. (A) J558HSP, J558HS and J558 tumour cells were stained with a panel of Abs (solid lines) or isotype-matched irrelevant Abs (dotted lines), and analysed by flow cytometry. (B) J558HSP, J558HS and J558 tumour cells as well as irradiated J558 tumour cells were stained with FITC-annexin V (solid lines) or FITC anti-rat IgG (dotted lines) and analysed by flow cytometry. (C) Electron micrograph of EXO. The image shows small cup-shaped vesicles of 60–100 nm in diameter, which are representative of EXO released by tumour cells. Bar, 100 nm. (D) Western blot analysis of tumour cell-released EXO and tumour cell lysates was performed with a panel of antibodies. (E) Flow cytometric analysis of EXO. EXO were grouped for analysis of expression of surface molecules. (F) EXOneo, EXOHS and EXOHSP released from J558neo, J558HS and J558HSP tumour cells were stained with a panel of FITC antibodies (solid lines) or isotype-matched irrelevant FITC antibodies (dotted lines), and analysed by flow cytometry. One representative experiment of two is displayed.

Mentions: The original J558 tumour cells expressed H-2Kd, CD54 and P1A molecules, but not Iad and HSP70 (Fig. 1A). In addition to the above molecules expressed on J558 tumour cells, the engineered J558HSP cells transfected with pcDNAHSP70 displayed cellular surface HSP70 expression, whereas the heat-shocked J558HS tumour cells did not express membrane-bound HSP70. The control J558neo tumour cells transfected with the control vector pcDNA expressed a similar pattern of cellular surface molecules as the original J558 (data not shown). The above molecules expressed on J558HSP were stable since a long-term culturing J558HSP cell line had a similar phenotype as the originally generated one (data not shown). Heat-shocked J558HS cells were generated by culturing J558 tumour cells at 42°C for 1 hr [22, 23]. As shown in Fig. 1(B), the original J558, transfected J558HSP and heat-shocked J558SH tumour cells were mostly live cells without staining of annexin V (early apoptosis marker) [22, 23], whereas irradiated J558 tumour cells (95%) became apoptosis.


Membrane-bound HSP70-engineered myeloma cell-derived exosomes stimulate more efficient CD8(+) CTL- and NK-mediated antitumour immunity than exosomes released from heat-shocked tumour cells expressing cytoplasmic HSP70.

Xie Y, Bai O, Zhang H, Yuan J, Zong S, Chibbar R, Slattery K, Qureshi M, Wei Y, Deng Y, Xiang J - J. Cell. Mol. Med. (2010)

Phenotypic analysis of J558HSP-released EXO. (A) J558HSP, J558HS and J558 tumour cells were stained with a panel of Abs (solid lines) or isotype-matched irrelevant Abs (dotted lines), and analysed by flow cytometry. (B) J558HSP, J558HS and J558 tumour cells as well as irradiated J558 tumour cells were stained with FITC-annexin V (solid lines) or FITC anti-rat IgG (dotted lines) and analysed by flow cytometry. (C) Electron micrograph of EXO. The image shows small cup-shaped vesicles of 60–100 nm in diameter, which are representative of EXO released by tumour cells. Bar, 100 nm. (D) Western blot analysis of tumour cell-released EXO and tumour cell lysates was performed with a panel of antibodies. (E) Flow cytometric analysis of EXO. EXO were grouped for analysis of expression of surface molecules. (F) EXOneo, EXOHS and EXOHSP released from J558neo, J558HS and J558HSP tumour cells were stained with a panel of FITC antibodies (solid lines) or isotype-matched irrelevant FITC antibodies (dotted lines), and analysed by flow cytometry. One representative experiment of two is displayed.
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Related In: Results  -  Collection

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fig01: Phenotypic analysis of J558HSP-released EXO. (A) J558HSP, J558HS and J558 tumour cells were stained with a panel of Abs (solid lines) or isotype-matched irrelevant Abs (dotted lines), and analysed by flow cytometry. (B) J558HSP, J558HS and J558 tumour cells as well as irradiated J558 tumour cells were stained with FITC-annexin V (solid lines) or FITC anti-rat IgG (dotted lines) and analysed by flow cytometry. (C) Electron micrograph of EXO. The image shows small cup-shaped vesicles of 60–100 nm in diameter, which are representative of EXO released by tumour cells. Bar, 100 nm. (D) Western blot analysis of tumour cell-released EXO and tumour cell lysates was performed with a panel of antibodies. (E) Flow cytometric analysis of EXO. EXO were grouped for analysis of expression of surface molecules. (F) EXOneo, EXOHS and EXOHSP released from J558neo, J558HS and J558HSP tumour cells were stained with a panel of FITC antibodies (solid lines) or isotype-matched irrelevant FITC antibodies (dotted lines), and analysed by flow cytometry. One representative experiment of two is displayed.
Mentions: The original J558 tumour cells expressed H-2Kd, CD54 and P1A molecules, but not Iad and HSP70 (Fig. 1A). In addition to the above molecules expressed on J558 tumour cells, the engineered J558HSP cells transfected with pcDNAHSP70 displayed cellular surface HSP70 expression, whereas the heat-shocked J558HS tumour cells did not express membrane-bound HSP70. The control J558neo tumour cells transfected with the control vector pcDNA expressed a similar pattern of cellular surface molecules as the original J558 (data not shown). The above molecules expressed on J558HSP were stable since a long-term culturing J558HSP cell line had a similar phenotype as the originally generated one (data not shown). Heat-shocked J558HS cells were generated by culturing J558 tumour cells at 42°C for 1 hr [22, 23]. As shown in Fig. 1(B), the original J558, transfected J558HSP and heat-shocked J558SH tumour cells were mostly live cells without staining of annexin V (early apoptosis marker) [22, 23], whereas irradiated J558 tumour cells (95%) became apoptosis.

Bottom Line: We found that EXO(HSP) were able to more efficiently stimulate maturation of DCs with up-regulation of Ia(b) , CD40, CD80 and inflammatory cytokines than EXO(HS) after overnight incubation of immature bone-marrow-derived DCs (5 × 10⁶ cells) with EXO (100 μg), respectively.We demonstrate that EXO(HSP) are able to stimulate type 1 CD4(+) helper T (Th1) cell responses, and more efficient P1A-specific CD8(+) cytotoxic T lymphocyte (CTL) responses and antitumour immunity than EXO(HS) .In addition, we further elucidate that EXO(HSP) -stimulated antitumour immunity is mediated by both P1A-specific CD8(+) CTL and non-P1A-specific natural killer (NK) responses.

View Article: PubMed Central - PubMed

Affiliation: Research Unit, Division of Health Research, Saskatchewan Cancer Agency, Department of Oncology, University of Saskatchewan, Saskatoon, Canada.

Show MeSH
Related in: MedlinePlus