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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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J558HSP-released EXOHSP stimulate T-cell responses. (A) A T-cell proliferation assay. Splenic CD4+ T cells from EXOneo- or EXOHS- or EXOHSP-immunized mice were incubated with irradiated J558 tumour cells for 3 days. 3H-thymidine incorporation was assessed by liquid scintillation counting. *, P < 0.05 versus cohorts in EXOneo or EXOHS group (Student’s t-test). (B) Cytokine secretion. The culture supernatants of CD4+ T cells derived from EXOHSP-immunized mice were tested using cytokine ELISA kits. Values represent the mean of triplicates from three experiments. (C) Tetramer staining assay. 6 days after immunization of mice with EXOneo, EXOHS and EXOHSP, the tail blood and splenocyte samples were taken from the immunized mice, stained with PE-H-2Ld/P1A peptide tetramer and FITC anti-CD8 Ab and analysed by flow cytomery. The value in each panel represents the percentage of tetramer+ CD8+ T cells versus the total blood CD8+ T-cell population or the total tetramer+ CD8+ T cells per spleen. *, P < 0.05 versus cohorts in EXOneo or EXOHS group (Student’s t-test). (D and E) In in vivo cytotoxicity assay. BALB/c splenocytes were harvested from naive mouse spleens and incubated with either high (3.0 μM, CFSEhigh) or low (0.6 μM, CFSElow) concentrations of CFSE, to generate differentially labelled target cells. The CFSEhigh cells were pulsed with P1A peptide, whereas the CFSElow cells were pulsed with the control peptide and served as internal controls. These peptide-pulsed target cells were i.v. injected at 1:1 ratio into (D) the above immunized mice 6 days after immunization of EXOneo, EXOHS and EXOHSP, respectively, or into (E) the mice immunized with EXOHSP but also treated with anti-CD8 Ab to deplete CD8+ T cells. 16 hrs later, the spleens of immunized mice were removed and the percentages of the residual P1A-specific CFSEhigh (H) and control CFSElow (L) target cells remaining in the recipients’ spleens were analysed by flow cytometry. The value in each panel represents the percentage of CFSEhighversus CFSElow target cells remaining in the spleen. The value in parenthesis represents the standard deviation. *, P < 0.05 versus cohorts in EXOneo or EXOHS group (Student’s t-test). One representative experiment of three is shown.
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fig04: J558HSP-released EXOHSP stimulate T-cell responses. (A) A T-cell proliferation assay. Splenic CD4+ T cells from EXOneo- or EXOHS- or EXOHSP-immunized mice were incubated with irradiated J558 tumour cells for 3 days. 3H-thymidine incorporation was assessed by liquid scintillation counting. *, P < 0.05 versus cohorts in EXOneo or EXOHS group (Student’s t-test). (B) Cytokine secretion. The culture supernatants of CD4+ T cells derived from EXOHSP-immunized mice were tested using cytokine ELISA kits. Values represent the mean of triplicates from three experiments. (C) Tetramer staining assay. 6 days after immunization of mice with EXOneo, EXOHS and EXOHSP, the tail blood and splenocyte samples were taken from the immunized mice, stained with PE-H-2Ld/P1A peptide tetramer and FITC anti-CD8 Ab and analysed by flow cytomery. The value in each panel represents the percentage of tetramer+ CD8+ T cells versus the total blood CD8+ T-cell population or the total tetramer+ CD8+ T cells per spleen. *, P < 0.05 versus cohorts in EXOneo or EXOHS group (Student’s t-test). (D and E) In in vivo cytotoxicity assay. BALB/c splenocytes were harvested from naive mouse spleens and incubated with either high (3.0 μM, CFSEhigh) or low (0.6 μM, CFSElow) concentrations of CFSE, to generate differentially labelled target cells. The CFSEhigh cells were pulsed with P1A peptide, whereas the CFSElow cells were pulsed with the control peptide and served as internal controls. These peptide-pulsed target cells were i.v. injected at 1:1 ratio into (D) the above immunized mice 6 days after immunization of EXOneo, EXOHS and EXOHSP, respectively, or into (E) the mice immunized with EXOHSP but also treated with anti-CD8 Ab to deplete CD8+ T cells. 16 hrs later, the spleens of immunized mice were removed and the percentages of the residual P1A-specific CFSEhigh (H) and control CFSElow (L) target cells remaining in the recipients’ spleens were analysed by flow cytometry. The value in each panel represents the percentage of CFSEhighversus CFSElow target cells remaining in the spleen. The value in parenthesis represents the standard deviation. *, P < 0.05 versus cohorts in EXOneo or EXOHS group (Student’s t-test). One representative experiment of three is shown.

Mentions: To assess whether EXOHSP can stimulate CD4+ T-cell responses, we performed in vitro T-cell proliferation assay by using CD4+ T cells derived from EXO-immunized mouse spleens. As shown in Fig. 4(A), EXOHSP immunization could mount a more efficient CD4+ T-cell proliferation compared to that of EXOHS-immunized group (P < 0.05). To assess the type of CD4+ T-cell response, we measured the cytokine secretion of CD4+ T cells derived from EXOHSP-immunized mice by ELISA. CD4+ T cells derived from EXOHSP immunized mice secreted IL-2 (1.4 ng/ml/106 cells/24 hrs) and IFN-γ (1.1 ng/ml/106 cells/24 hrs), but not IL-4 (Fig. 4B), indicating that EXOHSP stimulate type 1 CD4+ helper T (Th1) cell responses.


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)

J558HSP-released EXOHSP stimulate T-cell responses. (A) A T-cell proliferation assay. Splenic CD4+ T cells from EXOneo- or EXOHS- or EXOHSP-immunized mice were incubated with irradiated J558 tumour cells for 3 days. 3H-thymidine incorporation was assessed by liquid scintillation counting. *, P < 0.05 versus cohorts in EXOneo or EXOHS group (Student’s t-test). (B) Cytokine secretion. The culture supernatants of CD4+ T cells derived from EXOHSP-immunized mice were tested using cytokine ELISA kits. Values represent the mean of triplicates from three experiments. (C) Tetramer staining assay. 6 days after immunization of mice with EXOneo, EXOHS and EXOHSP, the tail blood and splenocyte samples were taken from the immunized mice, stained with PE-H-2Ld/P1A peptide tetramer and FITC anti-CD8 Ab and analysed by flow cytomery. The value in each panel represents the percentage of tetramer+ CD8+ T cells versus the total blood CD8+ T-cell population or the total tetramer+ CD8+ T cells per spleen. *, P < 0.05 versus cohorts in EXOneo or EXOHS group (Student’s t-test). (D and E) In in vivo cytotoxicity assay. BALB/c splenocytes were harvested from naive mouse spleens and incubated with either high (3.0 μM, CFSEhigh) or low (0.6 μM, CFSElow) concentrations of CFSE, to generate differentially labelled target cells. The CFSEhigh cells were pulsed with P1A peptide, whereas the CFSElow cells were pulsed with the control peptide and served as internal controls. These peptide-pulsed target cells were i.v. injected at 1:1 ratio into (D) the above immunized mice 6 days after immunization of EXOneo, EXOHS and EXOHSP, respectively, or into (E) the mice immunized with EXOHSP but also treated with anti-CD8 Ab to deplete CD8+ T cells. 16 hrs later, the spleens of immunized mice were removed and the percentages of the residual P1A-specific CFSEhigh (H) and control CFSElow (L) target cells remaining in the recipients’ spleens were analysed by flow cytometry. The value in each panel represents the percentage of CFSEhighversus CFSElow target cells remaining in the spleen. The value in parenthesis represents the standard deviation. *, P < 0.05 versus cohorts in EXOneo or EXOHS group (Student’s t-test). One representative experiment of three is shown.
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fig04: J558HSP-released EXOHSP stimulate T-cell responses. (A) A T-cell proliferation assay. Splenic CD4+ T cells from EXOneo- or EXOHS- or EXOHSP-immunized mice were incubated with irradiated J558 tumour cells for 3 days. 3H-thymidine incorporation was assessed by liquid scintillation counting. *, P < 0.05 versus cohorts in EXOneo or EXOHS group (Student’s t-test). (B) Cytokine secretion. The culture supernatants of CD4+ T cells derived from EXOHSP-immunized mice were tested using cytokine ELISA kits. Values represent the mean of triplicates from three experiments. (C) Tetramer staining assay. 6 days after immunization of mice with EXOneo, EXOHS and EXOHSP, the tail blood and splenocyte samples were taken from the immunized mice, stained with PE-H-2Ld/P1A peptide tetramer and FITC anti-CD8 Ab and analysed by flow cytomery. The value in each panel represents the percentage of tetramer+ CD8+ T cells versus the total blood CD8+ T-cell population or the total tetramer+ CD8+ T cells per spleen. *, P < 0.05 versus cohorts in EXOneo or EXOHS group (Student’s t-test). (D and E) In in vivo cytotoxicity assay. BALB/c splenocytes were harvested from naive mouse spleens and incubated with either high (3.0 μM, CFSEhigh) or low (0.6 μM, CFSElow) concentrations of CFSE, to generate differentially labelled target cells. The CFSEhigh cells were pulsed with P1A peptide, whereas the CFSElow cells were pulsed with the control peptide and served as internal controls. These peptide-pulsed target cells were i.v. injected at 1:1 ratio into (D) the above immunized mice 6 days after immunization of EXOneo, EXOHS and EXOHSP, respectively, or into (E) the mice immunized with EXOHSP but also treated with anti-CD8 Ab to deplete CD8+ T cells. 16 hrs later, the spleens of immunized mice were removed and the percentages of the residual P1A-specific CFSEhigh (H) and control CFSElow (L) target cells remaining in the recipients’ spleens were analysed by flow cytometry. The value in each panel represents the percentage of CFSEhighversus CFSElow target cells remaining in the spleen. The value in parenthesis represents the standard deviation. *, P < 0.05 versus cohorts in EXOneo or EXOHS group (Student’s t-test). One representative experiment of three is shown.
Mentions: To assess whether EXOHSP can stimulate CD4+ T-cell responses, we performed in vitro T-cell proliferation assay by using CD4+ T cells derived from EXO-immunized mouse spleens. As shown in Fig. 4(A), EXOHSP immunization could mount a more efficient CD4+ T-cell proliferation compared to that of EXOHS-immunized group (P < 0.05). To assess the type of CD4+ T-cell response, we measured the cytokine secretion of CD4+ T cells derived from EXOHSP-immunized mice by ELISA. CD4+ T cells derived from EXOHSP immunized mice secreted IL-2 (1.4 ng/ml/106 cells/24 hrs) and IFN-γ (1.1 ng/ml/106 cells/24 hrs), but not IL-4 (Fig. 4B), indicating that EXOHSP stimulate type 1 CD4+ helper T (Th1) cell responses.

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