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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 DC maturation. (A) Flow cytometric analysis of EXO-treated DCs. Immature DCs with or without incubation of EXOneo, EXOHS and EXOHSP were stained with a panel of antibodies (solid lines) or isotype-matched irrelevant antibodies (dotted lines), and analysed by flow cytometry. Mean fluorescence intensity of solid line/mean fluorescence intensity of dotted line was presented in each panel. (B) Cytokine secretion. Culture supernatants of DCs alone (DC), DC with EXOHS (DC + EXOHS) and DC with EXOHSP (DC + EXOHSP) were tested using cytokine ELISA kits. Values represent the mean of triplicates from three experiments. (C) Mixed T lymphocyte reaction assay. Irradiated DCs including DC 1 EXOHS and DC 1 EXOHSP and their two-fold dilutions were co-cultured in 96-well plates with a constant number of allogeneic C57BL/6 naïve T cells. After 3 days, T-cell proliferation was measured by adding 1 μCi 3H-thymidine to each well in an overnight 3H-thymidine uptake assay. The levels of 3H-thymidine incorporation into cellular DNA were determined by liquid scintillation counting. (D) BALB/c mice were s.c. vaccinated with irradiated (4000 rad) DC 1 EXOneo, DC 1 EXOHS and DC 1 EXOHSP (0.5 × 106 cells per mouse). 6 days after immunization, the immunized mice were s.c. inoculated with J558 tumour cells (0.5 × 106 cells per mouse). Animal mortality and tumour growth were monitored daily for up to 60 days. *, P < 0.05 versus cohorts in EXOHSP group (log-rank test). One representative experiment of two is displayed.
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fig03: J558HSP-released EXOHSP stimulate DC maturation. (A) Flow cytometric analysis of EXO-treated DCs. Immature DCs with or without incubation of EXOneo, EXOHS and EXOHSP were stained with a panel of antibodies (solid lines) or isotype-matched irrelevant antibodies (dotted lines), and analysed by flow cytometry. Mean fluorescence intensity of solid line/mean fluorescence intensity of dotted line was presented in each panel. (B) Cytokine secretion. Culture supernatants of DCs alone (DC), DC with EXOHS (DC + EXOHS) and DC with EXOHSP (DC + EXOHSP) were tested using cytokine ELISA kits. Values represent the mean of triplicates from three experiments. (C) Mixed T lymphocyte reaction assay. Irradiated DCs including DC 1 EXOHS and DC 1 EXOHSP and their two-fold dilutions were co-cultured in 96-well plates with a constant number of allogeneic C57BL/6 naïve T cells. After 3 days, T-cell proliferation was measured by adding 1 μCi 3H-thymidine to each well in an overnight 3H-thymidine uptake assay. The levels of 3H-thymidine incorporation into cellular DNA were determined by liquid scintillation counting. (D) BALB/c mice were s.c. vaccinated with irradiated (4000 rad) DC 1 EXOneo, DC 1 EXOHS and DC 1 EXOHSP (0.5 × 106 cells per mouse). 6 days after immunization, the immunized mice were s.c. inoculated with J558 tumour cells (0.5 × 106 cells per mouse). Animal mortality and tumour growth were monitored daily for up to 60 days. *, P < 0.05 versus cohorts in EXOHSP group (log-rank test). One representative experiment of two is displayed.

Mentions: It has been demonstrated that HSP70 can stimulate DC maturation [20]. To assess whether EXOHSP were also able to stimulate DC maturation, we analysed immature DCs cultured with EXOHSP expressing membrane-bound HSP70 or EXOHS expressing cytoplasmic HSP70 or EXOneo without HSP70 expression. EXOneo-stimulated DCs still showed a similar pattern of the expression of the above molecules on immature DCs (Fig. 3A), indicating that EXOneo does not modulate DC maturation. Interestingly, we found that EXOHS- and EXOHSP-stimulated DCs up-regulated expression of Iad, CD40 and CD80, indicating that both EXOHS and EXOHSP are able to stimulate DC maturation. However, the up-regulation of the above molecules stimulated by the later was more than the former, indicating that EXOHSP is a stronger stimulator for DC maturation than EXOHS. In addition, EXOHSP-stimulated mature DCs also secreted more amount of inflammatory cytokines such as IL-1β (1.8 ng/ml/106 cells/24 hrs), IL-12 (1.1 ng/ml/106 cells/24 hrs), IFN-γ (0.8 ng/ml/106 cells/24 hrs) and tumour necrosis factor (TNF)-α (0.9 ng/ml/106 cells/24 hrs) (Fig. 3B) than EXOHS-stimulated DCs. Since EXOHSP harboured the above immune molecules, they may have potent effect in stimulation of T-cell immune responses [43]. We first assessed whether DCs with uptake of EXO by incubation of immature DCs with EXO for overnight stimulate allogeneic T-cell proliferation in a mixed T lymphocyte reaction assay. As shown in Fig. 3C, DCs with uptake of EXOHSP (DC + EXOHSP) stimulated the strongest allogeneic T-cell proliferation than DCs with uptake of EXOneo (DC + EXOneo) and EXOHS (DC + EXOHS) (P < 0.05). We then assessed whether DCs with uptake of EXO by incubation of immature DCs with EXO for overnight induce P1A-specific antitumour immunity. As shown in Fig. 3(D), DCs with uptake of EXOneo (DC + EXOneo), EXOHS (DC + EXOHS) and EXOHSP (DC + EXOHSP) were able to stimulate P1A-specific antitumour immunity to protect one of eight, three of eight and eight of eight mice from tumour growth after the immunized mice were challenged with J558 tumour cells, respectively, indicating that EXOHSP-treated DCs are the most immunogenic among these three types of DCs.


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 DC maturation. (A) Flow cytometric analysis of EXO-treated DCs. Immature DCs with or without incubation of EXOneo, EXOHS and EXOHSP were stained with a panel of antibodies (solid lines) or isotype-matched irrelevant antibodies (dotted lines), and analysed by flow cytometry. Mean fluorescence intensity of solid line/mean fluorescence intensity of dotted line was presented in each panel. (B) Cytokine secretion. Culture supernatants of DCs alone (DC), DC with EXOHS (DC + EXOHS) and DC with EXOHSP (DC + EXOHSP) were tested using cytokine ELISA kits. Values represent the mean of triplicates from three experiments. (C) Mixed T lymphocyte reaction assay. Irradiated DCs including DC 1 EXOHS and DC 1 EXOHSP and their two-fold dilutions were co-cultured in 96-well plates with a constant number of allogeneic C57BL/6 naïve T cells. After 3 days, T-cell proliferation was measured by adding 1 μCi 3H-thymidine to each well in an overnight 3H-thymidine uptake assay. The levels of 3H-thymidine incorporation into cellular DNA were determined by liquid scintillation counting. (D) BALB/c mice were s.c. vaccinated with irradiated (4000 rad) DC 1 EXOneo, DC 1 EXOHS and DC 1 EXOHSP (0.5 × 106 cells per mouse). 6 days after immunization, the immunized mice were s.c. inoculated with J558 tumour cells (0.5 × 106 cells per mouse). Animal mortality and tumour growth were monitored daily for up to 60 days. *, P < 0.05 versus cohorts in EXOHSP group (log-rank test). One representative experiment of two is displayed.
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fig03: J558HSP-released EXOHSP stimulate DC maturation. (A) Flow cytometric analysis of EXO-treated DCs. Immature DCs with or without incubation of EXOneo, EXOHS and EXOHSP were stained with a panel of antibodies (solid lines) or isotype-matched irrelevant antibodies (dotted lines), and analysed by flow cytometry. Mean fluorescence intensity of solid line/mean fluorescence intensity of dotted line was presented in each panel. (B) Cytokine secretion. Culture supernatants of DCs alone (DC), DC with EXOHS (DC + EXOHS) and DC with EXOHSP (DC + EXOHSP) were tested using cytokine ELISA kits. Values represent the mean of triplicates from three experiments. (C) Mixed T lymphocyte reaction assay. Irradiated DCs including DC 1 EXOHS and DC 1 EXOHSP and their two-fold dilutions were co-cultured in 96-well plates with a constant number of allogeneic C57BL/6 naïve T cells. After 3 days, T-cell proliferation was measured by adding 1 μCi 3H-thymidine to each well in an overnight 3H-thymidine uptake assay. The levels of 3H-thymidine incorporation into cellular DNA were determined by liquid scintillation counting. (D) BALB/c mice were s.c. vaccinated with irradiated (4000 rad) DC 1 EXOneo, DC 1 EXOHS and DC 1 EXOHSP (0.5 × 106 cells per mouse). 6 days after immunization, the immunized mice were s.c. inoculated with J558 tumour cells (0.5 × 106 cells per mouse). Animal mortality and tumour growth were monitored daily for up to 60 days. *, P < 0.05 versus cohorts in EXOHSP group (log-rank test). One representative experiment of two is displayed.
Mentions: It has been demonstrated that HSP70 can stimulate DC maturation [20]. To assess whether EXOHSP were also able to stimulate DC maturation, we analysed immature DCs cultured with EXOHSP expressing membrane-bound HSP70 or EXOHS expressing cytoplasmic HSP70 or EXOneo without HSP70 expression. EXOneo-stimulated DCs still showed a similar pattern of the expression of the above molecules on immature DCs (Fig. 3A), indicating that EXOneo does not modulate DC maturation. Interestingly, we found that EXOHS- and EXOHSP-stimulated DCs up-regulated expression of Iad, CD40 and CD80, indicating that both EXOHS and EXOHSP are able to stimulate DC maturation. However, the up-regulation of the above molecules stimulated by the later was more than the former, indicating that EXOHSP is a stronger stimulator for DC maturation than EXOHS. In addition, EXOHSP-stimulated mature DCs also secreted more amount of inflammatory cytokines such as IL-1β (1.8 ng/ml/106 cells/24 hrs), IL-12 (1.1 ng/ml/106 cells/24 hrs), IFN-γ (0.8 ng/ml/106 cells/24 hrs) and tumour necrosis factor (TNF)-α (0.9 ng/ml/106 cells/24 hrs) (Fig. 3B) than EXOHS-stimulated DCs. Since EXOHSP harboured the above immune molecules, they may have potent effect in stimulation of T-cell immune responses [43]. We first assessed whether DCs with uptake of EXO by incubation of immature DCs with EXO for overnight stimulate allogeneic T-cell proliferation in a mixed T lymphocyte reaction assay. As shown in Fig. 3C, DCs with uptake of EXOHSP (DC + EXOHSP) stimulated the strongest allogeneic T-cell proliferation than DCs with uptake of EXOneo (DC + EXOneo) and EXOHS (DC + EXOHS) (P < 0.05). We then assessed whether DCs with uptake of EXO by incubation of immature DCs with EXO for overnight induce P1A-specific antitumour immunity. As shown in Fig. 3(D), DCs with uptake of EXOneo (DC + EXOneo), EXOHS (DC + EXOHS) and EXOHSP (DC + EXOHSP) were able to stimulate P1A-specific antitumour immunity to protect one of eight, three of eight and eight of eight mice from tumour growth after the immunized mice were challenged with J558 tumour cells, respectively, indicating that EXOHSP-treated DCs are the most immunogenic among these three types of DCs.

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