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.
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.
Affiliation: Research Unit, Division of Health Research, Saskatchewan Cancer Agency, Department of Oncology, University of Saskatchewan, Saskatoon, Canada.Show MeSH
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Mentions: We previously established a protocol for in vitro generation of immature DCs by culturing bone marrow cells in low amount of GM-CSF, but no IL-4  and demonstrated that immature DCs can uptake mature DC-released EXO . To assess the phenotype of immature bone marrow-derived DCs generated in presence of low amount of GM-CSF, we performed flow cytometric analysis. As shown in Fig. 2(A), DCs cultured in presence of low GM-CSF (1 ng/ml) expressed CD11c and CD54, but no Iad, CD40 and CD80 , indicating that they are immature DCs. To assess uptake of EXO, immature DCs were incubated with CFSE-labelled J558 tumour cell-released EXO (EXOCFSE) for different times and then analysed by flow cytometry and confocal fluorescence microscopic analysis. As shown in Fig. 2(B), immature DCs displayed CFSE expression after incubation with EXOCFSE, indicating that immature DC uptake J558 tumour cell-released EXO. As shown in Fig. 2(C) and (D), the amount of immature DCs with acquired CFSE increased with the time of incubation with EXOCFSE and reached a maximal level after a 4-hr incubation.
Affiliation: Research Unit, Division of Health Research, Saskatchewan Cancer Agency, Department of Oncology, University of Saskatchewan, Saskatoon, Canada.