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.

Exosomes (EXO) derived from tumour cells have been used to stimulate antitumour immune responses, but only resulting in prophylatic immunity. Tumour-derived heat shock protein 70 (HSP70) molecules are molecular chaperones with a broad repertoire of tumour antigen peptides capable of stimulating dendritic cell (DC) maturation and T-cell immune responses. To enhance EXO-based antitumour immunity, we generated an engineered myeloma cell line J558(HSP) expressing endogenous P1A tumour antigen and transgenic form of membrane-bound HSP70 and heat-shocked J558(HS) expressing cytoplasmic HSP70, and purified EXO(HSP) and EXO(HS) from J558(HSP) and J558(HS) tumour cell culture supernatants by ultracentrifugation. 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 also i.v. immunized BALB/c mice with EXO (30 µg/mouse) and assessed P1A-specific T-cell responses after immunization. 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. Therefore, membrane-bound HSP70-expressing tumour cell-released EXO may represent a more effective EXO-based vaccine in induction of antitumour immunity.

CD4+ T cell acquisition of the bystander pMHC I colocalizing in the same immunological synapse comprising pMHC II and costimulatory CD40, CD54, CD80, OX40L, and 41BBL.

We previously showed that CD4+ T cells acquired peptide/major histocompatibility complex (pMHC) I and costimulatory molecules by dendritic cell (DC) activation. However, the molecular mechanism for pMHC I acquisition is unclear. In this study, by using a panel of engineered DC2.4 cells or incubation of these cells with Con A-stimulated CD4+ T cells, we conducted capping and synapse formation assay and examined them by confocal fluorescence microscopy. We demonstrated that (i) CD54 and CD80 colocalized with pMHC I/II in the same lipid rafts, whereas CD40, OX40L, and 41BBL localized in the lipid rafts but separately from pMHC I/II, and (ii) MHC I/II colocalized with the costimulatory molecules in the same synapse formed between a DC and a CD4+ T cell, leading to expression of the acquired bystander pMHC I on CD4+ T cells via internalization/recycling pathway. These results provide some useful information in composition and dynamics of immunological synapses.