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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

Abstract 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 J558HSP expressing endogenous P1A tumour antigen and transgenic form of membrane-bound HSP70 and heat-shocked J558HS expressing cytoplasmic HSP70, and purified EXOHSP and EXOHS from J558HSP and J558HS tumour cell culture supernatants by ultracentrifugation. We found that EXOHSP were able to more efficiently stimulate maturation of DCs with up-regulation of Iab, CD40, CD80 and inflammatory cytokines than EXOHS 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 EXOHSP 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 EXOHS. In addition, we further elucidate that EXOHSP-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.

in molecules potentially involved in effector cell targeting, that is, CD11b, lactadherin and CD9 molecules [7,8]. These tumour-derived EXO isolated from malignant effusions can transfer tumour Ags to dendritic cells (DCs) and induce tumour-specific cytotoxic T lymphocyte (CTL) responses and antitumour immunity [9][10][11]. It has been reported that EXO need the host DC as an adjuvant for stimulation of CD8 ϩ CTL responses [12,13]. Zitvogel et al. first demonstrated eradication of tumours by EXO vaccination in animal models [10]. Subsequently, EXO-based vaccines have been confirmed to stimulate CD8 ϩ CTL responses and induce antitumour immunity [9,[14][15][16]. However, its efficiency is less effective because it only induces either prophylatic antitumour immunity in animal models or very weak antitumour immune responses in clinical trials [17,18].
Heat shock protein (HSP) molecules are stress-induced molecular chaperones that function to facilitate presentation of endogenous antigenic peptides [19] leading to potent adjuvant effect on stimulation of DC maturation and enhanced CD8 ϩ CTL responses [20]. Tumour-derived HSP have thus been used as adjuvant in cancer vaccines [21]. It has been demonstrated that enhanced expression of cytoplasmic HSP in EXO derived from heat-shocked tumour cells induced more efficient CD8 ϩ CTL responses and antitumour immunity than EXO derived from untreated tumour cells [22,23]. It has also been shown that expression of membrane-bound exosomal HSP stimulated cytolytic activity of natural killer (NK) cells [24]. Within HSP family, HSP70, the peptides of which can be quickly loaded onto MHC I and II complexes of DCs, can exhibit potent adjuvant effect in stimulation of the host immune responses and antitumour immunity [25][26][27].
In this study, we compared the efficiency of stimulation of T-cell responses and antitumour immunity between membrane-bound HSP70-expressing EXO derived from HSP70engineered tumour cells engineered and cytoplasmic HSP70expressing EXO derived from heat-shocked tumour cells. We first transfected a myeloma cell line J558 expressing its tumour Ag P1A [28] with pcDNAHSP70 vector expressing membrane-bound HSP70 and the control vector pcDNAneo without HSP70 expression, respectively [28]. We also incubated J558 tumour cells at 42ЊC for 1 hr for heat shock treatment to generate heat-shocked J558 (J558HS) tumour cells. We then purified EXOHSP, EXOneo and EXOHS from transfected J558HSP and J558neo and heat-shocked J558HS cell culture supernatants, respectively. To assess the antitumour immunity derived from EXO vaccination, we immunized wild-type BALB/c mice with membrane-bound HSP70-expressing EXOHSP or control EXOneo or cytoplasmic HSP70-expressing EXOHS. We demonstrate that EXOHSP vaccination is able to more efficiently induce DC maturation leading to stimulation of type 1 helper CD4 ϩ T (Th1) and more efficient CD8 ϩ T-cell responses and immunity against J558 tumour cells than EXPHS and EXOneo vaccination. We also elucidate that the antitumour immunity resulted from EXOHSP vaccination is mediated by both CD8 ϩ CTL and NK cells.

Generation of engineered J558 HSP cell line
Twenty million J558 tumour cells were resuspended in 0.7 ml phosphatebuffered saline (PBS) and mixed with 0.3 ml PBS containing 10 g pcDNA-HSP70 DNA expressing membrane-bound inducible HSP70 or the control pcDNAneo DNA [29]. Tumour cells were transfected with these DNA using a Bio-Rad gene pulser (Bio-Rad Lab, Mississauga, Ontario, Canada) with parameters of 250 V and 125 F capacitance as previously described [29]. Transfected cells were selected for growth in medium containing G418 (4 mg/ml). The selected J558HSP and the control J558neo clones maintained in culture medium containing 10% foetal calf serum (FCS) and G418 (0.5 mg/ml) were subjected to flow cytometric analysis and production of tumour-released EXO.

Tumour cell lysate preparation
Live J558 tumour cell lysates were prepared as previously described [32].

Flow cytometric analysis of tumour cells and tumour cell-released EXO
Tumour cells and tumour cell-derived EXO were analysed by flow cytometry as previously described [13,33]

Stimulation of immature DCs
The generation of bone marrow derived immature DCs from wild-type BALB/c mice in presence of GM-CSF (1 ng/ml) has been previously described [34]. To assess stimulatory effect, immature DCs (5 ϫ 10 6 cells) were incubated with EXOHSP or EXOneo or EXOHS (100 g) for overnight, then stained with a panel of Abs, and analysed by flow cytometry.  [35].

Cytotoxicity assay
The in vivo cytotoxicity assay was performed as previously described [35]. C57BL/6 splenocytes were harvested from naive mouse spleens and incubated with either high (3.0 M, CFSE high ) or low (0.6 M, CFSE low ) concentrations of CFSE, to generate differentially labelled target cells. The CFSE high cells were pulsed with P1A peptide, whereas the CFSE low cells were pulsed with an irrelevant control peptide and served as internal control. These peptide-pulsed target cells were i.v. injected at 1:1 ratio into the above mice 6 days after immunization. Sixteen hours later, the spleens of immunized mice were removed and residual CFSE high and CFSE low target cells remaining in the recipients' spleens were analysed by flow cytometry [35]. In NK cytotoxicity assay, spleen T cells of mice i.v. immunized with EXOHSP (30 g/mouse) at different times (2, 4 and 6 days) after immunization were prepared by passing splenocytes through nylon wool columns, and red cells were then lysed using 0.84% ammonium chloride. These T cells were used as the source of effector NK cells. ( 51 Cr)-chromatelabelled J558 tumour cells were used as target cells [38].  [38]. To assess whether CD8 ϩ T or NK cells were involved in EXO-induced antitumour immunity, BALB/c mice (n ϭ 8) were i.p. injected with anti-CD8 or anti-NK Ab (0.5 mg/mouse) 1 day before immunization, and the treatment was repeated every 3 days for a total of three time [38].  [39].  [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.

Phenotypical characterization of J558 tumour cell-released EXO
EXO derived from J558, J558neo and J558HSP tumour cells were purified from tumour cell culture supernatants by differential ultracentrifugation.  Fig. 1(C), EXO derived from either J558neo or J558HSP or J558HS had typical exosomal characteristic of 'saucer' or round shape with a diameter between 50 and 90 nm [40]. We also confirmed that EXO-associated proteins including LAMP-1, Alix/AIP1 and H-2K d were abundant by Western blot analysis (Fig. 1D) [41], whereas galectin, a protein found in the endoplasmic reticulum [42] was only detectable in tumour cell lysates but absent in the EXO samples, indicating that there is no contamination of other vesicles in EXO preparation. Both EXOHS and EXPHSP contained HSP70 by Western blot analysis (Fig. 1D). EXO were then stained with a panel of FITC-Abs and analysed by flow cytometry. As shown in Fig. 1

Immature DC uptake J558 tumour cell-released EXO
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 [34] and demonstrated that immature DCs can uptake mature DC-released EXO [39]. 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 Ia d , CD40 and CD80 [41], 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

EXO HSP efficiently stimulate DC maturation
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) (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 ϩ EXOHSP) stimulated the strongest allogeneic T-cell proliferation

Fig. 1 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.
than DCs with uptake of EXOneo (DC ϩ EXOneo) and EXOHS (DC ϩ EXOHS) (P Ͻ 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

EXO HSP stimulate type 1 CD4 ؉ T-cell responses
To assess whether EXOHSP can stimulate CD4 ϩ T-cell responses, we performed in vitro T-cell proliferation assay by using CD4 ϩ T cells derived from EXO-immunized mouse spleens. As shown in Fig. 4(A)

EXO HSP stimulate stronger CD8 ؉ effector CTL responses than EXO HS
To assess the stimulatory effect of EXOHSP on in vivo CD8 ϩ T-cell responses, we performed PE-H-2L d /P1A peptide tetramer staining assay [43]. As shown in Fig. 4(C)

The EXO HSP -induced antitumour immunity is mediated by CD8 ϩ CTL and NK cells
Since CD8 ϩ T cells have been defined as effector T cells with cytotoxic activity to tumour or virally infected cells [44] and the mem-brane-bound HSP70 molecules are identified as a target structure for cytolytic attack mediated by NK cells [45], we assessed whether CD8 ϩ T and NK cells are involved in EXOHSP-induced antitumour immunity. We first performed a kinetic study on CD8 ϩ CTL and NK cell killing activity derived from EXOHSP vaccination. The killing activity of NK derived from naïve BALB/c mice was 8% (data not shown). As shown in Fig. 5(B) and (C), P1A-specific CD8 ϩ CTL responses (0.64%) and non-P1A-specific NK killing activity (38%) was peaked at day 6 and day 2 after vaccination. At day 6 after immunization, NK killing activity was still 20%, indicating that both CD8 ϩ CTL and NK cells may be involved in the antitumour immunity seen at day 6 after EXOHSP vaccination (Fig. 5A) [12,50]. We have recently demonstrated   [49,51] and CD14 [23] through NF-B to up-regulate costimulatory molecules of DCs and induce DC maturation and secretion of pro-inflammatory cytokines [52] leading to stimulation of both CD8 ϩ CTL and NK cell responses. Thus, the dual role of HSP70 as antigenic peptide chaperone [53] and danger signal [52] thus makes them especially critical in EXObased antitumour vaccine. Taken together, our data showed that EXO derived from tumour cells engineered to express membrane-bound HSP70 can induce DC maturation and stimulate CD4 ϩ Th1, CD8 ϩ CTL and NK cell responses leading to more efficient antitumour immunity. Therefore, membrane-bound HSP70-expressing EXO may represent a more effective EXO-based vaccine in induction of antitumour immunity.