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Introduction

The incidence of RCC is increasing worldwide. RCC accounts for approximately 2–3% of tumor diagnoses and tumor mortality cases worldwide; most RCC cases are resistant to traditional radiotherapy and chemotherapy and follow an unpredictable disease course (1,2). In recent years, great progress has been made in the diagnosis and treatment of RCC, although the prognosis remains unsatisfactory. To achieve a better therapeutic effect, early anticipation or identification of patients with aggressive disease or poor prognosis is urgently needed. More importantly, a more efficient and safer therapeutic strategy for eliminating RCC is required to prevent relapse.

One general characteristic of tumor cells is the secretion of membrane microvesicles, which are called exosomes. Exosomes, especially tumor-derived exosomes (TEXs), which are membrane-bound extracellular vesicles 30–100 nm in size secreted by all tumor cells (3), have attracted considerable attention as potential tools for diagnosing cancer and delivering therapeutic anti-cancer drugs (4,5). However, some studies have shown that TEXs alone are less efficient in promoting T cell activation, and may even induce T cell apoptosis; moreover, tumor progression is enhanced and immune escape is promoted due to the expression of immunosuppressive mediators, such as FasL, TNF-associated apoptosis inducing ligand (TRAIL) (6), programmed death-ligand 1 (PD-L1) (7), transforming growth factor-β (TGF-β) (8–11) and HSP72 (12). TEXs are highly enriched in tumor-associated antigens (TAAs) and a series of immune-associated proteins involved in antigen presentation, such as major histocompatibility complex (MHC) molecules (13), inducible co-stimulatory molecules (ICOS) (14) and heat shock proteins (HSP) (15). Functional analyses have demonstrated that TEXs can trigger potent CD8+ T cell-dependent antitumor responses and induce antitumor immunity via the transfer of exosomal molecules to dendritic cells (DCs) (5,16–18). Therefore, TEXs are an attractive potential vehicle for immunotherapeutics due to their positive role in intercellular communication.

Cytotoxic T cells (CTLs) can mediate their lytic effects through the Fas ligand (L)-Fas pathway, in which the interaction between FasL expressed on CTLs and Fas expressed on target cells triggers apoptosis of the target cells (19). Notably, not all tumor cells, including RenCa cells, express high levels of the Fas protein on the cell surface; this expression is necessary for stimulated CTL-mediated killing (20). CTLs initially recognize target cells through the T cell receptor (TCR) and then strongly adhere to these cells via accessory molecules. After the TCR engages antigenic peptides presented by MHC molecules on target cells, CTLs ultimately induce apoptosis via the interaction between FasL and Fas proteins (21). However, it remains unknown whether RenCa cell-derived TAA-stimulated CTLs induce the FasL/Fas pathway on the surface of other types of tumor cells. Therefore, whether this process of recognition and apoptosis is antigen-specific requires further investigation.

Previous reports showed that TEX-stimulated CD8+ T cells exhibit stronger cytotoxicity and more efficient antitumor immunity against autologous tumor cells than against other types of tumor cells due to the presentation of immune-associated proteins (16–18). Our pre-experimental study found that RCC-associated antigen G250, which is highly expressed in renal cancer-derived exosomes and has limited expression in normal tissue, may play an important role in antigen-specific antitumor immunity (22). However, in general, few previous studies have investigated the cytotoxicity of RDE-stimulated CD8+ T cells. Therefore, in this study, for further verification, exosomes derived from RenCa cell supernatants after 48 h of culture without fetal bovine serum (FBS) were isolated and characterized, and the activating effect of RDEs combined with GM-CSF and IL-12 on CD8+ T cells and the subsequent cytotoxicity of RDE-stimulated CD8+ T cells was also determined. Finally, the potent antigen-specific immunity mediated by RDE-stimulated CD8+ T cells against RenCa cells in comparison with other types of tumor cells were investigated using a variety of methods both in vivo and in vitro.

Materials and methods

Mice

A total of 200 six-to-eight-week-old (17.40–20.25 g) BALB/c mice were supplied by the Animal Experimental Center of Chongqing Medical University (Chongqing, China) and housed under standard laboratory conditions in the Laboratory Animal Care Facility of Chongqing Medical University (Chongqing, China). Animal care was provided in accordance with institutional guidelines, and all experimental protocols involving animals were performed using a protocol approved by the Ethics Committee of Chongqing Medical University.

Cell lines and culture

The mouse renal cortical adenocarcinoma RenCa cell line, mouse breast cancer 4T1 cell line, and mouse colon cancer CT26 cell line were purchased from Shanghai Cell Bank (Shanghai, China). The cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc. Waltham, MA, USA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in an incubator with a humidified atmosphere of 5% CO2 at 37°C. When the RenCa cells had grown to 80–90% confluency in the plates, the supernatants were harvested for the isolation of RDEs after 48 h of culture without FBS.

Isolation of RDEs

RDEs were extracted and purified as described previously (22). Briefly, culture supernatants were collected and successively centrifuged at 300 × g for 10 min, 800 × g for 30 min, and 10,000 × g for 30 min, after which cells and debris were removed (Ultracentrifuge CP100WX; Hitachi Koki Himac, NuAire, Tokyo, Japan). After concentration by ultrafiltration using a 100 kDa molecular weight cutoff (MWCO) Centriplus centrifugal ultrafiltration tube (EMD Millipore, Billerica, MA, USA) at 1,000 × g for 30 min, the exosome-enriched ultrafiltrate was added to a 30% sucrose/D2O density cushion (Tenglong Weibo Technology, Qingdao, China), followed by ultracentrifugation at 100,000 × g for 60 min at 4°C. The bottom of the cushion was collected and diluted with phosphate-buffered saline (PBS). The exosomes were further concentrated by centrifugation for 30 min at 1,000 × g in 100 kDa MWCO Amicon Ultra-15 ultrafiltration tubes (EMD Millipore). The collected fluid passed through a membrane filter (0.22 µm) for sterilization, and the exosome sample was stored at −80°C.

Electron microscopy

The BCA method (Beyotime Institute of Biotechnology, Shanghai, China) was used to quantify the total protein in the RDEs. After quantification, a 20 µl drop of the suspension was loaded onto an electron microscopy grid. Heavy metal staining was performed with 2% phosphotungstic acid (pH 6.8) for 1 min and the morphological characteristics of the RDEs were visualized and observed under a transmission electron microscope (TEM) (JEM-2010, Jeol Ltd., Tokyo, Japan).

Flow cytometry

RDEs and PBS were injected into BALB/c mice via the caudal vein at 10 µg/mouse and 200 µl/mouse per injection, respectively, a total of three times per week for 4 weeks. For tumor inoculation, 200 µl of PBS containing 2×106 RenCa cells was injected subcutaneously into the right flanks of the mice. The mice were sacrificed under deep inhalation anesthesia (2–3% isoflurane; Zhenzhun Institute of Biotechnology, Shanghai, China) and local analgesia (oxybuprocaine hydrochloride; Zhenzhun Institute of Biotechnology) for isolation of splenocytes and tumor single cell suspensions one day after the last injection. Splenocytes and tumor single cell suspensions were generated from mice subjected to three different treatments on days 10, 20 and 30, as described previously (23,24). In brief, erythrocyte-free single cell suspensions were acquired by grinding the spleens several times in RPMI-1640 and incubating with red blood cell lysis buffer (Beyotime Institute of Biotechnology) on ice for 5 min. Tumor biopsies were cut into several small fragments (2–3 mm in length) and placed in an enzyme digest mix consisting of 3,000 U/ml DNAse, 10 mg/ml collagenase and 10 mg/ml hyaluronidase (all from Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) at 37°C for 2 h. Splenocytes and tumor single cell suspensions were collected, After several washes, membrane markers of T cell subsets were stained with anti-CD3-APC (Miltenyi Biotec, Bergisch Gladbach, Germany), anti-CD4-FITC (Miltenyi Biotec) and anti-CD8-PE (Miltenyi Biotec) antibodies. Then, the proportions of CD3+, CD4+ and CD8+ T cells in splenocytes and tumor single cell suspensions were analyzed using flow cytometry (Beckman Coulter, Pasadena, CA, USA).

IFN-γ measurements

Next, the mice were divided into three groups and vaccinated intravenously (i.v.) three times per week for 2 weeks. For each injection, three groups of mice received 5 µg RDEs/mouse, 10 µg RDEs/mouse and 200 µl PBS/mouse, respectively. Two weeks after the last immunization, splenic CD8+ T cells from PBS- or RDE-immunized mice were sorted by magnetic beads (Miltenyi Biotec) according to the manufacturer's protocol and as previously described (24), seeded at a concentration of 2.0×106 cells per well in 24-well plates and incubated for 48 h at 37°C. After incubation, the culture supernatants were collected for cytokine detection. IFN-γ levels in the culture supernatants were measured by enzyme-linked immunosorbent assays (ELISAs) using a commercial kit (R&D Systems, Minneapolis, MN, USA) based on the manufacturer's instructions.

Proliferation assay for CD8+ T cells

Splenocytes from the mice were harvested and depleted of red blood cells. Then, CD8+ T cells were sorted using magnetic beads according to the manufacturer's protocol as previously described (24). The isolated CD8+ T cells were resuspended in PBS at a concentration of 1×107 cells/ml and labeled with 2.5 µM CFSE (Macklin Biochemical Co., Ltd., Shanghai, China) for 10 min at room temperature in the dark. After quenching CFSE with five volumes of RPMI-1640 medium containing 10% FBS and washing several times with PBS containing 2% FBS, CD8+ T cells at a concentration of 1×106 cells/ml were co-cultured with 100 µl of PBS, 5 µg/ml phytohemagglutinin (PHA) (Sigma-Aldrich; Merck Millipore), 5 µg/ml PHA+20 µg/ml RDEs, 20 ng/ml GM-CSF (PeproTech, Shanghai, China), 20 ng/ml IL-12 (PeproTech), 5 µg/ml PHA+20 µg/ml RDEs+20 ng/ml GM-CSF, 5 µg/ml PHA+20 µg/ml RDEs+20 ng/ml IL-12 and 5 µg/ml PHA+20 µg/ml RDEs+20 ng/ml GM-CSF+20 ng/ml IL-12 for a period of 48 h. The cultured cells were harvested, and the proliferation of CD8+ T cells was evaluated by flow cytometry. The supernatant was harvested and analyzed for the release of IFN-γ.

Tumor growth assays

The RenCa cell line, 4T1 cell line, and CT26 cell line were prepared for injection from cultures grown to nearly 95% confluence. The mice were randomly divided into groups, and the three types of tumor cells were enumerated, adjusted to a concentration of ~2×106 in 200 µl of PBS as described, and injected subcutaneously into the right flanks of the mice to generate three different kinds of tumor model. Then, the mice from the experimental group and the control group were vaccinated three times per week for 4 weeks. For each injection, each mouse in the experimental group received 10 µg of RDEs via the caudal vein and 200 µl of PBS alone as a control. Tumor volume was measured by caliper every three days and calculated according to the formula V=π/6×LxW2 (L, length; W, width). The mice were sacrificed under deep inhalation anesthesia (2–3% isoflurane) and local analgesia (oxybuprocaine hydrochloride) for the isolation of tumor tissues.

Cytotoxicity assays

BALB/c mice were i.v. immunized with RDEs and PBS. Seven days later, the spleens were ground, and an erythrocyte dissolving agent was used to acquire splenocyte single cell suspensions after the final stimulation. To perform in vitro CD8+ T cell assessments, erythrocyte-free single cell suspensions were sorted by magnetic beads to obtain CD8+ T cells according to the manufacturer's protocol. Next, the isolated CD8+ T cells were re-stimulated with irradiated RenCa cells (4,000 rads; 40 Gy) in the presence of 20 ng/ml IL-2 (PeproTech, Shanghai, China) in vitro for three days. Then, the re-stimulated CD8+ T cells were treated with 20 ng/ml GM-CSF, 20 ng/ml IL-12 or 20 ng/ml GM-CSF in combination with 20 ng/ml IL-12 for 48 h. PBS-treated CD8+ T cells were treated with 20 ng/ml GM-CSF or 20 ng/ml IL-12.

Following five days of treatment, the CD8+ T cells derived from different treatments were harvested as effector cells and RenCa cells were used as target cells to establish a co-culture system with different E/T (effector/target) ratios for the LDH release assay via a CytoTox96 Non-Radioactive Cytotoxicity Assay Kit (Promega Biological Products, Ltd., Shanghai, China) and E/T=3 for the apoptosis assay via flow cytometry. Subsequently, RDE-stimulated CD8+ T cells were used as new effector cells, whereas 4T1 cells and CT26 cells served as control target cells for the LDH release assay, apoptosis assay and cell cycle analysis. Briefly, co-culture systems were established with different E/T ratios of RDE-stimulated CD8+ T cells and three types of tumor cells at 37°C for 4 h. For the inhibition of FasL-dependent cytotoxicity, co-cultures (RenCa cells and CD8+ T cells) were treated or not with 10 µg/ml anti-FasL antibody (MFL-3, 555291; BD Bioscience, San Jose, CA) or isotype control (eBioscience, San Diego, CA, USA) and maintained at 37°C. Specific lysis (%) was calculated as follows: (Experimental LDH release-effector cells-target spontaneous LDH release)/(target maximum LDH release) ×100 (10). Then, when E/T=10, the three types of tumor cells were digested by trypsinization (Beyotime Biotechnology, Shanghai, China) and washed several times for apoptosis assays and cell cycle analysis via flow cytometry. Subsequently, CytoFLEX software (Beckman Coulter, CA, USA) was used to analyze the flow cytometry data. The percentage of apoptosis was calculated from early apoptosis (Annexin V+/PI−) plus late apoptosis (Annexin V+/PI+), and the cell cycle analysis was compared with the S-phase ratio. Each experiment was performed in triplicate.

Western blot analysis

Western blotting was performed as described previously (25). A total of 20 µg of RDEs or crude protein extracted from RenCa cell lysates was fractionated by 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked in Tris-buffered saline with Tween (TBST) and 5% nonfat milk for 1 h at room temperature. The rabbit anti-mouse HSP70 monoclonal antibody (ab181606; Abcam, Cambridge, MA, USA), rabbit anti-mouse CD63 monoclonal antibody (ab217345; Abcam) and rabbit anti-mouse CD81 monoclonal antibody (ab109201; Abcam) diluted 1:1,000 were added and incubated at 4°C overnight. The next day, after incubation with the appropriate horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (ab6721; Abcam) (1:5,000 dilution), the bots were then detected using an Odyssey Infrared System (LI-COR Bioscience, Lincoln, NE, USA).

The cell fractions of three types of tumor cells from different treatments and CD8+ T cells from different treatments were prepared, and protein quantification was performed using the BCA method. Equal amounts of protein were separated via 10–15% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with TBST and 5% non-fat milk to block nonspecific binding at room temperature, and then they were incubated overnight at 4°C with rabbit anti-mouse FasL polyclonal antibody (ab15285; Abcam) (for CD8+ T cells), rabbit anti-mouse Fas polyclonal antibody (ab82419; Abcam) (for tumor cells) and rabbit anti-mouse β-actin polyclonal antibody (ab8227; Abcam) (1:1,000 dilution). The next day, after incubation with the HRP-conjugated goat anti-rabbit secondary antibodies (1:5,000 dilution), the immunoassociated protein bands on the membrane were visualized using a chemiluminescence kit (Beyotime Biotechnology, Shanghai, China) and detected using an Odyssey Infrared System. The density of each band was normalized to its loading control (β-actin).

Statistical analysis

All experiments were performed at least three times in triplicate, and all data represent the mean ± SEM. One-way ANOVA or two-way ANOVA followed by Tukey's test was used to analyze the data. Statistical analysis was performed using GraphPad Prism software 7.0 for Mac (GraphPad Software, Inc., San Diego, CA, USA).

Results

Isolation and characterization of RDEs

Whether RenCa cells produce and secrete exosomes was determined by TEM (Fig. 1A). We first isolated and purified exosomes from the culture supernatants of RenCa cells by ultrafiltration and sucrose density gradient centrifugation. Subsequently, the RDEs exhibited the expected morphology and were visualized as spherical membrane-bound vesicles with a diameter of 30–100 nm as determined by TEM. In addition, the western blot analysis results showed that the molecular markers HSP70, CD63 and CD81 were highly expressed in RDEs but relatively rare in the lysates of RenCa cells (Fig. 1B).

Figure 1. Identification of RDEs. (A) Electron microscopy results showing exosomal membrane-bound spherical vesicles isolated from RenCa cells. Purified RDEs were observed by TEM after ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The exosomes are dimpled microvesicles, ranging from 30–100 nm. The scale bar is 100 nm. (B) Western blot analysis of the expression of proteins, such as HSP70, CD63 and CD81, in RDEs and cell lysates from RenCa cells. RDEs, RenCa cell-derived exosomes; TEM, transmission electron microscopy; HSP70, heat shock protein 70; CD, cluster of differentiation.

RDEs alter T cell subset ratios and stimulate CD8+ T cells

Exosomes from tumor cells have been shown to either suppress or stimulate the immune response (26). Because exosomes have complex immune functions due to their composition, we initiated the study by detecting T cell subset ratios (CD3+ CD4+ and CD8+) in splenocytes from RDE-immunized mice and tumor single cell suspensions from PBS-treated mice via flow cytometry at different time points. An examination of the T cell subset ratios in splenocytes (Fig. 2C and D) from RDE-immunized mice indicated a significant increase in the ratio of CD8+/CD4+ T cells compared with that of the PBS treatment group, although no difference was observed in the number of CD3+ T cells in the splenocytes (Fig. 2A and B) on days 10, 20 and 30. However, the CD3+ T cells in splenocytes (Fig. 2A and B) from the tumor-bearing mice were significantly decreased. We also found that the proportion of CD8+/CD4+ T cells in RenCa tumor tissues gradually increased with tumor progression (Fig. 2E and F). To further investigate the immune response induced by RDEs, the levels of IFN-γ in culture supernatants of splenic CD8+ T cells obtained from RDE-immunized mice were measured via an ELISA kit, according to the manufacturer's instructions. As shown in Fig. 2G, compared with CD8+ T cells from PBS-immunized mice, RDEs induced splenic CD8+ T cells obtained from RDE-immunized mice to produce a large amount of IFN-γ.

Figure 2. Evaluation of the immune properties of RDEs. Seven-week-old BLAB/c mice were immunized intravenously with PBS and RDEs three times per week for 4 weeks, and RenCa cells were simultaneously injected subcutaneously to generate a tumor-bearing mouse model. One day after a 10-, 20- or 30-day immunization with RDEs, splenocytes without erythrocytes and tumor single-cell suspensions were isolated from mice in the different treatment groups to determine the percentages of T cell subsets [(A and B) CD3+ T cells in splenocytes, (C and D) CD8+/CD4+ T cells in splenocytes in tumor single cell suspensions] via flow cytometry. Similar data were obtained from three independent experiments. Asterisks indicate significant differences (**P<0.01). RDEs, RenCa cell-derived exosomes; CD, cluster of differentiation; IFN-γ, interferon-γ; ELISA, enzyme-linked immunosorbent assay; APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; SSC-A, side scatter area. Evaluation of the immune properties of RDEs. Seven-week-old BLAB/c mice were immunized intravenously with PBS and RDEs three times per week for 4 weeks, and RenCa cells were simultaneously injected subcutaneously to generate a tumor-bearing mouse model. One day after a 10-, 20- or 30-day immunization with RDEs, splenocytes without erythrocytes and tumor single-cell suspensions were isolated from mice in the different treatment groups to determine the percentages of T cell subsets [(E and F) CD8+/CD4+ T cells in tumor single cell suspensions] via flow cytometry. (G) Splenic CD8+ T cells obtained from mice immunized with PBS and RDEs were incubated under a humidified atmosphere of 5% CO2 at 37°C in vitro. After 48 h, the supernatants were cleared of dead cells and cell debris, collected and utilized for ELISA to measure the quantity of IFN-γ secreted from RDE-stimulated splenic CD8+ T cells. Similar data were obtained from three independent experiments. Asterisks indicate significant differences (**P<0.01). RDEs, RenCa cell-derived exosomes; CD, cluster of differentiation; IFN-γ, interferon-γ; ELISA, enzyme-linked immunosorbent assay; APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; SSC-A, side scatter area.

RDEs combined with GM-CSF and IL-12 promoted the proliferation of CD8+ T cells more effectively than RDEs alone with GM-CSF or IL-12 in vitro

To investigate whether RDE immunization could induce the proliferation of CD8+ T cells effectively in vitro, we initiated the study by determining the effect of RDEs on the proliferation rate of CD8+ T cells. Splenic CD8+ T cells obtained from the mice were labeled with CFSE and stimulated with PBS, PHA, PHA+RDEs, GM-CSF, IL-12, PHA+RDEs+GM-CSF, PHA+RDEs+IL-12 and PHA+RDEs+GM-CSF+IL-12. As shown in Fig. 3A and B, the proliferation index of CD8+ T cells was significantly enhanced by stimulation with PHA compared with PBS; enhanced by stimulation with PHA+RDEs compared with PHA; enhanced by stimulation with GM-CSF or IL-12 compared with PBS; and enhanced by stimulation with PHA+RDEs+GM-CSF or PHA+RDEs+IL-12 compared with PHA+RDEs. The maximum proliferative index was observed in CD8+ T cells stimulated with PHA+RDEs+GM-CSF+IL-12.

Figure 3. RDEs combined with GM-CSF and IL-12 induce the proliferation and stimulation of CD8+ T cells more effectively than RDEs alone with GM-CSF or IL-12 in vitro. Splenic CD8+ T cells were labeled with CFSE and treated with PBS, PHA, PHA+RDEs, GM-CSF, IL-12, PHA+RDEs+GM-CSF, PHA+RDEs+IL-12 and PHA+RDEs+GM-CSF+IL-12 for 48 h. (A) Number of cell divisions was assessed by successive halving of the fluorescence intensity of CFSE by flow cytometry. RDE-treated CD8+ T cells exhibited a smaller number of cells with lower (G1, G2 and G3) cell divisions and a greater number of cells with higher (G4+) cell divisions than non-RDE-treated CD8+ T cells. The application of GM-CSF and IL-12 enhanced this effect. (B) Relative proliferation index of each treatment group was also assessed by flow cytometry. (C) Supernatants of different treatment groups were harvested, and the release of IFN-γ was determined by ELISA. Each group was tested in triplicate wells, and the values were statistically analyzed by one-way ANOVA (*P<0.05). RDEs, RenCa cell-derived exosomes; ELISA, enzyme-linked immunosorbent assay; IFN-γ, interferon-γ; GM-CSF, granulocyte-macrophage colony stimulating factor; G1-4, generation 1–4; IL-12, interleukin-12; PHA, phytohemagglutinin; CFSE, carboxyfluorescein diacetate succinimidyl ester; ANOVA, analysis of variance; CD, cluster of differentiation.

To further investigate the ability of RDEs to induce the release of IFN-γ by CD8+ T cells, the cell supernatants of the different treatments described above were harvested and subjected to an IFN-γ release assay by ELISA. The results demonstrated that RDEs induced the production of IFN-γ by CD8+ T cells in vitro, and similar results can be observed above (Fig. 3C). This finding indicates that RDEs can effectively promote the proliferation and activation of CD8+ T cells. Furthermore, the combination of RDEs with GM-CSF and IL-12 may also facilitate a stronger positive effect than RDEs alone with GM-CSF or IL-12 on the stimulation and proliferation of CD8+ T cells.

More effective induction of cytotoxic effects in RDE-stimulated CD8+ T cells treated with GM-CSF and IL-12

To explore whether RDE-stimulated CD8+ T cells combined with GM-CSF and IL-12 could induce more potent cytotoxic effects, we harvested RDE-stimulated CD8+ T cells treated with GM-CSF, IL-12 or GM-CSF in combination with IL-12 to establish a co-culture system with different E/T ratios. GM-CSF, IL-12-treated CD8+ T cells served as the control. The results (Fig. 4A) showed that RDE-stimulated CD8+ T cells treated with GM-CSF or IL-12 could kill RenCa cells more effectively than RDE-stimulated CD8+ T cells alone, and GM-CSF in combination with IL-12 could achieve the maximum cytotoxic effect. However, GM-CSF or IL-12 alone failed to increase the cytotoxic effect of control CD8+ T cells. Similar results were observed when flow cytometry was used to measure the proportion of apoptotic RenCa cells when E/T=3 (Fig. 4B and C). The results demonstrated the antitumor effects of RDE-stimulated CD8+ T cells combined with GM-CSF and IL-12.

Figure 4. Increased cytotoxicity of RDE-stimulated CD8+ T cells in combination with GM-CSF and IL-12. Splenic CD8+ T cells from immunized mice were re-stimulated with irradiated RenCa cells in the presence of IL-2 in vitro. Three days later, re-stimulated CD8+ T cells were treated with GM-CSF, IL-12 and GM-CSF in combination with IL-12 for 48 h. Control CD8+ T cells were treated with GM-CSF or IL-12. CD8+ T cells derived from the different treatments were used as effectors, and the target cells were RenCa cells. (A) Cytotoxic assay of CD8+ T cells was performed using the LDH release assay with the following formula: Lysis (%)=(experimental LDH release-effector cells-target spontaneous LDH release)/(target maximum LDH release) ×100. (B and C) Apoptosis analysis was performed when E/T=3, and the proportion of apoptotic RenCa cells was detected by flow cytometry (**P<0.01). IL, interleukin; GM-CSF, granulocyte-macrophage colony stimulating factor; CD, cluster of differentiation; RDEs, RenCa cell-derived exosomes; LDH, lactate dehydrogenase; E/T, effector/target; FITC-A, fluorescein isothiocyanate-area; PI PC 5.5-A, propidium iodide PC 5.5-area.

RDEs specifically inhibit the growth of RenCa tumors

To determine whether RenCa tumors were more affected by RDEs than other types of tumors, we used RDEs and PBS to treat BALB/c mice bearing established subcutaneous RenCa, 4T1 and CT26 tumors and followed the tumor growth. Tumor size was measured using calipers, and overall survival was monitored and recorded. As shown in Fig. 5A and D, compared with PBS, RDE vaccination promoted a marked inhibition of RenCa tumor growth. In addition, a phenomenon occurred in which the difference in tumor growth between the RDE groups and the PBS groups was not observed in mice with 4T1 (Fig. 5B and D) and CT26 (Fig. 5C and D) tumors. Moreover, the survival rate of RenCa tumor-challenged mice was also significantly increased by RDE immunization, although mice with 4T1 and CT26 tumors did not exhibit corresponding differences between treatment groups (Fig. 5E). Taken together, the data indicate that RDEs induced more effective therapeutic immunity against renal cortical adenocarcinoma than against other cancer types.

Figure 5. Specific resistance effect of RDEs on RenCa tumor growth. BALB/c mice received a subcutaneous injection of 2×106 tumor cells and an intravenous injection of 10 µg of RDEs or 200 µl of PBS for a total of three treatments per week for nearly 4 weeks. Tumor growth curves and survival curves of RDE-treated mice bearing (A) RenCa tumors, (B) 4T1 tumors and (C) CT26 tumors and the corresponding control groups are shown. (D) Mean ± SD values for tumor size and (E) survival rate showing significant or non-significant differences between tumor-bearing mice treated with RDEs and PBS. The growth of the implanted tumor was measured over a 26-day period, and the survival rate of immunized mice was recorded until the 69th day. Each point represents the mean of the average tumor size (*P<0.05; **P<0.01). RDEs, RenCa cell-derived exosomes; SD, standard deviation.

RDE-stimulated CD8+ T cells induce stronger immunity against renal cancer than against other cancer types in vitro

Next, we investigated the specific cytotoxic effects of RDE-stimulated splenic CD8+ T cells against three types of tumor cells using the LDH release method. Seven days after intravenous immunization with RDEs, splenic CD8+ T cells re-stimulated with irradiated RenCa cells were sorted and co-cultured with different target cells at various E/T ratios. As shown in Fig. 6A, RDE-stimulated CD8+ T cells displayed potent cytotoxic ability against RenCa cells at all E/T ratios, while no detectable CD8+ T response was induced by PBS. A less dramatic increase in the killing rate occurred when 4T1 and CT26 cells were treated with RDE-stimulated CD8+ T cells at E/T ratios over 12:1.

Figure 6. Development of antigen-specific CD8+ T cell cytotoxic activity in vitro. (A) Splenic CD8+ T cells from immunized mice were re-stimulated with irradiated RenCa cells in the presence of IL-2 in vitro. Three days later, stimulated CD8+ T cells were harvested and used as effector (E) cells in the LDH release assay, whereas RenCa cells or control 4T1 and CT26 cells were used as the target (T) cells and mixed with effector cells at different ratios. Specific lysis (%) was calculated with the following formula on the basis of the amount of LDH release: (Experimental LDH release-effector cells-target spontaneous LDH release)/(target maximum LDH release) ×100. RDE-stimulated CD8+ T cells and control CD8+ T cells were co-cultured with three types of tumor cells at a 10:1 ratio. (B) Cell cycle distribution of the three types of tumor cells were detected by flow cytometry. The data are representative of three independent experiments and expressed as the mean ± SD (*P<0.05, **P<0.01). IL, interleukin; CD, cluster of differentiation; RDEs, RenCa cell-derived exosomes; FITC-A, fluorescein isothiocyanate-area; FL2-A PI PE-A, FL2-A propidium iodide PE 5.5-area; PI PC 5.5-A, propidium iodide PC 5.5-area; SD, standard deviation; LDH, lactate dehydrogenase; FasL, Fas ligand. Development of antigen-specific CD8+ T cell cytotoxic activity in vitro. RDE-stimulated CD8+ T cells and control CD8+ T cells were co-cultured with three types of tumor cells at a 10:1 ratio. (C and D) Percentage of apoptosis of the three types of tumor cells was detected by flow cytometry. (E) FasL protein levels on the surface of CD8+ T cells after different treatments and Fas levels on the surface of RenCa, 4T1 and CT26 cells were assessed by western blot analysis. The upper panel and lower panel show histogram analyses of the expression levels of the FasL and Fas proteins, respectively. β-actin was used as an internal control. (F) Cytotoxic assay performed in the presence of anti-FasL antibody or isotype control (10 µg/ml). The data are representative of three independent experiments and expressed as the mean ± SD (*P<0.05, **P<0.01). IL, interleukin; CD, cluster of differentiation; RDEs, RenCa cell-derived exosomes; FITC-A, fluorescein isothiocyanate-area; FL2-A PI PE-A, FL2-A propidium iodide PE 5.5-area; PI PC 5.5-A, propidium iodide PC 5.5-area; SD, standard deviation; LDH, lactate dehydrogenase; FasL, Fas ligand.

Subsequently, to determine the effect of effector cells on apoptosis and the cell cycle in three types of target cells, the percentage of cells undergoing apoptosis and the cell cycle distribution of target cells were determined and quantified using an apoptosis assay and a cell cycle assay, respectively, via flow cytometry. As shown in Fig. 6C and D, Annexin V-based apoptosis analysis revealed significant increases in the percentages of apoptotic cells in the lower right and upper right quadrants of the histograms, called early and late apoptotic cells, respectively, in RenCa cells treated with RDE-stimulated CD8+ T cells, compared with controls at an E/T ratio of 10:1. By contrast, compared with the controls, there was a less marked increase in the apoptosis rate when 4T1 and CT26 cells were tested with RDE-stimulated CD8+ T cells. Furthermore, in cell cycle assays (Fig. 6B), RenCa cells treated with RDE-stimulated CD8+ T cells showed a significantly decreased S-phase ratio compared with the controls, but the 4T1 and CT26 cells did not, indicating that the killing activity of these RDE-stimulated CD8+ T cells was exosome antigen-specific.

A previous study demonstrated that cytotoxic T cells can mediate their lytic effects through a distinct pathway called the Fas-based pathway, in which the interaction between FasL expressed on CTLs and Fas presented on target cells triggers apoptosis and cell death (20). Hence, in the present study, the expression of FasL on CD8+ T cells and Fas on tumor cells was detected by western blotting. As shown in Fig. 6E, the co-culture of RDE-stimulated CD8+ T cells and RenCa cells resulted in an increase in FasL expression on the CD8+ T cell surface and an increase in Fas expression on RenCa cells, compared with the control levels. However, in other types of tumor cells, similar results were not observed. The perforin inhibitor blocked the majority of RenCa cell CTL lysis, suggesting a major effector role for granule-mediated lysis in vitro (21). However, some inhibition of lysis was also observed when the anti-FasL antibody was present (Fig. 6F). These findings suggest that the FasL/Fas-mediated mechanism also plays a significant role in the cytotoxicity of RDE-stimulated CD8+ T cells.

Discussion

Research on exosomes has attracted substantial attention since their initial discovery. TEXs are not only involved in establishing a favorable microenvironment surrounding the tumor but also induce a comprehensive alteration of the immune system (27,28).

A full understanding of TEX immunization includes the observation that TEXs are a source of shared TAA and stimulatory molecules for the innate immune response, which can induce T cell-dependent immunity in mice or human tumor models under some conditions (8–11,16–18). This property makes TEXs novel potential vaccines for the treatment of tumors. However, according to previous studies, TEXs can be equipped with either immune-stimulatory ability or immunosuppressive ability due to the complexity of their components (26). The immunosuppressive abilities of TEXs have been characterized in numerous cancer types and in the context of several different mediators, such as the amplification of myeloid-derived suppressor cell (MDSC) (23), the induction of effector T cell apoptosis (29,30), the suppression of natural killer (NK) cell capability (31) and the disruption of dendritic cell (DC) differentiation and maturation (32). However, this effect has no negative impact on their availability to transmit TAA to mature DCs and induce an immune-stimulatory response that is superior to their immunosuppressive effect in tumor-bearing hosts. Therefore, we have demonstrated by means of T cell subset ratio analyses, tumor growth assays, IFN-γ secretion assays, proliferation assays of CD8+ T cells and cytotoxicity assays of RDE-stimulated CD8+ T cells that RDEs express immunologically relevant molecules to induce in vivo and in vitro immune activation and elicit a potent immune response. Additionally, when RDE-stimulated CD8+ T cells are combined with GM-CSF and IL-12, the immune response is further optimized. Furthermore, we can conclude from the tumor growth assay that in the case of RDEs, their immune-stimulatory ability is greater than their immunosuppressive ability.

Previous studies have reported that the injection of an increasing number of TEXs could provide a source of tumor-rejection antigens relevant for immunization (33) and the crucial components of the immune response are DCs that participate in antigen presentation and mediate antigen specificity of the immune response (34). Therefore, based on the positive effects of RDEs on immune cells, we investigated the specific cytotoxic function of RDE-stimulated splenic CD8+ T cells. As expected, RDE antigen-specific splenic CD8+ T cells displayed stronger cytotoxicity for the autologous tumor cells than the other types of tumor cells at any E/T ratio. In vitro experiments have shown that TEXs may need to rely on host DCs for the induction of an immune response (8–11,12–14); however, TEXs can be taken up by CD8+ T cells, although a lack of DC participation was reported in some experiments, and TEXs can directly induce specific killing activity of CD8+ T cells via exosomal MHC molecules (35). Notably, we observed a dramatic phenomenon in which RDE-stimulated CD8+ T cells displayed a slightly weaker lethality for CT26 cells and 4T1 cells. This effect could be caused by nonspecific cytotoxicity or a mixture of RDE-stimulated NK cells that lack specific cytotoxicity for tumor cells among the CD8+ T cells derived from the splenocytes of immunized mice (15).

Two major molecular pathways of CD8+ T cell-regulated cytotoxicity have been proven: i) the exocytosis of granules containing perforin and granzyme molecules and ii) the interaction of FasL on T cells with the apoptosis-inducing Fas molecule on target cells (19). CTLs initially recognize target cells using the TCR and strongly adhere to these cells via accessory molecules; finally, FasL partially transmits death signals to Fas-positive target cells and induces cell apoptosis. These anti-RenCa tumor CD8+ T cells can lyse RenCa cells in vitro via the perforin/granzyme and FasL/Fas pathways. Moreover, proinflammatory cytokines, such as IFN-γ and TNF-α could play a key role in the effector phase of the immune response because these cytokines sensitize tumor cells to lysis by both of the above pathways (21). This observation may explain why RDE-stimulated CD8+ T cell-treated RenCa cells highly expressed the Fas protein compared with RenCa cells treated with control CD8+ T cells or PBS. T-cell-mediated cytotoxicity in vitro was enhanced via triggering through the crossing-linking of TCR, which is known to be necessary for the up-regulation of cell surface FasL (36). However, similar results were not observed in other types of tumor cells, suggesting that the effect of pro-inflammatory cytokines is based on the specific recognition and combination of FasL and Fas and that apoptosis mediated by the FasL-Fas pathway is characterized by antigenic specificity. However, controlling tumor growth and metastasis using CTLs alone is difficult due to the complex mechanisms of tumor cell proliferation, progression and metastasis. CTLs expressing FasL can induce apoptosis in tumor cells, whereas tumor cells expressing FasL can also induce the elimination of CTLs according to the tumor anti-killing theory (37), which necessitates the optimization of RDE-based vaccines, such as the use of RDE-stimulated CD8+ T cells in combination with GM-CSF and IL-12.

Taken together, these data demonstrate that investigating TEX-associated mechanisms in the context of the immune response is vital for the development of novel therapies. Additional mechanisms that modify TEXs to suppress and evade the immune system have attracted attention. Immunosuppressive mediators that are present in TEXs, such as FasL, TRAIL (6), TGF-β (8–11), PD-L1 (7), and HSP72 (12), can exert adverse effects on the antitumor response. Furthermore, a negative effect of the immunogenicity of TEX-based vaccines arises when TEXs lack the expression of MHC complexes (13), adhesion molecule signals (38) and co-stimulatory molecules (14) that can enhance antitumor immunity. Therefore, extensive research has already demonstrated that downregulating these mediators of immunosuppression and avoiding the absence of these immune-stimulatory factors can lead to a more efficient induction of systemic antitumor immunity and CTL responses. Furthermore, the combination of TEXs with GM-CSF (39) and IL-12 (22) may also facilitate a stronger positive effect on the antitumor response. The activation of NK cells induced by TEXs from one of these tumors may have a relatively weak nonspecific killing effect on other tumors in patients who have two or more tumors simultaneously. In summary, exosome therapy offers a promising immune-based strategy for the development of cancer vaccines; however, we must still optimize the TEX-based treatment program in clinical trials due to the complex role of TEX components in immune regulation.

In conclusion, our study demonstrated that RDEs could be used as vaccines to effectively retard renal cancer growth and prolong survival in mice. RDEs can promote the proliferation and activation of CD8+ T cells, and the combination of RDE-stimulated CD8+ T cells with GM-CSF or IL-2 may achieve a more satisfactory antitumor immune response. Furthermore, RDE-stimulated CD8+ T cells can induce antigen-specific anti-renal cancer effects through the FasL/Fas signaling pathway. Therefore, our study provides a promising therapy for renal cancer using exosome-based antigen-specific vaccines.

Acknowledgements

Not applicable.

Funding

The National Natural Science Foundation of China (grant no. 81272572) provided financial support for this study.

Availability of data and materials

All data generated or analyzed during this study are included in this published article, and are available on reasonable request.

Authors contributions

HYX conducted experiments, data acquisition and analysis and was a major contributor in writing the manuscript; YZ made a significant contribution to the conception and design of the experiments; NL and NY were involved in cell culture and exosome extraction; XFX, HXW and XYL collected and analyzed the data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The Ethics Committee of Chongqing Medical University approved this study.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests, and all authors have confirmed the accuracy of this statement.

References