- Open Access
Dual effect of DLBCL-derived EXOs in lymphoma to improve DC vaccine efficacy in vitro while favor tumorgenesis in vivo
- Zhenzhen Chen†1, 2,
- Liangshun You†1, 3,
- Lei Wang1,
- Xianbo Huang1,
- Hui Liu1, 3,
- Ju ying Wei1, 2, 3,
- Li Zhu1, 2 and
- Wenbin Qian1, 2, 3Email author
© The Author(s). 2018
- Received: 27 May 2018
- Accepted: 2 August 2018
- Published: 13 August 2018
Exosomes derived from tumor cells (TEXs) are involved in both immune suppression, angiogenesis, metastasis and anticancer stimulatory, but the biological characteristics and role of diffuse large B cell lymphoma (DLBCL)-derived exosomes have been less investigated.
Exosomes (EXOs) were isolated from OCI-LY3, SU-DHL-16, and Raji cells and biological characteristics of EXOs were investigated using electron microscopy, flow cytometry analysis, and Western blot analysis. The protein expression of EXOs was determined by an antibody array. Next, the communication between EXOs and lymphoma cell, stromal cell, dendritic cells (DCs), and T cells was evaluated. Finally, effect of DLBCL TEXs on tumor growth in vivo was investigated.
We demonstrated that EXOs derived from DLBCL cell lines displayed malignancy molecules such as c-Myc, Bcl-2, Mcl-1, CD19, and CD20. There was a different protein expression pattern between DLBCL TEXs and Burkitt lymphoma TEXs. DLBCL TEXs were easily captured by DCs and lymphoma cells, and mainly acted as an immunosuppressive mediator, evidenced by induction of apoptosis and upregulation of PD-1 in T cells. Furthermore, the TEXs stimulated not only cell proliferation, migration of stromal cells but also angiogenesis. As a result, the TEXs promoted tumor growth in vivo. On other hand, DLBCL TEXs did not induce apoptosis of DCs. After pulsed with the TEXs, DCs could stimulate clonal expansion of T cells, increase the secretion of IL-6 and TNFα, and decrease the production of immunosuppressive cytokine IL-4 and IL-10. The T cells from tumor bearing mice immunized by TEX were shown to possess superior antilymphoma potency relative to immunization of tumor lysates.
This study provides the framework for novel immunotherapies targeting TEXs in DLBCL.
- Diffuse large B-cell lymphoma
- Dendritic cells
- Cancer immunotherapy
- Tumor microenvironment
Diffuse large B-cell lymphoma (DLBCL), a clinically and biologically heterogeneous tumor, is the most common subtype of lymphoma, representing of 20–30% of all lymphoproliferative disorders . Although 50–70% of DLBCL patients can be cured by current standard treatment with rituximab-based chemotherapy, about 50% of the patients are found to be inadequate by this treatment, in which 20% of patients suffer from primary refractory and others relapse after achieving complete remission . Most patients with refractory DLBCL have no effective treatment options. In the last years, immunotherapy, an alternative method, appears promising and probably will improve therapeutic strategy for the patients with DLBCL. Immunotherapies fall into four categories such as immune-checkpoint inhibitors, adoptive cellular therapy including chimeric antigen receptor T-cell, and therapeutic cancer vaccines [3–5]. Among them, dendritic cell (DC)-based vaccines offer a promising therapeutic platform for a variety of cancer including lymphoma. For example, a pilot study demonstrated that vaccination with DCs loaded with apoptotic and necrotic autologous tumor cells increased natural killer (NK) cell activation, reduced Treg frequency and induced both T- and B-cell anticancer responses associated with clinical efficacy in heavily pretreated B cell lymphoma patients .
Exosomes (EXOs) are membrane vesicles with a diameter of 30–100 nm originating from multivesicular bodies of many types of cells including cancer cells, which function as a mode of intercellular communication and molecular transfer [7, 8]. Recently, tumor-derived exosomes (TEXs) have been shown in various cancer models to actively promote tumorigenesis and metastasis through intricate mechanisms including transfer of oncogenic receptors, protein and RNA, suppression of the function of NK cells and T cells, promotion of T regulatory cell expansion, and mediation of tumor microenvironment (TME) via angiogenesis promotion, stromal remodeling, and signaling pathway activation [9–14]. Although TEXs are predominantly immunosuppressive, they can also enhance immunostimulation and therefore serve as cancer vaccines. TEXs bear major histocompatibility complex (MHC) protein, chaperones, such as heat shock protein-70 (HSP-70) and/or HSP-90, and tumor-associated antigens (TAAs) taken up by DCs, which are effective in mediating anti-tumor immunity in vitro and in vivo [7, 15, 16]. The anti-cancer efficacy of TEXs was also confirmed in lymphoma. Menayet al  demonstrated that T cells from T-cell lymphoma TEXs-immunized mice secrete interferon-γ in response to tumor stimulation and administration of the TEXs into mice induces a tumor-specific immune response. In a mouse model, EXOs obtained heat-shocked B lymphoma cells (HS-Exo) had been shown to contain HSP-60, HSP-90 and molecules involved in immunogenicity including MHC class I, MHC class II, CD40 and CD86, and to induce maturation of DCs. Furthermore, HS-Exo immunization strong activated T cell response . The dual role of EXOs from B-cell lymphoma already has been characterized extensively; however, there are only a few studies [19–21] that elucidate characteristic of the EXOs secreted by DLBCL cells.
In this study, we report a comprehensive analysis of EXOs derived from DLBCL cell lines and their role in the communication with T cells, DCs, and stromal cells including human umbilical vein endothelial cells (HUVEC) and human fibroblasts. More specifically, our results suggest a novel strategy by targeting TEXs in lymphoma therapeutic development.
The human DLBCL cell lines OCI-LY3 and SU-DHL-16 were kindly provided by Professor Jianyou Gu, Zhejiang Provincial Hospital of TCM (Hangzhou, China). The human Burkitt lymphoma cells Raji, HUVEC and the murine B lymphocyte cell line A20 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The human dendritic cell line DCS, the normal human T cell line Th2, the human skin fibroblasts HSF and the murine DC cell line D2SC/1 were purchased from Huazhong University of Science and Technology (Wuhan, China). The SU-DHL-16, Raji and A20 cells were cultured in RPMI 1640 medium supplemented with 10% depleted fetal bovine serum (FBS; Gibico, Grand Island, NY, USA), which obtained by ultracentrifugation at 100,000 g for 18 h to remove possible FBS-containing EXOs. The OCI-LY3 cells were cultured in Iscove’s Modified Dulbecco’s Medium with 15% FBS. The DCS, Th2, HUVEC, HSF and D2SC/1 cells were cultured in Dulbecco’s Modified Eagle’s Medium with 10% FBS.
Human DCs and fibroblasts (SFs)
Human peripheral blood mononuclear cells (PBMNCs) were obtained from healthy volunteers (provided by Zhejiang Blood Center, Zhejiang, China), and SFs were isolated from non-tumoral gastric walls of the patients who underwent surgery in our hospital. Informed consent was obtained from all volunteers and patients. PBMNCs were isolated using a human Lymphoprep solution (Axis-shield PoC AS, Oslo, Norway), and cultured in a 10-cm Petri dish and incubated for 24 h to allow them to adhere to the dish’s surface. Adherent cells were induced to form immature DCs, supplemented with 120 ng/mL recombinant human granulocyte macrophage colony-stimulating factor (PeproTech, Offenbach, Germany), and 60 ng/mL recombinant human interleukin-4 (PeproTech) to decrease contamination by macrophages for 5 days. Nonadherent cells were harvested and used as human lymphocytes. SFs were prepared by transferring the gastric tissue to a T25 flask and cutting into 1mm3 pieces. Incubate the chopped material with 5 ml of trypsin EDTA for 5 min at 37 °C, and then the trypsin was Inactivated by adding 1 ml FBS. The cell pellet was obtained by centrifuging of suspension at 200 g speed for 10 min, which was transferred to a T25 flask containing DMEM medium with 20% FBS and 2 × Penicillin/Streptomycin) after resuspend using DMEM.
Murine bone marrow stromal cells (BMSCs)
The BMSCs were isolated from the bone marrow of C57BL/6 mice. The medullar canal of the tibias and femurs was flushed with PBS. The resulting suspension was harvested and filtered through a 70 μm cell strainer. After centrifugation, BMSCs were resuspended in red blood cell lysis buffer to remove red blood cells.
Preparation of exosomes and cell lysates
The supernatant was sequentially centrifuged at 500 g for 10 min, followed by 2000 g for 30 min, and filtered with a 0.22 μm filter (Millipore, Bedford, MA, USA) to remove cells and cellular debris. The EXOs were isolated by ultracentrifugation at 110,000 g for 70 min at 4 °C. EXOs pellets were washed with phosphate-buffered saline (PBS) and ultracentrifugated at 110,000 g for 70 min. The cell lysates were obtained by five successive cycles of freeze-thawing. Cell lysates were then followed by centrifugation at 3000 g for 30 min, and filtered with a 0.22 μm filter. The protein concentration of EXOs and cell lysates were quantified by the Bradford assay (Sangon Biotech, shanghai, China).
Nanoparticle tracking analysis (NTA)
The size distributions and surface Zeta potential of DLBCL TEXs were analyzed by NanoSight NS300 (NanoSight, Amesbury, United Kingdom) using NTA 3.2 software, as described elsewhere [22–24]. EXOs were measured upon dilution into PBS at a concentration of 6 × 108 particles/ml in triplicates.
Phenotypic characterization of exosomes
A total of 30 μg EXOs were precoated with 10 μL aldehyde/sulfate latex beads (Invitrogen, Carlsbad, CA, USA) overnight at room temperature and stopped by 0.1%BSA. EXOs, coated on Beads were stained with a panel of fluorescein APC-or PE-conjugated antibodies, CD19, CD20, CD40, CD80, CD83, CD86 and HLA-DR (BioLegend, San Diego, CA, USA) and the corresponding isotype-matched antibodies, and then followed by flow cytometry analysis (FACS; Accuri C6, BD, Franklin Lakes, NJ, USA).
Transmission electron microscopy (TEM)
EXOs were loaded onto the shiny side of the copper grid and stained with 2% uranyl acetate for 3 min at room temperature (RT). Incubate the grid on top of some small drops of ultrapure water 2 min for each wash. Blotting the grid at 45-degree angle once from the side of the grid to remove excess solution by filter paper. The grid was observed with TEM (JEM-1200EX, JEOL, Japan).
T cell proliferation assay
T lymphocytes derived from human PBMCs were stained with carboxy fluorescein diacetatesuccinimidyl ester (CFSE; Life Technologies, Waltham, MA, USA). DCs were pretreated with 10 μg/mL mitomycin C (Sigma, St Louis, MO, USA) to inhibit cells division. CFSE-labeled T cells were cocultured with DCs, DClys and DCtex at different ratios for 4 days. Subsequently, T cells were counterstain with anti-CD3-APC (BioLegend) for 30 min, and then analyzed by FACS.
Cytokine release assay
Cytokines including IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α in supernatants of T lymphocytes co-cultured with DCs in vitro were detected using Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit (BD, Franklin Lakes, NJ, USA). The data were analyzed by the BD Biosciences CBA analysis software.
Cytotoxic lymphocyte activity was evaluated by a cytotoxicity detection kit (Promega, Madison, WI, USA), by measuring the cytolysis rate elicited by effector T lymphocytes against tumor cells. The indicator for cytotoxicity was the amount of lactate dehydrogenase released from lysed target cells. Red blood cell-depleted splenic lymphocytes and lymph nodes cells from the mice immunized with EXOs were harvested after 7 days of immunization as effector T lymphocytes. An MTT (3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide; Sigma) assay was used for evaluation of the effects of TEXs on proliferation of stromal cells as described previously . Briefly, cells were seeded in 96-well plates at a density of 2 × 104 cells/ml. After treated with different concentrations of TEXs for 96 h, the MTT assay was performed.
Annexin V/PI binding assays
Cells were cultured at a density of 105 cells/ml in a 6-well plate and treated with different concentrations of TEXs for 24 h. Apoptotic cells were quantified by propidium iodide (PI) and Annexin V-FITC double staining using a detection kit purchased from MultiSciences Biotech (Hangzhou, China) according to the manufacturer’s instructions, then analyzed by FACS analysis.
Western blot analysis
Western blotting was performed as described previously . EXOs and cell lysates were immunoblotted with these antibodies: anti-CD63, CD81, TSG101, Alix, Cytochrome C, Hsp70, MMP-2, MMP-9, CTLA-4, and c-Myc (Abcam, Cambridge, MA); anti-Bcl-2, Bcl-xl, Mcl-1, xIAP, TGF-β, TRAIL, GSK-3β, and FASL (Cell signaling technology, Beverly, MA); anti-FAS and BTLA (Proteintech, Rocky Hill, NJ, USA). The horseradish peroxidase-conjugated secondary antibody was obtained from MultiSciences Biotech, Hangzhou, China. Immunoblotting with anti-actin or anti GAPDH (Cell signaling technology) confirmed equivalent protein loading.
Confocal laser scanning microscopy
Cells was incubated in complete medium with 30 μg PKH67(sigma)-labeled EXOs for 0–24 h at 37 °C, and then were washed twice with PBS, fixed in 4% formaldehyde (sigma) for 10 min at RT. After washed twice with PBS, co-stained with PE-anti CD19 for 30 min and 1 μM DAPI (Southern Biotech, Birmingham, AL, USA) for 5 min, cells were coated on microscopy slides and analyzed by a confocal microscope (Nikon C1-Si, Japan).
Label-based human antibody array
The protein expressions of TEXs or cell lysates were analyzed by RayBio Biotin Label-based Human Antibody Array (Raybiotech, Norcross GA). This antibody array experiment was performed by Wayen Biotechnology (Shanghai, China) according to their established protocol. In brief, the samples were biotinylated and dialyzed, and then were added to the array and incubated overnight at 4 °C. After incubation with Cy3-Conjugated Streptavidin, the slides were scanned on a GenePix 4000 scanner and the images were analyzed with GenePix Pro 6.0 (Molecular Devices, Sunnyvale, CA).
Quantitative real-time PCR
Total RNA was isolated using TRIzol reagent (Invitrogen), according to manufacturer’s instructions. cDNA was made from total RNA using reverse transcriptase kit (TaKaRa Shuzo, Kyoto, Japan), which was amplified on an Applied Biosystems 7500 real-time PCR system (Foster City, CA, USA). The specific primers (Invitrogen) for MMP-2 were 5′- CATTTGGCGGACTGT-3′ (forward) and 5′- AGGGTGCTGGCTGA -3′ (reverse), and for MMP-9 were 5′- TTGACAGCGACAAGAAGT-3′ (forward) and 5′- GGGCGAGGACCATAGA -3′ (reverse). The specific primers for GAPDH were 5’-GTCATCACCATTGGCAATGAG-3′ (forward) and 5’-CGTCACACTTCATGATGGAGTT-3′ (reverse).
Transwell invasion assay
The effect of TEXs on cell invasion was determined by Transwell invasion assay as described previously . Briefly, cells in DMEM were stimulated with EXOs (100 μg/mL) or vehicle (PBS) alone for 4 h at 37 °C, and then were collected and seeded onto the Matrigel-coated transwell inserts (Corning Inc., Corning, NY, USA). The inserts were placed onto a 24-well plate that contained DMEM (5% FBS). After incubated for 24 h at 37 °C to facilitate invasion, cells were fixed in 4% formaldehyde for 15 min and stained with 0.1% crystal violet for 20 min at RT. The invaded cells were imaged using light microscope and counted. Five fields of view were obtained per insert (n = 3 biological replicates).
Matrixgel plug assay
Six-week-old NOD/SCID mice were injected subcutaneously along the abdominal midline with or withour 500 μL growth factor-reduced Matrigel containing OCI-LY3 EXOs (100 μg). Mice were sacrificed 14 days later, Matrigel plugs were removed, fixed in 4% formaldehyde, and embedded in paraffin. The paraffin sections were stained with hematoxylin/eosin (H & E) or stained with anti-CD31 (Abcam), followed by an Alexa Fluor 488-conjugated goat anti-mouse antibody (Invitrogen).
Scratch wound assay (SWA)
SWA was performed as described previously . Briefly, cells (2 × 104) were incubated at 37 °C for 24 h. A scratch (wound) was performed on monolayer of cells along the vertical axis of each well under a light microscope. All the experiments were carried out in three replicates and three measurements were taken for each wound.
All animal experiments were carried out in the animal research center of Zhejiang Chinese Medical University (Zhejiang, China).C57BL/6 mice, BALB/C mice and NOD-SCID mice were purchased from Shanghai Slac Laboratory Animal CO. LTD (Shanghai, China). To examine whether EXOs could induce protective antitumor immunity, BALB/C mice were intravenously immunized with EXOs (10 μg/mouse). The mice, injected with PBS were considered as control. The tail blood samples were harvested for FACS after 7 days of immunization. To investigate immunomodulatory effect of EXOs in DC-vaccination, DCs activated by EXOs or tumor cell lysates were injected intravenously into MHC-matched BALB/C mice 3 times at weekly interval. The immunized mice were then challenged with 5 × 106 tumor cells. In addition, for the mouse tumor model, six-week-old NOD-SCID mice were challenged subcutaneously in the flank with tumor cells, supplemented with 200 μg DLBCL EXOs. The tumors cells were injected subcutaneously with PBS as the control.
Experimental results were analyzed by one-way analysis of variance. The P-value, below 0.05 was considered as statistically significant. All data are presented as mean ± standard deviation.
The characterization of exosomes from B cell lymphoma
Phenotype characterization of TEXs of B-cell lymphoma
To investigate its marker, TEXs coated on beads were stained with a panel of FITC-labelled antibodies, and then followed by FACS analysis. There is high expression of exosomal marker proteins CD63 and CD81, with a range of 55.3% to 75.2% in beads (Fig. 1c). Western blotting analysis also showed that CD81 and CD63, as well as the endocytic pathway and formation associated proteins TSG-101 and Alix were expressed in OCI-LY3TEXs (Fig. 1d). Taken together, these data confirmed the presence of TEXs. We further compared the phenotype of OCI-LY3, SU-DHL-16, and Raji cells with their corresponding TEXs. EXOs of SU-DHL-16, and Raji cells expressed CD19 and CD20, whereas OCI-LY3 EXOs only expressed CD20 (Fig. 1e), which is consistent with the results of their parent cell lines (Additional file 1: Figure S1). In addition, all three cell lines showed positive expression of costimulatory molecules including CD40, CD80, CD83 and CD86 (Additional file 1: Figure S1); however, only HLA-DR expression was positive in OCI-LY3 EXOs, while expression of CD80, CD86, and HLA-DR was positive in SU-DHL-16TEXs (Fig. 1e). Interestingly, the expression of surface markers related to specific malignancy lineages, such as CD19 and CD20, and costimulatory molecules in DLBCL TEX was much weaker than that in their parent cell lines.
DLBCL-derived exosomes harbor TAAs and are easily taken up by DCs and lymphoma cells
In order to study the capture of TEXs by lymphoma cells, we treated Raji cells with the PKH-67 labeled Raji-derived EXOs. After incubation for 24 h, the cell membrane was stained with PE-anti CD19 antibody, and TEXs internalization was checked by confocal microscopy that showed apparent internalization (Fig. 3d). Time kinetics indicated TEXs accumulation in Raji cells in a time-dependent manner (Fig. 3e), consistent with published results of mantle cell lymphoma (MCL) .
Lymphoma TEXs stimulate T cell function and proliferation via the host DCs in vitro
B cell lymphoma-derived exosomes upregulated inhibitory receptors PD-1, CTLA-4 and BTLA, and induced apoptosis of T cells through activation of Fas/FasL pathway
B cell lymphoma-derived exosomes facilitate invasion of HUVEC and human normal fibroblasts
B cell lymphoma-derived exosomes not only enhance cell proliferation, migration and angiogenesis of stromal cells, but also promote tumor growth in vivo
Comparative studies of proteomic characterization of DLBCL-derived exosomes
Cancer cell-derived EXOs, also known as TEXs, have the ability to promote a favorable microenvironment that supports tumor growth, and to induce formation of new vessels and contribute to the metabolic reprogramming of cancer cells providing means for their sustained proliferation [36, 37]. Koch R, et al.  demonstrated that DLBCL possesses a self-organized infrastructure comprising side population (SP) and non-SP cells and that this transition between clonogenic states is regulated by EXO-mediated Wnt signaling. In addition, TEXs also have the ability to stimulate extracellular matrix remodeling, cancer cell migration and invasion [28, 36]. Importantly, TEXs play a crucial role in the escape of the cancer to immune surveillance [11–13]. However, relatively few studies have evaluated characterization of lymphoma cell-derived EXOs and its role in DLBCL. Our study now shows that EXOs derived from both OCI-LY3 and SU-DHL-16 cells are membrane-bound vesicles heterogeneous in size, with a mean diameter of 173.8 nm and 167.9 nm, respectively. Like other EXOs, they carry the exosomal markers CD81 and CD63, and endosome-associated proteins such TSG101 and ALIX. Importantly, using flow cytometry analysis, we observed the presence of surface markers related to malignant B-cell lineages such as CD19 and CD20, which is consistent with previous studies that show that different B-cell surface proteins (CD19, CD20, CD24, CD37 and HLA-DR) are expressed on EXOs from B-cell lymphoma cell lines [7, 39]. The presence of MHC and co-stimulatory molecules in EXOs is immunogenic; however, some kind of TEXs does not carry these molecules . Here we show that SU-DHL-16 EXOs displayed expression of CD80 and CD86 similar to, but in less extent to its parental cells. Whereas, OCI-LY3 EXOs did not show any CD40, CD80, CD86, and CD83 molecules, indicating the heterogeneity of lymphoma TEXs.
TEXs contain TAAs that can be efficiently taken up by DCs, thereby eliciting specific anticancer immunity [29, 30]. It was reported that T-cell lymphoma TEXs contain tumor antigens CD24 and HSP-70 . Our data show that DLBCL TEXs carry not only HSP-70 but also c-Myc, Bcl-2, Mcl-1, xIAP and Bcl-xL molecules. Furthermore, among the cargoes identified in DLBCL TEXs, molecules involved in phosphatidylinositol, ERK, MAPK, chemokine, cell surface receptor, and G-protein, etc., signaling pathway are related with cell proliferation, apoptosis resistance, and antitumor immunity. EXOs-derived from CLL and MCL cells have been demonstrated to enter and deliver their content such as miRNA and proteins to malignant B-cells and normal cells including mesenchymal stem cells and endotherlial cells [26, 28, 41]. Our results provide evidences for the uptake of DLBCL TEXs by DCs and B lymphoma cells. Given the fact that TEXs carry both TAAs and immunosuppressive mediators , we investigated the effect of TEXs-derived from DLBCL cells on DCs, T-cells, and TME. Using flow cytometric analyses and Western blotting, we demonstrated that OCI-LY3 EXOs are either able to up-regulate expression of PD-1 or to induce the apoptosis of Th2 cells. However, these effects were not observed in DCs. In agreement with previous studies [29, 42], our data show a dose-dependent increased expression of Fas, FasL, and TRAIL in Th2 cells treated with TEXs, which may contribute to induction of apoptosis. In addition to immunosuppressive effects of DLBCL TEXs, we also demonstrated that the TEXs play an important role in not only enhancing cell proliferation, invasion, and migration of stromal cells HUVEC and human fibroblasts, and angiogenesis, but also promoting tumor growth in vivo. Taken together, targeting lymphoma TEXs by silencing or inhibition of TEXs production may be a promising therapeutic approach.
DCs can be exploited for vaccination against cancer, which aims at stimulating tumor-specific immune responses to prevent, treat or eradicate tumors. However, therapeutic efficacy frequently remains below expectation . TEXs contain TAAs, even or tumor-specific antigens that can be transferred to DCs, thereby enhancing anticancer immune responses [7, 44]. In T-cell lymphoma, TEXs bearing the marker of malignancy CD24 and HSP-70elicited specific immune responses and immune memory that allowed the rejection of subsequent tumor challenges . Similar results have been reported in leukemia models [45, 46]. In this regard, our results show that TEXs did not induce apoptosis of DCs. Moreover, DCs pulsed with DLBCL TEXs have a higher stimulatory capacity in both inducing expansion of T-cells and inhibiting secretion of immunosuppressive cytokine by T helper 2 cells. The lymphocytes from mice treated with TEXs demonstrated a specific anti-lymphoma activity. Collectively, our results suggest that DLBCL TEXs can provide a source of TAAs to enhance a DC-based immunotherapeutic effect.
We thank the healthy volunteers for their peripheral blood samples and the medical staff of department of Hematology, the First Affiliated Hospital, College of Medicine, Zhejiang University.
The research was supported by National Natural Science Foundation of China (No. 81670178,81500110,81500111), The National Key Research and Development Program of China (No. 2016YFC090150X), Research Project for Practice Development of National TCM Clinical Research Bases (No. JDZX2015113), Zhejiang Provincial Natural Science Foundation (No.LY18H160008), and Funds of Science Technology Department of Zhejiang Province (No. 2018C03016–1).
Availability of data and materials
The datasets and material used and/or analyzed during the current study are available from the corresponding author upon request.
WBQ designed study; ZZC, LSY, LW, XBH, LZ and HL performed research and contributed new analytical tools and reagents; ZZC, LSY and WBQ analyzed data and wrote the paper. All authors participated in the drafting of the manuscript and approved its final.
For peripheral blood samples from healthy volunteers, this experiment was approved by an independent ethics committee of The First Affiliated Hospital, College of Medicine, Zhejiang University. All animal experiments were carried out in accordance with the National Institutes of Health’s Guidelines for the Care and Use of Laboratory Animals.
Consent for publication
This is not applicable for this study.
The authors declare that they have no competing interests.
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