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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

Streszczenie

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating naïve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Wprowadzenie

Dendritic cells (DCs) are an integral part of the innate immune system and were first discovered and characterized by Ralph Steinman and Zanvil Cohn in 1973 as primary professional antigen presenting cells1. DCs have been shown to play an important role in immune activation by presenting processed antigens to T cells and B cells via major histocompatibility complexes (MHC) in secondary lymphoid organs to link the innate and adaptive immune systems2. In the mammalian immune system, there are at least two categories of DCs which have been described as myeloid DCs and plasmacytoid DCs (pDCs)3. Myeloid DCs, also known as conventional DCs (cDCs), are characterized by the expression of CD11c and can be differentiated as immature DCs (iDCs) in vitro from bone marrow progenitor cells or peripheral blood monocytes using granulocyte-macrophage-colony-stimulating factor (GM-CSF) and IL-4 in murine or human species, respectively4.

Activating 'danger' signals, such as pathogen-associated molecular patterns (PAMP) or damage-associated molecular patterns (DAMP), will drive iDCs maturation toward immunogenic DCs as mature DCs (mDCs) via binding various pattern recognition receptors on the DC surface5. Immunogenic DCs further prime naïve T cell proliferation and differentiation through upregulation of MHCII2, costimulatory ligands (CD80, CD86, and CD40)6, cytokines, and other soluble mediators7. A cascade of pro-inflammatory mediator production from Immunogenic DCs is essential for cytokine-mediated T cell differentiation. For example, both IFN-γ and IL-12 are necessary for Th1 differentiation8 and IL-1, IL-6, and IL-23 are critical for naïve T cell polarization towards Th17 cells9. Although mature DCs react to foreign antigens, uncontrolled DC activation by self-antigens may cause tolerance ablation and foster the development of autoimmune diseases by generating autoreactive T cells whose activation leads to tissue destruction10.

Recent reports have provided clear evidence of DC plasticity, exemplified by their ability to interact with different cues within their tissue microenvironment and to differentiate into distinct effector/suppressor DC subsets. The anti-inflammatory mediators, such as IL-1011, TGF-β12, and HO-113 have been shown to play an important role in immune suppression by inducing tolerogenic DCs (TolDCs). These TolDCs acquire regulatory functions and suppress T cell proliferation14. Moreover, the lack of co-stimulation by DCs and the production of anti-inflammatory mediators from TolDCs both contribute to the induction of regulatory T cells (Tregs) and also effectively inhibit both Th1 and Th17 differentiation and expansion15. In past two decades, the therapeutic potential of TolDCs has been reported by several investigators. In these studies, the administration of ex-vivo generated TolDCs not only ameliorated pathological symptoms in different preclinical models of autoimmune diseases16 but also led to the development of immune tolerance in patients17,18. Interestingly, today the TolDCs therapy has been considered as an alternative or adjunctive approach for autoimmune diseases in several clinical trials, including type 1 diabetes mellitus19, rheumatoid arthritis20,21, multiple sclerosis (MS)22,23,24, and Crohn's disease25.

There are a variety of protocols that have been employed to develop TolDCs and several laboratories have reported methods for generation and phenotypic characterization of TolDCs. These methods can be used to reproducibly generate TolDCs in vitro from hematopoietic progenitors and to stably maintain them in a tolerogenic state in vivo26,27,28,29. The iDCs can be converted into TolDCs by exposure to various immunomodulatory pharmacological agents or anti-inflammatory cytokines. For example, Vitamin D3 is a well-known pharmacological agent known to augment IL-10 production and suppress IL-12 secretion from DCs and to thereby boost their immunosuppressive function30. Moreover, when DCs are exposed to potent inflammatory stimuli, such as lipopolysaccharides (LPS), several pharmacological agents such as dexamethasone31, rapamycin32, and corticosteroids33 have been shown to induce the TolDC phenotype by reducing DC surface expression of CD40, CD80, CD86, and MHCII34. IL-10 and TGF-β are the most-studied anti-inflammatory cytokines to induce DC tolerance35 and the concomitant exposure to both of these cytokines have been shown to induce a tolerogenic phenotype in DCs36.

Since the tolerogenic DC is defined by functional characteristics rather than by phenotypic markers, there is a great need to develop a consistent method for cellular and functional characterization of TolDCs. Moreover, a rigorous and consistent protocol must be established for the consistent evaluation and characterization of the tolerogenic DC phenotype if we are to effectively and reproducibly compare the ability of new agents to induce the TolDC phenotype in the laboratory. Here we provide a detailed protocol with step-by-step methods to isolate iDCs from hematopoietic progenitors of mice and to subsequently analyze the efficacy of new agents under evaluation for their capacity to convert iDCs into TolDCs, providing a robust functional and phenotypic characterization of TolDCs both in vitro and in vivo. This description includes an elaborate method to characterize the TolDCs by their surface ligands, cytokine profile, and immunosuppressive functions in vitro. We also provide an example of a method to explore the potential therapeutic application of these TolDCs in a pre-clinical model of MS, experimental autoimmune encephalomyelitis (EAE). This established protocol will help investigators to evaluate the capacity of new agents to promote the induction of TolDCs and will facilitate the effort to broaden the scope of TolDC therapeutic development.

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Protokół

All studies were performed in compliance with procedures approved by the Case Western Reserve University School of Medicine's Institutional Animal Care and Use Committee.

1. Prepare Bone Marrow-derived Dendritic Cells (BMDCs)

  1. Sterilize all the surgical instruments via autoclaving and perform the experiment in class II biological safety cabinet with appropriated safety procedures.
  2. Euthanize C57BL/6 mice 8 - 10 weeks of age by using CO2 chamber. Place the mouse on a dissecting board and rinse with 70% ethanol. Excise tibia-fibula and femur bones by using a surgical scissor and place them with 70% ethanol in a 10 cm culture dish.
  3. Use surgical blades and forceps to dissect tissue away as much as possible on the lid of the 10 cm culture dish and isolate tibias and femurs to place them with 70% ethanol in a 6 cm culture dish.
  4. Use surgical blades to trim both ends of tibias and femurs. Use 3 mL PBS in 3 ml syringe with a 23G needle to flush the contents of marrow from one end of bones to a conical tube containing 12 ml PBS. Repeat this step 3x for each end of bones.
  5. Centrifuge the cell suspension at 300 x g for 5 min.
  6. Remove the supernatant and resuspend the cell pellet with 1 ml ACK lysing buffer for 5 mins to remove red blood cells.
  7. Add 9 ml PBS to dilute ACK lysing buffer and centrifuge at 300 x g for 5 mins.
  8. Remove the supernatant and resuspend with 10 ml culture medium (RPMI-1640 plus L-glutamine, 10% FBS, 1% non-essential amino acid (100x), 10 mM HEPES, 50 nM β-mercaptoethanol, and 5% penicillin/streptomycin).
    NOTE: Endotoxin level has to be less than 0.1 EU/ml in FBS.
  9. Pass the cell suspension through a 40 μm cell strainer and take 20 µl of cell suspension and mix with 80 µl of trypan blue to count the number of live cells by using cytometer.
  10. Adjust the cell number to 1 x 106 cells/ml with 15 ng/ml GM-CSF and 10 ng/ml IL-4.
  11. Plate 3 ml of 1 x 106 cells/ml in each well of 6-well plate and incubate the cells at 37°C, 5% CO2, and 95% humidity in the CO2 incubator.
    NOTE: The bone marrow cells from one mouse is around 3 - 5 x 107 cells, indicating that it can be placed from 2 to 3 6-well plates.
  12. On day 3, remove all 3 ml culture medium from each well, add 2 ml fresh PBS in each well, and then gently swirl the plate to ensure removing all the non-adherent cells.
  13. Replace 3 ml fresh culture medium with 15 ng/ml GM-CSF and 10 ng/ml IL-4 in each well. Incubate the cells at 37 °C, 5% CO2, and 95% humidity in the CO2 incubator.
  14. On day 5, directly add another 3 ml fresh culture medium with 15 ng/ml GM-CSF and 10 ng/ml IL-4 in each well. Incubate the cells at 37°C, 5% CO2, and 95% humidity in the CO2 incubator.
    NOTE: The total volume in each well is now 6 ml.
  15. On day 7, put the entire plate on ice for 10 mins. Gently pipette the culture medium in each well to dislodge the loosely-adherent BMDCs as iDCs into suspension.
    NOTE: The adherent macrophages are still attached to the plate. Low temperature and gently procedures are the key to avoid DC activation to affect further experiments.
  16. Centrifuge the cell suspension at 300 x g for 5 mins and resuspend with fresh culture medium for further experiments.
    NOTE: BMDCs can be identified with fluorescence-labeled CD11c antibody by flow cytometry and we showed our gating strategy of BMDCs for further experiments in Figure 1.

2. Characterize TolDC Gene and Protein Profile

  1. Plate 2 ml of 1 x 106 BMDCs/ml per well in 6 well-plate with culture medium in the presence or absence of 100-400 nM CDDO-DFPA for incubating 1 h at 37°C, 5% CO2 and 95% humidity in the CO2 incubator.
    NOTE: CDDO-DFPA, a synthetic triterpenoid, is a nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2) inducer and nuclear factor-κB (NF-κB) inhibitor. Apply other agents for induction of TolDCs from iDCs, such as IL-10, vitamin D3, dexamethasone or BAY 11-7085, and modify this step to an optimal condition for each agent.
  2. Add 10 or 100 ng/ml of LPS for incubating 4-24 hrs to induce mDCs (incubation time is different due to mRNA or protein measurement).
  3. Gently pipette the culture medium in each well and harvest the cell suspension by using 1 ml pipette. Centrifuge at 300 x g for 5 mins to collect the cells and supernatant, respectively.
  4. Analyze the surface ligands from the cell pellets by flow cytometry, such as stimulatory ligands: CD40, CD80, CD86, MHC-II, OX40L, ICOSL, or inhibitory ligands: PD-L1, PD-L2, ILT3, ILT4.
  5. Isolate the RNA from the cell pellets and analyze the RNA and supernatant samples for the cytokine profile and gene and protein levels by quantitative real-time PCR (qRT-PCR) and ELISA, respectively.
    NOTE: For example, inflammatory cytokines: TNF-α, IFNγ, EDN-1, IL-6, IL-12, and IL-23, or anti-inflammatory cytokines: IL-4, IL-10, IL-15, TGF-β1, and HO-1.

3. Evaluate the Function of TolDCs In Vitro and In Vivo

  1. T-cell Syngeneic Proliferation Assay
    1. Sterilize all the surgical instruments via autoclaving and perform the experiment in class II biological safety cabinet with appropriated safety procedures.
    2. Prepare MACS buffer using 0.5% bovine serum albumin (BSA) and 2 mM EDTA in 500 ml PBS. Sterilize the buffer by filtering through a 0.2 µm filter.
      NOTE: Keep the buffer on the ice during the following experiment.
    3. To obtain CD4+ T cells, euthanize OT-II T-cell receptor (TCR) transgenic mice 8-10 weeks of age by using CO2 chamber. Place the mouse on a dissecting board and rinse with 70% ethanol. Isolate the spleen from the left side of the abdomen by using surgical scissors and forceps.
    4. Put the spleen with 2 ml PBS in a 6 cm culture dish and use the back of push-stick of 3 ml syringe to mince the spleen by passing a 40 μm cell strainer.
    5. Collect the cell suspension and centrifuge at 300 x g for 5 mins.
    6. Resuspend the cell pellet in 400 μl of MACS buffer.
    7. Add 100 μl of CD4+ T Cell Biotin-Antibody Cocktail at 4°C for 5 mins.
      NOTE: The antibody cocktail binds on other cell types except CD4+ T cells, such as CD8a, CD11b, CD11c, CD19, CD25, CD45R (B220), CD49b (DX5), CD105, Anti-MHC class II, Ter-119, and TCRγ/δ.
    8. Add 300 μl of MACS buffer and 200 μl of Anti-Biotin beads (Table of Materials) at 4°C for 10 mins.
    9. Place the column composed of ferromagnetic spheres (Table of Materials) and Pre-Separation Filter together in the magnetic field and rinse it with 3 ml of MACS buffer.
    10. Add 9 ml of MACS buffer in the cells and centrifuge at 300 x g for 5 mins.
    11. Resuspend the cell pellet in 3 ml of MACS buffer and apply onto the column. Collect flow-through containing CD4+ T cells.
    12. Wash column with another 3 ml of MACS buffer and also collect the flow-through.
    13. To obtain splenic pan DCs, euthanize C57BL/6 mice 8-10 weeks of age by using CO2 chamber. Place the mouse on a dissecting board and rinse with 70% ethanol. Isolate the spleen from the left side of the abdomen by using surgical scissors and forceps. Put the spleen in a 6cm culture dish containing 2 ml of collagenase D solution (2 mg/ml collagenase D dissolved in HBSS containing calcium, magnesium).
    14. Inject 1 ml of collagenase D solution to the spleen two times with a 1 ml syringe and a 25G needle. Cut the spleen into small pieces with small scissors.
    15. Shake and incubate at room temperature for 25 mins.
    16. Add 500 μl of 0.5 M EDTA at room temperature for 5 mins.
      NOTE: The steps from 3.1.13-3.1.14 are critical for increasing the yield of DCs.
    17. Now use the back of push-stick of 3 ml syringe to mince the spleen slurry by passing a 40 μm cell strainer and collect the cell suspension by centrifuging at 300 x g for 5 mins.
    18. Resuspend the cell pellet in 350 μl of MACS buffer, 50 μl of FcR Blocking Reagent, and 100 μl of Pan Dendritic Cell Biotin-Antibody Cocktail at 4°C for 10 mins.
      NOTE: The antibody cocktail against antigens that are not expressed by DCs.
    19. Wash the cells by adding 9 ml of MACS buffer and centrifuge at 300 x g for 5 mins.
    20. Resuspend the cell pellet in 800 μl of MACS buffer and add 200 μl of Anti-Biotin beads at 4°C for 10 mins.
    21. Repeat the steps from 3.1.9-3.1.11 to collect DCs.
    22. Wash column with another 3 ml of MACS buffer two times and also collect the flow-through.
    23. Treat the 2 x 105 DCs/ml in the presence or absence of 100-400 nM CDDO-DFPA at 37°C for 1 hr. Seperately, label the CD4+ T cells (1 x 107/ml) with 1 μM CFSE at 37°C for 15 mins, wash with PBS, and readjust the volume to get final concentration at 2 x 106 T cells/ml.
    24. Next, in a 96-well plate, co-culture 100 μl of the treated dendritic cells with the same volume of CFSE-labeled CD4+ T cells, both collected from the above steps to get 1:10 ratio. Now add 100 ng/mL ovalbumin (OVA) peptide 323-329 per well and measure the CFSE intensity of the T cells by flow cytometry after 2-3 days of incubation.
      NOTE: The cell numbers and ratio of DCs and T cells have been optimized from our previous work37.
  2. Induce Passive EAE by Injecting BMDCs Pulsed with Myelin Oligodendrocyte Glycoprotein (MOG) (35-55)
    1. Plate 2 ml of 1 x 106 BMDCs/ml per well in 6 well-plate with culture medium in the presence or absence of 100-400 nM CDDO-DFPA for incubating 1 hr.
    2. Add 10 ng/ml of LPS for incubating 24 hrs.
    3. Add 100 μg/ml of MOG (35-55) for incubating 4 hrs.
    4. Harvest the cell suspension and centrifuge at 300 x g for 5 mins.
    5. Resuspend the cell pellet with PBS and count the cells.
    6. Subcutaneously inject 200 μl of 2 x 106 cells to 8-10 weeks old female C57BL/6 mice (100 μl into each hind leg).
    7. On the day of BMDC injection and 48 hrs. later, inject 200 ng of pertussis toxin (PTX) in each mouse.
    8. Repeat steps 3.2.6-3.2.7 once each week for 4 consecutive weeks.
    9. Evaluate the clinical symptoms of EAE daily by using standard criteria37 (0.5-limp tail end, 1-limp tail, 2-moderate hind limb weakness, 3-severe hind limb weakness, 4-complete hind limb paralysis, 5-quadriplegia or moribund state, 6-death).

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Wyniki

The differentiation and selection of BMDCs:

Bone marrow progenitor cells were cultured in complete RPMI medium in the presence of GM-CSF and IL-4 to differentiate into iDCs for 7 days (Figure 1A). On day 1, cells were small in size and showed spherical morphology. Washing with PBS before the replacement of fresh medium on Day 3 helped cells to form clusters and also increased the p...

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Dyskusje

This paper describes an efficient protocol that may be used to reproducibly to generate iDCs and to subsequently differentiate them into TolDCs, and we propose that this may be applied to evaluate the capacity of new molecular target agents to induce the TolDC phenotype. As described in this report, we followed a sequence in which we first analyzed TolDC expression of surface ligands by flow cytometry, followed by an assessment of the DC cytokine profile as measured by qRT-PCR and ELISA. Finally, the immunoregulatory fun...

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Ujawnienia

None

Podziękowania

We thank Reata Pharmaceuticals for providing CDDO-DFPA. We also acknowledge the support of the Jane and Lee Seidman Chair in Pediatric Cancer Innovation (John Letterio). This work was supported by Department of Defense [W81XWH-12-1-0452]; the Angie Fowler Adolescent and Young Adult Cancer Research Initiative at the Case Comprehensive Cancer Center; and the Callahan Graduate Scholar Award for Hsi-Ju Wei from F.J. Callahan Foundation.

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Materiały

NameCompanyCatalog NumberComments
CDDO-DFPA (RTA-408)Reata Pharmaceuticalsin house synthesisCell culture
Mouse GM-CSFPeprotech Inc.315-03BMDC differentiation
Mouse IL-4Peprotech Inc.214-14BMDC differentiation
Lipopolysaccharides (LPS)Sigma Aldrich Inc.L2880Cell culture
β-mercaptoethanolSigma Aldrich Inc.516732Cell culture
Pertussis toxin (PTX)R&D systems3097EAE induction
MOG (35–55) peptide21stCentury Biochemicalsin house synthesisEAE induction
Trypan blueGibco, Life Technologies15250-061Cell culture
RPMI-1640 plus L-glutamineThermoFisher Scientific11875-093Cell culture
Non-essential amino acid (100X)ThermoFisher Scientific11140050Cell culture
HEPESThermoFisher Scientific15630080Cell culture
penicillin/streptomycinThermoFisher Scientific15140122Cell culture
40 μm cell strainerCorning352340Cell isolation
PE-conjugated CD80BD Biosciences557227Flow cytometry
PE-conjugated CD86BD Biosciences555665Flow cytometry
PE-conjugated PD-L1BioLegend124307Flow cytometry
APC-conjugated MHCIIMiltenyi Biotec Inc.130-112-388Flow cytometry
APC-conjugated CD11cBD Biosciences340544Flow cytometry
Isotype matched PEMiltenyi Biotec Inc.130-091-835Flow cytometry
Isotype matched APCMiltenyi Biotec Inc.130-091-836Flow cytometry
CFSEBioLegend423801T cell proliferation assay
Pan dendritic cell isolation kitMiltenyi Biotec Inc.130-100-875T cell proliferation assay
FcR Blocking ReagentMiltenyi Biotec Inc.130-100-875T cell proliferation assay
Pan Dendritic Cell Biotin-Antibody CocktailMiltenyi Biotec Inc.130-100-875T cell proliferation assay
Anti-Biotin MicroBeadsMiltenyi Biotec Inc.130-100-875T cell proliferation assay
CD4+ T cell isolation kitMiltenyi Biotec Inc.130-104-454T cell proliferation assay
CD4+ T cell Biotin-Antibody CocktailMiltenyi Biotec Inc.130-104-454T cell proliferation assay
Anti-Biotin MicroBeadsMiltenyi Biotec Inc.130-104-454T cell proliferation assay
ACK lysing bufferThermoFisher ScientificA1049201BMDC differentiation
1 ml syringeBD Biosciences309626T cell proliferation assay
3 ml syringeBD Biosciences309588BMDC differentiation
25G needleBD Biosciences309626T cell proliferation assay
23G needleBD Biosciences309588BMDC differentiation
BSASigma Aldrich Inc.A2058T cell proliferation assay
EDTAThermoFisher Scientific15575020T cell proliferation assay
LS ColumnMiltenyi Biotec Inc.130-042-401T cell proliferation assay
Pre-Separation FilterMiltenyi Biotec Inc.130-095-823T cell proliferation assay
collagenase DSigma Aldrich Inc.11088858001T cell proliferation assay
HBSSThermoFisher Scientific14025076T cell proliferation assay
ovalbumin (OVA) peptide 323–329Sigma Aldrich Inc.O1641T cell proliferation assay
Mouse IFN-γ TaqMan probeThermoFisher ScientificMm01168134_m1qRT-PCR
Mouse IL-12a TaqMan probeThermoFisher ScientificMm00434165qRT-PCR
Mouse IL-12 p70 DuoSet ELISAR&D systemsDY419-05ELISA
Mouse EDN-1 ELISARayBiotechELM-EDN1-1ELISA
TNF-α TaqMan probeThermoFisher ScientificMm00443258qRT-PCR
Mouse TNF-α Quantikine ELISA KitR&D systemsMTA00BELISA
IL-6 TaqMan probeThermoFisher ScientificMm00446190qRT-PCR
Mouse IL-6 Quantikine ELISA KitR&D systemsM6000BELISA
IL-23a TaqMan probeThermoFisher ScientificMm01160011qRT-PCR
Mouse IL-23 DuoSet ELISAR&D systemsDY1887-05ELISA
IL-4 TaqMan probeThermoFisher ScientificMm99999154_m1qRT-PCR
IL-10 TaqMan probeThermoFisher ScientificMm01288386_m1qRT-PCR
TGF-β TaqMan probeThermoFisher ScientificMm01178820_m1qRT-PCR
Anti-Heme Oxygenase 1 antibodyAbcamab13248Western blotting
Anti-β-actin antibodyAbcamab8226Western blotting
CFX96 Touch Real-Time PCR Detection SystemBio-Rad Inc.qRT-PCR
BD FACSCalibur Cell AnalyzerBD BiosciencesFlow cytometry

Odniesienia

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