Method Article

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

DOI:

10.3791/57188

⸱

May 31st, 2018

 ,  ,  ,  ,  ,  , 
1Department of Pathology, Sackler School of Medicine, Tel-Aviv University, 2Department of Pathology, School of Medicine, Stanford University

* These authors contributed equally

In This Article

Summary

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Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Abstract

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Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Introduction

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Since their discovery, dendritic cells (DC) have been a focus of extensive research due to their unique ability to skew T cell differentiation1. Over the past several decades, an extensive research effort has sought to define the various DC subsets and their function during tumor progression and immunity 2. DCs are composed of heterogeneous cell populations that differ from each other in their pattern-recognition receptors, tissue distribution, and migratory and antigen presentation capabilities3,4,5. Compared to other DC subsets, monocyte-derived DC (MoDC) are far more abundant in tumors and can be easily generated from circulating or tumor-infiltrating monocytes6,7. Therefore, many clinical trials seeking to take advantage of their relative prevalence are based on in vivo and ex vivo manipulation of autologous MoDC in order to elicit T cell immunity 8,9.

Similarly, DC-based vaccination of experimental tumor models requires 2-3 serial injections, 5-7 days apart, of 1-2 x 106 activated DC pulsed with tumor antigens. Therefore, to achieve this large number of DC, most mouse studies have primarily used MoDC cultured from bone marrow (BM) precursors in GM-CSF for 7-9 days (IL-4 is not needed in the mouse setting)10,11. Nonetheless, given that GM-CSF knockout mice have overall normal DC compartment 12,13, and given the mixed populations obtained from that culture,14 the physiological relevance of these DC has been called into question.

Alternatively, DC may be routinely isolated from spleen cells. However, DC comprise only about 0.3-0.8% of total spleen cells (resulting in approximately 7 x 105 DC/spleen), and of these cells, only CD103+ DC and MoDC can migrate back to lymphoid organs. Since MoDCs comprise approximately 10-15% of splenic DC populations15,16, most isolation protocols yield approximately 1 x 105 MoDC per spleen. Expansion of MoDC can be achieved by injecting transfected B16 cells that secrete GM-CSF, resulting in a 100-fold increase in splenic MoDC17. However, the use of MoDC for developing DC vaccines is limited since this procedure cannot be done in humans and the obtained MoDC are already highly activated.

In addition to obtaining adequate numbers of DC, another challenge for developing effective DC vaccines against autologous cancer cells involves the lack of sufficient danger signals in the tumor setting to fully activate DC. Induction of co-stimulatory signals is usually achieved through activation of pattern-recognition receptors (PRR), or c-type lectin signaling pathways18,19,20,21. A further approach for activating DC exploits their ability to take up antigens through interactions with surface Fcγ receptors (FcγR). Indeed, a number of important manuscripts have shown that injection of MoDC from BM precursors activated with tumor-IgG IC can prevent tumor growth in prophylactic settings, and can lead to the eradication of established tumors22,23.

In two recent papers, Carmi et al. discovered that in contrast to BMDC and spleen DC, MoDC from the blood and tumors cannot respond to IgG IC without additional stimuli. This was found to be due to the presence of high intracellular levels of tyrosine phosphatases regulating FcγR signaling24,25. By defining a critical checkpoint in DC, this work provided an important insight into the requirements for successful DC-based vaccination. The requirement for additional stimuli to enable FcγR signaling, and presumably signaling from other lectin receptors utilizing a similar phosphorylation cascade, thus underscores the need for avoiding the priming of DC during their isolation.

Therefore, the present protocol describes the isolation of MoDC from blood and tumors, which differ markedly from BM and spleen DC, and highlights precautions worth considering during the process.

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Protocol

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The protocols below refer to the isolation of mouse MoDC, yet the overall principles may apply to other DC subsets cells, as well. 12 - 16-week-old C57Bl/6j mice were maintained in an American Association for the Accreditation of Laboratory Animal Care–accredited animal facility. All protocols were approved by Stanford University and Tel-Aviv University Institutional Animal Care and Use Committee.

1. Isolation of Tumor Associated Monocyte-Derived DC

  1. In a laminar flow hood, remove tumors from CO2 euthanized mice and place tumors in RPMI medium without fetal bovine serum (FBS).
    NOTE: It is highly recommended to shave the mice and spray them with 70% ethanol before removing the tumors, to minimize potential contaminations from the fur. Be certain that the tumor does not exceed 25 - 40 mm2 since larger tumors have fewer DC that tend to be fragile and prone to undergo cell death. If larger numbers of DC are needed, each mouse can be injected with tumors cells at multiple sites.
  2. Under a sterile laminar hood, chop the tumors using surgery scissors into small pieces (approximately 1 x 1 mm2).
    1. Add all tumor fragments from one mouse into a 30-mL sterile flat-bottom tube with magnetic stir bars, which contains 5 mL of Hank's balanced salt solution (HBSS), 2 mg/mL collagenase IV, and 0.01 mg/mL DNase I.
      Caution: Myeloid cells produce energy through glycosylation, even under steady state. Therefore, only use media that contains glucose (e.g. HBSS, RPMI, and DMEM), as glucose starvation for as little as 10 - 15 min will result in cell death and apoptosis within 24 h. Use only a low endotoxin collagenase IV, as collagenase is produced by Gram-positive bacteria (Clostridium histolyticum).
  3. Stir at about 200 - 400 rpm in a 37 oC incubator with a magnetic stirrer for 20-30 min.
  4. Add 5 mL of complete media and re-suspend vigorously.
  5. Filter cells through a 70-µm cell strainer. Centrifuge at 4 oC 400 rcf for 5 - 10 min to pellet the cells.
    Caution: Do not attempt to isolate the cells directly following enzymatic digestion, as some tumors are necrotic and contain relatively large amounts of cell debris or extracellular matrix. Apply cells on 15% density gradient medium to obtain only the cells.
  6. Suspend 9 mL of density gradient medium with 1 mL of 10x PBS to adjust osmolality, and to obtain 100% Percoll stock solution. Mix 1.5 mL density gradient medium stock solution with 8.5 mL HBSS and mix it vigorously with tumor-derived cell pellet. Gently layer 2 mL of DMEM on the top of 15% density gradient medium and centrifuge at 400 rcf for 20 min at room temperature. Discard the supernatants, as the cells will form a pellet at the bottom of the tube.
    1. Wash the pelleted cells twice by re-suspending them in 10 mL HBSS containing 2% FBS, 1% penicillin/streptomycin, and 10 mM EDTA (Isolation Buffer), and centrifuge at 4 oC 400 rcf for 5-10 min to pellet the cells.
    2. Count cells on a hemocytometer using trypan blue under a light microscope.
  7. Re-suspend 1 x 108 cells in 1 mL isolation buffer and incubate them at 4 oC with 30 μL of CD11b+ conjugated magnetic beads for 15 min.
    Caution: Avoid the use of CD11c-conjugated beads, as this will activate the DC and induce cell death. Do not attempt to block FcγRII and FcγRIII with anti-CD16/32 antibodies. Blocking antibodies ligate FcγR and induce strong phosphorylation of MAP kinases and prime the DC.
    1. Remove the excess unbound beads by adding 9 mL of isolation buffer and centrifuge cells at 400 rcf for 5 - 10 min at 4oC.
  8. Aspirate the supernatant and re-suspend cells in 1 mL isolation buffer. Apply the cells on a prewashed magnetic column. Wash the column twice with 3 mL of isolation buffer.
    1. Remove the column from the magnet. Pipette 6 mL of isolation buffer onto the column and flush out the magnetically-labeled cells into a sterile collection tube by pushing the plunger. Centrifuge cells at 400 rcf for 5 - 10 min at 4 oC.
    2. Re-suspend cells at 100 μL per 1 x 107 cells and stain with the following fluorophore-conjugated antibodies: Linage negative (TCRb, Siglec F, B220, CD19, FceRI and Ly6G), MHCII, Ly-6C.
  9. Sort cells by gating the small cells, using side scatter (SSC) and forward scatter (FSC) and culture in complete medium supplemented with 5 ng/mL GM-CSF. Incubate cells for 1 hour at 3 7 oC in order to allow macrophages to adhere the plate. Then, transfer the loosely and non-adherent cells to a new culture dish.
    NOTE: Mouse MoDC are defined as CD11b+/CD11c+/MHCIIhi/Ly6Clo/int 7,26,27. Expression of Ly6C may vary dramatically between tumor models, and in some models (e.g. LMP) MoDCs completely lack Ly6C expression. Overall, one 100 mm3 B16F10 tumor typically contains 5 x 104 of DC, resulting in approximately 4 - 5 x 106 total cells. Of these cells, 10 - 15% are immune cells and 8 - 10% are MoDC. Increasing the DC numbers can be achieved by injecting mice with tumors at multiple sites. Sort only small SSC/FCS cells, as other myeloid cells can express markers such as CD11c and Ly6C. 
    Optional: Sort the tumor-infiltrating monocyte as small SSC/FSC cells that express Ly6Chi and are negative for MHCII. Afterward, culture monocytes in vitro with 50 ng/mL GM-CSF to obtain DC.

2. Isolation of Monocyte-derived DC from Peripheral Blood

NOTE: Since mature MoDCs are relatively rare in mouse blood, the protocol below refers to their derivation in vitro from sorted monocytes.

  1. To increase the levels of monocytes in blood, anesthetize mice with 4% isoflurane and inject them subcutaneously (s.c.) with 1 μg of GM-CSF in 50 μL PBS.
  2. After 1-2 h, sacrifice mice using CO2.
    Caution: Do not sacrifice mice by cervical dislocation, as this will cause internal bleeding.
  3. Immediately after euthanization,spray the mouse with 70% ethanol and remove the skin that covers the heart using surgical scissors, under a sterile laminar flow hood. Clean the scissors with ethanol and cut the right atrium of the heart.Slowly, flush the heart through the right ventricle with 20 mM EDTA HBSS using a 10-mL syringe and 25-G needle.
    1. Collect the blood with a sterile syringe from the pleural cavity into a sterile tube containing heparan sulfate and EDTA. Continue to flush the heart until the liver and lungs become pink or white.
  4. Apply blood onto a density gradient mediumand centrifuge at 400 rcf at room temperature for 15 min with low brake.
    Caution: Use endotoxin-free density gradient medium (usually less than 0.12 EU per mL).
    1. Collect mononuclear cells into a new tube. Wash the cells by re-suspending in 10 mL HBSS containing 2% FBS, 1% penicillin/streptomycin and 10 mM EDTA (isolation buffer), and then centrifuging at 4 oC 400 rcf for 5-10 min to pellet the cells.
      Caution: Use only media that contains at least 1 g/L of glucose.
  5. For CD11b positive selection, re-suspend 1 x 108 cells in 1 mL isolation buffer and incubate them for 15 min with 50 μL of CD11b-conjugated magnetic beads at 4 oC.
    1. Wash excess beads by adding 9 mL of isolation buffer and centrifuging at 400 rcf for 5-10 min at 4 oC. Aspirate the supernatants and re-suspend the cells in 1 mL isolation buffer. Apply the cells on a prewashed magnetic column according to manufacturer's instructions.
    2. Remove the column from the magnet, pipette 6 mL of isolation buffer onto the column, and flush out the magnetically-labeled cells into a sterile collection tube by pushing the plunger. Centrifuge cells at 400 rcf for 5-10 min at 4 oC. Wash the column twice with 3 mL of isolation buffer.
    3. Re-suspend cells at 1 x 107 cells/100 μL isolation buffer and stain with the following fluorophore-conjugated antibodies: 0.1 μg CD115, 0.25 μg MHCII, and 0.1 μg Ly-6C/1 x 106 cells.
  6. Sort cells by gating on small side scatter (SSC) and small forward scatter (FSC) cells.
    NOTE: Mouse inflammatory monocytes are generally defined as CD115+/MHCIIlo/neg/Ly6Chi, and patrolling monocytes are defined as CD115+/MHCIIlo/neg/Ly6Cneg 4,5. In cases where no separation between the two subsets is needed, total SSClo/FCSlo/CD115+/MHCIIlo cells can be sorted. Both naïve and tumor-bearing mice contain approximately 5 - 8 x 104 MoDC per 1 mL of blood and up to 4 - 5 x 105 MoDC following injection of GM-CSF. Of them, approximately 70% are inflammatory monocytes and 30% are patrolling monocytes.
  7. Plate the cells at a concentration of 1 x 106 cells/mL in a complete media supplemented with 20 ng/mL GM-CSF. After 1 day, transfer the non-adherent and loosely adherent cells to a new plate and culture for an additional 4-5 days.
    Caution: DC media should not be supplemented with pyruvate.

3. Preparation of Tumor-IgG Immune Complexes

  1. Culture tumor cells in 75 cm2 culture flask to 70% confluence in complete DMEM media.
    1. Add 2 mL of 0.25% trypsin/EDTA to detach cells from the culture flask and monitor cell morphology under a light microscope to avoid over-trypsinization.
    2. Add 8 mL of complete culture media (per 2 mL of trypsin) to inhibit trypsin digestion, and centrifuge at 4 oC, 400 rcf for 5-10 min to pellet the cells.
      NOTE: Make sure to check tumor cells for Mycoplasma using a commercial PCR kit. Test for the presence of Gram-negative bacteria and fungal endotoxins using Limulus Amebocyte Lysate (LAL) assay28). Serum must be filtered through 0.22 μM and tested for the 9 Code of Federal Regulations viruses. Additionally, test the culture media and serums by dropping 100 - 200 μL onto LB agar or broth, and culture for 2 days at 37 oC.
    3. Wash cells again from trypsin and serum remains by re-suspending them in 10 mL PBS and centrifuge at 400 rcf for 5 - 10 min. Aspirate supernatant and repeat the wash 2 more times.
  2. Fix cells in 1.8% buffered paraformaldehyde for 10 min at room temperature.
    1. Wash the cells by re-suspending them in 10 mL PBS and centrifuge at 4 oC 400 rcf for 5 - 10 min.
    2. Aspirate supernatants and repeat wash two more times.
      Optional: For tumor uptake assays, cells can be labeled by incubating them for 5 min at 37 oC in PBS containing 1 μM carboxyfluorescein succinimidyl ester (CFSE). CFSE is then quenched with complete media for 10 min in ice. The cells should be washed extensively in PBS containing 2% serum to remove residual dye.
  3. Re-suspend cells in FACS buffer (PBS supplemented with 2% FCS + 5 mM EDTA) and 0.5 μg/mL of anti-CD16/32 in order to block potential non-specific protein-protein interactions. Plate in a U-shape 96 wells/plate at a concentration of 1 x 105 cells per 100 μL.
    1. Add different dilutions of tumor-binding antibodies ranging from 5 μg - 5 ng/1 x 105 cells. Incubate the plate on ice for 15-20 min.
      NOTE: IgG antibodies were isolated from the serum of naïve 20-24 weeks old female mice on protein A columns, as described24.
  4. Wash cells by adding 150 μL of PBS and centrifuging the plate at 4 oC 400 rcf for 5 - 10 min.
    1. Discard the supernatants and repeat the wash twice. Re-suspend cells in 100 μL FACS buffer containing fluorophore-conjugated secondary antibody. Incubate plate on ice for 20 min.
    2. Wash cells by adding 200 μL of FACS buffer and centrifuging the plate at 400 rcf for 5 - 10 min. Discard the supernatants and repeat wash.
    3. Analyze tumor binding by flow cytometry and determine the minimal concentration required to coat the cells.
      NOTE: Antibodies that do not increase mean fluorescence intensity (MFI) of stained tumor cells by at least fivefold over isotype control should not be used in subsequent functional assays.

4. Activation of MoDC with Tumor-IgG IC

  1. Coat tumor cells with minimal IgG concentration, as described in sections 3.1 through 3.4.3
    1. One day before activating MoDC with tumor IC, replace MoDC culture media, which contains GM-CSF. To do so, gently aspirate the media and wash the cells once with pre-warmed complete culture media.
      NOTE: Isolated mature tumor-associated MoDC should be cultured for at least 2 - 3 h (or even overnight) after sorting in complete media without GM-CSF, and before activating them with IC.
    2. For tumor uptake analyses, add the CFSE-labeled tumor IC to MoDC at a ratio of 1:5 (IC:MoDC) and incubate overnight for 12 - 16 h in 1 mL of complete media per 1 x 106 DC.
    3. For FACS analyses of MoDC activation experiments, add tumor-IC at 1:1 (IC:MoDC) ratio and incubate overnight for 12 - 16 h.
      NOTE: It is highly recommended to include a positive control well, in which MoDC are stimulated with 1 μg/mL of LPS or other TLR agonists.
    4. Following overnight activation, aspirate the supernatants and wash cells gently three times with isolation buffer, or 10 mM EDTA HBSS.
    5. To detach DC from the plate, incubate cells for 2 - 3 min in 1 mL HBSS containing 10 mM EDTA, and detach cells by vigorous pipetting.
    6. Centrifuge cells at 400 rcf and re-suspend 1 x 106 DC in 90 μL PBS supplemented with 2% FCS, 5 mM EDTA (FACS buffer) and 0.5 μg of blocking antibodies. Incubate on ice for 5 - 10 min.
    7. Add 10 μL of staining antibodies mixture to the cells and incubate on ice for 15 min.
    8. Add 2 mL FACS buffer to cells and centrifuge at 4 oC 400 rcf for 5 - 10 min.
  2. Re-suspend cells in 200 μL FACS buffer.
    1. Add 0.5 - 1 μg/mL of DAPI 1 - 2 min before running the samples, to exclude dead cells from analyses. Do not over-incubate DAPI, as MoDC will take it up within 10 - 15 min.

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Results

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We initially compared the capacity of antibodies from naïve syngeneic and allogeneic mice to bind to tumor cells. To this end, B16F10 and LMP tumor cell lines were fixed in paraformaldehyde and washed extensively. B16F10 is a melanoma cell line, which was originally isolated from lung metastases in C57Bl/6 mice. LMP is a pancreatic tumor cell that was isolated from KrasG12D/+, LSL-Trp53R172H/+, and Pdx-1-Cre mice, and grows steadily in 129F1 mice. To obtain IC, tumor cells were incubated ...

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Discussion

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Given the large number of DC required for vaccinating mice (approximately 2-4 x 106 DC per one mouse), most of the vaccination strategies in mice are based on isolation of DC from BM and spleen followed by their ex vivo activation. However, attempts to activate tumor DC in vivo, using the same conditions for activating spleen and BM DC, have often been unsuccessful in producing effective immunity. In two subsequent publications, Carmi et al. have found that blood and tumor MoDC diffe...

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Disclosures

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All authors declare they have no conflict of interest and that they have nothing to disclose.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Ficoll-Paque PREMIUMGE-Healthcare17-5442-02
OptiPrepStemCell Technologies07820
CD45 MicroBeadsMiltenyi130-052-301
EasySep Monocyte Isolation KitStemCell Technologies19861
Collagenase IVSigmaC9697-50MGTest each lot for endotoxin
DNase ISigmaDN25-10MG
HBSSThermoFisher14025092
FBSThermoFisher16140071Test each lot for endotoxin
PE-CD11cBiolegend117307
APC-CD11bBiolegend101211
Brilliant Violet 650 MHCIIBiolegend107641
AF48- CD86Biolegend105017
APC/Cy7-Ly-C6Biolegend108423
PE/Cy7-CD15Biolegend135523

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Monocyte derived Dendritic CellsImmune Complex ActivationTumor Cell IsolationBlood Cell IsolationMagnetic Bead SeparationDensity Gradient CentrifugationFlow Cytometry AnalysisCD11b Cell SelectionCFSE labeled Tumor ComplexDendritic Cell Activation

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