An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Summary

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Abstract

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8α^Pos and CD8α^Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8α^Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8α^Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8α^Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Introduction

Dendritic cells (DC) were discovered almost forty years ago as the "large stellate (Greek dendron) cell" found in lymphoid organs¹. Many studies have shown that DCs are the only antigen presenting cells that can effectively stimulate naïve T cells². A major function of these cells is the uptake and presentation of antigens and their efficient processing and loading of these onto antigen presenting molecules. In mouse spleen, DCs can be separated into plasmacytoid and conventional subsets. The plasmacytoid DCs are characterized by low expression of CD11c and high levels of B220 and Gr-1. They are also positive for the surface marker mPDCA1 and secrete type I interferon in response to toll like receptor 9 (TLR9) ligands. The conventional DCs are high for CD11c and MHC class II expression. They can be split into three distinct subsets based on the surface expression of phenotypic markers such as CD4, CD8α, DEC205, CD11b and dendritic cell inhibitory receptor 2 (DCIR2, recognized by the 33D1 antibody) proteins ^3,4. The CD8α^Pos DCs are also known as cDC1, are positive for DEC205, but negative for myeloid markers such as CD11b and 33D1. The CD8α^Neg DCs, also called cDC2, are positive for 33D1, CD11b and CD4 but lack DEC205. The double negative subset (i.e., negative for both CD4 and CD8α) is relatively rare, and is negative for DEC205 and 33D1. It is the least characterized subset and may be a less differentiated form of CD8α^Neg DC.

Phenotypic differences in the various DC subsets also extend to their in vivo functions. The CD8α^Neg DCs are highly phagocytic and are thought to present exogenous antigen mainly via MHC class II to CD4 T cells ³. In contrast, the CD8α^Pos DCs are specialized for presentation of soluble protein antigen on MHC class I in a mechanism called cross-presentation. The outcome of cross-presentation depends on the activation status of these DCs⁵, and can either lead to expansion of cytotoxic T cells (CTLs) or development of regulatory T cells ^62,7. Targeting of antigen to CD8α^Pos DCs using anti-DEC205-antibody-mediated delivery results largely in the deletion of T cells⁸, whereas presentation of antigens derived from infected apoptotic cells induces a strong CTL response ⁹.

In addition to recognition of peptide antigens, the mammalian immune system has evolved to recognize lipid and glycolipid antigens. These antigens are presented by CD1 molecules, which are MHC class I-like cell surface proteins that exist in multiple related forms in various mammals. In mice, a single type of highly conserved CD1 molecule called CD1d is responsible for presentation of glycolipid antigens ¹⁰. The major population of T cells that recognize CD1d/glycolipid complexes is called invariant NKT cells (iNKT cells). These cells express a semi-invariant T cell receptor (TCR) composed of an invariant TCRα chain that is paired with TCRβ chains that have limited diversity ¹¹. Unlike conventional T cells that need to proliferate and differentiate to become activated effector T cells, iNKT cells exist as an effector population and start responding rapidly after glycolipid administration ¹². Identification of physiologically relevant lipid antigen presenting cells is an active area of research, and several distinct cell types such as B cells, macrophages and DCs have been suggested to perform this function. However, it was demonstrated that the CD8α^Pos subset of DCs is the primary cell mediating uptake and presentation of lipid antigens to mouse iNKT cells ¹³ and glycolipid mediated cross-priming of CD8 T cells ¹⁴.

To compare the efficiency of antigen presentation by different antigen presenting cells, a straightforward approach is to transfer different types of purified APCs pulsed with equivalent amounts of antigen into naïve hosts. Cell transfer experiments of this type are often performed for immunological studies. However, performing transfer studies with ex vivo antigen treated DCs is challenging, since these cells exist as rare populations in lymphoid organs where they constitute less than 2% of total cells¹⁵. It is therefore necessary to enhance the development of these cells in donor animals to increase the efficiency of isolation protocols.

It is known that the common lymphoid and common myeloid progenitors, which are required for generation of pDC, CD8^Pos and CD8^Neg DC subsets, express fms-related receptor tyrosine kinase 3 (Flt-3). Upon in vivo Flt-3 ligand (Flt-3L) administration, emigration of Flt-3 expressing progenitor cells from bone marrow is increased, resulting in the increased seeding of peripheral lymphoid organs and the expansion of their mature DC progeny¹⁶. Expression of Flt-3 is lost during commitment to the B, T or NK cell differentiation pathways. Therefore, only minimal alterations are observed in these cells upon Flt-3L administration. Similar expansion in DC populations is observed in mice bearing tumors generated by implantation of a B16-melanoma cell line secreting murine Flt-3L, which provides a simple and economical method for providing sustained systemic levels of Flt-3L^17,18. Using this approach, we have developed a protocol based on the implantation of B16-melanoma cells secreting Flt-3L to stimulate the expansion of all normal DC subsets in mouse spleen, thus greatly increasing the yields of these cells that can be isolated for subsequent experiments. We consistently find that within 10 - 14 days following subcutaneous implantation of the Flt-3 secreting tumor, mice develop splenomegaly with marked enrichment of DCs to constitute 40 - 60% of total spleen cells. From these spleens, different DC subsets can be isolated with high purity using standard commercially available cell purification kits that employ subset-specific phenotypic markers.

Protocol

Animal experiments are done in accordance with approved guidelines from the institutional animal care and usage committee (IACUC). All procedures requiring sterility are performed in a biosafety cabinet.

1. Implantation of B16.Flt3L Melanoma in Mice

1. Culture the Flt-3 expressing B16 melanoma cell line in T25 tissue culture flasks at a density of 0.5 x 10⁶ cells/ml in complete DMEM medium with 10% CO₂ at 37 °C. Grow until the cells reach ~90% confluence (~20 hr).
2. Harvest the cells by trypsin digestion. Aspirate cell culture media and wash the adherent cells with PBS. Add 5 ml of 0.05% trypsin and incubate for 5 min at 37 °C. Add cold 20 ml complete DMEM medium (4 °C) containing fetal calf serum (FCS) to quench the protease digestion. Lift the cells off by lightly tapping followed by gently pipetting up and down.
3. Collect the cells by centrifugation at 300 x g for 10 min at 4 °C. Count the cells using a hemocytometer or an automated cell viability counter. Resuspend to a density of 10⁸ cells/ml in PBS.
4. Implant mice (C57BL/6 or another histocompatible strain) with tumor cells by subcutaneous injection as described by Machholz et al.¹⁹ at the base of the neck with 100 µl of cell suspension (10⁷ cells).
5. Allow the tumor to grow until palpable or visible (2 - 10 mm in diameter, usually 7 - 12 days) before sacrificing animals for harvesting organs.

2. Preparation of Splenic Single Cell Suspension from Tumor Bearing Mice

1. Anesthetize mice with an overdose of isoflurane (adjust flow rate of isoflurane to >5% and continue exposure at least 2 min after breathing stops). Sacrifice the Flt-3 melanoma tumor bearing mouse by cervical dislocation according to institutional guidelines for euthanasia.
2. Disinfect the skin with 70% ethanol and harvest spleen using aseptic technique with the aid of sterile scissors ²⁰.
3. Place the spleen into a sterile Petri dish for transport to the biosafety cabinet. Perform all steps from here on under sterile conditions.
4. Transfer spleen to a fresh Petri dish and wash with RPMI medium. Cut the spleen into small (~0.2 cm²) pieces using a scalpel. Incubate for 30 min at 37 °C with 10 ml of collagenase/DNase solution.
5. Pour the partially digested suspension of splenic tissue onto a 70 µm cell strainer. Compress the remaining tissue fragments through the mesh of the strainer using a 5 ml syringe plunger.
6. Wash the mesh filter with 10 ml of fresh medium and discard the filter. Centrifuge the suspension at 300 x g for 10 min at 4 °C to pellet the cells.
7. Aspirate the supernatant and resuspend the cell pellet in 2 ml of RBC lysis buffer. Incubate at RT for 10 min. Quench the RBC lysis buffer by adding 10 ml of complete RPMI medium.
8. Pellet the cells by centrifugation at 300 x g for 5 min at 4 °C. Resuspend in appropriate volume to achieve cell density of 10⁸ cells/ml in cell sorting buffer (e.g., MACS) containing 20 µg/ml of Fc-block antibody (2.4G2).

3. Purification of Plasmacytoid DCs, CD8α^Pos DCs and CD8α^Neg DCs from Splenic Single Cell Suspension

Note: Isolation of the splenic DC subsets from single cell suspension is a multi-step procedure as illustrated in Figure 1. Perform all the steps using pre-cooled buffer and an ice water bath to maintain temperature at 4 - 8 °C. All reagent volumes in the following section of the protocol are calculated for an initial input of 200 µl of splenic cell suspension at 10⁸ cells/ml). This can be scaled up or down based on your need by changing the volume of cell suspension (always at a density of 1 X 10⁸ cells/ml) and other reagents proportionately in the initial step (3.1.1 and 3.2.1). Adjust reagent volumes in all later steps accordingly. For example, if starting with 100 µl of splenic cell suspension at 1 x 10⁸/ml, use half the stated reagent volumes in all steps.

1. Isolation of Plasmacytoid DCs (pDC)
   1. Add 300 µl of biotin-labeled antibody cocktail provided in the plasmacytoid DC isolation kit to 200 µl of splenic cell suspension.
   2. Mix thoroughly and incubate in ice water bath for 15 min. Tap the tube every 3 min to keep cells suspended, and to maximize antibody binding.
   3. Add 12 ml cell sorting buffer and pellet the cells by centrifugation at 300 x g for 5 min at 4 °C. Aspirate supernatant to remove unbound biotinylated antibodies.
   4. Resuspend the cell pellet in 500 µl of cell sorting buffer. Add 300 µl of anti-biotin antibody-conjugated beads. Repeat step 3.1.2 and 3.1.3.
   5. Discard supernatant and resuspend the cell pellet in 2 ml of cell sorting buffer. Perform all steps from this point on at RT using pre-cooled buffers.
   6. Place a fresh magnetic activated cell sorting LS column in the magnetic stand. Wash and equilibrate column with 5 ml of cell sorting buffer.
   7. Pipette the cell suspension onto the column, applying it gently to the center of the surface of the column bed. Collect the flow through.
   8. Wash the column with 10 ml of cell sorting buffer. Collect this flow through and combine with the flow through from step 3.1.7. The pDCs are enriched in this column flow through (FT1).
   9. Pellet the cells from FT1 by centrifugation at 300 x g for 5 min at 4 °C, resuspend in 2 ml cell sorting buffer and pass the cells through a fresh LS column by repeating steps 3.1.6 - 3.1.8. This use of two sequential columns yields enhanced purity of isolated cells.
   10. Pellet cells by centrifugation at 300 x g for 5 min at 4 °C, and resuspend with 2 ml complete RPMI. Place on ice until needed.
2. Isolation of CD8α^Pos and CD8α^Neg DCs
   Note: The first step in this procedure is depletion of B, pDC, T and NK cells.
   1. Starting with the whole spleen cell suspension (1 ml of 10⁸ cells/ml obtained in step 2.8), add 100 µl of biotin-conjugated antibody cocktail provided in the CD8α^Pos DC isolation kit.
   2. Mix well and incubate in ice water bath for 15 min. Tap the tube gently every 3 min to maximize binding of biotin-labeled antibodies to their target cells.
   3. Add 12 ml cell sorting buffer and pellet the cells by centrifugation at 300 x g for 5 min at 4 °C. Aspirate supernatant to remove unbound biotinylated antibodies.
   4. Add 150 µl of cell sorting buffer and 100 µl of anti-biotin beads provided in the CD8α^Pos DC isolation kit. Mix well and incubate in ice water bath for 15 min.
   5. Add 10 ml cell sorting buffer and pellet the cells by centrifugation at 300 x g for 5 min at 4 °C.
   6. Resuspend cells in 1 ml cell sorting buffer and pass over a fresh LS column according to procedure in steps 3.1.6 through 3.1.8.
   7. Collect the unlabeled flow through 2 (FT2) and discard the column retentate. This unlabeled fraction is enriched for CD8α^Pos and CD8α^Neg DCs.
   8. Pellet the cells in FT2 by centrifugation at 300 x g for 5 min at 4 °C.
   9. Resuspend the cells in 1 ml cell sorting buffer and add 200 µl of anti-CD8α conjugated magnetic beads provided in the CD8α^Pos DC isolation kit.
   10. Repeat steps 3.2.2 through 3.2.6. The flow through of the column is enriched for CD8α^Neg DCs and depleted for CD8α^Pos DCs. This is labeled flow through 3 (FT3).
   11. To elute the CD8α^Pos DCs retained in the column, remove the column from the magnet, add 5 ml of cell sorting buffer and purge the column matrix using the plunger provided with the LS column.
   12. Pellet the cells by centrifugation at 300 x g for 5 min at 4 °C. Aspirate supernatant and resuspend in 1 ml cell sorting buffer. To increase the purity, run the eluted cells again through a fresh LS column by repeating steps 3.1.6 to 3.1.8, except discard the flow through and collect retained cells as in 3.2.11.
   13. To purify the CD8α^Neg DCs from the FT3 suspension, pellet the FT3 by centrifugation at 300 x g for 5 min at 4 °C and resuspend in 300 µl of cell sorting buffer.
   14. Add 100 µl of anti-CD11c magnetic beads. Mix well and incubate in ice water bath for 15 min.
   15. Add 10 ml cell sorting buffer and pellet cells by centrifugation at 300 x g for 5 min at 4 °C.
   16. Repeat steps 3.1.5 through 3.1.8. The CD8α^Neg DCs are positively selected with CD11c beads and are bound to the column. The flow through can be discarded.
   17. Elute the cells retained in the column as described in step 3.2.11 to collect the purified CD8α^Neg DCs.

4. Pulsing APCs with Antigens and Cell Transfer

1. Count the live cells after purification using trypan blue exclusion ²¹ to distinguish live and dead cells.
2. Incubate the cells with the antigen of choice. Use analogues of glycolipid antigen called alpha-galactosyl ceramide (αGalCer) at 100 nM concentration¹³. Typically, plate 10⁶ viable cells/well in complete RPMI medium using ultra-low attachment U-bottom 96 well plates to minimize cell attachment. Incubate the cells in a 5% CO₂ atmosphere at 37 °C for 1 - 4 hr.
3. Harvest cells by gently pipetting several times. Use wide bore pipette tips to minimize cell damage from shear forces. Wash the wells with PBS and pool these washes to maximize cell harvest.
4. Pellet the cells at 300 x g for 5 min at 4 °C and resuspend to desired density in PBS. Typically, inject 1 million cells in 200 µl per recipient animal. Keep the cells on ice to maximize viability before administration into host animals.
5. Inject cells intravenously into mice either through the lateral tail vain or the retro-orbital venous plexus ¹⁹. Use a 27 G needle on a 1 ml syringe, and inject a maximum volume of 0.1 ml per mouse.

Results

The outcome of this procedure relies on the expansion of DC subsets by murine Flt-3L expressed by the implanted melanoma cells. The B16.Flt3L tumor was derived from a C57BL/6 mouse, and should be implanted into animals with that strain background in order to avoid failure of the tumor to become established due to rejection. In some cases, it may be desirable to use genetically modified mice to derive DCs with known defects in signaling pathways or receptors of interest. It is important to...

Discussion

Dendritic cells are accepted to be the major professional antigen presenting cells involved in the priming of T cell responses. Their main function is to survey the tissue microenvironment by taking up and processing antigens for presentation to T cells. In order to study the function of specific DC subsets, these need to be isolated in sufficient numbers using an approach that maintains their normal phenotype and functions. Most protocols rely on the isolation of specific DC subset from naïve mice. Since these cell...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA	Life Technologies, Gibco	25300-054
Isoflurane	Sigma-Aldrich	CDS019936-250MG
Collagenase D	Roche Diagnostics	11088858001
DNase I (dry powder)	QIAGEN	79254
200 proof ethanol	Thermo Fisher Scientific	9-6705-004-220	Used to prepare 70% ethanol
RBC lysis buffer	Sigma-Aldrich	R7757
RPMI-1640 medium with L-glutamine	Life Technologies, Gibco	11875-119
DMEM medium with L-glutamine	Life Technologies, Gibco	11995-073
200 mM L-glutamine	Life Technologies	25030081
MEM non-essential amino acids	Life Technologies, Gibco	11140-050
MEM essential amino acids	Life Technologies	11130-051
β-mercaptoethanol	Life Technologies, Invitrogen	21985-023
Sodium pyruvate	Life Technologies	11360-070
HEPES	Life Technologies, Invitrogen	15630
Phosphate buffered saline (PBS Ca2+ and Mg2+ free pH 7.2)	Life Technologies, Invitrogen	20012-050
Dulbecco’s PBS (DPBS with Ca2+ and Mg2+)	Life Technologies, Gibco	14040-182
0.5 M Ethylenediaminetetraacetate (EDTA) solution	Life Technologies	15575-020
Bovine serum albumin (BSA)	Sigma-Aldrich	A2153
Fetal calf serum	Atlanta Biologicals	S11050
Penicillin/streptomycin	Life Technologies, Invitrogen	15140-163
Trypan blue (dry powder)	Sigma-Aldrich	T6146-5G	Prepare 0.08% with PBS
Plasmacytoid Dendritic Cell Isolation Kit II, mouse	Miltenyi Biotech	130-092-786
Magnetic beads conjugated with anti-mouse CD11c	Miltenyi Biotech	130-152-001
CD8αPos mouse DC isolation kit	Miltenyi Biotech	130-091-169
Fc-gamma receptor blocking antibody (Clone 2.4G2)	BD Biosciences	553141
Anti-mouse CD11c-FITC	BD Biosciences	553801
Anti-mouse CD8α-PerCP	BD Biosciences	553036
Anti-mouse B220-PE	BD Biosciences	553090
1 ml syringes	BD	26048
23 G1 needle	BD	305145
100 mm Petri dishes	Thermo Fisher Scientific	875712
Surgical instruments	Kent Scientific	INSMOUSEKIT
Cell strainer (70 µm)	BD	352350
Large Petri plates	Thermo Fisher Scientific	FB0875712
Vacuum filtration system (500 ml (0.22 µm))	Corning	431097
LS columns	Miltenyi	130-042-401
Magnetic stand MACS separator	Miltenyi Biotec	130-042-302
Wide-bore 200 μl pipette tips	PerkinElmer	111623
Corning ultra-low attachment 96-well plates	Corning	CLS3474-24EA
6-8 week old female C57BL/6 mice	Jackson Research Laboratories, Jax
Murine B16.Flt3L melanoma cell line			described by Mach et al., 2000
Serum free DMEM and RPMI media 500 ml DMEM with L-glutamine, or 500 ml RPMI-1640 with L-glutamine 5 ml MEM nonessential amino acids (100x, 10 mM) 5 ml HEPES Buffer (1 M) 5 ml L-glutamine (200 mM) 0.5 ml 2-mercaptoethanol (5.5 x 10-2 M)			Mix all the ingredients in a biosafety cabinet with either DMEM or RPMI media depending on your need. Filter sterilize the media by passing through a 0.22 µm vacuum filtration system.
Complete RPMI and DMEM media			Add 50 ml of heat inactivated fetal calf serum to serum free RPMI or DMEM media to obtain complete media.
MACS buffer			Add 2 ml of 0.5 M EDTA and 10 ml of heat inactivated fetal calf serum to 500 ml of Phosphate-buffered saline (PBS, Ca2+ and Mg2+ Free, pH 7.2) and 2 µg/ml 2.4G2 (Fc-Block antibody).
FACS staining buffer			Dissolve sodium azide to 0.05% (0.25 g per 500 ml) in MACS buffer to obtain FACS staining buffer
10x Collagenase D and DNase 1 stock solution Dissolve 1 g of collagenase D and 0.2 ml of DNase 1 stock (1 mg/ml, 100x) in 20 ml of PBS containing Ca2+ and Mg2+ to obtain a solution of approximately 1,000-2,000 Units of collagenase activity per ml.			This 10x stock solution can be prepared ahead of time and stored at -20 °C for several weeks. Dilute 1 ml of this with 9 ml of serum free RPMI immediately before use.