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Dendritic Cell Subset Purification from Human Tonsils and Lymph Nodes

  • Mélanie Durand
  • Elodie SeguraEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1423)

Abstract

Dendritic cells (DCs) are a rare population of antigen-presenting cells that initiate immune responses in secondary lymphoid organs. In order to better understand the properties of DC in humans, it is essential to analyze DC subsets directly purified from tissues. Here, we describe a protocol allowing the purification of DC subsets from human tonsils and human lymph nodes.

Key words

Dendritic cells DC subsets Human Tonsil Lymph node 

1 Introduction

Dendritic cells (DCs) represent a complex population of antigen-presenting cells. Numerous studies in mice and humans have revealed DC heterogeneity and the existence of phenotypically and ontogenically distinct DC subsets. Two main categories of DC can be distinguished: plasmacytoid DC (pDC) and classical DC (cDC). cDC can be further classified according to their migratory capacity. Migratory DCs are first localized in peripheral tissues and can migrate through the lymph to the draining lymph nodes to initiate immune responses. They represent roughly 50 % of draining lymph node DC and are absent from non-draining lymphoid organs (tonsils, spleen, and thymus) [1]. By contrast, resident DCs spend their entire life cycle in lymphoid organs and represent all the DCs found in non-draining lymphoid organs and 50 % of the DCs in draining lymph nodes.

Three resident DC subsets are present in human non-draining lymphoid organs: CD1c/BDCA1+ DC, CD141/BDCA3+ DC, and pDC [2, 3]. In the tonsils, pDCs are the most abundant DC subset, while CD141+ DCs are the rarest. It has been shown that resident DCs from the tonsils are immature, as they do not express co-stimulatory molecules [4].

In skin-draining lymph nodes, the three resident DC subsets were identified (CD1c+ DC, CD141+ DC, and pDC) along with three subsets of migratory DC (Langerhans cells (LCs), CD1a+ DC, CD206+ DC) [5]. Phenotypic and functional evidence suggests that the CD206+ DC subset is the lymph node counterpart of CD14+ DC from the dermis [5]. During migration, migratory DCs upregulate MHC II molecules and co-stimulatory molecules [5, 6]. Recent studies describe the existence of CD141+ DC in peripheral tissues [7, 8], suggesting that lymph nodes contain a proportion of previously unnoticed migratory CD141+ DC.

The protocol described here is designed for the purification of the different DC subsets from tonsils and lymph nodes. This protocol consists of three steps: digestion of the organs, DC enrichment, and cell sorting.

2 Materials

2.1 Mechanical and Enzymatic Tissue Digestion

  1. 1.

    Scalpel.

     
  2. 2.

    14 and 50 mL tubes.

     
  3. 3.

    Iscove’s Modified Dulbecco’s Medium (IMDM).

     
  4. 4.

    DNase (Roche).

     
  5. 5.

    Liberase TL Research Grade low Thermolysin concentration (Roche).

     
  6. 6.

    Multi-Axle-Rotating Mixer.

     
  7. 7.

    EDTA 0.1 M pH 7.2.

     
  8. 8.

    40 μm cell strainer.

     
  9. 9.

    Digestion medium: IMDM, 0.1 mg/mL of liberase TL, and 0.1 mg/mL of DNase.

     

2.2 DC Enrichment

  1. 1.

    Iscove’s Modified Dulbecco’s Medium (IMDM).

     
  2. 2.

    Lymphoprep (Greiner Bio-One).

     
  3. 3.

    Staining buffer: PBS, 0.5 % human serum, and 2 mM EDTA (see Note 1 ).

     
  4. 4.
    Antibodies for T and B lymphocytes, NK cells, and erythrocyte depletion (see Table 1).
    Table 1

    Antibodies and microbeads for DC enrichment

    Name

    Fluorochrome

    Clone

    Company

    Suggested dilution

    Mouse anti-CD3

    Purified

    HIT3a

    eBioscience

    1/100

    Mouse anti-CD19

    Purified

    HIB19

    eBioscience

    1/100

    Mouse anti-CD235a

    Purified

    HIR2

    eBioscience

    1/100

    Mouse anti-CD56

    Purified

    CMSSB

    eBioscience

    1/100

    Anti-mouse IgG1

    Microbeads

     

    Miltenyi Biotec

    1/10

    Anti-mouse IgG2a + b

    Microbeads

     

    Miltenyi Biotec

    1/20

    Antihuman CD15

    Microbeads

     

    Miltenyi Biotec

    1/20

     
  5. 5.

    Magnetic microbeads for T and B lymphocytes, NK cells, erythrocytes, and myelomonocytic cell depletion (see Table 1).

     
  6. 6.

    LD columns (Miltenyi Biotec).

     
  7. 7.

    MACS separator magnet (Miltenyi Biotec).

     

2.3 Cell Sorting of the DC Subsets

  1. 1.

    Staining buffer.

     
  2. 2.

    Fc receptor blocking reagent (TruStain, BioLegend).

     
  3. 3.
    Antibodies for tonsil DC (see Table 2) or for lymph node DC (see Table 3).
    Table 2

    Antibodies for cell sorting of tonsil DC

    Name

    Fluorochrome

    Clone

    Company

    Suggested dilution

    Mouse anti-HLADR

    APC efluor780

    LN3

    eBioscience

    1/100

    Mouse anti-CD11c

    PE Cy7

    Bu15

    eBioscience

    1/200

    Mouse anti-CD14

    FITC

    61D3

    eBioscience

    1/100

    Mouse anti-CD1c

    PerCP efluor710

    L161

    eBioscience

    1/100

    Mouse anti-CD141

    PE

    A05-14H2

    Miltenyi Biotec

    1/200

    Mouse anti-CD304

    APC

    REA380

    Miltenyi Biotec

    1/20

    Table 3

    Antibodies for cell sorting of lymph node DC

    Name

    Fluorochrome

    Clone

    Company

    Suggested dilution

    Mouse anti-HLADR

    APC efluor780

    LN3

    eBioscience

    1/100

    Mouse anti-CD11c

    PE Cy7

    Bu15

    Biolegend

    1/200

    Mouse anti-CD14

    PE

    61D3

    eBioscience

    1/100

    Mouse anti-CD1c

    PerCP efluor710

    L161

    eBioscience

    1/100

    Mouse anti-CD206

    Alexa Fluor 647

    15-2

    Biolegend

    1/50

    Mouse anti-CD1a

    Alexa Fluor 488

    703217

    R&D Systems

    1/100

    Mouse anti-Clec9a

    Biotin

    8F9

    Miltenyi Biotec

    1/50

     
  4. 4.

    DAPI: stock solution at 1 mg/mL.

     
  5. 5.

    5 mL polystyrene tubes with a 40 μm cell strainer cap.

     
  6. 6.

    Cell sorter cytometer.

     
  7. 7.

    5 mL polypropylene tubes.

     
  8. 8.

    Decomplemented fetal bovine serum (FBS).

     

3 Methods

The use of human samples must be approved by an institutional ethics committee. All steps must be performed in sterile conditions.

3.1 Purification of DC from Tonsils

3.1.1 Mechanical and Enzymatic Tissue Digestion

To dissociate thoroughly the tissue and be able to isolate DC subsets, we perform first a mechanical dissociation followed by an enzymatic digestion with constant agitation.
  1. 1.

    Use a scalpel to chop the tissue into small pieces until to get a “mash” of tissue.

     
  2. 2.

    Prepare 40 mL of digestion medium per pair of tonsils (see Note 2 ).

     
  3. 3.

    Place the “mash” of tissue into 40 mL of digestion medium and incubate for 40–45 min at room temperature (RT) (see Note 3 ) and with constant agitation (see Note 4 ).

     
  4. 4.

    Add 2 mL of EDTA 0.1 M to the digested cell suspension and incubate for 2 min at RT (see Note 5 ).

     
  5. 5.

    To remove undigested debris, filter the cell suspension into a 40 μm cell strainer.

     
  6. 6.

    Centrifuge the cell suspension for 10 min at 450 × g at RT.

     
  7. 7.

    Resuspend the cells into 20 mL of IMDM.

     

3.1.2 DC Enrichment

Due to the scarcity of DC, it is necessary to enrich the sample for DC by depleting T and B lymphocytes, NK cells, erythrocytes, and myelomonocytic cells (see Figs. 1 and 2).
Fig. 1

Gating strategy of the cell sorting of the three tonsil resident DC subsets after DC enrichment. After tissue digestion and DC enrichment, cells were stained for CD11c, HLADR, CD14, CD1c, CD141, and CD304. Dead cells and doublets were excluded by using DAPI staining and SSC-W SSC-A lasers, respectively. Macrophages and cDC are first gated on CD11c and HLA DR (top left panel). Macrophages are CD14+, and CD1c DCs are CD1c+CD14, whereas the double negative population contains CD141 DC (top right panel). CD141 DCs are identified using CD141 staining (bottom right panel). pDCs are first gated as HLADR+ CD11c− (top left panel) and subsequently selected using CD304 staining (bottom left panel)

Fig. 2

Staining of tonsil resident DC subsets without pre-enrichment. After tissue digestion, cells were stained for CD11c, HLADR, CD14, CD1c, CD141, and CD304. Macrophages and cDC are contained within CD11c + HLADR+ cells (top left panel). Macrophages are CD14+, and CD1c DCs are CD1c + CD14−, whereas the double negative population contains CD141 DC (top right panel). CD141 DCs are identified as CD141+ (bottom right panel). pDCs are contained within HLADR+ CD11c− cells (top left panel) and are identified as CD304+ (bottom left panel)

  1. 1.

    Put 17 mL of Lymphoprep into a 50 mL tube, and then slowly layer the 20 mL cell suspension on top of the Lymphoprep solution (see Note 6 ).

     
  2. 2.

    Centrifuge for 15 min at 800 × g at RT with the decelerating brake off (see Note 7 ).

     
  3. 3.

    Collect the cells at the interface with a 5 mL pipette, and wash the cells three times with 50 mL of cold IMDM (see Note 8 ).

     
  4. 4.

    Count the cells, centrifuge for 5 min at 450 × g at 4 °C, and resuspend the cells at 200 × 106 cells/mL in the antibodies solution (anti-CD3, anti-CD19, anti-CD56, and anti-CD235a antibodies at 10 μg/mL in staining buffer (see Table 1)) (see Note 9 ).

     
  5. 5.

    Incubate for 30 min at 4 °C.

     
  6. 6.

    Wash the cells with 10–20 mL of cold staining buffer.

     
  7. 7.

    Resuspend the cells in staining buffer at 400 × 106 cells/mL, and add the microbeads (mouse IgG1 microbeads, mouse IgG2a + b, and CD15 microbeads) (see suggested dilution in Table 1).

     
  8. 8.

    Incubate for 20 min at 4 °C.

     
  9. 9.

    Place the LD columns on the separator magnet and wash them with 3 mL of staining buffer (see Note 10 ).

     
  10. 10.

    Wash the cells with 10 mL of staining buffer.

     
  11. 11.

    Resuspend the cells at 350 × 106 cells/mL in staining buffer.

     
  12. 12.

    Distribute 2 mL of cell suspension per LD column.

     
  13. 13.

    Collect the eluate (negative fraction that is enriched in DC).

     
  14. 14.

    Wash twice the columns with 1 mL of staining buffer and collect the eluate.

     
  15. 15.

    Centrifuge the eluate for 5 min at 450 × g at 4 °C (see Note 11 ).

     
  16. 16.

    Remove the column from the separator magnet, add 3 mL of staining buffer at the top of the column, and use the syringe to push the positive fraction out. These cells can be used for unstained and DAPI control tubes for cell sorting.

     

3.1.3 Cell Sorting of the DC Subsets

  1. 1.

    Resuspend the cells enriched in DC at 10 × 106 cells/100 μL in antibodies solution containing Fc receptor blocking reagent (see Table 2 for antibodies and suggested dilutions).

     
  2. 2.

    Incubate for 30 min at 4 °C.

     
  3. 3.

    Wash the cells with 10 mL of cold staining buffer.

     
  4. 4.

    Resuspend the cells in staining buffer containing DAPI (200 ng/mL final dilution) (see Note 12 ).

     
  5. 5.

    Prepare single-stain tubes for the different fluorochromes. Cells from the positive fraction of the DC enrichment can be used to compensate the DAPI (see Note 13 ).

     
  6. 6.

    Prepare collecting tubes: put 1 mL of FBS in 5 mL polypropylene tubes (see Note 14 ).

     
  7. 7.

    Sort the cells using a cell sorter cytometer. CD1c DCs are CD11c+ HLA DR+,CD14 CD1c+ cells. CD141 DCs are CD11c+ HLA DR+ CD14, CD1c CD141+ cells. pDCs are CD11c HLA DR+ CD14 CD1c CD304+ cells (see Fig. 1).

     

3.2 Purification of DC from Lymph Nodes

3.2.1 Mechanical and Enzymatic Tissue Digestion

To dissociate thoroughly the tissue and be able to isolate DC subsets, we perform first a mechanical dissociation followed by an enzymatic digestion with constant agitation.
  1. 1.

    Use a scalpel to chop the lymph nodes into small pieces until to get a “mash” of tissue.

     
  2. 2.

    Prepare 5 mL of digestion medium per lymph node sample (see Note 2 ).

     
  3. 3.

    Place the “mash” of tissue into 5 mL of digestion medium, and incubate for 20 min at room temperature (RT) (see Note 3 ) and with constant agitation (see Note 4 ).

     
  4. 4.

    Add 0.25 mL of EDTA 0.1 M to the digested cell suspension and incubate for 2 min at RT (see Note 5 ).

     
  5. 5.

    To remove undigested debris, filter the cell suspension into a 40 μm cell strainer.

     
  6. 6.

    Centrifuge the cell suspension for 10 min at 450 × g at 4 °C.

     
  7. 7.

    Resuspend the cells into 5 mL of cold IMDM.

     

3.2.2 DC Enrichment

Due to the scarcity of DC, it is necessary to enrich the sample for DC by depleting T and B lymphocytes, NK cells, erythrocytes, and myelomonocytic cells.
  1. 1.

    Count the cells, centrifuge, and resuspend the cells at 200 × 106 cells/mL in the antibodies solution (anti-CD3, anti-CD19, anti-CD56, and anti-CD235a antibodies) at 10 μg/mL in staining buffer (see Table 1) (see Note 9 ).

     
  2. 2.

    Incubate for 30 min at 4 °C.

     
  3. 3.

    Wash the cells with 5 mL of cold staining buffer.

     
  4. 4.

    Resuspend the cells in staining buffer at 400 × 106 cells/mL, and add the microbeads (mouse IgG1 microbeads, mouse IgG2a + b, and CD15 microbeads) (see suggested dilution in Table 1).

     
  5. 5.

    Incubate for 20 min at 4 °C.

     
  6. 6.

    Place the LD columns on the separator magnet and wash them with 3 mL of staining buffer (see Note 10 ).

     
  7. 7.

    Wash the cells with 10 mL of staining buffer.

     
  8. 8.

    Resuspend the cells at a maximum concentration of 350 × 106 cells/mL in staining buffer. Minimum volume for the cell suspension is 2 mL.

     
  9. 9.

    Apply the cell suspension on LD columns.

     
  10. 10.

    Collect the eluate (negative fraction that is enriched in DC).

     
  11. 11.

    Wash twice the columns with 1 mL of staining buffer and collect the eluate.

     
  12. 12.

    Centrifuge the eluate for 5 min at 450 × g (see Note 11 ).

     
  13. 13.

    Remove the column from the separator magnet, add 3 mL of staining buffer at the top of the column, and use the syringe to push the positive fraction out. These cells can be used for unstained and DAPI control tubes for cell sorting.

     

3.2.3 Cell Sorting of the DC Subsets

  1. 1.

    Resuspend the cells at 10 × 106 cells/100 μL in antibodies solution containing Fc receptor blocking reagent (see Table 3 for antibodies and suggested dilutions).

     
  2. 2.

    Incubate for 30 min at 4 °C.

     
  3. 3.

    Wash the cells with 10 mL of staining buffer.

     
  4. 4.

    If necessary, incubate the cells 20 min at 4 °C with fluorochrome-conjugated streptavidin and repeat step 3.

     
  5. 5.

    Resuspend the cells in staining buffer with DAPI (200 ng/mL final dilution) (see Note 12 ).

     
  6. 6.

    Prepare single-color stained tubes for the different fluorochrome; you can use the positive fraction of the DC enrichment to compensate the DAPI (see Note 13 ).

     
  7. 7.

    Prepare collecting tubes: put 1 mL of FBS in 5 mL polypropylene tubes (see Note 14 ).

     
  8. 8.
    Sort the cells using a cell sorter cytometer. CD1a DCs are CD11c+ HLADR+ CD1a+ CD14 Epcam cells. LCs are CD11c+ HLADR+ CD1a+ CD14 Epcam+ cells. Macrophages are CD11c+ HLADR+ CD1a+ CD14+ cells. CD141 DCs are CD11c+ HLADR+ CD1a CD14 CD1c Clec9a+ cells. CD1c DCs are CD11c+ HLADR+ CD1a CD14 CD1c+ Clec9a CD206 cells. CD206 DCs are CD11c+ HLADR+ CD1a CD14 CD1c+ Clec9a CD206+ cells. pDCs are CD11c HLADR+ CD304+ cells (see Fig. 3 for gating strategy).
    Fig. 3

    Gating strategy of the cell sorting of DC subsets from lymph node after DC enrichment. (a) After tissue digestion and DC enrichment, cells were stained for CD11c, HLADR, CD1a, Clec9a, CD1c, and CD206. Resident cDC and migratory DC are gated as CD11c + HLADR+ (top left panel a). Cells are then separated according to the expression of CD1a and CD14: macrophages are CD1aCD14+ and CD1a+CD14 contain both Langerhans cells and CD1a + DC (top right panel a). CD1a− CD14− are further separated according to the expression of Clec9a and CD1c (bottom left panel a). CD141 DCs are identified as Clec9a+CD1c. CD1c + cells are finally separated using CD206 staining allowing the separation of migratory CD206 DC and resident CD1c DC (bottom right panel a). (b) Additional stainings allow the isolation of pDC. pDCs are first gated as CD11cHLA DR+, then identified by the expression of CD304. (c) Epcam staining allows the separation of CD1a DC (Epcam) and Langerhans cells (Epcam+)

     

4 Notes

  1. 1.

    To minimize DC activation, the staining buffer has to remain at 4 °C during the whole protocol.

     
  2. 2.

    Liberase is used instead of collagenase because of higher efficiency and lower stripping of cell surface molecules.

     
  3. 3.

    The digestion is done at RT to minimize DC activation.

     
  4. 4.

    The digestion has to be performed in constant agitation for improved efficiency.

     
  5. 5.

    The EDTA stops the enzymatic digestion and allows the dissociation of the cell conjugates (in particular T cell-DC conjugates).

     
  6. 6.

    Other Ficoll reagents can be used.

     
  7. 7.

    The use of the function “brake off” is essential for the isolation of low-density cells on Ficoll gradient.

     
  8. 8.

    At this step you can resuspend the cells in culture medium containing 10 % of FBS, store the cells at 4 °C for 8–12 h, and resume the protocol later. We recommend using Yssel medium for improved DC viability.

     
  9. 9.

    Include an antihuman CD14 antibody if you wish to remove macrophages.

     
  10. 10.

    Determine how many LD columns are needed. For optimum enrichment, use one LD column for a maximum of 700 × 106 cells.

     
  11. 11.

    At this step you can resuspend the cells in culture medium containing 10 % of FBS, store the cells at 4 °C for 8–12 h, and resume the protocol later. We recommend using Yssel medium for improved DC viability.

     
  12. 12.

    Other viability dyes can be used.

     
  13. 13.

    Using beads for single-stain compensation controls (for instance, BD CompBeads from BD Biosciences) allows saving cells from the samples.

     
  14. 14.

    Using polypropylene tubes avoids the attachment of DC onto the tube wall during cell sorting.

     

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Institut Curie, Centre de rechercheParis Cedex 05France
  2. 2.INSERM U932Paris Cedex 05France

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