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MACS Handbook

NK cells

 

1 Introduction

Natural Killer (NK) cells are part of the innate immune system. First described for their capacity to directly kill tumor cells without prior sensitization, NK cells are now known to participate in a variety of immune responses, including host protection against viruses, parasites, bacteria and tumor cells. They also play an important role in stem cell transplantation and pregnancy. NK cells perform their effector functions by either mediating direct cell lysis or by producing a plethora of cytokines. Although typically studied in the mouse spleen, NK cells are also found in other organs, such as the thymus, lung and liver.

2 NK cells from spleen and lymph nodes

NK cells develop mainly in bone marrow (BM).  The NK cell developmental process has been progressively dissected, starting from committed precursors (NKP: Lin−/CD122+/NK1.1−/CD49b−) and immature cells (iNK: NK1.1+/CD49b−CD11blow/CD51low), to a mature NK subpopulation (mNK: CD49b+/Ly49s+). NK cells make up 3–4% of total lymphocytes in spleen tissue and less than 1% in lymph nodes.
MACS Handbook:

Spleen (mouse)

Lymph node (mouse)

2.1 Cell subsets, frequencies, and marker expression

At   a glance: Natural Killer (NK) cells in various tissues (PMID: 19234143, 16424180) 
Frequencies: (PMID: 23034281, 23649470); Function: (PMID: 28261203)

 Cell subsetFrequencyMarkersFunction
CD11blowCD27low NK cells1.1-2.75%CD27, CD11bFound predominantly in bone marrow and very low frequency in all lymphoid and nonlymphoid organs
CD11blowCD27high NK cells11.5-16.5%CD27, CD11bFound predominantly in bone marrow, lymph nodes and in a greater proportion in the liver
Exhibit greater effector function and responsiveness to chemokines
Produce larger amounts of cytokines
Show decreased cytotoxicity
Interact with dendritic cells
CD11bhighCD27high NK cells23.2-49.3%CD27, CD11bHomogeneously distributed
Exhibit greater effector function and responsiveness to chemokines 
Express higher levels of killer cell lectin-like receptor subfamily G, member 1 (KLRG1)
Interact with dendritic cells
CD11bhighCD27low NK cells31.4-58.3%CD27, CD11bMost abundant in blood, spleen, lung and liver
Increased cytotoxicity against target cells
A key marker of the NK cell lineage is CD27, a member of the TNF receptor superfamily. Its expression combined with the integrin chain CD11b, defines four sequential developmental stages of NK cells: CD11blowCD27low, CD11blowCD27high, CD11bhighCD27high, and CD11bhighCD27low subsets (PMID: 19234143).
These NK cell subsets differ widely in tissue distribution (PMID: 16424180).  CD11blowCD27high NK cells are predominantly found in bone marrow and lymph node, whereas CD11bhighCD27low NK cells are more abundant in blood, spleen, lung and liver, and double-positive CD11bhighCD27high NK cells are more homogeneously distributed.

2.2 Miltenyi Biotec application protocols for NK cells from spleen

Miltenyi Biotec has created dedicated application protocols to isolate and analyze NK cells

2.3 Sample Preparation of spleen 

The main source for mouse NK cells is single-cell suspensions from spleen and lymph nodes, which are used for many downstream applications, including isolation of cell subpopulations, cell culture, or flow cytometry analysis.
Dissociation can be fully automated using the gentleMACS™ Dissociators and dedicated tissue dissociation kits (e.g., Spleen Dissociation Kit, mouse). A special protocol for the preparation of single-cell suspensions from mouse spleen without enzymatic treatment can be downloaded from the Related Resources panel to the right. Alternatively, tissues can be dissociated manually. For details, see related chapters.

MACS Handbook:

Sample preparation

Spleen (mouse)

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2.4 Magnetic Cell Separation of NK cells from spleen and lymph nodes

Miltenyi Biotec has developed numerous products for the magnetic separation of the various B cell types and subsets that can be found in mouse lymphoid tissue. For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic Cell Separation .
MACS Handbook:

Magnetic cell separation

2.4.1 Isolation of NK and NKT cells from spleen and lymph nodes
Starting materialIsolation strategyCommentsAutomationProduct
Single-cell suspensions from spleen and lymph nodesDepletion of non-target cellsIsolation of all NK cells via depletion of all non-NK cellsYes*NK Cell Isolation Kit, mouse
Positive selection of target cellsCD49b is expressed on NK cells and a small population of T cells (CD4+CD3+ TCRα/β+). CD49b is less mouse strain–specific than other NK cell markers (e.g. NK1.1) and is expressed by the most common inbred mouse strainsYes*CD49b (DX5) MicroBeads, mouse
Positive selection of target cellsNKp46 seems to be exclusively expressed on NK cells. Expression has been shown on BALB/c, SJL, CBA/CA, C57Bl/6, NOD, DBA/2, and B6.129 miceYes*Anti-NKp46 MicroBead Kit, mouse
Sequential separationIsolation of NK1.1+ iNKT cells via depletion of all NK1.1+ iNKT cells followed by enrichment via anti-NK1.1-APC and Anti-APC MicroBeadsYes*NK1.1+ iNKT Cell Isolation Kit, mouse
*Automation options range from fully automated benchtop solutions such as the autoMACS® Pro Separator to high-throughput platforms such as the MultiMACS™ Cell24 Separator Plus or MultiMACS X. 
The NK Cell Isolation Kit, mouse enables isolating untouched NK cells from single-cell suspensions of murine spleen. Non-NK cells (i.e., T cells, dendritic cells, B cells, granulocytes, macrophages, and erythroid cells) are magnetically labeled with a cocktail of biotin-conjugated antibodies and Anti-Biotin MicroBeads, and then depleted to generate highly pure unlabeled NK cells. The kit is optimized to give outstanding purities in C57BL/6J and BALBc mice.
NK cell isolation from single-cell suspensions of spleen.
Before separation
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Isolated NK cells
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NK cell isolation from single-cell suspensions of spleen. NK cells were isolated from BALB/c mice using the NK Cell Isolation Kit, a LS Column, and a MidiMACS™ Separator. The cells were fluorescently stained with CD3-FITC, CD49b-APC, and Anti-NKp46-PE. Cells were analyzed by flow cytometry using the MACSQuant® Analyzer 10. Cell debris and dead cells were excluded from the analysis based on scatter signals, CD45-VioGreen™, and 4’,6-diamidino-2-phenylindole (DAPI) fluorescence.

Before separation
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Isolated NK cells
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NK cell isolation from single-cell suspensions of spleen. NK cells were isolated from C57BL/6 mice using the NK Cell Isolation Kit, a LS Column, and a MidiMACS™ Separator. The cells were fluorescently stained with CD3-FITC, CD49b-APC, and Anti-NK1.1-PE . Cells were analyzed by flow cytometry using the MACSQuant® Analyzer 10. Cell debris and dead cells were excluded from the analysis based on scatter signals, CD45-VioGreen™, and 4’,6-diamidino-2-phenylindole (DAPI) fluorescence.

The Anti-NKp46 MicroBead Kit and the CD49b (DX5) MicroBeads, mouse are used for the positive selection or depletion of mouse NK cells from single-cell suspensions of lymphoid and non-lymphoid tissues or from peripheral blood.
Positive selection of NK cells
Unseparated fraction
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After separation
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Positive selection of NK cells. NKp46+ NK cells were isolated from BALB/c mouse spleen using the Anti-NKp46 MicroBead Kit, two consecutive MS Columns, and a MiniMACS™ Separator. Cells were fluorescently stained with CD3ε-APC and Anti-NKp46-PE and analyzed by flow cytometry using the MACSQuant Analyzer.

Distinct separation of CD49b (DX5)+ cells
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Distinct separation of CD49b (DX5)+ cells. Isolation of mouse NK cells from a single-cell suspension of mouse spleen using CD49b (DX5) MicroBeads, a MiniMACS Separator, and an MS Column.

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2.5 Characterization of NK cells from spleen and lymph nodes by flow cytometry

NK cell subsets and their differentiation status can be determined by flow cytometry based on their expression of cell surface markers, transcription factors, and their secretion of cytokines. Miltenyi Biotec offers a vast portfolio of conventional and recombinant REAfinity™ antibodies for comprehensive analysis.
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2.5.1 Flow cytometry panels
 
Basic NK lineageActivating receptors, adhesion and effector functionsInhibitory receptorsTranscription FactorsLy49Secreted factorsOther interesting markers
NK1.1β-2 Integrins 
(CD11a-CD18, CD11b-CD18, CD11c-CD18) 		LAIR-1CD278 (ICOS)Ly49AIFN-γCD25
CD127CD335 (NKp46)CD94/NKG2AEOMESLy49A/DTNF-αCD27
CD49bDNAM-1SIGLEC-AGATA3Ly-49C/F/I/HIL-12 (p40/p70)Tim-3
CD1172B4/CD244KLRG1RORγ (t)Ly-49C/IGM-CSFTIGIT
CD11bCD314 (NKG2D)NKRP1-B, -DT-betLy-49E/F CD279 (PD1)
CD122NKG2C2B4 (CD244)TOXLy-49F PDL2
 NKG2E HeliosLy-49G2 PLZF
 CD25 IcarosLy-49H FcεR1γ
 TRAIL (CD253)  Ly-49I FcεRIα
 Granzyme B  Ly-49D  
 CD69  Ly-49G  
 CD62L     
 CD178 (FasL)     
 CD107a (LAMP-1)     
 Perforin     
 LAG-3     
Miltenyi Biotec has developed numerous products for the analysis of the NK cells and cell subsets found in mouse spleen and lymph nodes. The REAfinity antibodies combined with a wide selection of Vio® Dyes characterized by high fluorescence intensities and low spillover, enable successful NK cell analysis. 

The following panel was tested on a single-cell suspension from splenocytes from C57BL/6 mice:  
 
MarkerClone
Viability 405/520 Fixable Dye-
Anti-NK1.1-Pe-Vio770PK136 (soon REA632)
CD11b-VioBlueREA592
CD335 (NKp46)-FITCREA815
CD27-APCREA499
CD45-PE-Vio615REA737
Anti-KLRG1-PEREA1016
CD3-APC-vio770REA641
CD19-APC-vio770REA749
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Analysis of NK cell from mouse splenocytes

Single cell suspension from C57BL/6 mouse spleen was fluorescently stained with Anti-NK1.1-Pe-Vio770, CD11b-VioBlue CD335 (NKp46)-FITC, CD27-APC, CD45-PE-Vio615, Anti-KLRG1-PE, CD3-APC-vio770, CD19-APC-vio770 and analyzed by flow cytometry using the MACSQuant ® Analyzer 10. 

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3 NK cells from other tissues (lymphoid, non-lymphoid)

NK cells are widespread throughout lymphoid and nonlymphoid tissues. In most, NK cells represent only a minor fraction of total lymphocytes, from 2% in mouse spleen to 10% in mouse lung (PMID: 18425107), and there are differences among mice strains.
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Measured frequency of NK cells in various tissues of BALBc and C57BL/6 mice strains.

3.1 Tissue-specific marker expression 

Presence of NK cells in peripheral organs is presumably the result of chemokine-mediated migration from peripheral blood (PMID: 17979846). NK cells express several chemokine receptors, including CCR2, CCR5, CXCR3, and CX3CR1, which facilitate targeting specific NK cells to the relevant chemokine-producing organ.
 
 SpleenLiverLung Bone marrow   Uterus  
CD3-----
CD49b (DX5)+-++-/+
CD49a-+  +
CD27 -low-/+-
NKp46+++++
CD11b+-/lowhigh-/++
TRAIL #   
Ly49+-/lowhigh+high
CD94/NKG2A++high+ 
KLRG1low + +

3.2 Sample preparation of different tissues

The complexity of NK cell phenotyping makes it essential that all epitopes needed to identify subsets are preserved during tissue digestion. Miltenyi Biotec offers kits for the dissociation of various tissues — including lamina propria, liver, lung and skin — and the preparation of single-cell suspensions optimized to preserve epitopes. For details on sample preparation, see MACS handbook chapters on mouse cell sources and our specific tissue dissociation kits.
MACS Handbook:

Sample preparation

3.3 Magnetic Cell Separation of NK cells from other tissues (lymphoid, non-lymphoid)

Miltenyi Biotec has developed numerous products for the magnetic separation of cell types and cell subsets found in various mouse tissues. 
For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic Cell Separation .
MACS Handbook:

Magnetic cell separation

3.3.1 Isolation of NK cells from other tissues (lymphoid, non-lymphoid)
Depending on mouse strain and on NK cell phenotype, NK cells can be magnetically enriched after tissue dissociation with dedicated tissue dissociation kits, by positive selection with CD49b (DX5) MicroBeads, mouse or the Anti-NKp46 MicroBead Kit, mouse.
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NK cells isolated from mouse liver tissue

Perfused liver tissue was dissociated using the Liver Dissociation Kit, mouse followed by enrichment of NK cells using CD49b (DX5) MicroBeads.

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