Dendritic cells

MACS Handbook

1 Introduction

As sentinels of the immune system, dendritic cells (DCs) are naturally located in all tissues, especially those at the interface between the body and the external environment, such as skin, lung, and intestine. They are also present in lymphoid organs where they activate T cells, thus initiating immune responses.  Due to limited access to human solid tissues, most of the studies on DCs from lymphoid and non-lymphoid organs have been performed on tissues obtained from mouse models.

2 DCs from spleen and lymph nodes

2.1 Cell subsets, frequencies, and marker expression

At a glance: DC cell subsets from lymphoid tissue (spleen and lymph nodes)

Cell subsetFrequencyMarkersFunction
Plasmacytoid DCs (pDCs)0.5% in spleen; 0.2% in lymph nodesCD45, CD11c, MHC class II, mPDCA-1 (CD317 or BST2), Siglec-H, CD45R (B220), Ly-6CUpon pathogen encounter, pDCs produce large amounts of type I IFN and acquire antigen-presenting capacity.
Conventional DC 1 (cDC1)0.5% in spleen; 0.1% in lymph nodesCD45, CD11c, MHC class II, CD8a, XCR1, CD24, CLEC9AcDC1 perform cross-presentation of antigens to MHC class I and start type I cytotoxic immune responses.
Conventional DC 2 (cDC2)1% in spleen; 0.2% in lymph nodesCD45, CD11c, MHC class II, CD4, SIRPα, CD11bcDC2 activate innate lymphoid cells 2 (ILC2) and TH2 cells and Induce ILC3 and TH17 immune responses.

Different myeloid cell types have many markers and functions in common, such as expression of MHC class II, CD11c, CD11b, and antigen presentation. Therefore, the classification and identification of myeloid cells remain evolving subjects in the scientific community. Moreover, cell subsets and markers are not always consistent between mouse and human, leading to debates over the correspondence of mouse and human subsets.

The latest classification defines three mouse DC subsets in lymphoid organs, which have counterparts in human blood, namely pDCs and the two conventional or myeloid DC (cDC) subsets cDC1 and cDC2.

pDCs are primarily located in blood and lymphoid tissues. They depend on the E2-2 transcription factor and express B220, Siglec-H, mPDCA-1 (CD317 or Bst2), as well as intermediate levels of MHC class II, CD11c, and costimulatory molecules. pDCs are poor stimulators of T helper (TH) cells, but upon stimulation with bacterial DNA containing particular unmethylated CpG motifs or upon viral challenge, they produce large amounts of type I IFN and acquire antigen-presenting capacity (PMID: 16172135, 15728491).

cDC1 are located in both lymphoid and non-lymphoid tissues, and express higher levels of MHC class II and CD11c compared to pDCs. They show dependence on the Batf3 transcription factor and are characterized by different sets of markers depending on their location, origin, and function. Resident spleen and lymph node cDC1 express CD8a, XCR1, and CD24. When cDC1 migrate from non-lymphoid tissue to a draining lymph node, they can be distinguished from resident cDC by markers expressed in the periphery such as CD103, by activation molecules, by higher levels of MHC class II, and lower levels of CD11c. However, when resident cDCs are activated during inflammation, these markers cannot be used to discriminate between migratory and resident cDCs.

cDC1 recognize intracellular pathogens and start type 1 immune responses including ILC1, NK cells, and T helper cell 1 induction. Moreover, cDC1 efficiently cross-present extracellular antigens to CD8+ T cells and secrete IL-12, making them important for cytotoxic responses to viral infections and tumors (PMID: 23516985).

cDC2 exhibit the same MHC class II and CD11c expression pattern as cDC1, but express additional markers not present on cDC1 and depend on a different transcription factor, i.e., IRF4. Resident spleen and lymph node cDC2 express CD4 and SIRPα. Migratory cDC2 that infiltrate lymph nodes can be distinguished from resident cDCs under non-inflammatory conditions by the expression of MHC class II, CD11c, and peripheral and migratory markers (e.g. CCR7 and maturation markers).

cDC2 induce different responses, such as activation of ILC2 and TH2 cells against parasites and during asthma, and induction of ILC3 and TH17 immune responses to extracellular bacteria (PMID:    27760337).

2.2 Miltenyi Biotec application protocols for DCs from lymphoid tissue

2.3 Sample preparation of spleen and lymph nodes

Research on mouse DCs is hampered by difficulties in isolating viable cells from solid tissues in numbers that allow comprehensive downstream investigation, such as flow cytometry analysis, cell sorting, and transcriptional studies. One critical aspect is the reliable dissociation of tissues, yielding cells with high viability and epitope integrity. Both enzymatic and mechanical dissociation of spleen is necessary to achieve a high recovery of DCs which are trapped in connective tissues and are inadequately released by simple mechanical meshing (PMID: 23516985).

The Spleen Dissociation Kit, mouse in combination with the gentleMACS™ Octo Dissociator with Heaters enables efficient, automated, and hands-free enzymatic and mechanical dissociation of several spleens in parallel. Both cell recovery and the level of epitope preservation are high in the resulting single-cell suspensions. For more details, see the corresponding spleen chapter.

2.4 Magnetic separation of DCs from lymphoid tissue

Miltenyi Biotec has developed numerous products for the straightforward magnetic separation of DCs and distinct DC subsets.

For details on MACS® Cell Separation Technology, see the MACS Handbook chapter Magnetic cell separation .

2.4.1 Isolation of dendritic cells from lymphoid tissue

At a glance: Kits and reagents for the isolation of various DC subsets

Cell subsetIsolation strategyCommentsAutomationProduct
Pan DCs Positive selection of target cells Yes*CD11c MicroBeads UltraPure, mouse
Pan DCs Depletion of non-target cellsIsolation of all untouched DC subpopulations from spleenYes*Pan Dendritic Cell Isolation Kit, mouse
Pan DCs Positive selection of target cellsMixture of CD11c and Anti-mPDCA-1 MicroBeadsYes*Pan DC MicroBeads, mouse
CD4+ DCs Depletion of non-DC cells followed by positive selection of target cellsIsolation of cDC2 subset from spleenYes*CD4+ Dendritic Cell Isolation Kit, mouse
CD8+ DCs Positive selection of target cellsIsolation of cDC1 subset from spleenYes*Anti-XCR1 MicroBead Kit (Spleen), mouse
CD8+ DCs Depletion of non-DC cells followed by positive selection of target cellsIsolation of cDC1 subset from spleenYes*CD8+ Dendritic Cell Isolation Kit, mouse
pDCs Positive selection of target cellsIsolation of pDCs in non-inflammed tissuesYes*Anti-mPDCA-1 MicroBeads, mouse
pDCs Depletion of non-target cellsIsolation of untouched pDCsYes*Plasmacytoid Dendritic 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.

DCs constitutively express the hematopoietic surface markers CD45, MHC class II, Flt3, and CD11c, and lack T cell, natural killer (NK) cell, B cell, granulocyte, and erythrocyte lineage markers (PMID: 23516985). In mice, CD11c is a well-established, yet not exclusive DC marker. Pan DCs can be enriched by positive selection from spleen using CD11c MicroBeads UltraPure, mouse or isolated by depletion of non-target cells using the Pan Dendritic Cell Isolation Kit, mouse. Subset populations can be further enriched by a second separation step using MicroBeads or flow cytometry–based cell sorting.

Pan Dendritic Cell Isolation Kit, mouse
Before separation
Isolated total DCs

Isolation of total untouched DCs. Pan DCs were isolated from a mouse spleen cell suspension using the Pan Dendritic Cell Isolation Kit, mouse, an LS Column, and a MidiMACS™ Separator. Cells were fluorescently stained with CD11c-APC and mPDCA-1-FITC and analyzed by flow cytometry using the MACSQuant® Analyzer. Cell debris, dead cells, and lineage-positive cells were excluded from the analysis based on scatter signals, propidium iodide fluorescence, and lineage antigen expression.

The cDC2 subset of DCs co-expresses CD11c, CD11b, MHC class II, CD40, CD80, and CD86 and is negative for CD8 and CD205. cDC2 represent the major population of DCs in spleen (56% of all CD11c+ cells) and constitute only a small population in peripheral lymph nodes (4% of all CD11c+ DCs). The CD4+ Dendritic Cell Isolation Kit, mouse isolates CD4+ cells from an enriched DC sample, after depletion of non-DC cells.

CD4+ Dendritic Cell Isolation Kit, mouse
Spleen cells before separation
Enriched CD4+CD11c+ DCs
Isolated CD4+CD11c+ DCs

Two-step isolation of CD4+ DCs from a mouse spleen. CD4+ DCs were isolated from a spleen cell suspension using the CD4+ Dendritic Cell Isolation Kit, mouse, an LD and two MS Columns, a MidiMACS™ and a MiniMACS™ Separator. Cells were fluorescently stained with the antibodies as indicated and analyzed by flow cytometry. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

In the past, studies of cDC1 were hampered by the low frequency of this cell type and a lack of specific cell surface markers. Recently, however, the receptors Clec9A and XCR1 have been identified as specific markers for cross-presenting cDCs in lymphoid and non-lymphoid tissue. The Anti-XCR1 MicroBead Kit (Spleen), mouse represents a novel method for the fast and easy isolation of cross-presenting DCs, that allows routine enrichment of XCR1+ DCs with high recovery and purity, without pre-depletion of non-target cells or laborious cell sorting.

Anti-XCR1 MicroBead Kit (Spleen), mouse
Before separation
Before separation
After separation
After separation

Fast isolation of cross-presenting DCs from mouse spleen. XCR1+ DCs were isolated from a single-cell suspension of dissociated spleen tissue using the Anti-XCR1 MicroBead Kit (Spleen) with two MS Columns and a MiniMACS™ Separator. Cells were fluorescently stained with Anti-MHC Class II-VioGreen™, CD11c-VioBlue®, Anti-XCR1-PE, and CD8a-APC and analyzed using the MACSQuant Analyzer 10. Cell debris, dead cells, and autofluorescent cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence. The dot plots on the right show conventional DCs gated on CD11c+MHC class II+ cells, stained for markers of cross-presenting DCs (CD8a and XCR1).

pDCs can be isolated from spleen by positive selection using the Anti mPDCA-1 MicroBeads, mouse or by depletion of unwanted cells with the Plasmacytoid Dendritic Cell Isolation Kit, mouse, which was designed for the rapid enrichment of pDCs.

 Plasmacytoid Dendritic Cell Isolation Kit, mouse
Before separation
Isolated plasmacytoid dendritic cells

Depletion of non-target cells generates a highly enriched population of pDCs. pDCs were isolated from splenocytes using the Plasmacytoid Dendritic Cell Isolation Kit, a MidiMACS Separator, and an LS Column. Cells were fluorescently stained with Anti-mPDCA-1-FITC and Anti-Siglec-H-PE and analyzed by flow cytometry on the MACSQuant Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

2.5 Characterization of DCs from lymphoid tissue by flow cytometry

Phenotyping or enriching DCs based on surface markers can be challenging because several myeloid cells share similar marker patterns, and different DC subsets can strongly modify their surface marker expression depending on their location and activation status. Furthermore, enzymatic tissue dissociation can affect surface epitopes. Optimization of antibodies with a specific and consistent enzyme cocktail is important to avoid false negative staining that compromises the identification of specific DC subsets.

2.5.1 Flow cytometry panels

At a glance: Markers for the detection of DCs from lymphoid tissue by flow cytometry

Pan DCscDC1cDC2pDCs
CD11c+CD11c+CD11c+CD11cint
MHC class IIhighMHC class II+MHC class II+MHC class IIint
CD11b+/–CD8a+CD8a+CD317 (BST2)+
CD172a (SIRPα)+/–CD370 (CLEC9A)CD4+Siglec-H+
CD4+/–XCR1+CD11b+CD45R (B220)+
CD8+/–CD4CD172a (SIRPα)+CD11b
XCR1+/–CD11bLy-6Chigh
F4/80CD172a (SIRPα)
CD205 (DEC205) +
Related PDFs:
Related documents:
Related products:

2.6 Cell Culture of dendritic cells from lymphoid tissue

DCs can be cultured in vitro and stimulated by antigen or toll-like receptor (TLR) ligand presentation.

2.6.1 Activation of dendritic cells from lymphoid tissue

At a glance: Kits and reagents for the stimulation of DCs from lymphoid tissue

Use Comments Product
StimulationAntigen-specific activation of DCs; available in research, premium, and GMP GradesPepTivator Peptide Pools
StimulationTLR ligand presentationTLR7/8 Agonists

Effective stimulation of antigen-specific T cells is achieved reliably with PepTivator® Peptide Pools.  This extensive panel of virus- or tumor-specific antigens consists of 15-mer peptides with 11-amino-acid overlaps, covering the complete sequence of the respective antigen.

Antigen targeting of antigen-presenting cells (APCs) via specific receptors has been used to induce effective antigen-specific cell responses and characterize the function of new receptors on APCs and compare these with well-known receptors. The Ova Antigen Delivery Module Set was developed for in vitro targeting of ovalbumin to APCs such as DCs. The set includes all reagents needed for the isolation of DCs, for antigen delivery, and for subsequent analysis of antigen presentation.

Toll-like receptors (TLRs) recognize highly conserved structural motifs of a wide variety of ligands expressed exclusively by microbial pathogens, called pathogen-associated molecular patterns (PAMPs). This function is part of an early innate immune response. TLR7 and 8 are involved in responses to viral infection and recognize single-stranded RNAs as their natural ligand as well as small synthetic molecules. 

The TLR7/8 Agonists ORN R-0006, ORN R-2336, ORN RNA 40, and ORN R-2176-dT activate murine TLR7 and induce secretion of a broad spectrum of cytokines, including IFN-α, IFN-γ, IL-12, and TNF-α.

MACS Handbook:

Cell culture

2.7 Generation and cell culture of DCs derived from bone marrow

At a glance: Kits and reagents for the generation of BM-DCs

Use Comments Product
DC generationCan be combined with IL-4Mouse GM-CSF
DC generationMouse Flt3-Ligand

Murine bone marrow-derived dendritic cells (BM-DCs) are generated from bone marrow progenitor cells by culturing a total cell suspension from mouse bone marrow with specific cytokines. For example, BM-DCs that resemble conventional DCs can be generated using granulocyte/macrophage colony-stimulating factor (GM-CSF) alone, or GM-CSF in conjunction with interleukin-4 (IL-4). Alternatively, pDCs can be generated using Flt3-Ligand. These culture methods generate large numbers of DCs, but also result in heterogeneous DC populations.

Related documents:

3 DCs from non-lymphoid tissues

Each DC subset resident in spleen and lymph nodes (pDCs, cDC1, and cDC2) has a corresponding subset in non-lymphoid tissues that shares the same ontogeny and presents a similar but not identical phenotype (PMID: 23516985). This peripheral phenotype, in addition to certain activation markers, often allows the distinction of counterpart cells migrating into non-inflamed lymphoid tissues.

MACS Handbook:

Sample preparation

3.1 Cell subsets, frequencies, marker expression

At a glance: DC cell subsets from non-lymphoid tissues

Cell subsetFrequency 
(percentages refer to total CD45+ cells)
MarkersFunction
Plasmacytoid DCs (pDCs)Rare at steady state, variable during inflammationCD45, CD11c, MHC class II, mPDCA-1 (CD317 or BST2),Siglec-H, CD45R (B220), Ly-6CUpon pathogen encounter pDCs produce large amounts of type I IFN and acquire antigen-presenting capacity
Conventional DC 1 (cDC1)1% in skin, 0.5% in lungs, 1% in intestinesCD45, CD11c, MHC class II, CD103, XCR1, CD24, CLEC9APerform cross-presentation of antigens to MHC class I and start type I cytotoxic immune responses 
Conventional DC 2 (cDC2)25% in skin, 1.5% in lungs, 0.45% in intestinesCD45, CD11c, MHC class II, SIRPα, CD11bActivate ILC2 and TH2 cells and induce ILC3 and TH17 immune responses
Inflammatory DCsVariable, depending on inflammationCD11c, MHC class II
Mo-DCs: CD64, F4/80, MER (MERTK), CD11b
Induce activation of naive T cells 
Langerhans cells30–50% in skin Located in skin to take up microbial antigens and become APCs

3.2 Sample Preparation of non-lymphoid tissues

The complexity of DC phenotyping makes it essential that all epitopes that are needed to identify subsets are preserved during tissue dissociation. Miltenyi Biotec offers optimized kits for the preparation of single-cell suspensions from several tissues, including lamina propria, lung, and skin. These kits together with the  gentleMACS™ Octo Dissociator with Heaters provide optimal conditions for a combined enzymatic and mechanical tissue dissociation. 
 

MACS Handbook:

Sample preparation

Related products:

3.3 Magnetic separation of DCs from non-lymphoid tissues

At a glance: Kits and reagents for the separation of DCs from non-lymphoid tissues

Cell subsetIsolation strategyCommentsAutomationProduct
Pan DCs Positive selection of target cellsCan be used for various tissues, including lung and lamina propriaYesCD11c MicroBeads UltraPure, mouse
Langerhans cells Positive selection of target cellsYesEpidermal Langerhans Cell MicroBead 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 CD11c MicroBeads UltraPure, mouse are optimized for the rapid and easy isolation of mouse DCs from single-cell suspensions of lymphoid and non-lymphoid tissues, such as lung and lamina propria. These MicroBeads greatly improve recovery and purity of the sorted population by specifically enriching viable cells.

Langerhans cells are best enriched by the Epidermal Langerhans Cell MicroBead Kit, mouse.

Epidermal Langerhans Cell MicroBead Kit, mouse
Before separation
After separation

Langerhans cells isolated from a mouse epidermis single-cell suspension. A single-cell suspension was prepared using the Epidermis Dissociation Kit, mouse. The Epidermal Langerhans Cell MicroBead Kit, one MS Column, and a MiniMACS Separator were used to isolate the Langerhans cells, which were fluorescently stained with CD207 and CD11c and analyzed by flow cytometry on the MACSQuant Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

Related PDFs:

3.4 Characterization of DCs from non-lymphoid tissues by flow cytometry

3.4.1 Flow cytometry panels

For a comprehensive analysis of DCs in non-lymphoid tissues, the list of markers described for the characterization of DCs from lymphoid tissue (see 2.5. Characterization of DCs from lymphoid tissue by flow cytometry) can be expanded by including CD64 and F4/80. These markers should not be expressed in the lymphoid counterpart subsets.


 

At a glance: Markers for the identification of DCs from lymphoid and non-lymphoid tissues

MarkerpDCsLymphoid tissue cDCsNon-lymphoid tissue cDCsLangerhans cells
  CD8cDCCD11bcDCCD103+ CD11b+ cDCCD103+ CD11b+ intestinal cDCCD103CD11b+ cDC 
CD45+++++++
CD11c+++++++++++++++
MHC class II+++++++++++++
CD8subset+
CD4++/–ND
CD11b++++
CD103subset++++
Langerinsubset+++
EpCAM++
B220+-
CD24ND++++++++/–++
Btla++++++++/–ND
c-kit+++++/–ND
CD26++++++/–ND
Xer1++
CD36++ND
Cystatin C++++NDNDNDND
Clec9a (DNGR1)+++++
Cadm1 (Necl2)+NDNDNDND
CD205+++++NDND++
CX3CR1subset+++
CD209 (dc-sign)+++++/–
F4/80+++
CD172a (Sirpa)++++++
CD64 (Fc r1)++ND
Ly-6C++ +/–
View details

Flow cytometry analysis of DCs from spleen, lung, and lamina propria. Mouse spleens, lungs, and small intestines were dissociated with the gentleMACS Octo Dissociator with Heaters and the respective MACS Tissue Dissociation Kit. (A) Dissociated cells were stained with the specified antibodies and analyzed by flow cytometry using FlowLogic™ Software. Cells were gated on the PI population. (B) Enrichment of pan DCs and CD11c+ macrophage populations was done with CD11c MicroBeads Ultrapure, mouse. Enriched cells were stained with the specified antibodies and analyzed by flow cytometry. Cells were gated on the PI population.

Related PDFs:
Related documents:
Related products: