Dendritic cells

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 (T_H) 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 T_H2 cells against parasites and during asthma, and induction of ILC3 and T_H17 immune responses to extracellular bacteria (PMID:    27760337).

Miltenyi Biotec has created dedicated application protocols for working with mouse DCs.

• Isolation and analysis of tissue-derived CD11c⁺ mouse dendritic cells
• Multicolor flow cytometric analysis of dendritic cells (DCs) from mouse spleen

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.

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 .

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.

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

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.

cDC1 represent a very small population in non-lymphoid tissues and make up typically less than 1% of all hematopoietic cells. Similar to their lymphoid counterparts, they depend on BAFT3 and proliferate quickly in response to the Flt3-Ligand. cDC1 in non-lymphoid tissues are identified based on several markers they have in common with their lymphoid counterparts, such as CLEC9A and XCR1. However, instead of CD8a non-lymphoid cDC1 express CD103, which is absent on DCs in lymphoid organs. cDC1 in non-lymphoid tissues do not express CD11b, SIRPα, F4/80, or CD115, which helps to distinguish them from macrophages, cDC2, and monocytes (PMID: 19176316). 

Although CD103 is an important marker for cDC1, its presence is not crucial for cDC1 development and survival, and its expression can be down-regulated in certain tissues and conditions. Moreover, CD103 is not an exclusive marker, but can also be expressed by T cells. Two subsets of CD103⁺ DCs have been identified in lamina propria: CD103⁺CD11b^– and double-positive CD103⁺CD11b⁺. The latter subset is ontogenically close to cDC2, but expresses CD103.

Like lymphoid cDC1, non-lymphoid cDC1 have a superior ability to cross-present antigens and activate CD8⁺ cytotoxic T cell responses compared to cDC2 (PMID: 19176316).

The study of cDC2 in non-lymphoid tissues is challenging. cDC2 lack a specific marker, and the most commonly used defining marker, CD11b, is also expressed by other myeloid cells, including macrophages and monocyte-derived DCs infiltrating tissues. cDC2 are not very efficient in cross-presentation, but show prominent capacity to activate CD4⁺ T helper cells (PMID: 23516985).

Two other important subsets of ontogenically distinguishable DCs are present in non-lymphoid tissue: inflammatory/precursor-derived-DCs and Langerhans cells (PMID: 23516985).

Initially, DCs were believed to arise strictly from monocytes. Recent research however demonstrated that DCs generated from blood monocytes or other hematopoietic precursors under inflammatory conditions have a different developmental origin than pDCs and cDCs. Circulating Ly-6C^high monocytes are considered the direct precursors of monocyte-derived DCs (Mo-DCs). However, DCs can also arise from other precursors, including bone marrow cells. The exact ontogeny of blood precursor–derived DCs is not completely clear, but it is generally accepted that these cells are generated under inflammatory conditions via activating stimuli such as TLR ligands or cytokines. Inflammatory DCs express the typical DC markers CD11c and MHC class II, and efficiently present antigens to induce activation of naive T cells. Mo-DCs, however, share certain markers with macrophages and cDC2, such as CD64, F4/80, MER (MERTK), and CD11b, making it difficult to distinguish this subset from other tissue-resident and tissue-infiltrating myeloid cells.

Langerhans cells are located in the epidermis and can be distinguished from cDC and pDC populations. Langerhans cells derive from precursors of fetal origin and thus do not depend on bone marrow for regeneration, but self-renew throughout life.

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. 
 

• Lamina Propria Dissociation Kit, mouse
• Epidermis Dissociation Kit, mouse
• Lung Dissociation Kit, mouse
   

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.