Dendritic Cells

Not only are DCs potent initiators of immune responses, they also play important regulatory roles in determining the type, magnitude and duration of immune responses that ensue.^1,4,10,13 DCs accomplish this by their differential expression of cell surface ligands and receptors as well as by secreting distinct profiles of cytokines, chemokines and inflammatory mediators. For example, DCs that release IL-12p70 may preferentially promote type-1 CD4 ⁺ helper T cells (Th1) or cytolytic CD8 ⁺ T cells. Other DC types may promote T cell–dependent humoral or cell-mediated immune responses characteristic of Th2, Th9, Th17, Th22, T follicular helper (Tfh) or regulatory T (Treg) cells. The issue of exactly which DCs orchestrate these types of T cell–dependent immune responses, and how they do it, remains open and intensively investigated.

Some studies point to the DC’s maturity level as crucial, whereas others point to the major influence of the pathogen type or the tissue site involved. These are all critical parameters that require careful study. The truth may lie somewhere in between since there is such a large degree of functional plasticity within the DC pathway.^14,15,16 The essential link that DCs provide between innate and adaptive immunity is also becoming more appreciated. Not only do DCs mature in response to danger signals, thus becoming capable of inducing a productive T cell response, they also trigger natural responses to invading infectious agents by activating macrophages, natural killer (NK) cells, natural killer T cells (NKT cells), granulocytes and mast cells.¹⁴ The discovery that plasmacytoid DCs (pDCs) are a major source of IFNs, quickly secreting them in response to certain viruses,⁹ serves as an important example of the multifunctional role played by DCs in both innate and adaptive immune responses.

Dendritic cell heterogeneity

Multiple types of precursor, immature and mature DCs (for example, Langerhans cells, dermal or interstitial DCs, blood DCs) that differ in origin, morphology, localization, maturation state, phenotype and function^10,14 have been described. Despite some cell surface phenotypic differences between the two species, two generally accepted types of DCs have been described in human and mouse model systems that appear to represent different lineages: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs), also known as classical or conventional DCs (cDCs).¹ pDCs have a tremendous capacity to produce IFNs but may not present antigens as efficiently as mDCs.^1,3 Human pDCs are distinguished by their coexpression of CD123 and CD304 whereas mouse pDCs express CD45R/B220 and Ly-6C.^1,9,10 Two major classes of mDCs have been further classified in the human and mouse species, which are defined by the alternative expression of either IFN regulatory factor 4 (IRF4⁺ DCs) or IRF-8 (IRF-8⁺ DCs).¹³ IRF4⁺ DCs in humans characteristically express CD1c, whereas mouse counterparts express either CD4 (lymphoid resident DCs) or CD11b (migratory DCs). The IRF4⁺ DCs from both species coexpress CD172a/Sirp-α and can efficiently present antigens to naïve CD4⁺ T cells. Conversely, human IRF8⁺ DCs typically express CD141, while mouse equivalents express CD8a (lymphoid resident DCs) or CD103 (migratory DCs) with all subsets expressing the XCR1 chemokine receptor, CD370/Clec9a, and capable of presenting antigen to CD4⁺ T cells and CD8⁺ T cells. Human and mouse Langerhans cells (LCs) likewise coexpress several distinguishing markers in common including CD207/Langerin, CD326/EpCAM and CD324/E-Cadherin.^3,10 DC subsets residing in the dermis and intestines of both species have also been described.³  For a summary of human and mouse DC counterparts, see the table below.

Mouse dendritic cell (DC) subsets can be characterized by a number of surface and intracellular markers through flow cytometry.

The following examples on mouse spleen illustrate DC subset identification based on available BD OptiBuild™ Custom Reagents.

Immunophenotyping of mouse dendritic cell subsets. Ten-color immunophenotypic characterization of the three main subsets of mouse DCs (myeloid, lymphoid and plasmacytoid) in the mouse spleen. BALB/c mouse spleen was enzymatically digested, stained with a cocktail of antibodies (including BD OptiBuild™ Reagents Sirpα BV650 and Clec12A BV786) and analyzed on a BD FACSCelesta™ Flow Cytometer Blue/Violet/ Red (BVR) laser configuration.

Certain research approaches require the use of isolated populations of dendritic cells (DCs) for further characterization, culture or functional studies. Isolation of these very rare cell types, which make up only 0.1–1% of blood mononuclear cells, has been a limitation historically.¹ Advances in flow cytometry and magnetic-bead separation technologies have made DC isolation easier. Flow cytometry–based cell sorting enables the isolation of single cells for further analysis.

Dendritic cell populations

Isolated DC populations are often used to carry out functional assays, such as co-culture with T cells, or for other downstream applications. BD Biosciences offers approaches for the successful isolation of both human and mouse DCs.

The BD IMag™ Cell Separation System is based on a simple yet highly effective direct magnet technology. Positive or negative selection of DCs, for example, is feasible with researchers selecting from a range of BD IMag™ Particle-formatted cell surface binding antibodies. Easy-to-use human and mouse DC enrichments sets, employing a negative selection of non-DC populations in a few short steps, are also available.

Flow cytometric sorting is a very effective way to isolate these extremely rare cells, since it delivers high purity and recovery of cells and is well suited for the isolation of cells defined by a multimarker phenotype. Many researchers have relied on BD FACSAria™ Cell Sorters to isolate specific DC populations.²