What are dendritic cells?
Dendritic cells (DCs) play a pivotal role in the innate detection of pathogens and the subsequent activation of the adaptive immune response. DCs initiate the adaptive response by presenting antigenic peptides on major histocompatibility complex (MHC) molecules to induce T cell activation and differentiation. DCs also secrete cytokines and growth factors that enhance and modulate immune responses. In addition to their role in activating naïve T cells, DCs are thought to play a critical role in guiding the differentiation of regulatory T cells as well as the development of T cell tolerance. As key sentinel cells, they reside throughout the body, particularly in lymphoid organs and at environmental interfaces such as the intestine and skin.
Dendritic cell activation—bridging innate and adaptive immunity
DCs continuously sample their environment and, in the absence of inflammatory signals, are thought to reinforce peripheral T cell tolerance [1,2]. Activation, or maturation, of DCs occurs when pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) are recognized by pattern recognition receptors (PRRs). DCs express a wide array of PRRs, including surface and endosomal Toll-like receptors (TLRs), C-type lectins, and cytosolic sensors . Upon PRR activation, DCs undergo metabolic, cellular, and gene transcription changes that allow them to mature into efficient activators of T lymphocytes. They upregulate antigen presentation machinery, including MHC class II, co-stimulatory molecules, and pro-inflammatory cytokines, and migrate to the T cell areas of secondary lymphoid tissues where they prime antigen-specific T cells.
Figure 1. Immature dendritic cells vs mature dendritic cells functions. Mature dendritic cells undergo morphological, metabolic, and functional changes that allow them to migrate to lymphoid tissues and initiate antigen-specific T cell responses.
Dendritic cell development
DC development is governed by a multistep differentiation cascade that begins in the bone marrow. Although the precise upstream precursors of DCs have been debated for many years, it is generally agreed that DCs derive from hematopoietic stem cells through the myeloid progenitor lineage in a fms-like tyrosine kinase 3 ligand (Flt3L)-dependent manner . Most DCs arise from a common DC precursor (CDP) of classical/conventional DCs (cDCs) and plasmacytoid DCs (pDCs). pDC development continues in the bone marrow, while cDCs differentiate from pre-DCs in the periphery . Additional monocyte-derived cells (MCs) with DC-like properties (moDCs) can be generated during pathogen-initiated inflammation via the differentiation of monocytes .
Figure 2. Dendritic cell development. Dendritic cells (DCs) develop from hematopoietic stem cells (HSCs) in a multistep process that begins in the bone marrow. Most DCs arise through a common DC progenitor (CDP) that differentiates into conventional DC precursors (pre-cDCs) and plasmacytoid DC precursors (pre-pDCs) in a fms-like tyrosine kinase 3 (FLT3) ligand-dependent manner. E2-2–dependent pDC differentiation continues in the bone marrow, while the two conventional DCs emerge from pre-cDCs in peripheral lymphoid tissues. The development of cDC1 is dependent on basic leucine zipper ATF-like transcription factor 3 (BATF3), and cDC2 development is regulated by several transcription factors, including interferon regulatory factor 4 (IRF4). Under certain conditions, MCs differentiate from monocytes to supplement the activity of classical DCs.
Classical dendritic cells (cDCs)
In 2014, a unified classification system for conventional, or classical, (cDC) subsets was proposed to disambiguate an expanding plethora of tissue- and species-specific subset names . Under this convention, two major subsets are described as classical type 1 DCs (cDC1) and classical type 2 DCs (cDC2). cDC2 cells engage in the general functions normally ascribed to the DC family, the priming of naive CD4+ T cells through antigen presentation on MHC class II, and co-stimulation. cDC1 cells are specialized in cross-presentation, or the presentation of exogenous antigen on MHC class I to induce naive CD8+ T cells to acquire cytotoxic T lymphocyte (CTL) effector function . Consistent with their specialization, cDC1 and cDC2 cells also express the cytokines required to elicit CD4+ or CD8+ effector T cell functions. cDC2-driven helper T cell polarization leads to robust adaptive immune responses to extracellular pathogens, including microbial and helminth infections. The ability to cross-prime CD8+ T cells allows cDC1 to direct CTLs to respond to intracellular pathogens and tumors. Notably, Langerhans cells (LCs), the prototypical DCs described by Paul Langerhans in 1868, have been reclassified as macrophages in this system based on revelations about their macrophage-like embryonic origin .
cDCs can be distinguished from other mononuclear phagocytes by their branched “dendritic” morphology and high surface expression of cDC signature genes, including CD11c and MHC class II (HLA-DR in humans). Murine cDC1 cells are identified by CD8 or CD103 depending on their resident tissue, and murine cDC2 cells are distinguished by their expression of CD11b in addition to common cDC markers (Table 1). Humans cDC1 cells can be distinguished from cDC2 by their respective expression of BDCA1 and BDCA3, along with additional markers. Since moDCs and some macrophages can be difficult to distinguish from cDCs, exclusion of classic monocyte/macrophage markers, such as Ly6C and F4/80, and differential expression of certain markers such as CD64, MAR-1, MerTK, and CD88, may help to distinguish cDCs from other cell types .
The development of cDC1 cells require BATF3 . Selective depletion studies have demonstrated the importance of cDC1 in viral immunity and defense from intracellular infection . cDC1 derivation is an aim of DC-based cancer therapies because of their potential in driving CTL-mediated anti-tumor responses . Several transcription factors, including RelB, RBP-J, IRF4, and IRF2, are critical for the development of cDC2 .
Figure 3. Classical dendritic (cDC) cell stimulation of CD4+ T cells and CD8+ T cells. Activated classical dendritic cells (cDCs) travel to the T cell areas of secondary lymphoid tissues to stimulate CD4+ T cells through classical antigen presentation on MHC class II. cDCs can also stimulate CD8+ T cells through classical and cross-presentation pathways.
Plasmacytoid dendritic cells (pDCs)
Plasmacytoid DCs are a unique subset of DCs with the dedicated function of secreting type I interferon (IFN) [13,14]. They express high basal levels of the transcription factor IRF7 and endosomal TLRs, which allows them to quickly secrete massive amounts of IFN alpha and beta in response to viral infection . pDCs can be phenotypically distinguished from cDCs by their secretory morphology, which is eponymously reminiscent of plasma cells, atypical expression of cDC signature genes such as CD11c and MHC class II, and detection of specific pDC markers (Table 1). Although an intermediate level of CD11c is detected on murine pDCs, CD11c is not found on human pDCs . Rather, pDCs are distinguishable from cDCs by CD123 and CD303 expression in humans and Bst2 and B220 in mice [16,17].
The development of pDCs is dependent on regulation by the E-protein transcription factor E2-2 . Like cDCs, pDCs and their upstream precursors have been demonstrated to depend on Flt3 ligand signaling. In addition to their unique phenotype, pDCs express several hallmarks of the lymphoid lineage, which has been attributed to their dependence on the transcription factor E2-2 . E2-2 belongs to the E-protein family of regulators known to be indispensable for lymphoid lineage commitment. More recently, single-cell transcriptional analysis has again suggested that some pDCs develop through a lymphoid precursor . Regardless of their origin, pDCs clearly share attributes of both myeloid and lymphoid cells, a defining feature of this cell type since its original description.
Selective depletion studies have demonstrated the importance of pDCs in controlling viral infections. For example, pDCs are required to control infection with mouse hepatitis virus (MHV), and murine coronavirus . In contrast, pDCs play a pathogenic role in diseases that involve rampant IFN production, including autoimmune disorders such as systemic lupus erythematosus (SLE) . A rare and aggressive leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), originates from pDCs .
Table 1. Key transcription factors, surface markers, and functions associated with dendritic cell subsets.
|Cell type||Key transcription factors||Surface markers||Function|
|cDC1||BATF3, IRF8||Human:CD11c, HLA-DR, CD141 (BCDA3)|
|Cross-presentation to CD8+ T cells, CTL priming|
|Mouse:CD11c, MHCII, CD8 (l), CD103 (n)|
|cDC2||IRF2, IRF4, RelB, RBP-J||Human:CD11c, HLA-DR, CD1c (BDCA1), CD11b|
Additional:FCER1A, CLEC10A, CD2, CD172A, ILT1
|Antigen presentation CD4+ T cells, helper T cell priming|
|Mouse:CD11c, MHCII, CD11b|
|pDC||E2-2||Human:HLA-DR, CD303 (BDCA2), CD123||Type I IFN secretion|
|Mouse:CD11cint, MHCIIlo, Bst2, B220|
|moDC||KLF4, MAFB||Human:CD11c, CD11b, CD1a, CD1c (BDCA‐1)|
Additional:CD206, CD209, CD172A
|Recruited during inflammation|
Additional:CD64, MAR-1, MerTK, CD88
|Abbreviations: (l), expressed in lymphoid tissue; (n), expressed in nonlymphoid tissue; (s), expressed in subset; cDC1, classical type 1 dendritic cell; cDC2, classical type 2 dendritic cell; CTL, cytotoxic T lymphocyte; IFN, interferon; MoDC, monocyte-derived dendritic cells; pDC, plasmacytoid dendritic cell.|
Dendritic cell heterogeneity
Single-cell analyses combined with traditional approaches have revealed considerable heterogeneity within each classically defined DC subset. For example, a noncanonical AXL+ human DC that shares properties of both cDCs and pDCs has been identified, along with a murine counterpart that shares a similar expression profile [22,23,24,25]. These DCs present antigen but do not produce type I IFN, and they are thought to represent a “transitional” DC subtype. DC heterogeneity can also be described in terms of immunomodulatory states rather than developmental lineages. For example, a conserved program termed “mature DCs enriched in immunoregulatory molecules” (mregDCs) identifies human and mouse DCs with dampened functionality in lung cancer tissues .
Dendritic cells in cancer research
DCs are critical determinants in the immune response to cancer. The relative level of circulating DCs serves as a marker of cancer progression, as diminished levels have been observed in late-stage melanoma and breast cancer patients . DCs promote cancer immunity by identifying and infiltrating tumors, secreting soluble factors that condition the tumor microenvironment, and presenting tumor-associated antigen to prime T cell responses . In particular, the ability of DCs, especially cDC1, to cross-present antigen and promote CTL activity aids in the detection of tumors that downregulate MHC class I. In fact, cDC1 expansion is linked with increased response to therapy and patient survival in several cancers . In contrast, dysfunctional or tolerogenic DCs within the tumor microenvironment can drive immunosuppressive functions that promote tumor growth .
Tools to study dendritic cells
Culture methods for expanding DCs from a variety of sources, including bone marrow, circulating monocytes, and induced pluripotent stem cells (iPSCs) have been described . A major limitation of in vitro culture systems is their bias toward generating monocyte-derived cells over cDCs and pDCs. Two main methods for deriving DCs from mouse bone marrow are widely used. 1) GM-CSF–derived cultures can be used but result in a heterogeneous mixture of cells, including granulocytes, MCs, and cDC-like cells . 2) Flt3L-derived culture can be used to generate both cDC-like and pDC-like cells and is often preferred for its ability to generate relatively pure and functional DC populations. GM-CSF–derived culture from human PBMCs remains a predominant method of generating moDCs in the absence of alternative human hematopoietic sources. Although few human or mouse cDC cell lines have found widespread use, several human cell lines with pDC-characteristics have been employed to study features of human pDCs. Caution should be used, however, when interpreting results from human and mouse cultured DCs, which only approximate aspects of in vivo biology.
Activation: A variety of molecules can be used to activate or mature DCs, including LPS, CpG-containing nucleic acids, and other pathogen- or damage-associated stimulants and mimics. Maturation can be measured by upregulation of MHC class I and II and co-stimulatory molecules and cytokine secretion.
DCs and other cell types express Fc receptors that can non-specifically bind to antibodies. Fc receptor blocking agents should be used in conjunction with antibody-based flow analysis to avoid nonspecific binding.
OMIP-044 and OMIP-061 for human and mouse DC gating strategies: OMIP (optimized multicolor immunofluorescence panel), refers to a thoroughly tested and validated set of antibodies and reagents that can be used together for the multicolor characterization of a specific cell state or response. Published in the journal Cytometry Part A (Wiley Online Library), these OMIPs are designed for flow cytometry, but OMIPs may potentially be defined for image cytometry, fluorescence microscopy, and other polychromatic fluorescence-based methods.
Table 2. Cytometry Part A OMIPs designed for dendritic cell characterization.
|OMIP ID||OMIP name and link||Immune context (keywords)|
|OMIP-044||OMIP-044: 28‐color immunophenotyping of the human dendritic cell compartment|
|Antigen-presenting cells, dendritic cells, myeloid cells|
|OMIP-061||OMIP-061: 20‐color flow cytometry panel for high‐dimensional characterization of murine antigen‐presenting cells|
|Antigen-presenting cells, dendritic cells, myeloid cells, macrophages|
The cytokines Flt3L, SCF (stem cell factor), and GM-CSF (granulocyte-macrophage colony stimulating factor) play a definitive role in the development of DC subsets (both conventional and plasmacytoid) found in blood. These cytokines are produced by various tissue stroma including fibroblasts, endothelial cells, macrophages, mast cells, NK cells, and activated T Cells. DCs can also be derived from monocytes in vitro by treating them with IL-4 and GM-CSF. On exposure to activating stimuli such as TLR ligands or microbes, resting DC undergo a variety of functional and phenotypic changes as part of a maturation process which can be further influenced in an autocrine or paracrine fashion by cytokines such as IL-1, IL-4, TNFs, type 1 interferons, and TSLP.
Table 3: Key cytokines involved in dendritic cell differentiation, activation, and secretion.
|Cytokines, chemokines, and growth factors||SCF, Flt3L, GM-CSF, CSF-1||IL-1, IL-4, type 1 interferons, TNFs, TSLP||IL-12, IL-23, IL-10 IL-1 alpha, IL-1 beta, IL-15, IL-18, IFN-alpha, IFN-beta, IFN-gamma, IL-8 (CXCL8) IL-4, IL-10, IL-6, IL-17, IL-16, MIF, IL12p40, TNF-alpha, CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10|
Mature DC function as pivotal antigen presenting cells that can present distinct cytokine secretion patterns based on stimulation that can lead to inflammatory or immunosuppressive environments through the polarization of naïve T cells down a Th1 or a Th2 path. For example, IL-12 secreted by DCs can induce Th1 differentiation, which can then promote an immunostimulatory environment through cytokines such as IL-6 and IL-1β. DCs also secrete a number of chemokines such as CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10 that attract a variety of immune cell types (immature DCs, monocytes, T cells) at different times of the immune response to inflamed tissue. The Invitrogen 65-plex Human Immune monitoring panel provides a comprehensive way to profile cytokines and chemokines secreted by mature DC.
|Human||Immune Monitoring 65-Plex Human Panel||G-CSF (CSF-3), GM-CSF, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-16, IL-17A (CTLA-8), IL-18, IL-20, IL-21, IL-22, IL-23, IL-27, IL-31, LIF, M-CSF, MIF, TNF alpha, TNF beta, TSLP, BLC (CXCL13), ENA-78 (CXCL5), Eotaxin (CCL11), Eotaxin-2 (CCL24), Eotaxin-3 (CCL26), Fractalkine (CX3CL1), Gro-alpha (CXCL1), IP-10 (CXCL10), I-TAC (CXCL11), MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MDC (CCL22), MIG (CXCL9), MIP-1 alpha (CCL3), MIP-1 beta (CCL4), IP-3 alpha (CCL20), SDF-1 alpha (CXCL12), FGF-2, HGF, MMP-1, NGF beta, SCF, VEGF-A, APRIL, BAFF, CD30, CD40L (CD154), IL-2R (CD25), TNF-RII, TRAIL (CD253), TWEAK||EPX650-10065-901|
|Mouse||Immune Monitoring 48-Plex Mouse ProcartaPlex Panel||BAFF, G-CSF (CSF-3), GM-CSF, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p70, IL-13, IL-15/IL-15R, IL-17A (CTLA-8), IL-18, IL-19, IL-22, IL-23, IL-25 (IL-17E), IL-27, IL-28, IL-31, IL-33, LIF, M-CSF, RANKL, TNF alpha, ENA-78 (CXCL5), Eotaxin (CCL11), GRO alpha (CXCL1), IP-10 (CXCL10), MCP-1 (CCL2), MCP-3 (CCL7), MIP-1 alpha (CCL3), MIP-1 beta (CCL4), MIP-2, RANTES (CCL5), Betacellulin (BTC), Leptin, VEGF-A, IL-2R, IL-7R alpha, IL-33R (ST2)||EPX480-20834-901|
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