Dendritic Cell Maturation and Differentiation

See also Drugs/agents which inhibit DC maturation

Monocytes              IL-4 GM-CSF            Immature DCs                    TNF-alpha        Mature DCs

                                    (5 days)                                                                (2 days)

Banchereau et al., (2000)

DC are derived form bone-marrow progenitors (CD34+) and are present at two stages in the body. DC differentiation involves the change from multipotential hematopoietic progenitor cells (HPCs) to immature DCs (iDCs). Immature DC are present in most tissues and have the role of sentinel. Immature DC have a high capacity to capture antigens and transport them from peripheral tissues to the secondary lymphoid organs. Thus iDCs have a big capacity for Ag-uptake but a relatively poor ability to activate T cells. This differentiation is induced in vitro by exogenous cytokines such as GM-CSF and TNF-α for CD34+ HPCs, GM-CSF and IL-4 for monocytes, CD40 receptor cross-linking for CD34+ HPCs, or calcium ionophore alone for monocytes.

The next stage involves maturation of iDCs to mature DCs (mDCs), which exhibit enhanced Ag-presentation, up-regulated surface major histocompatibility comnplex (MHC) molecules, as well as co-stimulatory and adhesion molecules. Mature DC are present in secondary lymphoid organs and present antigens to T cells. Antigen presentation by DC to T cells occurs in the context of cell surface MHC class II molecules and co-stimulation signals, and to become fully potent APC, DC must thus undergo maturation. I

After internalization, most exogenous antigens are processed through an endosomal and lysosomal pathway in which proteins are cleaved into peptides and loaded onto MHC class II molecules. Alternatively, exogenous antigens can be released into the cytosol, gaining access the the proteasome, the main non-lysosomal protease, that generates peptides and transfers them to the endoplasmic reticulum, where they are loaded onto MHC class I molecules. This exogenous MHC class I presentation is also refered to as "cross-presentation".

What Drives DCs to Mature

In vivo, maturation (second stage) is probably triggered by "danger signals" which may include  a variety of stimuli including live bacteria and components (lipopolysaccharide (LPS), DNA), viral infection and inflammatory cytokines. I

DC differentiation is plastic. Different cytokine microenvironments, which can relate to tissue localization or to prior interaction with microorganisms, induce DCs to differentiate towards different stats that can polarize T cells towards different functions. Studies have shown that immature DCs mature in the following ways:

• Microbial signaling through toll-like receptors (TLRs) is an effective way to mature DCs to their immunogenic state.

Immature DCs can be considered as immunological sensors that analyze the nature of the microorganisms and other danger signals through cross-linking of their pattern recognition receptors (PRR). The majority of the known PRR recognize bacterial pathogen associated molecular patter (PAMP), such as Toll-like receptor (TLR) 2 for peptidoglycan, TLR4 for lipopolysaccharide, TLR5 for flagellin or TLR9 for CpG.

• Different cytokine microenvironments, which can be related to the tissue localization or to prior interaction with microogranisms induce DCs to differentiate towards different states that can polarize T cell towards different functions. Different microbial stimuli induce different responses from the same DC population.

Several soluble immunosuppressive factors on the maturation of DC form monocytes or CD34+ myeloid stem cell progenitors have been shown. Among these factors are a number of cytokines that are often produced by malignant cells such as TGF-β1, IL-6, vascular endothelial growth factor (VEGF) and IL-10.

• NF-kB: TLR ligation results in NF-kB activation and this pathway is important in DC maturation.

Translocation of the NFkB family members Rel B and p50 from cytoplasm to nucleus is required for myeloid DC maturation. Antigen-exposed myeloid DCs, in which RelB function is inhibited, lack cell surface CD40 expressionk prevent priming of immunity, and suppress a previously primed imune response. DCs in which RelB nuclear translocation is inhibted through prevention of IkB phosphorylation, DCs generated from RelB deficient mice, and DCs generated from CD40 deficient mice similarly confer suppression.

TLRs trigger DC maturation using a NFkB dependent pathway. Indirect activation of DCs by proinflammatory cytokines (Il-1, Il-18, TNF-alpha) and chemokines is also NFkB dependent.

• T cell-derived signals. Activated CD4+ T-helper cells upregulate CD40 ligand. Signaling through the CD40 receptor activates DCs.

• Antigens with which DCs interact:

• Recent studies in mice indicate that DCs also mature through interaction with natural killer T cells. This process is dependent on NK T-cell activation with the synthetic glycolipid antigen alpha-galacto-sylceramide, presented by CD1d molecules on the DCs.

Molecular Mechanisms of Maturation

The molecular mechanisms of DC maturation are not well understood but involve several routes such as NF-kB pathway and the p38 mitogen-activated protein kinase (MAPK) pathway. In vitro, bacteria-induced DC maturation involves (a) ERK kinase, allowing for DC survival, and (b) NF-kB, allowing for DC maturation characterized by increased expression of costimulatory and MHC-class II molecules, release of chemokines, and migration. This coordinated process leads to high T cell stimulatory capacity as well as IL-12 release, all of which result in the induction of protective immune responses.

Numerous publications have shown that all TLR agonists tested to date can lead to increased expression of CD40, CD80 and CD86 in at least one DC subset. NFқβ pathway  is a major transcription factor controlling the expression of these markers and thus it has been reasonable to assume that NF-kB activation following TLR signalling is sufficient to promote DC maturation. This has been put into question by the work of Hoshino et al., however, who showed a marked decrease in CD40 upregulation in STAT-1 -/- BM-DC treated with CpG or LPS compared to wild type DC. This result suggests that DC maturation in response to TLR ligation is, to a large extent, dependent on secondary production of cytokines such as type I interferons (IFN-I), which signal in an autocrine or paracine manner. Consistent with these results, IFN-I is a potent stimulus for DC maturation and IFN-I receptor (IFN-IR) is required for the adjuvanticity of Complete Freund's adjuvant which contains several TLR ligands in the form of killed Mycobacteria. TLR agonists and/or IFN-I may further promote DC maturation via induction of TNF, IL-15 or other inflammatory cytokines, which also promote upregulation of B7 and CD40 expression on DC.

The loss of receptors specific for inflammatory chemokines is mediated by distinct mechanisms. Following stimulation with LPS, the loss is very rapid (up to 80% and 60% of surface CCR1 and CCR5 expression in 3 h). Thus this rapid down regualtion cannot result from changes in transcription or mRNA stability. One proposal is that the rapid loss is mediated by a novel mechanisms that involves the production of chemokines by maturing DC, leading to homologous desensitization of the cognate receptors. Evidence for this mechanisms includes (1) maturing DC produce a number of chemokines that act on their own receptors, (2) receptor down regulation is prevented by brefeldin A and cycloeximide and (4) receptor levels can be reconstituted in mature DC by reculturing them in fresh medium without stimuli.

Importance of relB: On a molecular level DC maturation is guided by relB, a subunit of the NFkB transcription factor. RebB has been shown to play a mjor role in DC function by regulating CD40 and MHC rexpression. Upon stimulie exerted by TNF-alpha, LPS or virus-derived IL-1, relB translocates to the nucleus and promotes transcription of CD40, CD80/86 and MHC genes, all of which are indicators of DC activation. Blockage of this translocation can lock DCs in an immature state, as indicated by results using REelB-deficient mice. Most of the pharmaceuticals that inhibit DC maturation also interact with the relB pathway. For example, there is evidence that mycophenolate, mofetil, glucocorticoids and vitamin D3 all downregulate NFkB expression. RelB has even been suggested as a useful marker to qualify DC as Treg-inducing DCs. Evidence derives from observations showing that nuclear relB is absent in steady-state DCs located in peripheral tissues, whereas relB becomes upregulated in the nucleus in DCs residing in inlamed or lymphoid tissues.

P38 MAPK: LPS reportedly activates the p38 MAPK pathway in DC and this coincides with DC maturation. However, activation of p38, although necessary, is not sufficient to stimulate DC maturation, indicating that alternative pathways are required. See also P38 as an inhibitor of DC maturation The activation of the NF-kB pathway is a likely candidate. Activation of p38 may result in the modulation of chromatin structure and enhanced accessibility of NF-kB to the relevant elements in the promoters of cytokine genes and other genes presumably involved in DC maturation.

JNK: was reportedly not to be involved in controlling DC maturation.

Changes which occur as DCs mature

Maturation of DC results in a complete reprogramming of the cell, with downregulation of endocytic activity, upregulation of MHC, adhesion and costimulatory molecules, as well as a striking switch in chemokine receptor usage. Some changes that take place on DC maturation include the following:

• increased formation of stable MHC-peptide complexes. There is upregulation of cell surface MHC molecules, which in the case of both MHC I and II is due to increased biosynthesis, and in the case of MHC II is due to a prolongation of the half-life of MHC peptide complexes.
• higher expression of membrane molecules: DCs express on their plasma membrane high levels of coreceptors such as CD80 (B7-1), CD86(B7-2), CD40, CD54, OX40L, 4-1BBL, etc. that bind costimulatory receptors on T-cells.  CD95 is also up-regulated on mature DC.  For example, LPS has been shown to up-regulate expression of cell-surface MHC class II (Iab), CD40, CE54, CDE80 and CD86 (Chen, 2002). DC-LAMP is also known to be up-regulated upon differentiation/maturation of DCs.
• synthesis of cytokines that influence T cell proliferation and differentiation such as IL-12.
• altered production of chemokines    Maturing DCs entering the draiming lymph nodes will be driven into the paracortical area in response to the production of MIP-3β and/or 6Ckine by cells spread over the T cell zone. The newly arriving DCs might themselves become a source of these chemokines, allowing an amplifciation or persistence of the chemotactic signal. Because these two chemokines can attract mature DCs and naive T lymphocytes, they are likely to play a key role in helping Ag bearing DCs to encounter specific T cells. Upon encounters with T cells, which can take place not only in the secondary lymphoid organ but also the site of tissue injury, DCs receive additional maturation signals from CD40 ligand, RANK/TRANCE, 4-1BB and OX40 ligand molecules which induce the release of chemokines such as IL-8, factalkine, and macrophage derived chemokines that attract lymphocytes.
• Alterned production of chemokine receptors that intensify movement of DCs into lymphatic vessels and lymphoid organs. Depending on their tissue of origin, immature DC express a variety of chemokine receptors (CR), most of which bind to inflammatory chemokines. For example, infiltration of graft tissues by immature DC allows these DC to take up and process graft Ag for presentation to naive T cells in draining secondary lymph nodes (DLN). The internalization and processing of Ag initiates DC maturation, which involves decrease of CR for the inflammatory chemokines and increase of the CR for the constitutive chemokines.

Immature DCs express CC-chemokine receptor 1 (CCR1), CCR2, CCR5 and CXC-chemokine receptor 1 (CXCR1) and are attracted to non-lymphoid tissues by their respective ligands, which are expressed constitutively or at inflammatory sites. DC maturation results in the downregulation of expression of these chemokine receptors and the upregulation of CCR7 expression. Mature DC also upregulate CXCR4. Expression of CCR7 switches DC responsiveness to its ligands, CC-chemokine ligand 19 (CCL19) and CCL21, that guide migration to secondary lymphoid organs.

• Migration: Upon maturation DC appear to migrate into T cell dependent areas of secondary lymphoid tissues where they act as professional APC activating naive T cells. Recruitment of DCs from the BM into peripheral non-lymphoid tissues and subsequent migration of mature DCs from the periphery into lymphoid tissues are coordinated by chemokines that interact with corresponding recetpors on DCs. CCR7 up-regulation homes mature DCs since the CCR7 ligands, secondary lymphoid tissue chemokine (SLC) and ELC-MIP-3β are produced in secondary lymphoid organs. The significance of the up-regulation of CXCR4 and CCR4 is less clear since the cognate ligands, SDF-1 and TARC are constitutively produced in lymphoid tissues. Interestingly, CXCR4 and CCR7 are expressed not only on mature DC but also on naive T cells, a fact that will favor co-localization of these cells at sites where SDF-1, SLC and ELC/MIP-3β are produced.

Chemokine prouduction and receptor desensitization might serve two distinct functions in DC physiology. First, it may allow prompt down regulation of the receptors, which may be instrumental for migration out of inflamed tissues. Second, it may enhance recruitment of immature DC or DC precursors, which may sustain antigen sampling at later time points.

• Antigen-uptake mechanisms are down-regulated (mannose receptor and Fcγ receptor-mediated uptake, macropinocytosis, and phagocytosis.
• Changes in expression of adhesion molecules: When Langerhans cells are activated and migrate out of the skin, E-cadherin is down-regulated and laminin receptors are up-regualted.