DENDRITIC CELL MATURATION

Contents

• Introduction
• Antigen Uptake
• Antigen Presentation
• Migration to Lymphoid Organs
• Interaction with T cells
• Summary
• References

Introduction

The most efficient antigen-presenting cells (APCs) are mature, immunologically competent debdritic cells (DCs).^1-7 DCs are capable of evolving from immature, antigen-capturing cells to mature, antigen-presenting, T cell-priming cells; converting antigens into immunogens and expressing molecules such as cytokines, chemokines, costimulatory molecules and proteases to initiate an immune response. The types of T cell-mediated immune responses (tolerance vs. immunity, Th1 vs. Th2) induced can vary, however, depending on the specific DC lineage (myeloid DC1s or lymphoid DC2s) and maturation stage in addition to the activation signals received from the surrounding microenvironment.^5,6,8-12

The ability of DCs to regulate immunity is dependent on DC maturation. A variety of factors can induce maturation following antigen uptake and processing within DCs, including: whole bacteria or bacterial-derived antigens (e.g. lipopolysaccharide, LPS), inflammatory cytokines, ligation of select cell surface receptors (e.g. CD40) and viral products (e.g. double-stranded RNA). During their conversion from immature to mature cells, DCs undergo a number of phenotypical and functional changes. The process of DC maturation, in general, involves a redistribution of major histocompatibility complex (MHC) molecules from intracellular endocytic compartments to the DC surface, down-regulation of antigen internalization, an increase in the surface expression of costimulatory molecules, morphological changes (e.g. formation of dendrites), cytoskeleton re-organization, secretion of chemokines, cytokines and proteases, and surface expression of adhesion molecules and chemokine receptors.

Note: The following mini-review will provide a very brief overview of a variety of the DC-derived molecules involved in the process of DC maturation (e.g. cell surface receptors, costimulatory molecules, intracellular proteins, cytokines, chemokines and their corresponding receptors, and proteases). Additional details regarding the process of DC maturation and induction of T cell-mediated immune responses can be found in reviews such as references 1-7 and 9-13.

Fig. 1. (Panels A and B) Exogenous and endogenous antigens can be processed by DCs and presented in the context of either major histocompatibility complex (MHC) class I or II molecules. Heat shock protein (Hsp)/antigen complexes (e.g. viral) can bind CD91 on immature DCs and be delivered by TAP-1 and TAP-2 to the endoplasmic reticulum for antigen presentation via the MHC I pathway (panel A). A variety of cell surface receptors are expressed by DCs that can participate in receptor-mediated endocytosis and antigen presentation via the MHC II pathway (panel B). As DCs mature, they acquire the properties necessary to form and transport peptide-loaded MHC class II complexes to the cell surface. Antigen transport to the cell surface coincides with increased expression of costimulatory molecules, such as B7-1/CD80 and B7-2/CD86. These molecules amplify T cell receptor (TCR) signaling and promote T cell activation.

Antigen Uptake

DCs are derived from hematopoietic stem cells in the bone marrow and are widely distributed as immature cells within all tissues, particularly those that interface with the environment (e.g. skin, mucosal surfaces) and in lymphoid organs. Immature DCs are recruited to sites of inflammation in peripheral tissues following pathogen invasion. Internalization of foreign antigens can subsequently trigger their maturation and migration from peripheral tissues to lymphoid organs. Chemokine responsiveness and chemokine receptor expression are essential components of the DC recruitment process to sites of inflammation and migration to lymphoid organs. Immature DCs may express the chemokine receptors CCR1, CCR2, CCR5, CCR6 and CXCR1.^14,15 They can thus be chemoattracted to areas of inflammation primarily by MIP-3a/CCL20, but also in response to RANTES/CCL5 and MIP-1a/CCL3.¹⁶ Following antigen acquisition and processing, DCs migrate to T cell-rich areas within lymphoid organs via blood or lymph, simultaneously undergoing maturation and modulation of chemokine and chemokine receptor expression profiles. A change in expression levels of the chemokine receptors CCR6 and CCR7 contributes to the functional shifts observed during DC maturation.

Immature DCs capture antigens by phagocytosis, macropinocytosis or via interaction with a variety of cell surface receptors and endocytosis (see Table 1). The most prevalent antigen receptors expressed by DCs include members of the C-type lectin family. For example, DEC-205, a type I C-type lectin containing multiple calcium-dependent binding domains and a unique cytoplasmic tail, may function in directing captured antigens to specialized antigen-processing compartments within DCs.¹⁷ Three additional type II C-type lectins important for receptor-mediated antigen uptake include DC immunoreceptor (DCIR),¹⁸ DC-associated C-type lectin-2 (dectin-2)¹⁹ and C-type lectin receptor 1 (CLEC-1).²⁰ DCIR contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and can bind glycosylated ligands. Natural ligands for dectin-2 and CLEC-1 have not been identified as of yet. The macrophage mannose receptor (MR) and Fc receptors for immunoglobulins, Fc?R and FceR, are also involved in antigen handling by immature DCs.^21-25 DCIR, MR, Fc?R and FceR are all down-regulated upon DC maturation, further emphasizing their specific roles in antigen uptake in immature DCs.

A variety of additional cell surface receptors are expressed by DCs that may also play roles in antigen uptake and/or regulating DC activation. For example, Down-Regulated by Activation (DORA), a member of the immunoglobulin superfamily (IgSF), has been implicated in antigen uptake as well as in homing and recirculation of DCs.²⁶ DORA is down-regulated following CD40L engagement. Immunoglobulin-like transcript 3 (ILT3, another IgSF member) contains an ITIM in its cytoplasmic domain, which may potentially negatively regulate DC activation and maturation.²⁷ ILT3 is involved in antigen uptake and processing by DCs; it can be efficiently internalized upon cross-linking and can deliver its ligand to an intracellular compartment for processing and presentation to T cells. Viral, bacterial or tumor-specific antigens bound to heat shock proteins (hsps) can be captured via hsp receptors expressed at the cell surface of immature DCs.²⁸ Microbial lipopeptides can be taken up by Toll-like receptors (TLRs) expressed on immature DCs (e.g. TLR3) and as a result, stimulate DC maturation.^29-32 By contrast, ligation of CD47, a thrombospondin receptor, inhibits both cytokine production and maturation of DCs.³³

Intracellular proteins are also involved in antigen uptake and processing by DCs. The p55/fascin protein is an actin-bundling protein that may be involved in the organization of a specialized cytoskeleton for promoting antigen capture in immature DCs and the migration of DCs to lymphoid organs.³⁴ Members of the Rho family of GTPases are involved in regulating macropinocytosis and can be down-regulated during maturation and antigen presentation in DCs.^35-37 The ubiquitin protein family member, di-ubiquitin, contains two ubiquitin moieties and potentially functions in directing internalized antigens towards DC proteasomes.³⁸ Following antigen processing, antigenic peptides may then be presented via MHC molecules on the DC surface to CD4⁺, CD8⁺ or memory T cells.

Antigen Presentation

DCs are capable of processing both exogenous and endogenous antigens and present peptide in the context of either MHC class I or II molecules (see Figure 1, panel A and B). As DCs mature, they acquire the properties necessary to form and transport peptide-loaded MHC class II complexes to the cell surface.⁴ Antigen transport to the cell surface coincides with increased expression of costimulatory molecules, such as B7-1/CD80 and B7-2/CD86. These molecules amplify T cell receptor (TCR) signaling and promote T cell activation. DC-LAMP, a lysosomal-associated glycoprotein, is specifically expressed in the lysosomal MHC II compartment and is up-regulated following CD40 ligation on DCs. It may function in promoting the processing of exogenous antigen or in facilitating transport of antigen-loaded MHC II complexes to the cell surface.³⁹ Proteases, such as cathepsin S, have also been shown to be involved in regulating the intracellular trafficking of MHC II molecules within DCs.⁴⁰ Immature DCs may also express empty MHC II and HLA-DM at their cell surface which later disappear upon maturation.^41-43 Secreted DC proteases can process antigenic peptides and together with cell surface HLA-DM and empty MHC II molecules, antigen processing and loading events may thus occur extracellularly in addition to intracellularly within immature DCs.

DCs present antigenic peptides complexed with MHC class I molecules to CD8-expressing T cells in order to generate cytotoxic cells.44 In the event that DCs are themselves infected with a virus, proteasomes can simply degrade the viral proteins into peptides and transport them from the cytosol to the endoplasmic reticulum. The transporter associated with protein processing (TAP-1 and -2) is a dedicated peptide transporter that facilitates the transfer of cytosolic peptides to the endoplasmic reticulum where they can then bind to MHC class I molecules (see Figure 1, panel A). DCs thus "cross-prime" T cells.⁴⁵ A variety of cell surface receptors expressed by immature DCs may function in antigen uptake and also present antigen via the MHC I pathway. DCs use av-containing integrins and CD36, for example, to phagocytose apoptotic cells.^46,47 Hsp/antigen (e.g. bacterial, viral or tumor-derived) complexes can bind CD91 on DCs and be delivered via TAP-1 and -2 to the endoplasmic reticulum for MHC I-antigen presentation.^48-50 Fc?R-mediated internalization of immune complexes also results in presentation of exogenous antigen with MHC I.²⁴

Migration to Lymphoid Organs

Following antigen exposure and activation, DCs migrate into T cell areas of lymphoid organs, a process regulated by chemokine/chemokine receptor interaction and aided by a variety of proteases and corresponding receptors [e.g. urokinase plasminogen activator (uPA)/uPAR system⁵¹]. Mature DCs lose responsiveness to MIP-3a/CCL20, RANTES/CCL5 and MIP-1a/CCL3 and become particularly sensitive to MIP-3ß/CCL19.^16,52 They also lose cell surface expression of CCR1, CCR5 and CCR6, down-regulate CXCR1 and up-regulate expression of CXCR4 and CCR7.^14-16,53-55 The up-regulation of CCR7 promotes responsiveness to MIP-3ß/CCL19 and 6ckine/CCL21.^56,57 6Ckine/CCL21, a potent chemokine for mature DCs and naïve T cells, colocalizes these two cell types leading to cognate T cell activation in secondary lymphoid organs.⁵⁸ Maturation of DCs also induces the production of MDC/CCL22, TARC/CCL17 (i.e. chemokines that attract CCR4-expressing T cells), and PARC/CCL18.^14,59 DC production of the chemokine CXCL16, in T cell-rich areas of lymphoid organs, may also function in promoting interaction between DCs and cytotoxic T cells.⁶⁰

Interaction with T cells

Cell surface receptors not only facilitate antigen uptake, but also mediate physical contact between DCs and T cells (see Table 1). DC-specific intercellular adhesion molecule (ICAM)-3 grabbing non-integrin (DC-SIGN), a calcium-dependent, type II C-type lectin, is a DC-specific ligand for ICAM-3 expressed on naïve T cells. DC-SIGN can promote a transient clustering between a DC and T cell, thus allowing the DC to screen numerous T cells for an appropriately matched TCR.⁶¹ Dectin-1, a DC-specific type II C-type lectin that contains a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM), binds to a currently unidentified ligand on T cells and promotes T cell proliferation.⁶² Adhesion receptors such as lymphocyte function-associated antigen (LFA)-1, ICAM-1, LFA-3 and CD44 may also be expressed on mature DCs and promote adhesion to T cells.⁶³

The soluble cytokine profile secreted by DCs varies with the different stages of DC development and maturation thus influencing the different effector functions characteristic of immature vs. mature DCs. A wide variety of cytokines may be expressed (not necessarily simultaneously) by mature DCs including IL-12, IL-1a, IL-1ß, IL-15, IL-18, IFN-a, IFN-ß, IFN-?, IL-4, IL-10, IL-6, IL-17, IL-16, TNF-a, and MIF. The exact cytokine repertoire expressed will depend on the nature of the stimulus, maturation stage of the DC and the existing cytokine microenvironment. The distinct cytokine patterns released by mature DCs ultimately determine their Th1/Th2 polarizing capacities.^5,6,9-12,64 Antigens that prime DCs to secrete IL-12 will typically induce Th1 differentiation, whereas antigens that do not elicit or, on the contrary, inhibit IL-12 production (e.g. IL-10, prostaglandin E2, cholera toxin)^65-67 will promote Th2 differentiation.

Receptor-ligand interaction between various TNF superfamily receptors (TNFSFRs) and their corresponding ligands influences both DC maturation and T cell priming. Fas engagement on immature DCs, for example, induces both their maturation and release of IL-1ß and IFN-?.⁶⁸ Ligation of CD40 promotes an up-regulation of the costimulatory molecules B7-1/CD80 and B7-2/CD86,⁶⁹ IL-12 secretion^70,71 and release of chemokines (e.g. IL-8, MIP-1a, MIP-1ß).⁶⁹ DCs also up-regulate OX40L, another member of the TNFSF, in response to CD40 ligation.^72,73 OX40L binding to OX40 enhances cytokine production by DCs (e.g. TNF-a, IL-12, IL-1ß and IL-6) and also induces expression of costimulatory molecules.⁷² OX40L plays a critical role in DC-T cell interactions as evidenced by a study with mice deficient in OX40L exhibiting impaired contact hypersensitivity response due to defects in T cell priming and cytokine production.⁷⁴ Mature DC expression of the TNFSF member 4-1BBL promotes stimulation of CD8⁺ T cells.^75,76 Mature DCs have a finite life expectancy, however, ligation of TRANCE R/RANK on their cell surfaces leads to increased survival.^69,77 Signaling through these TNFSF/TNFRSF members involves NF-kB activation. Mature DCs express high levels of NF-?B transcriptional control proteins such as Rel A/p65, Rel B, Rel C, p50 and p52.^78,79 These transcriptional control proteins regulate the expression of genes encoding various immune and inflammatory proteins. Inhibition of NF-?B translocation will block DC maturation and thus prevent their induction of T cell differentiation.⁸⁰

The activity of various cytokines and chemokines secreted by mature DCs and T cells can be modulated by DC-associated proteases and protease inhibitors. A novel serpin (serine protease inhibitor) secreted by DCs, PI-11, may function in promotion of the immune response by protecting cytokines from degradation.⁸¹ Two different disintegrin proteases, decysin and MADDAM/ADAM19 (metalloprotease and disintegrin dendritic antigen marker/a metalloprotease and disintegrin 19), have been identified as markers for DC differentiation.^82-84 Disintegrins may potentially influence proteolysis, adhesion, fusion and intracellular signaling.⁸⁵ TACE/ADAM17, for example, has been shown to regulate the activity of TNF-a by cleaving the membrane-bound form to produce soluble TNF-a.⁸⁶ Decysin and MADDAM/ADAM19 may also play similar roles in modulating the cytokine microenvironment of DCs and T cells.

Summary

DCs are unique APCs as they can both initiate and modulate immune responses. Even small numbers of DCs and low levels of antigen can elicit strong immune responses. Maturation stage, antigen concentration and route of entry, as well as the cytokine microenvironment all influence DC effector functions. Further studies are necessary to fully elucidate the role DC maturation plays in regulating immune responses. These studies will hopefully facilitate the development of new modalities for manipulation of DCs and translate into effective therapeutic interventions.

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