1 células dendríticas - primeira apresentação

Dendritic cells (DCs) are essential for antigen presen-tation and the initiation of protective T cell responses and, thus, constitute a front-line defence against invad-ing pathogens. DCs are located throughout the body and form a sophisticated and complex network that allows them to communicate with different popula-tions of lymphocytes, thereby forming an interface between the external environment and the adaptive immune system. To provide this protection, different subsets of DCs have evolved, and these DC subsets are specialized to exist in distinct locations, where they acquire antigens and transport them to draining lymph nodes for T cell priming. The DC network is programmed by a group of transcription factors that determine the specification and differentiation of the different subsets of DCs. Recently, it has been shown that defects in transcription factor expression under-pin developmental defects in DCs and other immune cells, and these defects result in severe immuno-deficiencies and enhanced susceptibility to bacterial, fungal and viral infections in humans1–3. Thus, disrup-tion of transcription factor expression and selective loss of DC subsets is likely to have important implications for human disease.

In this Review, we focus on the role of transcription factors in generating different DC subsets and highlight the synergistic functions of cytokines in shaping DC fate decisions. Furthermore, we discuss the molecular pathways that may allow plasticity in DC fate decisions and that enable the rapid recruitment and differen-tiation of DCs in response to diverse environmental stimuli.

Unravelling the complexity of the DC networkDCs are a heterogeneous group of cells that have been divided into different subsets. This segregation was initially based on their distinct patterns of cell-surface molecule expression. The four major categories of DCs are conventional DCs, which predominate in the steady state; Langerhans cells; plasmacytoid DCs (pDCs); and monocyte-derived DCs, which are induced in response to inflammation.

Conventional DCs. Conventional DCs are special-ized for antigen processing and presentation. They can be grouped into two main classes based on their localization in tissues and their migratory pathways as they circulate in the body (FIG. 1a; TABLE 1). The first category of conventional DCs is generally referred to as the migratory DCs. These DCs develop from early precursors in the peripheral tissues, where they act as antigen-sampling sentinels. From the peripheral tissues, they migrate to the regional lymph nodes via afferent lymphatics, a process that is accelerated in response to danger signals, such as those that occur during pathogen infection. Migratory DCs are not found in the spleen and are restricted to the lymph nodes4, where they constitute a variable proportion of the steady-state DC population; this proportion depends on the specific tissues that are drained by the lymph node5 (FIG. 1). Migratory DCs can be broadly divided into CD11b+ DCs (also known as dermal or interstitial DCs) and CD11b– DCs6, which have more recently been shown to express CD103 (also known as integrin αE)4,7.

Division of Molecular Immunology, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Melbourne, Victoria 3052, Australia.e-mails: [email protected]; [email protected]:10.1038/nri3149

Transcriptional programming of the dendritic cell networkGabrielle T. Belz and Stephen L. Nutt

Abstract | Specialized subsets of dendritic cells (DCs) provide a crucial link between the innate and adaptive immune responses. The genetic programme that coordinates these distinct DC subsets is controlled by both cytokines and transcription factors. The initial steps in DC specification occur in the bone marrow and result in the generation of precursors committed to either the plasmacytoid or conventional DC pathways. DCs undergo further differentiation and lineage diversification in peripheral organs in response to local environmental cues. In this Review, we discuss new evidence regarding the coordination of the specification and commitment of precursor cells to different DC subsets and highlight the ensemble of transcription factors that control these processes.

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NATURE REVIEWS | IMMUNOLOGY VOLUME 12 | FEBRUARY 2012 | 101

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mailto:[email protected]

Nature Reviews | Immunology

Monocyte-derived DCs

Blood-derived DCs

Spleen

Lymphoid tissue-resident DCs

Spleen

Blood

Lymph node

Skin

Dermis

Langerhans cell

Non-lymphoid tissue

Migratory DCs

pDCs

Inflammation

CD4–CD8α–

(DN) DCsCD4+ DCs

CD4+ DC

CD8α+ DCs

CD8α+ DC

CD8α+ DC

DN DC

CD103+ DCs

CD103+ DC

CD103+ DC

CD103+ DC

CD11b+ (interstitial ordermal) DCs

CD11b+ DC

CD11b+ DC

CD11b+ DC-SIGN+

monocyte-derived DC

CD11b+ DC-SIGN+

monocyte-derived DC

CD11b+ DC

Langerhans cells

Langerhans cell

Lymph nodes

MDPMonocyte

pDC

pDC

Pre-DC

Pre-DC

HSC

FLT3+ CMP

CDP

Steady state

Epidermis

Bone marrow

b

a

DN DC

Figure 1 | Differentiation and trafficking of DC subsets. a | The figure shows the organization of the dendritic cell (DC) network, and includes the key surface phenotype markers of different DC subsets, which are delineated on the basis of their localization in secondary lymphoid tissues. Gut-associated DCs that express both CD103 and CD11b have been included in the CD11b+ DC subset. Inflammatory monocyte-derived DCs are rapidly recruited to sites of inflammation, whereas other DC subsets are normally present in the steady state. The relationship between inflammatory and steady-state DCs remains an open issue. Moreover, it is unclear whether monocyte-derived DCs can arise through in situ proliferation in addition to arriving at tissues via the circulation. b | In the mouse bone marrow, haematopoietic stem cells (HSCs) differentiate into common myeloid progenitors (CMPs), a fraction of which express FMS-related tyrosine kinase 3 (FLT3) and differentiate into more-restricted macrophage and DC progenitors (MDPs). MDPs appear to be the direct precursor to common DC progenitors (CDPs), which give rise to the DC lineages. CDPs produce precursor DCs (pre-DCs) and plasmacytoid DCs (pDCs) that exit the bone marrow and travel through the blood to secondary lymphoid organs and non-haematopoietic tissues. A small proportion of DCs may also be derived from CLPs in the bone marrow and from early T cell progenitors in the thymus. Under steady-state conditions, lymphoid tissue-resident DCs that arise from pre-DCs are the only subsets found in the spleen. This population is comprised of three conventional DC subsets, namely CD4+ DCs, CD8α+ DCs and CD8α–CD4– double-negative (DN) DCs. Peripheral lymph nodes contain CD8α+ and CD8α– DC populations but are also populated by two groups of migratory DCs. Langerhans cells develop in the epidermis and migrate through the basement membrane to the draining lymph nodes via terminal lymphatic vessels that arise in the dermis. The dermal DC population is broadly composed of CD11b+ and CD103+ DCs, and these cells migrate through the lymphatics to the lymph node. Monocytes arrive at tissues from the blood. In response to inflammation, they can develop into monocyte-derived DCs, which adopt many of the characteristics of conventional DCs. DC-SIGN, DC-specific ICAM3-grabbing non-integrin.

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The second major category of conventional DCs is the lymphoid tissue-resident DCs that are found in the major lymphoid organs, such as the lymph nodes, spleen and thymus. These DCs can be further classified by their expression of the surface markers CD4 and CD8α into CD4+ DCs, CD8α+ DCs and CD4–CD8α– DCs (typically referred to as double-negative DCs)8,9 (TABLE 1). CD8α+ DCs are noted for their capacity to cross-present antigens10 and for their major role in priming cytotoxic CD8+ T cell responses11–16 (BOX 1). CD4+ DCs and CD4–CD8α– DCs can also present MHC class I-restricted antigens in some settings15,17, but appear to be more efficient at presenting MHC class II-associated antigens to CD4+ T cells18–20. Lymphoid tissue-resident DCs do not traffic from other tissues but develop from precursor DCs found in the lymphoid tissues themselves21. In the absence of infection, they exist in an immature state (which is char-acterized by a high endocytic capacity and lower MHC class II expression compared with activated DCs), and their residency in lymphoid tissues makes them ideally placed to sense antigens or pathogens that are transported in the blood12,22,23.

Langerhans cells. Langerhans cells are resident in the skin and, like migratory DCs, migrate to the lymph nodes to present antigens (FIG. 1). However, unlike con-ventional DCs, which arise from a bone marrow precur-sor cell, Langerhans cells are derived from a local LY6C+ myelomonocytic precursor cell population in the skin. This precursor population originates from macrophages

that are present early in embryonic development and that undergo a proliferative burst in the epidermis in the first few days after birth24.

pDCs. pDCs are quiescent cells that are broadly distrib-uted in the body. They are characterized by their ability to rapidly produce large amounts of type I interferons (IFNs)25,26, a feature most evident during viral infection. pDCs express several characteristic markers, includ-ing sialic acid-binding immunoglobulin-like lectin H (SIGLEC-H) and bone marrow stromal antigen 2 (BST2) in mice and blood DC antigen 2 (BDCA2; also known as CLEC4C) and leukocyte immunoglobulin-like recep-tor, subfamily A, member 4 (LILRA4; also known as ILT7) in humans. In addition, both mouse and human pDCs express CD45RA27. pDCs have poor antigen- presenting capacity, and their precise contribution to immune responses is still unclear28.

Monocyte-derived DCs. Under inflammatory con-ditions, circulating blood monocytes can be rapidly mobilized and can differentiate into cells that possess many prototypical features of DCs21,29–32 (FIG. 1). In the steady state, monocytes express the macrophage colony- stimulating factor receptor (M-CSFR; also known as CD115), which is essential for their development, as well as other markers, such as LY6C and CX3C-chemokine receptor 1 (CX3CR1). In response to growth factors such as granulocyte–macrophage colony-stimulating factor (GM-CSF) in vitro or to Toll-like receptor 4 (TLR4) ligands or bacteria in vivo, fully differentiated monocyte-derived

Table 1 | Phenotypic markers of DC subsets

DC subset DC type CD8α CD103 CD205 EPCAM (CD326)

CD11b B220 or CD45RA

DC-SIGN Langerin (CD207)

Antigen presentation

Major cytokine produced

pDCs Lymphoid- resident DCs

+/– – – – – + ++ – Poor IFNα

CD8α+ DCs Lymphoid- resident DCs

+ low + – + – – +/– Cross- presentation on MHC class I; expression of cystatin C

IL-12p70, IFNλ

CD4+ DCs Lymphoid- resident DCs

– – – – + – – – Presentation on MHC class II

DN DCs Lymphoid- resident DCs

– – – – + – – – Presentation on MHC class II

CD11b+ DCs

Migratory DCs

– +/– + – + – ND – Presentation on MHC class II

CD103+ DCs•Lung•Intestine

Migratory DCs

– –

+ +

++ –

+/– –

– +

– –

– –

+ –

Cross- presentation on MHC class I

Langerhans cells

Migratory DCs

– – ++ + + – – ++ Presentation of self antigens for tolerance induction

IL-10

Monocyte- derived DCs

Induced by inflammation

– – – – + – + – Cross- presentation

TNF

DC, dendritic cell; DC-SIGN, DC-specific ICAM3-grabbing non-integrin; DN, double-negative; EPCAM, epithelial cell adhesion molecule; IFN, interferon; IL, interleukin; ND, not determined; pDC, plasmacytoid DC.

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DCs emerge. Similarly to conventional DCs, monocyte-derived DCs express CD11c, MHC class II molecules, CD24 and SIRPα (also known as CD172a), and they upreg-ulate their expression of DC-specific ICAM3-grabbing non-integrin (DC-SIGN; also known as CD209a) but lose expression of both M-CSFR and LY6C33 (TABLE 1). Monocyte-derived DCs also express the macrophage marker MAC3 (also known as CD107b and LAMP2)21,32. In addition, these cells acquire potent antigen-presenting capacity, including the ability to cross-present antigens33–35 (BOX 1). Thus, it is emerging that monocyte-derived DCs are a crucial reservoir of professional antigen-presenting cells (APCs) that are recruited into immune responses to certain microorganisms and potentially have an emergency back-up role in cases of acute inflammation.

Cytokines regulate DC developmentThe differentiation of DCs from haematopoietic progenitor cells relies on the activity of cytokines, in particular FMS-related tyrosine kinase 3 ligand (FLT3L), M-CSF and GM-CSF. These cytokines con-trol the initial production and lineage diversification of DCs, although the factors that regulate the expres-sion of the receptors for these key cytokines and the downstream transcriptional programmes instigated by FLT3L, M-CSF and GM-CSF are only now emerging. The ability of these cytokines to stimulate the differ-entiation of DCs in vitro (BOX 2) provides a tractable model system to address the influence of extrinsic factors on the DC transcriptional network.

FLT3L and FLT3 constitute the best-characterized growth factor–receptor axis for DCs, as mouse haemato-poietic progenitor cells cultured with FLT3L generate a diverse array of conventional DC subsets and pDCs36,37. In agreement with the important role of FLT3L in DC differentiation, DCs can be generated from essentially any FLT3+ progenitor cell either in vitro or following adoptive transfer in vivo38–40 (FIG. 2). In addition, enforced expression of FLT3 in megakaryocyte–erythrocyte progenitors (MEPs), which are normally FLT3–, results in the acquisition of DC potential41. An instructive role for FLT3 in DC development is further supported by the finding that in all cell lineages except the DC lineage, FLT3 is downregulated following differentiation and, at least in the case of B cells, this repression of FLT3 is essential for further development42.

Mice deficient for FLT3L or signal transducer and activator of transcription 3 (STAT3; a signalling mol-ecule downstream of FLT3) have markedly reduced numbers of lymphoid-resident conventional DCs and pDCs, whereas mice lacking FLT3 have a milder pheno-type, which suggests the presence of a second ligand for this receptor43–46. FLT3 signalling is also crucial for the development of migratory DCs, as the numbers of both pre-DCs and CD103+ DCs were found to be reduced in a range of tissues from Flt3l–/– mice compared with the

Box 1 | Direct presentation, cross-presentation and cross-dressing

EfficientpresentationofantigenstoCD8+T cellsdependsonthegenerationofpeptidesforloadingintoMHCclass Icomplexes.SeveralpathwayshavebeenuncoveredtoachievethisactivationofCD8+T cells.DirectantigenpresentationbyinfectedormalignantcellsensuresthedestructionofthesecellsbycytotoxicCD8+T cells(CTLs),withoutharmingadjacenthealthycells.Therestrictionofantigenpresentationtodirectlyinfectedcells,however,isnotsufficienttoensuretheactivationofCD8+T cells,particularlywhenthepathogendoesnotinfectprofessionalantigen-presentingcells,suchasdendriticcells(DCs).Inthiscase,CD8+T cellscanbeactivatedbyDCsthatpresentextracellularantigensontheirMHCclass Imoleculesviatheprocessofcross-presentation.ThispathwaycanresultinthegenerationofCTLsthatarereactivetoforeignantigens,orintheinductionoftolerancethroughthedeletionofautoreactiveCD8+T cellsfollowingthecross-presentationofselfantigens.Inmice,mostfocushasbeenonthecross-presentingcapacitiesofCD8α+DCs,andmorerecentlyCD103+DCs6,7,10,138,althoughotherpopulationsalsopossessthisability.Forexample,fullydifferentiatedmonocyte-derivedDCsthatexpressDC-specificICAM3-grabbingnon-integrin(DC-SIGN)arepotentcross-presentingcells33.Analternativepathwayfortheacquisitionofpeptide–MHCcomplexesisknownas

‘cross-dressing’139,140(originallyknownastrogocytosis141).Throughthispathway,DCsacquirepreformedpeptide–MHCclass Icomplexesfrominfectedcells.Complexesacquiredinthiswaycandrivetheactivationofmemory,butnotnaive,CD8+T cellsduringviralinfection,perhapsowingtotheloweractivationthresholdofmemorycells.Themolecularmechanismsinvolvedincross-presentationandcross-dressingareonlybeginningtobeunravelled.

Box 2 | In vitro models for investigating DC development and behaviour

Theestablishmentofwell-definedcellculturesystemsthatallowthegenerationoflargenumbersofdendriticcells(DCs)frombonemarrowhasbeeninstrumentalforunderstandingDCbiology.RecentrefinementofthetoolsandsurfacemarkersusedtoanalyseculturesnowallowstheresolutionofDCprecursorsequivalenttothosefoundin vivo,togetherwithfullydifferentiatedDCsubsets,inthetissuecultureflask.

Generation of steady-state DC subsets in vitroThedevelopmentofsteady-stateDCsdependsonsignallingthroughFMS-relatedtyrosinekinase 3(FLT3),whichisexpressedonthesurfaceofDCprecursors.BonemarrowprecursorsculturedwithFLT3ligand(FLT3L)giverisetoplasmacytoidDCs(pDCs)andmultiplelymphoidtissue-residentconventionalDCsubsets36,37,89.Intriguingly,in vitro-generatedDCsdonotexpressCD4orCD8,buttheirpatternsofexpressionofthemarkersCD103,CD11b,CD172aandCD24indicatethepresenceofconventionalDCsubsetsinadditiontoDCprecursors.

Generation of monocyte-derived DCs in vitroPerhapsthemostcommonlyusedapproachtogenerateDCsinvolvesthecultureofbonemarrowprecursorsinamediumsupplementedwithgranulocyte–macrophagecolony-stimulatingfactor(GM-CSF).AstheseDCscanalsobeproducedbyculturingmonocytesinGM-CSFandinterleukin-4(IL-4),theyarereferredtoasmonocyte-derivedDCsandcorrespondtothedominantinflammatoryDCtypethatismobilizedduringsomebacterialinfections33.ThesehighlyrefinedculturesystemsallowcomparativestudiesbetweendifferentDCsubsets.Furthermore,the

abilitytogeneratelargenumbersofDCsin vitroshouldgreatlyfacilitatemolecularexplorationofthegenomicandtranscriptionalmachinerythatleadstothegenerationofdifferentDCsubsets.

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CD8α+ DC

CD103+ DC

CD11b+ DC

Monocyte

Macrophage

M-CSF GM-CSF

GM-CSFM-CSF

Monocyte-derived DC

CLP

LMPP FLT3+

CMP

FLT3L

FLT3L

FLT3L

E4BP4ID2 BATF3Lymphocytes

MDP?PU.1 PU.1

IRF8

E2-2

PU.1hi

ID2hi

Lossof E2-2

PU.1low

ID2–

E2-2hi

GFI1

CDP Pre-DC

Transitional pre-DC

Immature pDC

Mature pDC

Pre-CD8α+ DC

RELBIRF2IRF4 Ikaros (null)

Ikaros (L) IRF8

BATF3

Figure 2 | Growth factors and transcription factors that regulate DC differentiation. The developmental pathways from myeloid and lymphoid progenitors to precursor dendritic cells (pre-DCs) in the bone marrow and the peripheral diversification of DC subsets are shown (see FIG. 1 legend for details). The approximate points at which key transcription factors are first required for DC development are indicated by vertical lines. Stages at which key growth factors have been determined to be essential are indicated. The development of both DCs and monocytes depends on high concentrations of PU.1, which regulates the expression of the cytokine receptors FMS-related tyrosine kinase 3 (FLT3), macrophage colony-stimulating factor receptor (M-CSFR) and granulocyte–macrophage colony-stimulating factor receptor (GM-CSFR). The development of CD8α+ and CD103+ DCs relies on the stepwise activity of interferon-regulatory factor 8 (IRF8), inhibitor of DNA binding 2 (ID2), E4 promoter-binding protein 4 (E4BP4) and basic leucine zipper transcription factor, ATF-like 3 (BATF3), as well as on FLT3 signalling. CD11b+ DCs depend on a unique set of transcription factors, including RELB, IRF2, IRF4 and Ikaros, and to some extent on the cytokines M-CSF and GM-CSF. The plasmacytoid DC (pDC) lineage requires IRF8, a low level of PU.1 and the absence of ID2. The differentiation of pDCs from an immature precursor requires E2-2 and Ikaros, with induced loss of E2-2 converting pDCs into cells that closely resemble CD8α+ conventional DCs. CDP, common DC progenitor; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; FLT3L, FLT3 ligand; GFI1, growth factor independent 1; LMPP, lymphoid-primed multipotent progenitor; MDP, macrophage and DC progenitor.

numbers in wild-type mice. By contrast, the development of CD11b+ DCs and Langerhans cells is largely independ-ent of FLT3L47. Thus, FLT3L has two distinct roles in DC biology: it is required for the early development of DCs from haematopoietic progenitors; and later it functions to maintain DC homeostasis by promoting limited levels of proliferation of DCs in peripheral tissues43.

In addition to FLT3L, GM-CSF has long been known to stimulate DC differentiation in culture (BOX 2). However, GM-CSF is not essential for DC differentia-tion in the steady state, as mice that lack the GM-CSF receptor (GM-CSFR) have only mildly reduced num-bers of DCs48. Nonetheless, GM-CSF is not completely redundant in DC production, as mice deficient in both GM-CSF and FLT3L have a greater loss of DCs than either single-knockout strain44. Moreover, other recent studies have demonstrated that GM-CSF is necessary for there to be normal numbers of CD103+CD11b+ DCs in the lamina propria49,50. The addition of GM-CSF to cultures of bone marrow cells promotes the development of cells

that resemble monocyte-derived DCs, while repress-ing the development of pDCs in a STAT5-dependent manner46,51. This finding has generally been interpreted to show that GM-CSF has a greater role in the produc-tion of monocyte-derived DCs than in the generation of other DC subsets21,52, although the importance of this process in vivo is still to be established.

M-CSF is the major cytokine involved in the produc-tion of monocytes and macrophages53. A role for this cytokine in DC biology was suggested by the expression of M-CSFR by DCs54,55 but came to prominence with the finding that M-CSFR-deficient mice lack Langerhans cells56. Surprisingly, mice lacking M-CSF (op/op mice) have normal numbers of Langerhans cells, a quandary that was resolved by the identification of interleukin-34 (IL-34) as a second ligand for M-CSFR57. The relatively normal DC numbers in mice lacking M-CSF, despite the profound reduction in monocytes, demonstrates that the monocytic system is not the major source of steady-state DCs. M-CSF is, however, required for the

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normal development of CD103–CD11b+ DCs in non-lymphoid tissues49 and is able to support conventional DC and pDC differentiation in cell culture in the absence of FLT3 (REF. 58).

Stages of programming DC identityDC ontogeny. Although it is well established that all DCs, with the exception of Langerhans cells, are derived from bone marrow-resident haematopoietic stem cells (HSCs), mapping the origins of the DC lineages has proven to be both difficult and controversial. Early transfer experiments led to the surprising conclusion that DCs can develop with approximately equal effi-ciency from both lymphoid and myeloid pro genitors59,60, whereas in vitro cultures with GM-CSF show that DCs can arise from monocytic precursors, as mentioned above. However, monocytes are not likely to be a major source of steady-state DCs in lymphoid organs, as lineage-tracing experiments have shown that monocytic cells give rise to neutrophils and macrophages but not DCs5. More recently, the adoption of a ‘FLT3-centric’ view of haematopoiesis38–40,61 has established that most steady-state DCs arise from FLT3+ progenitors (FIG. 2).

The pathway of DC differentiation from primitive bone marrow progenitors has been extensively reviewed elsewhere4,53,62 and is only briefly summarized here. FLT3 expression is first induced in a subset of the HSC com-partment that has only short-term pan-haematopoietic repopulating activity, and the expression of this recep-tor is then maintained in lymphoid-primed multipotent progenitors (LMPPs)63 and in a subpopulation of com-mon myeloid progenitors (CMPs)38. CMPs are thought to differentiate into macrophage and DC progenitors (MDPs)64, which appear to be the direct precursors of common DC progenitors (CDPs)40,61. Both MDPs and CDPs are proliferating cells that reside in the bone mar-row and express FLT3 and M-CSFR. CDPs differentiate directly into pDCs and into the precursors of conven-tional DC subsets, termed pre-DCs, but they lack the potential to give rise to macrophages21,65. Pre-DCs then leave the bone marrow and are found in blood, second-ary lymphoid organs and some tissues21,49,52,65, where they mature into the conventional DC subsets (FIG. 1). Differentiation into different conventional DC subsets appears to be a late step in DC development that is per-haps important in maintaining the stability or plasticity of the peripheral DC compartment. The key features and mechanisms involved in the plasticity of the DC network are likely to include the short lifespan of mature conven-tional DCs (5–7 days, although up to 25 days in some cir-cumstances4,47); the rapid recruitment and proliferation of pre-DCs and their capacity to respond to extrinsic signals (such as TLR ligands and pro-inflammatory cytokines); and the active expression by DCs of transcription factors such as E2-2 (also known as TCF4).

Initiating the DC programme in haematopoietic progeni-tors. The information outlined above demonstrates the rapid progress that is being made in understanding the developmental stages and cell biology of the DC lineages. Much less is known about the transcriptional programme

that specifies the DC lineage in more-immature pro genitors and then drives differentiation into the DC subsets. Three transcription factors — PU.1 (encoded by Sfpi1), Ikaros and growth factor independent 1 (GFI1) — appear to be prime candidates for DC-specifying fac-tors. In addition, the signalling and transcription factors STAT3 and STAT5 are known to have a role in DC differ-entiation, as they mediate the signals transduced through FLT3 and GM-CSFR, respectively (TABLE 2).

PU.1 belongs to the ETS family of transcription factors, which has multiple context-specific roles in haemato poiesis. PU.1 is an attractive candidate for being a crucial regulator of the DC lineages, as it is expressed by all DCs and by CDPs66–68. A role for PU.1 in DC develop-ment was initially suggested by the analysis of mice with a germline deficiency of PU.1. Indeed, one such study concluded that PU.1 was necessary for all embryonic DC development69, although a second study reported that Sfpi1–/– fetal thymi could generate DCs70. However, these approaches could not distinguish between the require-ments for PU.1 in multipotent progenitors and the role of PU.1 specifically in the DC lineages. Moreover, the impact of enforced expression of PU.1 in haematopoietic progenitors suggests an instructive and concentration-dependent role for PU.1 in promoting macrophage and DC development71–73.

A recent study used conditional gene deletion in defined haematopoietic progenitors and CDPs to show that PU.1 is absolutely essential for the generation of all conventional DCs and pDCs both in vivo and in FLT3L-containing cultures in vitro66. Moreover, in line with its established role in regulating GM-CSFR expression, PU.1 is required for GM-CSF-induced DC differentiation from early haematopoietic progenitors. Among the many genes that are potentially regulated by PU.1, Flt3 was demonstrated by molecular studies to be directly regulated by PU.1 in DCs and haemato-poietic progenitors66. This regulation occurred in a concentration-dependent manner, as Sfpi1+/– cells had reduced FLT3 expression and an impaired ability to generate conventional DCs. Interestingly, previous stud-ies have shown that FLT3 signalling is able to activate PU.1 expression in MEPs, suggesting a self-reinforcing loop between PU.1 and FLT3 in DCs41. Whether PU.1 is required for monocyte-derived DC formation in vivo remains to be determined.

Ikaros is a zinc-finger transcription factor that has important roles in haematopoiesis74. Expression of a dominant-negative form of Ikaros that also impairs the function of other Ikaros family members, such as Aiolos, resulted in a complete loss of all conventional DC sub-sets. By contrast, a null mutation in the gene encoding Ikaros led to the selective loss of CD11b+ DCs, with some CD8α+ DCs being retained75. Whether Ikaros directly regulates DC differentiation, as opposed to having a role in early myeloid progenitors, is at present unclear76, as mice homozygous for a severely hypomorphic allele of the Ikaros gene (IkL/L mice) lack mature pDCs but con-tain relatively normal numbers of conventional DCs77. DC-specific conditional mutagenesis is now required to decipher the exact function of Ikaros in DCs (FIG. 2).

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E proteinThe E proteins (including E12, E47, HEB and E2‑2) have emerged as key regulators of the immune system. They are a family of basic helix‑loop‑helix factors that work together with their antagonists, the ID proteins (ID1–ID4), to regulate lymphocyte development.

GFI1 is a small, zinc-finger-containing transcriptional repressor that is important for early haemato poiesis78. GFI1 is expressed in DC precursors, and Gfi1–/– mice have reduced numbers of all lymphoid-resident DC subsets, whereas Langerhans cell numbers were actu-ally increased79. Interestingly, GFI1-deficient haemato-poietic progenitor cells were unable to develop into DCs in vitro in the presence of either FLT3L or GM-CSF and instead differentiated into macrophages, suggesting that GFI1 is a crucial modulator of DC versus macrophage development (FIG. 2).

Establishing pDC and conventional DC identity. Restriction of the developmental programme of DC progenitors to the conventional DC and pDC lineages occurs at the CDP stage40,61 (FIG. 2). Conventional DCs and pDCs differ markedly in their appearance, functions and transcriptional programmes, and thus how a CDP is influenced to develop into either a pDC or a conven-tional DC is a question of major importance for under-standing and manipulating DC biology. In this context,

it is surprising how little we actually understand about this process. Two developmental systems appear to be in place to separate pDCs and conventional DCs (FIG. 2). First, pDCs absolutely rely on the expression of the E protein E2-2 (REF. 80) and the absence of the E protein antagonist inhibitor of DNA binding 2 (ID2)81. Second, pDCs have a uniquely low level of PU.1 (REF. 68) and an extremely high concentration of interferon-regulatory factor 8 (IRF8), a transcription factor that can form a complex with PU.1 on a class of ‘composite’ DNA elements82.

One model to explain the pDC versus conventional DC lineage split is to assume that the conventional DC is the default setting and that progenitors have to be diverted to the pDC lineage83. E2-2 fits the bill for a fac-tor that could control this diversion, as it is abundantly expressed by pDCs and is required for pDC lineage specification80. Once progenitors have committed to a pDC fate, it appears that continuous expression of E2-2 is essential to maintain the mature pDC phenotype84. In mice, a specific deletion of the gene encoding E2-2

Table 2 | Transcription factors guiding steady-state DC subset development

Transcription factor

Transcription factor family Function Refs

PU.1 (SFPI1, SPI1)

ETS-domain transcription factor; binds to PU box sequences

Required for the development of all DC subsets 66, 69,70

IRF2 Interferon-regulatory factor; inhibits the IRF1-mediated transcription of type I IFNs

Alters pDC ratios; in its absence the numbers of CD8α– DCs and Langerhans cells are reduced

122

IRF4 Interferon-regulatory factor Required for non-CD8α+ DC lineage development 126,127

IRF8 (ICSBP) Interferon-regulatory factor Required for the development of pDCs and most conventional DCs

90,92, 93,104

GFI1 Zinc-finger protein; transcriptional repressor

GFI1 deficiency results in a 50% reduction in the numbers of conventional DCs and pDCs and increased numbers of Langerhans cells

79

ID2 Inhibitor of DNA binding family protein containing HLH domains

Required for the development of CD103+ DCs and CD8α+ DCs in PLNs and spleen; not required for DCs in MLNs

47, 87,89

E4BP4 (NFIL3) PAR-related bZIP transcription factor Required for the development of CD8α+ DCs 108

E2-2 (TCF4) E protein containing bHLH domains Required for the development and maintenance of pDCs

80,84

STAT3 Signal transducer and activator of transcription

STAT3 deficiency results in a substantial reduction in conventional DC numbers

46

STAT5A and STAT5B

Signal transducer and activator of transcription

Inhibit pDC development by interacting with IRF8; deficiency results in reduced conventional DC and increased pDC numbers

51

Ikaros (IKZF1) Zinc-finger DNA-binding protein Ikaros deficiency results in the absence of most DCs; a hypomorphic mutation leads a specific loss of pDCs

75,77

BATF3 bZIP family; heterodimerizes with JUN

BATF3-deficient mice fail to develop CD103+ DCs and show impaired survival of precursor CD8α+ DCs

13, 89,111

RELB REL-homology domain family; interacts with NF-κB family members

RELB deficiency results in the loss of CD8α– DCs 129,130

SPIB ETS-domain transcription factor Required for human pDC differentiation 91

BATF3, basic leucine zipper transcription factor, ATF-like 3; bHLH, basic HLH; bZIP, basic leucine zipper; DC, dendritic cell; E4BP4, E4 promoter-binding protein 4; GFI1, growth factor independent 1; HLH, helix-loop-helix; ID2, inhibitor of DNA binding 2; IFN, interferon; IRF, interferon-regulatory factor; MLN, mesenteric lymph node; NF-κB, nuclear factor-κB; PAR, proline- and acidic-rich region; pDC, plasmacytoid DC; PLN, peripheral lymph node; STAT, signal transducer and activator of transcription.

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Lymphoid tissue-inducer cells(LTi cells). A cell type that is present in developing lymph nodes, Peyer’s patches and nasopharynx‑associated lymphoid tissue (NALT). LTi cells are required for the development of these lymphoid organs. The inductive capacity of these cells for the generation of Peyer’s patches and NALT has been shown by adoptive transfer, and it is generally assumed that they have a similar function in the formation of lymph nodes.

Nucleosome remodellingChanges in the nucleosome structure are mediated by dedicated nuclear enzymes (for example, ATP‑dependent nucleosome‑remodelling enzymes) that change the accessibility of DNA and the expression of genes.

Histone modificationsHistones are essential to maintain DNA organization and may be modified by methylation and acetylation — changes that are thought to keep genes active or silent, respectively — thereby altering the genetic code read by transcriptional regulators.

in pDCs led to the expansion of a population of DCs that exhibit many characteristics of conventional DCs. In the in vivo setting, however, it is particularly dif-ficult to dissect whether E2-2-deficient pDCs undergo phenotypic conversion to conventional DCs owing to a reduction in E2-2-mediated repression of ID2, as pro-posed, or whether normal conventional DC numbers are increased in the absence of a full pDC compart-ment, as occurs in other settings. Nevertheless, pDCs are particularly sensitive to E2-2 concentration, as both E2-2-deficient mice and patients with a rare mono-allelic loss of E2-2 (Pitt–Hopkins syndrome) show impaired pDC formation and function80. E2-2 binds directly to the promoters of several pDC-expressed genes, including BDCA2, LILRA4, IRF7, the pre-TCR α-chain gene, IRF8 and SPIB (which encodes a close relative of PU.1 that is expressed by human and mouse pDCs)80. The reliance of pDCs on an E protein such as E2-2 may explain the observation that pDCs express many lymphocyte-associated transcripts (including SPIB, RAG1, IL7R and TDT), as E proteins are central to many aspects of lymphopoiesis27.

ID proteins are direct inhibitors of DNA bind-ing by E proteins. ID2 is the predominant ID protein expressed in the DC lineage and is also involved in the development of multiple lineages during haemato-poiesis, particularly that of lymphoid tissue‑inducer cells (LTi cells) and natural killer (NK) cells85–88. ID2 expression is extremely low in CDPs, pre-DCs and pDCs, whereas all conventional DC populations express high levels of ID2 (REF. 89) (FIG. 3). In line with this expression pattern, ID2-deficient mice have a profoundly altered conventional DC compartment but still produce pDCs (see below)87. This leads to a model whereby the acquisition of high levels of ID2 and subsequent suppression of E2-2 activity blocks pDC development and allows progression along the conventional DC pathway. This model is clearly an oversimplification, as E2-2-deficient mice still produce some pDC progenitors and ID2 is not essential for the differentiation of all conventional DCs.

The development of pDCs also depends on PU.1, IRF8 and potentially SPIB66,90,91. pDCs are absent in PU.1- or IRF8-deficient mice66,90,92, as well as in humans with a mutation in IRF8 (REF. 3), although it remains to be proven whether these factors function specifically in the pDC lineage or have a role in CDPs (FIG. 2). However, circumstantial evidence favours a specific function for these factors in determining pDC versus conventional DC fate. PU.1 is expressed at a uniformly high level in CDPs, but at a lower level in pDCs, with the timing of downregulation coinciding with pDC formation66, whereas IRF8 is expressed at very high levels in both CDPs and pDCs (FIG. 3). The stoichiometric relationship between PU.1 and IRF8 is likely to be important for the pDC versus conventional DC branch point, as both of these factors are known to function in a dose-dependent manner66,93 and can bind to distinct DNA sequences both individually and in a complex82. The extent to which PU.1 and IRF8 share target genes in pDCs is currently unknown (although

they have been shown to share a few targets, such as Ciita94, Tlr9 (REF. 95) and Ifna96). However, BXH2 mice, which harbour a spontaneous point mutation in Irf8, have defects in CD8α+ DC development but not in pDC generation. This mutation is thought to ablate the interaction of PU.1 and IRF8, raising the possibil-ity that the PU.1–IRF8 complex is not as crucial for the differentiation of pDCs as for the development of conventional DCs93. There are, as yet, no genome-wide DNA-binding datasets for IRF8 and PU.1 in DCs, although similar data from macrophages sug-gest that PU.1 might occupy most of the active regula-tory regions in DCs97,98 and that IRF8 might bind to a subset of these sites99. Importantly, in macrophages and myeloid progenitors, PU.1 is directly involved in nucleosome remodelling, and this leads to the generation of an open chromatin conformation and histone modi‑fications, suggesting that PU.1 can directly programme the fate of myeloid cells97,98.

The ETS-family transcription factor SPIB — the closest homologue of PU.1 in the mammalian genome — is expressed, within the DC lineages, specifically by pDCs100. Knockdown of the expression of either SPIB or PU.1 in human haematopoietic progenitors strongly inhibits pDC formation, suggesting that both factors function in human pDCs91. The extent of any functional redundancy between PU.1 and SPIB in pDCs has so far not been addressed in mice.

In summary, the separation of pDC and conven-tional DC lineages represents the first major division in the DC pathway and requires the concerted action of both E proteins and the PU.1–IRF8 complex (FIGS 2,3). Although the activation of STAT5 inhibits pDC for-mation in vitro51, the exact signals that initiate this process are not known. In addition, the ways in which the E2-2–ID2 and PU.1–IRF8 axes interact are still unclear, as are the identities of most of the genes tar-geted by these transcription factors. pDCs represent the end point of their lineage; however, the production of conventional DCs is only the first step in their further diversification, which is outlined in the next section.

Genetic programming of conventional DC subsetsAlthough it is fairly clear that conventional DCs, pDCs, Langerhans cells and monocyte-derived DCs represent developmentally distinct lineages, the relationships between the various anatomically, phenotypically and functionally distinct conventional DC populations remain to be fully elucidated. Conventional DCs are thought to be derived from circulating pre-DCs21,65. Although it remains possible that multiple develop-mentally distinct types of pre-DC exist and act as the precursors for individual conventional DC lineages, we favour a model that considers all the populations of conventional DCs as related subsets derived from a single pre-DC population. In this model, the conven-tional DC subsets are induced by the environmental milieu that they reside in, and thus it is the interaction of these extrinsic signals with the core transcriptional programme of conventional DCs that dictates the outcome of DC terminal differentiation.

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CD8α+ DC

CD103+ DC

CD11b+ DC

CDP Pre-DC

Transitional pre-DC

Immature pDC

Mature pDC

Pre-CD8α+ DC

IRF8 +++ID2 +/–PU.1 +++

IRF8 +++ID2 +/–PU.1 +++

IRF8 +++ID2 +/–PU.1 +E2-2 +

IRF8 +++ID2 +/–PU.1 +++

IRF8 +++ID2 ++PU.1 +++

BATF3 ++IRF8 +++ID2 +++IRF4 +E4BP4 ++PU.1 +++E2-2 +

BATF3 ++IRF8 +ID2 ++IRF4 +++E4BP4 ++PU.1 +++E2-2 +

BATF3 +IRF8 +++ID2 +/–IRF4 ++E4BP4 ++PU.1 +E2-2 +++

BATF3 +++IRF8 +++ID2 +++IRF4 ++E4BP4 ++PU.1 +++E2-2 +

Figure 3 | Differential expression of transcription factors regulating DC differentiation. The stages of differentiation of conventional dendritic cells (DCs) and plasmacytoid DCs (pDCs) from the common DC progenitor (CDP) are shown, together with the relative levels of expression of key transcription factors in each cell type indicated on an arbitrary scale. The expression pattern of some of the factors has not been determined at the earliest stages of DC ontogeny. –, no expression; +, low expression; ++, intermediate expression; +++, maximal expression; BATF3, basic leucine zipper transcription factor, ATF-like 3; E4BP4, E4 promoter-binding protein 4; ID2, inhibitor of DNA binding 2; IRF, interferon-regulatory factor; pre-DC, precursor DC. Figure is modified, with permission, from REF. 89 © Macmillan Publishers Ltd. All rights reserved.

NFAT(Nuclear factor of activated T cells). A family of transcription factors that are regulated by calcium signalling and expressed by a variety of immune cells.

AP1(Activator protein 1). A heterodimeric transcription factor that is composed of proteins belonging to the FOS, JUN and JUN‑dimerization protein families. AP1 controls various cellular processes, including differentiation, proliferation and apoptosis.

Cross-primingA mechanism by which immunogenic CD8+ T cells are activated by the presentation of an antigen that was not synthesized by the antigen‑presenting cell itself.

Transcriptional regulators of CD8α+ and CD103+ DCs. CD8α+ and CD103+ DCs have gained consider-able attention owing to their specialized roles as induc-ers of MHC class I-restricted immune responses to pathogens. Moreover, a human BDCA3+ DC subset has recently been identified that shares features with both of these mouse DC subsets; such features include cross-presenting capacity and the expression of XC-chemokine receptor 1 (XCR1)101–103. The differentiation of DC pre-cursors into the CD8α+ and CD103+ DC lineages appears to depend on the integration of four key transcription factors — namely, IRF8, E4BP4 (E4 promoter-binding protein 4; also known as NFIL3), ID2 and BATF3 (basic leucine zipper transcription factor, ATF-like 3).

IRF8 is highly expressed in CDPs, pDCs (dis-cussed in detail above) and conventional DCs, par-ticularly the CD8α+ and CD103+ DC subsets (FIG. 3). IRF8-deficient mice lack many mature DC subsets, including Langerhans cells90,104. In addition to regulating the generation of pDCs and conventional DC subsets, IRF8 controls various functional features of DCs, such as the expression of TLR9 and IFNα by pDCs and the production of IL-12 by CD8α+ DCs93. Although IRF8

clearly has a key role in the function of conventional DCs, Irf8–/– mice develop a myeloproliferative syndrome that is characterized by the overproduction of granulocytes105. This implies that IRF8 may also be required for the gen-eration or maintenance of MDPs, which can give rise to monocytes, conventional DCs and pDCs. The exact developmental stage at which IRF8 exerts its activity on conventional DCs remains to be fully determined.

E4BP4 is a mammalian basic leucine zipper (bZIP) transcription factor that is required for the development of NK cells but not of other lymphocyte lineages106,107. In NK cells, E4BP4 acts in a dose-dependent man-ner downstream of the IL-15 receptor to regulate ID2 expression106,107. More recently, the induction of E4BP4 has been shown to be important for the development of CD8α+ DCs108. E4BP4-deficient CDPs had lower levels of BATF3 expression than control CDPs, and enforced expression of BATF3 in the mutant cells rescued CD8α+ DC development in vitro108. Thus, E4BP4 is emerging as a key regulator of conventional DC diversity. Whether E4BP4 also acts through ID2 to mediate these effects in DCs has not yet been addressed.

ID2 is expressed by all conventional DC subsets, with the highest levels of expression in CD8α+ and CD103+CD11b– DCs47,89. Loss of ID2 prevents the devel-opment of these two subsets in the skin-draining lymph nodes and spleen. However, CD103+CD11b+ DCs in the mesenteric lymph nodes, together with CD4+ and CD4–

CD8α– DCs in lymphoid tissues, appear to develop nor-mally in the absence of ID2 (REFS 13,47,87). Although it is at present unclear which E protein (or E proteins) — E2A (also known as TCF3), E2-2 or HEB (also known as TCF12) — is the crucial target of ID2 in conventional DCs, selective deletion of the gene encoding E2-2 in mature pDCs results in the spontaneous differentiation of pDCs into cells that exhibit conventional DC properties, perhaps through the induction of ID2 (REF. 84). Progress in dis-cerning the key target genes of E proteins in conventional DCs will be required to understand why ID2 has such an important role in CD8α+ and CD103+CD11b– DCs.

BATF3, which is also known as JUN-dimerization protein p21SNFT, is a bZIP transcription factor that acts to repress the activity of NFAT–AP1 complexes by competing with FOS for JUN dimerization109,110. BATF3 was the first transcription factor that appeared to have an exclusive role in the development of the CD8α+ DCs111, although it has since been shown to be involved in the development of CD103+CD11b– DCs in peripheral lymphoid tissues13, but not that of CD103+CD11b+ DCs isolated from gut lymphoid tis-sues112. More recently, detailed analyses of Batf3–/– mice have shown that despite the reduction in the frequency of CD8α+ DCs, particularly in the spleen, CD8α+ DCs are still present in the absence of BATF3 (REFS 89,113). Nevertheless, Batf3–/– mice exhibit severe defects in their capacity to respond to pathogen infections — includ-ing West Nile virus, influenza virus and Toxoplasma gondii infections111,114–116 — and an impaired ability to mediate cross‑priming. This suggests that the main role of BATF3 may be in regulating the cross-presentation of exogenous antigens to CD8+ T cells (BOX 1).

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The transcriptional network in CD8α+ and CD103+ DCs. As outlined above, a deficiency of IRF8, E4BP4, ID2 or BATF3 results in a lack of both the CD8α+ and CD103+ DC subsets. This dependency on the same transcriptional regulators, as well as similar func-tional and localization characteristics13,47,111, suggests that CD8α+ and CD103+ DCs represent a single sub-set. However, similar developmental requirements do not necessarily imply close lineage relationships; for example, PU.1 is required for the development of multiple distinct lineages, including macrophages, granulo cytes and DCs, but very different mechanisms are involved66,117.

One approach used to investigate the relative con-tributions of IRF8, ID2 and BATF3 to conventional DC differentiation has been to engineer a fluorescent reporter into the Id2 locus (to generate Id2gfp mice)89. Analyses of Id2gfp mice showed that neither CDPs nor pre-DCs expressed ID2. Thus, it is more likely that ID2 drives the terminal differentiation of different DC sub-sets rather than influencing early lineage-commitment decisions. Using Irf8–/– or Batf3–/– mice crossed with Id2gfp mice, it became clear that IRF8 is required for the generation of DC precursors from a very early time point, whereas BATF3 has a role later in conven-tional DC development, downstream of ID2 (REF. 89). BATF3-deficient progenitors gave rise to precursors of SIRPα– DCs (which are the precursors of CD8α+ and CD103+ DCs) in FLT3L-containing cultures and to a lesser extent in vivo, although both the frequency of these DCs and their expression of CD8α were reduced compared with wild-type CD8α+ DCs89 (FIG. 2). Thus, it is clear that IRF8, ID2 and BATF3 each functions at a distinct point in the differentiation of CD103+ and CD8α+ DCs. What is lacking, however, is an under-standing of how the programmes that are activated by each of these transcription factors interact to give rise to the diversity of DC subsets with unique functions. At present, the major approaches for dissecting this net-work are the deletion of individual transcription factor genes from the entire haematopoietic compartment, and/or limited sampling of tissues and inference with the on/off regulatory switches. But these approaches provide only ‘black and white snapshots’ of transcription factor involvement in DC development. The establishment of the identity of DC subsets, their differentiation and their maintenance are unlikely to be so simple and may require combinatorial interactions between transcrip-tion factors to guide fate decisions118,119. Genome-wide DNA-binding data will be crucial for the precise eluci-dation of the manner in which different transcription factors work together to define different DC subsets.

Transcriptional regulation of CD8α– DCs. Much less is known about the transcription factors that regulate the differentiation decisions of CD8α– conventional DC lin-eages, despite the dominant role of these cells in present-ing antigens to CD4+ T cells. The transcription factors IRF2, IRF4 and RELB have been shown to be important in the development of these subsets and their subsequent maturation (FIG. 2; TABLE 2).

IRF2 acts as a transcriptional repressor of genes encoding type I IFNs (IFNα and IFNβ) and thus limits inflammation120,121. Mice lacking IRF2 exhibit a selective, cell-autonomous loss of CD4+ DCs in the spleen and epi-dermis, and these subsets are restored when type I IFN signalling is eliminated122,123. Precisely how IRF2 is regu-lated during DC development is not clear, but one role of IRF2 may be to protect developing DCs from maturation arrest when the levels of type I IFNs are elevated. IRF2 has also been reported to form complexes with IRF8 and to act cooperatively with this factor in regulating the expression of IL12 (REF. 124).

IRF4, a crucial regulator of many aspects of lympho-cyte differentiation125, is most highly expressed in CD4+ DCs (in which IRF8 expression is lowest). In line with this expression profile, CD4+ DCs are largely absent from mice lacking IRF4, but these mice also show defects in pDCs in the spleen126,127. Moreover, DCs generated in vitro through the stimulation of bone marrow precursors with FLT3L appear to rely on IRF8 rather than IRF4, but those generated in the presence of GM-CSF depend on IRF4 (REFS 126,127). Thus, it has been proposed that the main action of IRF4 is to coordinate signals from GM-CSF stimulation through the nuclear factor-κB (NF-κB) path-way128. The extent to which IRF4 and IRF8 regulate simi-lar or distinct sets of target genes, with or without PU.1, remains an open question that needs to be addressed.

RELB is a member of the NF-κB family and can func-tion either as an activator or as a repressor of transcrip-tion by forming heterodimers with the p50 and p52 NF-κB family members. RELB is most highly expressed in the CD8α– and CD11b+ DC subsets, and these DC subsets are absent in RELB-deficient mice129,130. To date, there have been no studies addressing the mechanism by which RELB controls DC differentiation. It is pos-sible that IRF4 — the deletion of which also results in a substantial loss of CD4+ DCs127 — may be a key target of the RELB pathway. RELB has a crucial role in the upregulation of the signalling molecule CD40, which is required to induce immunogenic DCs and for the induction of IFNα131,132. Loss of IFNα results in impaired cross-priming of exogenous antigens132,133. CD40 acts through TNF receptor-associated factor 6 (TRAF6) to activate the NF-κB cascade134. Strikingly, Traf6–/– mice lack CD8α– DCs and have an impairment in DC matu-ration similar to that observed in Relb–/– mice. It seems likely that TRAF6 regulates CD4+ DC development in a RELB-dependent manner.

Much attention has been focused on delineating the development of CD8α+ and CD103+ DCs. However, despite the crucial role of CD8α– DCs in immune responses — particularly in the activation of CD4+ T cells18,19 and possibly of follicular helper T cells135–137 — a detailed understanding of the transcription factor networks that drive CD8α– DC development is lacking. Furthermore, transcription factors normally associ-ated with CD8α+ and CD103+ DC lineages, such as ID2 (REF. 89) and IRF8, are differentially expressed among CD8α– DCs, suggesting that they may have additional roles in the generation of fully matured DCs that do not express CD8α.

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Future directionsIt is now clear that an extraordinarily complex net-work of conventional DC, pDC and monocyte-derived DC subsets contributes to the induction of immune responses and the maintenance of tolerance. A major challenge now will be to discern the overall contribu-tions of different DC subsets to these processes, and to understand the changes in the transcriptional network that are induced by inflammatory states to ensure the mobilization of DC precursors. Although it is gener-ally assumed that surface markers expressed by DCs in the steady state are faithfully retained during inflam-matory conditions, it is not yet clear whether this is indeed the case. Thus, in the inflammatory setting, fate-mapping experiments based solely on surface markers may not result in the identification of DCs with a phenotype similar to that of steady-state DCs. However, it is known that conventional DC precursors differentiate into defined subsets late during infec-tion. This may reflect the ability of their transcrip-tional machinery to remain poised, allowing for rapid

responses to different pathogens and for the mainte-nance of highly flexible genetic programmes within DCs, but the factors that guide such decisions are still to be determined. It seems likely that a combination of experimental approaches will be required to estab-lish the broader transcriptional network that controls DC differentiation. Possible approaches include single-cell differentiation, flow cytometry, profiling of global gene expression or chromatin modifications, and the use of innovative mouse models that allow gene expression to be tracked and enable the conditional inactivation of subset-specific genes.

DCs have long been considered ideal candidates for targeted vaccine approaches, but this promise remains largely unfulfilled. Recent progress in understanding the cell biology of DC lineages, complemented by an emerg-ing understanding of the transcription programmes that drive their differentiation, has greatly facilitated the iden-tification of the human counterparts of mouse DC sub-sets. Translating these findings into the clinic may allow the development of more-effective vaccine approaches.

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AcknowledgementsThe authors thank R. Allan, A. Kallies, M. Chopin and M. Pellegrini for helpful discussions and critical reading of the manuscript. This work is supported by the National Health and Medical Research Council (NHMRC) of Australia and the Wellcome Trust. G.T.B. is supported by a Sylvia and Charles Viertel Foundation Fellowship and S.L.N. is supported by an Australian Research Council Future Fellowship. This work was made possible by Victorian State Government Operational Infrastructure Support and the Australian Government NHMRC Independent Research Institute Infrastructure Support Scheme.

Competing interests statementThe authors declare no competing financial interests.

FURTHER INFORMATIONGabrielle T. Belz’s homepage: http://www.wehi.edu.au/faculty_members/dr_gabrielle_belzStephen L. Nutt’s homepage: http://www.wehi.edu.au/faculty_members/dr_stephen_nuttDendritic Cell Research Knowledge Portal: http://dc-research.eu/Immunological Genome Project: http://www.immgen.orgInternational Society for Dendritic Cell and Vaccine Science: http://www.dc-vaccine.org/

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1 células dendríticas - primeira apresentação

Health & Medicine

dc cd11b

dcs cd103

dcs cd8

dc dc dermishsc cd103

dc specification

distinct dc subsets

firstdifferent subsets

dc networktation