Mouse and human dendritic cell subtypes

REVIEWS

Dendritic cells (DCs) are a sparsely distributed, migra-tory group of bone-marrow-derived leukocytes that arespecialized for the uptake, transport, processing and pre-sentation of antigens to T cells1–3. At an ‘immature’ stageof development, DCs act as sentinels in peripheral tis-sues, continuously sampling the antigenic environment(FIG. 1). Any encounter with microbial products or tissuedamage initiates the migration of the DCs to lymphnodes (LNs). The antigenic sample at the time of theDANGER, including any microbial products, is processedand fixed on the DC surface as peptides that are pre-sented by major histocompatibility complex (MHC)molecules. The DCs also upregulate the co-stimulatorymolecules that are required for effective interaction withT cells. In the LNs, the now-mature DCs efficientlytrigger an immune response by any T cells with areceptor that is specific for the foreign-peptide–MHCcomplexes on the DC surface.

Immunoregulation by DCsDCs as determinants of tolerance. The model of DCs asnatural ADJUVANTS that promote the immune response toforeign antigens has been modified after the realizationthat the antigen-presenting cells (APCs) that areinvolved in immunity must also be involved in toleranceto self-antigens. Central tolerance of T cells in the thy-mus, which is achieved by inducing the apoptotic deathof potentially self-reactive T cells, is mediated by thymicDCs4. Are these DCs specially armed to induce T-cell

death rather than proliferation? The evidence so far indi-cates that it is the stage of T-cell differentiation, ratherthan the type of DC, that determines the negative out-come of this interaction5. Nevertheless, thymic DCs dohave a life history that differs from the standard model,because most of them derive from an intrathymic pre-cursor and develop and die within the thymus6. A non-migratory behaviour seems to be more appropriate to such DCs, the function of which is to present self-antigens rather than to collect foreign antigens.

However, not all self-reactivity is eliminated fromthe T-cell receptor repertoire by central tolerance7.Because the DCs that carry foreign antigens into theLNs must also be carrying self-antigens8, the DCsthemselves are likely to be responsible for tolerance aswell as immunity. Tolerance to self-antigens in vivoseems to require an active, proliferative response by theself-antigen-reactive T cells9. Accordingly, the prolifera-tive responses of T cells to DCs in culture are as likelyto model the initiation of tolerance as the initiation of aprotective immune response.

Two general mechanisms have been proposed bywhich DCs might maintain peripheral tolerance. Thefirst is that a subtype of specialized regulatory DCs isinvolved; there is some evidence for such DCs, but noconsensus10–12. The second is that all DCs have a capac-ity for initiating tolerance or immunity, the distinctiondepending on the maturation or activation state of theDC (FIG. 1). The original concept was that IMMATURE DCS

MOUSE AND HUMAN DENDRITICCELL SUBTYPESKen Shortman* and Yong-Jun Liu‡

Dendritic cells (DCs) collect and process antigens for presentation to T cells, but there are manyvariations on this basic theme. DCs differ in the regulatory signals they transmit, directing T cellsto different types of immune response or to tolerance. Although many DC subtypes arise fromseparate developmental pathways, their development and function are modulated by exogenousfactors. Therefore, we must study the dynamics of the DC network in response to microbialinvasion. Despite the difficulty of comparing the DC systems of humans and mice, recent workhas revealed much common ground.

NATURE REVIEWS | IMMUNOLOGY VOLUME 2 | MARCH 2002 | 151

*The Walter and Eliza HallInstitute of Medical Research,Victoria 3050, Australia.‡DNAX Research Institute,Palo Alto,California 94304, USA.e-mails: [email protected], [email protected]: 10.1038/nri746

DANGER

A term, popularized by P. Matzinger, that covers thevarious signals from damagedtissues or from microbialproducts that trigger dendriticcell activation.

ADJUVANT

A substance that enhances theimmune response to antigens.

IMMATURE DCS

(iDCS.) These dendritic cells aretypically found in non-lymphoid tissues and are capableof antigen uptake andprocessing, but as yet are unableto interact with T cells. Theyhave intracellular but low or nosurface MHC II, and do not yetexpress co-stimulator molecules.

ACTIVATED DCS

These dendritic cells are fullymature and express high surfacelevels of MHC II and co-stimulator molecules. Theyinitiate T-cell immuneresponses.

QUIESCENT DCS

These dendritic cells are typicallyfound in the lymphoid tissues ofuninfected laboratory mice andare sufficiently mature to expressmoderate surface levels of MHCII and co-stimulator molecules,but they are not yet fullyactivated and maintain somepotential for antigen uptake andprocessing. They might mediateT-cell tolerance.

152 | MARCH 2002 | VOLUME 2 www.nature.com/reviews/immunol

R E V I E W S

example, CPG motifs in bacterial DNA, double-strandedviral RNA, lipopolysaccharides (LPS) and necrotic cellproducts (such as heat-shock proteins) activate DCs18–21.In addition, DCs distinguish between tissue cells that dieby the normal process of apoptosis and those that die byexternally generated necrosis — antigens from bothtypes of dead cell are taken up and presented to T cells,but only the latter activate the DC22,23. The receptors thatrecognize these diverse stimuli vary from lectin-domainscavenger receptors that are similar to those on phago-cytes to the Toll-like receptors (TLRs), which are relatedto proteins from the defence systems of plants andinsects24,25. DCs link these evolutionarily conserved pat-tern-recognition systems of the innate immune systemto the variable pattern-recognition receptors of theadaptive immune system: the T-cell receptors.

DCs as cross-presenting APCs. DCs are equipped withthe biochemical machinery for processing and present-ing peptide fragments of protein antigens on MHCmolecules, rather than merely digesting them26,27. APCsusually present exogenous antigens on MHC class II(MHC II) molecules, whereas they usually presentendogenous antigens (from self-components or a viralinfection) on MHC class I (MHC I) molecules.However, it is clear that certain antigens outside theAPC system somehow enter the MHC-I processingpathway, both to generate cytotoxic T cells in responseto viruses that do not infect the APCs themselves, andto maintain self-tolerance to non-APC components7,28.Many experiments now point to DCs being the previ-ously elusive cross-presenting APCs29–33. The receptorsand biochemical machinery that lead exogenous anti-gens into the MHC-I presentation pathway of DCs nowneed to be elucidated.

DCs direct the T-cell response. The experimental out-come of a DC–T-cell interaction is often simply T-cellproliferation. Although triggering of T cells into cell-cycle progression is a central function of DCs, it is nowclear that DCs can also influence, and perhaps dictate,the subsequent development of these dividing T cells.T-cell activation and proliferation might lead to immu-nity or to tolerance, to the generation/activation ofeffector T cells or regulatory T cells, and to T cells thatsecrete different patterns of cytokines, including theextreme cytokine-polarized T helper 1 (T

H1) AND T

H2

responses (FIG. 1).Although the signals from DCs that govern T-cell

cytokine polarization have not all been determined, theproduction of the T

H1-inducing cytokine interleukin-12

(IL-12) is an important factor34. The production of thebioactive p70 form of IL-12 by DCs is tightly regulatedand involves separate control of the production of thep40 and p35 components. Several factors are needed toinduce IL-12 production by DCs, including microbialproducts, the CD40 ligand CD154, stimulation fromactivated T cells and the appropriate cytokinemilieu35,36. Notable examples of controls on IL-12 pro-duction include the negative-feedback effect (in whichthe T

H2 cytokine IL-4 acts as a strong promoter of IL-12

(iDCs) produced tolerance, whereas mature DCs pro-duced immunity13,14. The iDCs might induce toleranceby killing T cells, by paralysing them (anergy) or, par-ticularly, by inducing the generation of regulatory T cells15. However, the idea has been revised to suggestthat tolerance is induced by DCs that are mature butquiescent, whereas immunity is induced by DCs thatare fully activated16,17. These ACTIVATED DCS then provideT cells with additional signals or much stronger ver-sions of the same signals. The emerging model is thatQUIESCENT DCS that bear self-components are required forthe continuous maintenance of self-tolerance, whereasimmune responses only occur when invading pathogensprovide danger signals to trigger DC activation. Wesuggest that the effects of microbial infections extendfurther, to inducing not just the final activation but alsothe production of certain DC subtypes.

Recognition receptors on DCs. These models require theDCs to sense ‘danger’ in the form of microbes or tissuedamage. Indeed, a series of microbial products (for

ActivatedDC

QuiescentDC

Immaturedendritic cell

TH1

TH2

TH1 bias

TH2 bias

CD4T cell

CD8T cell

Non-lymphoid tissues Draining lymph nodes

Immunity

Tolerance

Danger-signal-induced migration

Steady-statemigration

Danger

Dangersignal A

Dangersignal B

MHC IIpresentation

MHC Icross-presentation

Deletion

Regulatorycells

Deletion

Regulatorycells

Foreign antigens

Self-antigens

Figure 1 | Dendritic cells and immunoregulation. The current view of the ways in which theactivation states of dendritic cells (DCs) can determine the nature of T-cell responses. In theabsence of microbial infections and related ‘danger’ signals, there is a low-level, steady-stateentry of DCs into lymphoid tissues, in which quiescent DCs help to maintain a state of peripheralT-cell tolerance to self-antigens. Microbial infection, inflammation and tissue damage all activatethe DCs and increase their rate of migration into lymphoid tissue, where they signal to T cells thatare specific for the foreign antigens that are presented by the DCs to initiate immune responses.The idea that microbial invasion might also trigger the production of new DC subtypes that are notpresent at all in uninfected animals is not shown here. MHC, major histocompatibility complex;TH1, T helper 1 cell; TH2, T helper 2 cell.


R E V I E W S

Mouse DCsSubtypes of mature DC. Distinct subtypes were initiallymore evident among mouse DCs than among humanDCs, because of the ready availability of different murinelymphoid tissues and the expression on mouse DCs ofmarkers not present on human DCs. Mouse DCs thatare classed as ‘mature’ express CD11c (the integrin-α

x

chain) and the co-stimulator molecules CD80, CD86and CD40, and have moderate to high surface levels ofMHC II, although the levels of all of these can be furtherelevated on activation. These features always correlatewith a striking ability to induce the proliferation of allo-geneic T cells. Such mature DCs are heterogeneous innormal, uninfected laboratory mice39–42. Surprisingly,the T-cell markers CD4 and CD8 are expressed onmouse DCs and are useful for segregating subtypes. CD8on DCs is in the form of an αα-homodimer rather thanthe αβ-heterodimer that is typical of T cells. AlthoughCD4 is also present on human DCs, CD8α is not. So far,there is no evidence that either marker is functionallysignificant on mouse DCs. Other markers that are use-ful for segregating mouse DC subtypes include CD11b(the integrin α

Mchain of Mac-1) and the interdigitating

DC marker CD205 (the multilectin domain moleculeDEC205, originally known as NLDC-145).

Using these surface markers, five DC subtypes areconsistently found in the lymphoid tissues of unin-fected laboratory mice (TABLE 1). Spleen contains threeof these: the CD4−CD8+, the CD4+CD8− and the CD4−

CD8− DCs42. The CD8+ DCs are concentrated in the T-cell areas and the CD8− DCs in the marginal zones ofmost laboratory mice, but the CD8− DCs migrate intothe T-cell zones on stimulation with microbial prod-ucts39,43,44. The CD4−CD8+ DC subtype (FIG. 3), which isCD205+CD11b−, is also found at moderate levels inLNs, but is the dominant subtype among thymic DCs.LNs contain two extra DC subtypes that are not nor-mally found in spleen, which have apparently arrived inthe LNs through the lymphatic system39–41. Found in allLNs, CD4−CD8−CD11b+ DCs express moderate levels ofCD205, in contrast to spleen CD8− DCs. This LN DCsubtype is believed to be the mature form of tissueinterstitial DCs. Another distinctive DC subtype, foundonly in skin-draining LNs, expresses high levels of lan-gerin, a characteristic marker of epidermal Langerhanscells, and is believed to be the mature form of thisLangerhans cell41. It also expresses a range of myeloidmarkers, including CD11b, stains at low levels for CD8αand has high surface levels of CD205, as high as on theCD8hi DC subtype. These Langerhans DCs in LNs arealso distinguished by their high surface levels of MHCII and high expression of CD40, CD80 and CD86(showing many characteristics of fully activated DCs).

Developmental origins. It is important to know whetherDC subtypes are products of separate developmentallineages or whether they are different activation states ofa single lineage (FIG. 2). Past studies on the mouse DCsubtypes focused on their possible origin from sepa-rate early haematopoietic precursors. Although mostDCs were thought to be of myeloid origin45, there was

production by DCs36), temporal regulation (wherebyDCs that have produced a pulse of IL-12 become‘exhausted’ and do not respond to subsequent stim-uli37), and the subtle discrimination by DCs of micro-bial stimuli (such that Candida albicans stimulates IL-12production in its yeast stage but IL-4 production in itshyphal stage38). It is therefore not surprising that DCsshow considerable plasticity in their ability to induceT

H1 or T

H2 responses.

DC subsetsIn addition to these interactions with T cells in the lym-phoid organs, DCs interact with other cell types, includ-ing B cells and natural killer (NK) cells. They might alsoremain in peripheral tissues as inflammatory mediatorsrather than migrating to LNs. The several and oftenopposing roles now ascribed to DCs cannot all be car-ried out at once by the same cell, so there should be dif-ferent sets of DCs that perform different functions. Suchspecialized DC subtypes might represent different acti-vation states of a single lineage, the functional differ-ences depending entirely on local environmental signals(the functional plasticity model; FIG. 2). Alternatively, thespecialized DC subtypes could be the products ofentirely separate developmental lineages. The signalsthat determine lineage segregation would then act ear-lier and the immediate precursors of the DCs (PDCS)would already be separate and functionally committed(the specialized lineage model; FIG. 2). The reality is aconfusing mixture of these two models, and a largedegree of functional plasticity seems to be a general feature of both DCs and pDCs.

CPG

Denotes a deoxynucleotidemotif in bacterial DNA that actsas an adjuvant. It is recognizedby, and activates, certaindendritic cells. The nucleotidesequence and methylation stateof this portion of bacterial DNAdiffers from those ofmammalian DNA.

TH1 AND T

H2

T-helper cells have the potentialto produce many cytokines.If suitably and extensivelystimulated, two polarizedpatterns of cytokine productioncan emerge. T

H1 cells

characteristically produce highlevels of interferon-γ andpromote macrophage-mediatedinflammatory responses. T

H2

cells characteristically produceinterleukins-4 and -5, andpromote mast cell andeosinophil functions. In balancedimmune responses, individual T cells show a range of cytokineproduction potentials, not justthese extreme forms.

Immature DCs

Mature DCs

Haematopoieticprecursors

DC precursors

a Functional plasticity modelSingle DC lineage, functional plasticity

b Specialized lineage modelMultiple DC lineages, functional diversity

pDC1 pDC2

iDC1 iDC2

DC1 DC2DC1 DC2

Figure 2 | Alternative models for the generation of functionally distinct dendritic cell (DC)subtypes. a | The functional plasticity model proposes that all DCs belong to a singlehaematopoietic lineage, the different subtypes of DCs being generated by local environmentalinfluences on a relatively mature but plastic end-product cell. b | The specialized lineage modelproposes that the different subtypes of DCs derive from early divergences in the developmentalpathway, producing several distinct haematopoietic sublineages. We propose that the actualsituation is a mixture of these extreme models. iDC, immature DC; pDC, precursor of DC.


R E V I E W S

Does this developmental plasticity at the haematopoi-etic precursor stage extend to the entire DC developmentpathway? This question is difficult to answer directlybecause the downstream precursor cells are only begin-ning to be identified. The finding that the differentiationof CD8− and CD8+ DCs requires different cytokines andinvolves different transcription factors indicates thattheir developmental pathways differ53–57. Because matureDCs are not dividing cells, direct information on theirprecursor–product relationships can be obtained fromthe order of uptake of a DNA precursor, bromod-eoxyuridine (BrdU), into the DC subsets. The kinetics ofDC-subset labelling after continuous BrdU administra-tion to mice indicated that none of the five DC subtypesare products of the others and that all are short-livedproducts of separate developmental streams, at least asfar back as the last dividing precursor58 (A. Kamath, S.Henri and K. S., unpublished observations). A degree ofDC sublineage commitment must therefore occurdownstream of the early haematopoietic precursors.

Immediate precursors. The isolation from mice of cellswith immediate DC precursor potential (pDCs) hasprovided further evidence of separate DC developmen-tal sublineages and made some crucial connectionswith studies on human DCs (TABLE 2). CertainCD45RA−CD11c+CD11b+ monocytes isolated frommouse blood can produce mature DCs in culture withgranulocyte–macrophage colony-stimulating factor(GM-CSF) and tumour-necrosis factor-α (TNF-α) (M.O’Keeffe, personal communication). This correspondsto the production of myeloid DC1 cells by humanblood monocytes (see below). The monocyte-to-DCpathway has also been tracked in intact mice after theinjection of fluorescent latex microspheres into mouseskin tissue59. Inflammatory monocytes that had ingestedthe microspheres crossed the endothelial barrier into thelymph, migrated to LNs and were identified there asmature CD8−CD11clo DCs. These were a minority sub-group, apparently distinguished by these surface mark-ers from most of the LN DCs found in the steady state.The development of these mouse monocyte-derivedDCs in vivo was therefore strongly dependent on anexogenous stimulus of phagocytosis.

A second mouse DC precursor has recently been iso-lated from mouse lymphoid tissues60–62 and mouse

evidence that some DCs shared early steps of develop-ment with B cells46,47 and that the CD8+ DCs of the thy-mus shared early steps of development with T cells6. AllCD8+ mouse DCs were thought to derive from lym-phoid-restricted precursors, and all CD8− DCs to derivefrom myeloid-restricted precursors, leading to the terms‘lymphoid’ and ‘myeloid’ DCs.

However, this concept is not correct. The linkbetween thymic T-cell and DC development was ques-tioned when certain mutant mice (c-kit−γc− and a condi-tional Notch1 knockout), which cannot form T cells, stillproduced thymic CD8+ DCs48,49. Direct tests of the lin-eage segregation model became possible when bothmyeloid-restricted and lymphoid-restricted precursorcells were isolated from bone marrow. This confirmedthat both lymphoid-restricted and myeloid-restrictedprecursors could produce DCs. However, each precur-sor could produce all the mature splenic and thymic DCsubtypes (TABLE 1), albeit with some bias in the subsetbalance50–52. The final surface phenotype of DCs and allthe functional capabilities tested to date are not prede-termined at this early haematopoietic precursor level.

PRECURSOR DCS

(pDCs.) These dendritic cells aredownstream of multipotenthaematopoietic progenitors andare largely committed todendritic cell production, but donot have the morphologicalappearance or functions ofdendritic cells.

Figure 3 | CD8+ dendritic cells (DCs) isolated from mousespleen. The DCs were extracted and enriched from mousespleen42 and then sorted as CD11c+CD8α+ cells. The sortedcells were incubated overnight in culture medium, whichactivates the DCs. Surface major histocompatibility complexclass-II (MHC-II) molecules were then stained red (with anti-MHC-II antibody coupled to Texas Red) and the DNA of thenucleus was stained purple (with DAPI; 4′,6-diamidino-2-phenylindole). Confocal image provided by J. Villadangos (The Walter and Eliza Hall Institute).

Table 1 | Lymphoid tissue distribution of mouse dendritic cell (DC) subtypes

‘Lymphoid’ DCs ‘Myeloid’ DCs ‘Myeloid’ DCs ‘Myeloid’ DCs ‘Langerhans’ DCsCD4–CD8hi CD4+CD8– CD4–CD8– CD4–CD8– CD4–CD8lo

CD205hiCD11b– CD205–CD11b+ CD205–CD11b+ CD205+CD11b+ CD205hiCD11b+

Percentage total DCs in

Spleen 23 56 19 < 4 < 1

Thymus 70

Mesenteric lymph nodes 19 4 37 26 < 4

Skin-draining lymph nodes 17 4 17 20 33

The terms ‘lymphoid’ and ‘myeloid’ DC no longer denote haematopoietic origin but are still commonly used. These values for steady-state, uninfected laboratory mice arebased on data from references 37 and 38. Pickup of CD4 and CD8αβ from thymocytes makes thymic DCs difficult to assess. The remaining 30% of the DCs seem to bemainly CD4−CD8−DEC205+CD11b−, with a few CD11b+.


R E V I E W S

Functional specialization. The different subtypes ofmouse DC share a common capacity to present anti-gens to T cells and to promote cell-cycle progression.However, they differ in the other aspects of DC–T-cellsignalling that determine the subsequent fate of the T cells that they activate. The most striking biologicaldifference so far discovered is the ability of the CD8+

DCs to induce a TH1-biased cytokine response in reac-

tive CD4+ T cells, whereas CD8− DCs tend to induce aT

H2-biased response34,63,64. The main factor in this dif-

ference is the much higher level of IL-12 p70 producedby CD8+ DCs under the experimental conditions, butother factors might be involved, and there might be aspecific signal for inducing the T

H2 cytokine pattern.

CD4−CD8− splenic DCs can also produce some IL-12p70 and this might be sufficient to induce T

H1 cytokines

during a close DC–T-cell interaction.The production of IL-12 p70 is regulated by many

factors. Although the appropriately activated CD8+ DCshave the greatest capacity for IL-12 production, differ-ent stimuli can change the balance. So, even though LPSfrom Escherichia coli stimulates CD8+ DCs to produceIL-12 and to induce T

H1 responses, LPS from

Porphyromonas gingavalis does not stimulate IL-12 pro-duction and preferentially induces T

H2 responses65. The

three DC subtypes found in uninfected laboratorymouse spleen differ in their capacity to producecytokines when stimulated under conditions designedto induce a maximal response66. The CD8+ DCs canproduce a small amount of IFN-α and IFN-β, as well asproducing the highest levels of IL-12. By contrast, CD4−

CD8− DCs produce the most IFN-γ in the presence ofexogenous IL-12 and IL-18. The CD4+CD8− DCs seemto be poor producers of all three cytokines.

In addition to their effects on the type of cytokineproduced by the activated T cells, the DC subtype caninfluence the amount of cytokine these T cells secrete.CD4−CD8− DCs induce more IL-2, IFN-γ, GM-CSF andIL-3 from CD8+ and CD4+ T cells than do CD4−CD8+

DCs67–70. An intriguing point is that the enhancedcytokine production on stimulation with CD4−CD8−

DCs compared with CD4−CD8+ DCs is obtained evenwhen the T-cell proliferation is equivalent, indicatingthat there are signals from the DCs that govern T-cellcytokine output separately from T-cell proliferation.

A further functional difference between the variousDCs of uninfected mouse spleen is that CD4−CD8+ DCscause early apoptotic death in a proportion of the CD4+

T cells activated in culture, whereas CD4−CD8− DCs donot11. The basis of this effect and its in vivo significance

blood (M. O’Keeffe, personal communication). It seemsto be the equivalent of the human DC precursor pDC2,which has PLASMACYTOID morphology, can generate DCsin culture and is a chief producer of class I interferons.This mouse pDC2 population is found in all lymphoidtissues and consists of large, round cells that resemble itshuman counterpart. As with the human plasmacytoidpopulation, it is a potent producer of both interferon-α(IFN-α) and IFN-β when stimulated by bacterial CpG orby viral infection. This population had been missed inmouse cell suspensions because it expresses B220 (CD45)(and so might be eliminated along with B cells duringthe FACS (fluorescence-activated cell sorter) analysisselection procedure) and stains with anti-GR-1 antibody(and so might be eliminated along with granulocytes).The GR-1 staining intensity is low and apparently repre-sents crossreactivity between the anti-GR-1 antibody(anti-Ly6G) and Ly6C (a glycosyl phophatidylinositol(GPI)-linked membrane protein).

The surface phenotype of murine pDC2 cells resem-bles in many respects that of their human counterparts(TABLE 2), but differs from mature DCs in beingCD45RAhighCD11clo and expressing only low surfacelevels of MHC II. On activation in culture with CpGand IL-3, mouse pDC2 cells change from plasmacytoidto DC morphology and, without evident cell prolifera-tion, become fully mature DCs. The plasmacytoid pre-cursors already contain intracellular MHC II, similar toiDCs, and this shifts to surface MHC-II expression onactivation. The resultant culture-generated DCs expressCD8α and resemble in many respects the normalmouse CD4−CD8+ DC subtype.

Is this mouse pDC2 subtype, therefore, the precursorof the CD4−CD8+ DC subtype that is found in normal,uninfected mice? There are two arguments against this.First, although many of these putative precursors expressCD4, continuous elimination of CD4+ cells in vivo withan anti-CD4 antibody removes only the CD4+CD8− DCwithout affecting the levels of the CD4−CD8+ DCs58.Second, BrdU labelling studies show that the half-life ofCD4−CD8+ DCs is very short (~1.5 days)58, whereas thatof the mouse plasmacytoid cells is much longer (~9days) (M. O’Keeffe and K. S., unpublished observations).Because these populations are similar in number andneither is dividing, the slower-turnover plasmacytoidcells cannot be producing the normal CD8+ DCs inuninfected mice. Production of DCs from the mousepDC2 cells seems to require an exogenous stimulus, suchas a viral or bacterial infection, so these DC progenymight not be present in uninfected laboratory mice.

PLASMACYTOID

Having the morphologicalappearance of B-lineageantibody-producing plasmacells, including the presence ofrough endoplasmic reticulum.Plasmacytoid dendritic cells donot, however, produceimmunoglobulin and lack mostB-lineage markers.

Table 2 | Interferon-producing, plasmacytoid dendritic cell (DC) precursors

Mouse Human

Surface phenotype CD11clo CD45RAhi MHC IIlo CD11clo CD45RAhi MHC IIlo

CD4hi and CD4−, CD8+ and CD8− CD4hi CD8−

IL-3R+ CD19− CD2+ CD38+ CD43+ IL-3Rhi CD19− CD2+ CD38+ CD43+

Surface phenotype CD11c+ CD45RAlo MHC IIhi CD11c+ CD45RAlo MHC IIhi

of DC product CD4loCD8+ CD4loCD8−

Cytokine production IFN-α,-β+++ IL-6++ IL-10− IFN-α,-β+++ IL-6++ IL-10−

IFN, interferon; IL, interleukin; IL-3R, IL-3 receptor.


R E V I E W S

Human DCsMature DC subtypes in vivo. In contrast to the manystudies on mouse DCs, there are relatively few studies onmature human DCs freshly isolated from tissue. Blood isthe only readily available source and, although somehuman blood DCs are sufficiently mature to give a pro-liferative response in mixed leukocyte cultures, blood ismore a source of iDCs and pDCs. Human blood DCsare heterogeneous in their expression of a range ofmarkers, but many of these reflect differences in thematuration or activation states of DCs rather than sepa-rate sublineages2. A final barrier to comparing themdirectly with mouse DCs is the lack of expression ofCD8 on human DCs, so the human DC equivalent ofmature mouse CD8+ DC remains elusive. However, as inthe mouse, the human Langerhans-cell DC is recog-nized as a separate DC subtype with distinct markers,including the presence of BIRBECK GRANULES and theexpression of CD1a and langerin.

In a few cases, human DCs have been isolated fromlymphoid tissues without any incubation steps to pro-mote differentiation, and the mature DCs analysed.Only in these cases can a direct comparison with mouseDC subtypes be made. Splenic and tonsillar DCs iso-lated in this way show heterogeneity in the expression ofCD4, CD11b and CD11c, indicating a level of complex-ity resembling mouse splenic DCs72 (N. Mavaddat andK. S., unpublished observations). The relationshipbetween these subtypes is not yet clear. Recent studieson human thymic DCs make a stronger case for subsetsegregation, similar to that in the mouse73,74. Mosthuman thymic DCs are CD11c+CD11b−CD45ROlo andlack myeloid markers, so resemble mouse thymic CD8+

DCs. A minority of human thymic DCs areCD11chiCD11b+CD45ROhi and express many myeloidmarkers, so resemble mouse CD8α− ‘myeloid’ DCs. Thissupports the view that a direct comparison of humanand mouse DCs, using similar isolation conditions andstaining reagents, will lead to similar conclusions.

Mature DC subtypes in vitro. Most of the insights intohuman DC subsets and their developmental originshave come not from direct isolation of the mature DCsfrom tissues but from studies of their development inculture from iDCs or pDCs. These studies have led tothe concept of distinct pathways of human DC develop-ment (FIG. 4), although the correspondence between theDCs generated in culture and naturally occurring DCssubtypes in human or mouse is not clear.

Three different precursor-cell starting points havebeen used to generate human DCs in culture (TABLE 3).The earliest precursor used is a CD34+ fraction isolatedfrom bone marrow or umbilical-cord blood. As well ascontaining haematopoietic stem cells and progenitorcells, this fraction contains some committed DC precur-sors capable of forming pure DC colonies in semisolidmedia75. Liquid culture with GM-CSF and TNF-α leadsto two types of intermediate precursor and to twoapparently separate pathways of DC development76,77.

One pathway leads to DCs resembling Langerhanscells, in that they have Birbeck granules and express the

are uncertain. However, CD8+ DCs could be a special-ized subset of tolerogenic or regulatory DCs. There arearguments for such a role in vivo12, and some experi-mental systems show a negative effect on anti-allograftand delayed-hypersensitivity responses10,71. However,this conflicts with the ability of CD8+ DCs to promoteT

H1 responses.

Antigen-processing differences. The ability of DCs todirect or regulate a T-cell response should be linked tothe nature of the antigens they present, whether theseare from self-components or from different invadingpathogens. One relevant finding is that the CD8+ DCs ofnormal, uninfected mice seem to be specialized for theuptake and cross-presentation of exogenous antigens onMHC I. When soluble or cell-bound ovalbumin wasinjected into mice, CD8+ DCs but not CD8− DCs cross-presented the antigen on MHC I and stimulated CD8+ Tcells32,33. By contrast, when soluble ovalbumin wasinjected, the CD8− DC but not the CD8+ DC efficientlypresented the antigen on MHC I and stimulated CD4+ Tcells33. At this stage, it is not clear whether the biochemi-cal machinery for antigen processing differs betweenCD8+ and CD8− DCs, whether the difference lies in thereceptors used for antigen uptake or whether a complexmulticellular process is required to produce selectiveantigen processing.

BIRBECK GRANULES

A characteristic structure that isvisualized by electronmicroscopy in sections ofLangerhans epidermal dendriticcells. They are believed to be acomponent of the antigen-processing system of these cells.

Activated Langerhans DC

Activated Interstitial DC

Langerhans DC Interstitial DC

CD34+ CLA+ CD34+ CLA–

DC1 DC2

Haematopoieticstem cell

Precursor 1(myeloid?)

Precursor 2(lymphoid?)

Monocyte/pDC1 PlasmacytoidIPC/pDC2

?

Equivalent?

Endogenousstimulus(cytokines)

Exogenousstimulus(microbial products)

Figure 4 | Pathways of human dendritic cell (DC) development. These pathways werededuced from the culture studies of TABLE 3. Some DC lineages progress to a mature butquiescent state under the influence of cytokines alone, requiring exogenous stimuli only for fullactivation. Other DC lineages, however, probably remain at a precursor state in an uninfectedindividual, because they require stimulation by microbial products to produce mature DCs. CLA,cutaneous lymphocyte-associated antigen; IPC, interferon-producing cell; pDC, precursor of DC.


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clear, but a similar monocyte pDC1 transformation toDC1 has been obtained in culture with precursorsobtained from mouse blood.

A model of human monocyte-to-DC transforma-tion has been devised that, although it still involves cellculture, is faster and might be closer to events in vivo85.This model involves a layer of endothelial cells over acollagen matrix. Monocytes migrate across the endothe-lial barrier into the collagen matrix, mimicking theentry of monocytes into tissues. Many remain andbecome the equivalent of tissue macrophages. However,some reverse direction and migrate back across theendothelial barrier, mimicking the transit from tissuesto lymph; these transmigrating monocytes becomeDCs. The process is markedly enhanced if particulatematerial is phagocytosed in the collagen matrix. Noexogenous cytokines are added, although factors suchas GM-CSF could be produced by the endothelialcells. The in vivo studies in the mouse lend support tothis model59.

The precursor for the final pathway of human DCdevelopment in culture, termed pDC2, is the interferon-α/β-producing plasmacytoid cell86,87 (TABLES 2,4). Humanplasmacytoid cells were recognized well before theirmouse counterparts, initially by their plasma-cell-likemorphology and their unique surface phenotype(CD4+IL-3-receptor+CD11c−, negative for most lineagemarkers) (TABLE 2). They are found in blood and manylymphoid tissues, entering LNs by L-selectin-dependenttransfer across high endothelial venules. It has been sug-gested that at least some of these plasmacytoid cells are oflymphoid origin: they express many lymphoid markers,lack surface myeloid markers and produce mRNA forgerm-line IgK and for PRE-T-CELL RECEPTOR-α 88,89. The plas-macytoid cells respond to viral and microbial stimuli by

Langerhans-cell-associated antigens (Lag), langerin andE-cadherin. The intermediates along this pathwayexpress the cutaneous lymphocyte-associated antigen(CLA, a skin-homing receptor)78, CD11c and CD1a.This pathway depends on the presence of transforminggrowth factor β (TGFβ)79, which agrees with evidencethat Langerhans cells are absent in mice lackingTGFβ80. The second pathway of development fromCD34+ precursors leads to DCs resembling intestinalDCs, lacking Birbeck granules and the Langerhans cellantigens but expressing CD9 (a tetraspan molecule),CD68 (macrosialin) and coagulation factor XIIIa. Theintermediates along this pathway lack CLA and CD1abut express the GPI-linked myeloid differentiationantigen CD14 and resemble blood monocytes in manyrespects. The third pathway of DC development, whichrequires a different combination of cytokines and isunder different transcription-factor regulation, pro-duces DCs from lymphoid-restricted precursors withinthe CD34+ population81. This might be analogous tothe production of DCs in culture from mouse-thymuslymphoid-restricted precursors53.

Blood monocytes, termed pDC1, are the most com-monly used precursor cells for generating human DCsin culture (TABLE 4). In the presence of macrophagecolony-stimulating factor (M-CSF), they will generatemacrophages but, in the presence of GM-CSF and IL-4,DCs termed DC1 are produced after six days82–84 andlittle or no proliferative expansion is involved. Finalmaturation to CD14−CD38+CD86+-surface MHC-IIhi

DCs is achieved by stimulating with proinflammatorycytokines such as TNF-α or microbial products such asLPS. Helper CD4+ T cells, signalling by means ofCD154, can also mature and activate these DCs16. Thein vivo significance of this route of DC generation is not

Table 3 | Culture models of human dendritic-cell (DC) development

Precursor Culture system and cytokines DC product Comments References

CD34+ precursors from Liquid culture Interstitial-DC-like and CD1a−CD14+ monocyte-like intermediate 76,77,79umbilical-cord blood and GM-CSF and TFN-α Langerhans-cell-like CD1a+CD14− intermediate TGFβ dependantadult bone marrow

CD34+ precursors from Semi-solid colony-formation Mature DCs Segregation of DC precursors frombone marrow culture granulocyte/macrophage precursors

GM-CSF and TFN-α; 75pre-expansion with c-kit ligand

CD34+Lin−CD10+ Liquid culture Mature and immature Equivalent of murine lymphoid-derived DCs 81lymphoid-restricted Mixture of nine cytokines DCs with a range ofprecursors from morphologiesbone marrow

Blood monocytes (pDC1) Liquid culture Immature DCs and Most common procedure for generating DCs 82–84GM-CSF and IL-4; maturation mature DC1 for immunotherapywith TFN-α

Blood monocytes (pDC1) Endothelial cell layer over a Mature DCs Might model the entry of precursor DCs into 85collagen matrix. Cells first migrate tissue then the migration of DCs into lymphinto matrix. Those that then after microbial stimulationtransmigrate back across theendothelium are DCsNo exogenous cytokines

CD45RAhiCD11cloIL-3Rhi Liquid culture Distinct CD11clo mature Precursors show evidence of a lymphoid origin 86–89plasmacytoid precursors IL-3; microbial products DC2 cells(pDC2) from blood,tonsil, thymus

GM-CSF, granulocyte–macrophage colony-stimulating factor; IL, interleukin; TGF-β, transforming growth factor-β; TNF-α, tumour-necrosis-factor-α.


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rapidly produce IFN-α and IFN-β in response to theappropriate microbial stimuli, whereas monocyte pDC1cells produce TNF-α and IL-6. The pDC1 and pDC2cells express different sets of pattern-recognition recep-tors25 (TABLE 4) and show corresponding differences inreactivity to different microbial products. Monocytes(which are pDC1 cells) therefore express TLR1, TLR2,TLR4, TLR5 and TLR8, and respond to the appropriatemicrobial ligands, including peptidoglycan, lipoteichoicacid and LPS, whereas pDC2 cells express TLR7 andTLR9, and respond to CpG oligonucleotides. TheCD11c+ iDCs found in blood express yet another set ofTLRs, and their corresponding reactivity differs. In par-ticular, they selectively express TLR3 and, accordingly,selectively respond to double-stranded RNA25,95. Theexpression of these TLRs is downregulated as the pDCsdevelop into mature DCs, in accordance with a shift infunction from reactivity to microbial stimuli to interac-tion with T cells. These differences in pDC expression ofthe primitive TLRs might be pointers to the evolutionaryorigin of the different DC sublineages.

The mature DCs generated from these precursors inculture also show functional specialization. CD154-acti-vated DC1 cells were found to prime T cells to produce aT

H1 response, whereas CD154-activated DC2 cells

induced a TH

2 response86. The ability of DC1 cells toinduce a T

H1 response was associated with a high level

of production of IL-12 p70, in response to the appropri-ate stimuli. This result has led to considerable contro-versy, first because of contradictory results with DCsgenerated from human cell cultures and, second,because of an apparent discrepancy with the results formouse DCs. First, Cella et al.96 isolated plasmacytoidpDCs using different markers and found that their DC2

producing class I interferons. They express intracellularbut not surface MHC II, similar to iDCs. When culturedwith IL-3 and CD154, or with microbial stimuli such asbacterial CpG or human herpes simplex virus, theybecome mature DCs without apparent proliferativeexpansion. The progeny DCs, termed DC2, express lowlevels of CD11c and lack typical myeloid markers, liketheir precursors, but otherwise display the characteristicsof mature DCs86,87. It is not clear which, if any, of themature DC subtypes in human lymphoid tissue corre-spond to DC2 cells, but a similar plasmacytoid pDC2population can be isolated from mouse blood and lym-phoid tissue, and transformed to DC2 cells in culture.The production of DC2 cells in vivo might require amicrobial infection.

Functional specialization. As with mouse DC subtypes,the functional plasticity of a given human DC subtypewhen exposed to different cytokines or pathogensmakes it difficult to ascribe a fixed function to a particu-lar DC lineage. For example, the ability of humanmonocyte-derived DC1 cells to produce IL-12 and so todirect a T

H1 rather than a T

H2 response varies with the

conditions of DC generation and stimulation90,91.Exposure of such monocyte-derived DCs to differentpathogens has been shown to cause extensive changes ingene-expression patterns, emphasizing the extent of DCadaptability92.

Despite this plasticity, each human DC subtype doesseem to have a different functional bias. The evidencefor this comes mainly from studies on the DC1 andDC2 populations generated from precursors in cul-ture93,94. The precursors themselves show differences intheir biological responses. The plasmacytoid pDC2 cells

PRE-T-CELL RECEPTOR-α

The form of T-cell receptor-αthat is expressed at early stagesof T-cell development.It temporarily substitutes forthe mature T-cell receptor-α-chain during a developmentalcheckpoint.

Table 4 | Human dendritic cell (DC) precursors

pDC1 (monocytes) pDC2 (plasmacytoid interferon producers)

Markers

CD11c + –CD11b + –CD14 + –pTα – +CD4 + +++CD45RA – ++CD45RO + –IL-3 receptor + +++GM-CSF receptor ++ +

Pattern-recognition receptors

BDCA2 – +TLR1 ++ +TLR2 ++ –TLR3 – –TLR4 ++ –TLR5 ++ –TLR6 + +TLR7 – ++TLR8 ++ –TLR9 – ++TLR10 – +/–

Function

IFN-α/β production + +++Phagocytosis ++ –

BDCA2, a C-type lectin; IFN, interferon; pTα, pre-T-cell receptor-α; TLR, Toll-like receptor.


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had similar characteristics. It is also clear that the DCsystems of the two species are not only heterogeneous insubtype and developmental origin but are also dynamicand rapidly influenced by microbial products and otherdanger signals.

Tolerance versus immunityThe dynamics of such a flexible and responsive DC sys-tem might determine the balance between immunityand tolerance. Each mature DC in an uninfected indi-vidual has a short lifespan58, so a changeover of thetypes of DC interacting with T cells in lymphoid tissuecan be rapid. In an uninfected individual, and especiallyin the artificial conditions of laboratory mice, most DCsare likely to be presenting self-antigens and, if interactingwith T cells, to be engaged in promoting tolerance toself-components. Some DC lineages develop to a maturebut quiescent stage under these conditions, driven byendogenous factors including cytokines. These DCsmight mediate self-tolerance. Other DC lineages remainat a pDC stage, not resembling DCs at all and unable tointeract with T cells. The input of danger signals, partic-ularly the products of microbial invasion, would rapidlychange the balance of the DC populations in three ways.First, DCs not initially in contact with T cells would besignalled to migrate to the T-cell areas of lymphoidorgans. Second, in some DC lineages, this would signal aswitch from a quiescent to an activated state, corre-sponding to a shift from tolerance-inducing to immuno-genic DCs. Third, we introduce the new concept that, inDC lineages in which the development of DCs does notoccur at all without exogenous microbial stimuli, a waveof new immunogenic DCs of the DC1 or DC2 subtypeswould develop from precursor cells and displace thetolerogenic DC subtypes.

This model involves exogenous stimuli that changethe type of DC that interacts with T cells from one thatinduces steady-state self-tolerance to one that inducesthe generation of immature effector cells. This mightrequire a shift in the type of signal transmitted from T cells to DCs (a new immunogenic signal overridingtolerance) or might simply cause a shift in signal inten-sity (weak signals produce tolerance, stronger or moreprolonged signals produce immunity). Whether or notthis proves to be a valid picture of the functioning ofthe DC network, it is clear that we must move fromconsidering DC subtypes as being static elements inhealthy individuals to considering the dynamic behav-iour of the entire DC system in response to infectionsor pathological states.

preparation, in contrast to the results of Rissoan et al.86,produced IL-12 and induced a T

H1 response in response

to CD154 and LPS. This fitted with the concept of DCfunctional plasticity, whereby most DCs have somepotential to produce IL-12 (different isolation and stim-ulation conditions, leading to different results). However,recent studies25 now indicate that the production of IL-12 in response to LPS by this DC2 preparation was dueto a separate DC1-like contaminant, because theresponse to LPS requires TLR4 and this is not normallyexpressed by the plasmacytoid DC2 precursor. Theredoes seem to be substantial lineage specialization if theDC lineages are clearly segregated.

The second discrepancy was that freshly isolatedmouse CD8+ DCs, originally thought to be equivalentto the human DC2 population rather than the DC1population, are the most efficient producers of IL-12p70. It is not appropriate to compare DCs freshly iso-lated from uninfected mice with culture-generatedhuman DC2 cells but, nevertheless, CD8+ DCs gener-ated in culture from mouse plasmacytoid precursors(mouse DC2 cells) are also moderate producers of IL-12 p70 (M. O’Keeffe, personal communication) andso there might well be a species difference. No suchdifference was apparent, however, when DC subtypeswere compared that had been freshly isolated fromhuman and mouse thymus73. In both cases, the mainthymic DC population (CD11b− for human DCs andCD11b−CD8+ for mouse DCs) produced much moreIL-12 p70 when appropriately stimulated than did theCD11b+ DC subtypes.

Of mice and menIn this review of the mouse and human DC popula-tions, we have emphasized the basic similarities betweenthe two systems, despite differences in some surfacemarkers and cytokine production patterns. Most appar-ent discrepancies have reflected the differences betweenculture-generated and freshly isolated DCs or betweenblood versus lymphoid-tissue DCs, rather than funda-mental species differences. Culture-generated DCsmight be more like the immunogenic type of DC that isgenerated in vivo in response to microbial infections,whereas DCs freshly isolated from uninfected micemight be involved in the steady-state maintenance ofself-tolerance. Direct comparisons between the twomight therefore be misleading. When mature DCsfreshly isolated from the same lymphoid organ werecompared, or when pDC subtypes were isolated fromthe blood of both species and matured in culture, they

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AcknowledgementsWe thank M. O’Keeffe, E. Maraskovsky and W. Heath for their sug-gestions, and A. Alafaci for preparing the manuscript.

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DATABASESThe following terms in this article are linked online to:LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/CD1a | CD4 | CD8 | CD8α | CD9 | CD11b | CD11c | CD14 | CD34 |CD40 | CD45 | CD68 | CD80 | CD86 | CD154 | CD205 | c-kit |coagulation factor XIIIa | E-cadherin | GM-CSF | GR-1 | IFN-γ | IL-3 |IL-4 | IL-6 | IL-18 | langerin | L-selectin | Ly6C | Notch1 | TLR1 |TLR2 | TLR3 | TLR4 | TLR5 | TLR7 | TLR8 | TLR9 | TNF-αAccess to this interactive links box is free online.

Mouse and human dendritic cell subtypes

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