Dendritic cells in a mature age

Dendritic cells (DCs) have a role in both the priming of adaptive immune responses and the induction of self-tolerance. Two impor-tant properties of DCs underlie their ability to exert these activities. First, the antigen sampling and migratory capacities of DCs effectively allow naive T cells to come into contact with peripheral antigens that they would otherwise not have encountered. Second, the ability of DCs to sense and translate environmental cues dictates, to a large extent, the fate of T cells that respond to such antigens. The mechanisms that are used by DCs to translate their environment for the benefit of lymphocytes are embodied in the concept of DC maturation proposed by Steinman and colleagues. This states that DCs can exist in two functional states, immature and mature, with only a mature DC having the ability to prime an immune response1,2. However, the term mature is also commonly used by other investigators as a phenotypic rather than a functional description, for example to denote DCs expressing high cell-surface levels of MHC molecules, CD40, CD80, CD83 and CD86. Because expression of these molecules often correlates with T-cell-priming ability, it is generally assumed that DCs that are mature by pheno typic criteria are also functionally mature, that is, immunogenic. However, this has been put in question by recent observations that pheno-typically mature DCs do not always promote

T-cell immunity and can, in fact, induce toler-ance. Here, I argue that although the concept of DC maturation has been crucial in shaping contemporary immunology, it has also been misunderstood. This has led to some confus-ing terminology, which, unless revised, could hinder further progress and the translation of basic DC research into immunotherapy.

Classical model of DC maturationThe superior ability of DCs to stimulate T cells became clear in the late 1970s and early 1980s, from studies that compared the ability of DCs, B cells and macrophages to stimulate a primary mixed leukocyte reaction (MLR)3–6. For example, Inaba et al. showed that only mouse splenic DCs and, to a lesser extent, B-cell blasts, but not rest-ing B cells or interferon-γ (IFNγ)-treated macrophages, could induce substantial levels of allogeneic T-cell proliferation6. However, when the T-cell blasts that developed in these MLRs were re-stimulated, the antigen-presenting cells (APCs) used in the initial stimulation all promoted proliferation6. This indicated a previously unappreciated functional distinction between the require-ments for priming quiescent T cells and those for re-stimulating activated T cells. This led to the idea that DCs initiate de novo immune responses, whereas other APCs (that is, B cells and macrophages) participate in amplifying those responses.

This conceptual separation between antigen presentation and ‘immunogenicity’ became increasingly apparent in the late 1980s, when it was realized that T-cell-stimulatory capacity is not a constitutive property of DCs. Schuler and Steinman7 discovered that although freshly isolated Langerhans cells (LCs) — a type of DC found in the epidermis and mucosa — expressed high levels of MHC class II molecules, they were poor stimulators of T-cell proliferation. By contrast, LCs purified from epidermal cell suspensions that were cultured for 2 days in vitro were exceedingly potent T-cell stimu-lators in the same assays7. Subsequent work by Romani et al. showed that cultured LCs were less efficient than freshly isolated LCs at pro-cessing intact antigens, formally separating T-cell stimulation from antigen acquisition and processing8. This was further confirmed by showing that cultured LCs could stimulate T-cell proliferation more efficiently than freshly isolated LCs, even when freshly isolated LCs presented an excess of CD3-specific antibody9. Overall, these observa-tions led to the concept that antigen-presenting DCs can exist in two states, ‘off ’ (immature) or ‘on’ (mature), with only mature DCs able to drive T-cell clonal expansion and prime immune responses.

Studies by Austyn et al. showed that DCs can migrate from the blood to the spleen10 and that LCs emigrate from skin transplants and explants, maturing in the process11 and losing their capacity to phago-cytose particulate antigens12. Taken together, these observations led to the view that imma-ture DCs residing in the periphery captured and processed antigens, after which they migrated to secondary lymphoid tissues and gave rise to mature DCs that had downregu-lated antigen-sampling functions but were exceedingly powerful at priming T cells (see REF. 13 and BOX 1 for some common miscon-ceptions of this model). Phenotypic analysis was consistent with this dichotomy between immature and mature DCs; compared with freshly isolated LCs, cultured LCs express lower levels of receptors involved in antigen uptake (such as receptors for IgG (FcγRs)) but higher levels of molecules necessary for T-cell priming (including MHC molecules, the integrin lymphocyte function-associated

E S S AY

Dendritic cells in a mature ageCaetano Reis e Sousa

Abstract | A common view supposes that dendritic cells (DCs) exist in two basic functional states: immature DCs induce tolerance to self, whereas mature DCs induce immunity to foreign antigens. However, the term ‘mature’ is often used not only functionally to designate immunogenic DCs but also as a phenotypic description of DCs expressing high levels of MHC, adhesion and co-stimulatory molecules. The recent realization that DCs can express such markers under non-immunogenic conditions raises the question of whether the two connotations of the term ‘mature’ should continue to be used interchangeably. Here, I discuss the origins of the maturation model and how terminology is evolving to better accommodate our current understanding of the function of DCs.

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PERSPECTIVES

© 2006 Nature Publishing Group

antigen 1 (LFA1), and the co-stimulatory molecules CD80 and CD86)7,14,15. These phenotypic distinctions have since been extended to other mouse and human DCs, and high levels of MHC, adhesion and co-stimulatory molecules are now widely considered to be markers of DC maturation and, by inference (albeit wrongly), as markers for immunogenic DCs.

Experiments using dissociated epidermal cell suspensions or skin explants did not dis-criminate whether maturation was an induc-ible or spontaneous process7,11,16. However, the advent of culture systems in which DCs could be grown from progenitors and main-tained in a relatively quiescent state allowed careful dissection of the signals involved in DC maturation. It was then shown that addition of inflammatory cytokines (such as tumour-necrosis factor (TNF)), microbial products (such as lipopolysaccharide (LPS)) or CD40 ligand (CD40L) could induce an acute decrease in antigen uptake, concomi-tant with the upregulation of maturation markers and an increase in the ability to stimulate T cells17,18. These observations were confirmed in vivo, when injection of LPS was shown to similarly trigger features of DC maturation19,20. Therefore, it became clear that DC maturation is regulated by exogenous signals.

The observation that DC maturation is inducible fitted nicely with theories about innate regulation of adaptive immunity, including Janeway’s ‘stranger’ model21 and Matzinger’s ‘danger’ model22, which stated that environmental signals control APC delivery of ‘signal 2’ (BOX 2). This helped to integrate DC research with lymphocyte biol-ogy, leading to the paradigm that is currently found in many textbooks: immature DCs

in the periphery receive maturation signals in the form of microbial patterns, danger signals or inflammatory cytokines, and these signals promote migration to secondary lymphoid tissues and DC transition from an antigen-sampling mode to a mature APC that can prime T cells (FIG. 1). More recently, several observations have indicated that the antigen uptake and processing functions of immature DCs are actually transiently increased by maturation signals23–29. In other words, exposure of immature DCs to a matu-ration stimulus leads to an initial upregula-tion of antigen sampling that is followed by a rapid shutdown of this function and transition to the mature immunostimulatory state (FIG. 1).

This paradigm of DC function has been a guiding force in immunology research for the past 15 years. Below, I discuss how, with few modifications, it can explain the main features of adaptive immunity and tolerance as we know them today. However, before proceed-ing, I want to point out two important issues. First, there are many misconceptions regard-ing the maturation model, some of which are listed in BOX 1. These misconceptions do not form part of the model as originally proposed by Steinman and colleagues (discussed ear-lier), and do not invalidate it in any way, but they have led to a substantial degree of confu-sion that needs to be dispelled. Second, much of the model derives from in vitro observa-tions using a limited number of DC types and, given the heterogeneity of DCs and the differences between in vitro and in vivo mod-els, it is possible that not all DC populations behave in vivo strictly as per the maturation paradigm30. For example, in several studies LCs migrate to lymph nodes but do not seem to directly present antigens for priming T-cell responses31–33 and might, instead, deliver antigens to lymph-node-resident DCs34. In addition, it is unclear whether all the putative maturation signals defined in vitro have qualitatively similar effects in vivo, and which signals truly initiate DC maturation, as opposed to acting in secondary amplification loops35. Finally, the efficiency with which immature DCs can process exogenous anti-gens for MHC-class-II-mediated presentation in the absence of any stimulus in vivo remains the subject of debate and might vary depending on the DC subset analysed23,24,36.

Box 1 | Misconceptions of the dendritic-cell-maturation paradigm

Four common misconceptions of the dendritic cell (DC)-maturation paradigm are stated here and the evidence indicating that these statements are misconceptions is discussed.

DC maturation accompanies migration. DCs continuously arrive in secondary lymphoid tissues from the periphery bearing fragments of apoptotic cells93. These cells can stimulate allogeneic T cells but do not induce autoimmune T-cell responses93; instead they promote tolerance94.

DCs migrate from the periphery to secondary lymphoid tissues; therefore, DCs in secondary lymphoid tissues are the progeny of those in the periphery. Most DCs resident in the spleen and half the DCs in lymph nodes seem to be derived from a blood-borne progenitor13. In the steady state, only a fraction of DCs in lymph nodes are derived from cells previously resident in peripheral tissues13.

DCs in secondary lymphoid tissues are mature. Many DCs in secondary lymphoid tissues, especially those derived from blood-borne progenitors, are in an immature state30,95. Notably, these cells are not devoid of migratory activity: in the spleen of ‘clean’ mice, they are more prevalent in the marginal zone and red pulp, and migrate to the T-cell area after exposure to maturation stimuli20,96,97.

DCs that express maturation markers are immunogenic. In several instances such cells have been shown not to prime T-cell responses and instead to induce tolerance50,66,67,70.

Box 2 | Signal 1, signal 2 and signal 3

Signal 1, signal 2 and signal 3 are the signals delivered by an antigen-presenting cell (APC) that are thought to determine the fate of naive T cells98. Signal 1 is delivered through the T-cell receptor (TCR) when it engages an appropriate peptide–MHC complex. Signal 1 alone is thought to promote naive T-cell inactivation by anergy, deletion or co-option into a regulatory cell fate, thereby leading to ‘tolerance’. Signal 2 is referred to as ‘co-stimulation’ and is taken to mean an accessory signal(s) that, together with signal 1, induces ‘immunity’. This is often measured as T-cell clonal expansion, differentiation into effector cells and a long-term increase in precursor frequency (‘memory’)74. Signal 2 is often equated with signalling through CD28 when it engages CD80 and/or CD86 (REF.72). However, the actual ‘signal 2’ that favours immunity is likely to be a fine balance of positive and negative co-stimulatory signals emanating from many receptors98. Signal 3 is a recent addition to the terminology and refers to signals delivered from the APC to the T cell that determine its differentiation into an effector cell (for example, differentiation into T helper 1 (TH1) cells, TH2 cells or cytotoxic T lymphocytes (CTLs))49,99. Interleukin-12 (IL-12) is an example of a mediator that delivers a signal 3 that can promote TH1-cell or CTL development100. The signal 3 for TH2-cell development could be a Notch ligand44,101.

Strictly speaking, the ‘signal’ refers to what happens biochemically to the T cell. However, many people commonly use the terminology to refer to the APC ligands that deliver such signals. Therefore, in common parlance, CD80 and CD86 are referred to as ‘signal 2’ and IL-12 as ‘signal 3’ rather than as ‘the APC-derived mediators that deliver signal 2 or signal 3’. There is a danger in this terminology: even if CD28 is a receptor that can transmit ‘signal 2’, its ligands CD80 and CD86 might not always deliver ‘signal 2’ as they can also engage cytotoxic T-lymphocyte antigen 4 (CTLA4) and deliver tolerogenic signals.

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Antigen captureand antigen-processing abilityor immunogenicity

Immature DC: does not providesignals to prime T-cell responses

Mature DC: provides signal 1and signal 2 to prime T-cell responses

Time

Antigen captureand antigen-processing ability

Immunogenicity

Maturationstimulus

DC maturation and the class of immunityMuch has been written about the ability of DCs to direct different classes of immu-nity37,38 and this is only briefly summarized here. It is now clear that cytokines such as interleukin-12 (IL-12), IL-18 and IFNα, which are produced by DCs, can bias CD4+ T-cell priming towards a pro-inflammatory T helper 1 (TH1)-cell fate. These cytokines can act directly on newly activated T cells and, indeed, their production by DCs is often amplified by positive-feedback signals that are provided by the differentiating T cells35,39. These cytokines can also activate natural killer (NK) cells, which produce IFNγ and indirectly promote the same type of immu-nity40. It is also thought that DCs can promote TH2-cell responses41–43, perhaps by selectively expressing members of the Jagged family of Notch ligands44. In addition, DCs have been implicated in the induction of CD4+ T-cell differentiation into alternative cell fates, including regulatory cells45 or the newly discovered IL-17-producing CD4+ T cells46.

In this article, the point of interest is not so much how DCs instruct different modes of CD4+ T-cell differentiation, but how this can be reconciled with a linear maturation model in which DCs are either on (mature)

or off (immature). One solution is to invoke different DC subsets, each specialized to induce a different class of immune response after maturation47. This proposal is linear and therefore true to the original model of DC maturation (FIG. 2a). Alternatives include the temporal model (FIG. 2b), in which an immature DC first gives rise to a mature DC that makes high levels of IL-12 and induces TH1-cell responses, and then further matures into a DC that is incapable of producing IL-12 and that promotes TH2-cell priming48; or the flexible model (FIG. 2c), in which an immature DC can give rise to either a mature DC that can induce TH1-cell responses or one favouring TH2-cell priming38,49. The temporal and flexible models of DC maturation mark a subtle, but important, departure from the earlier linear model of DC maturation in which DCs were either immature or mature. Both models imply that there can be distinct types of mature DC, which are defined by their ability to deliver distinctive ‘signal 3s’ (BOX 1). These signals direct the class of effector T-cell response and are, therefore, an integral determinant of immunity50.

The need to invoke different types of mature DC is also apparent when one con-siders the priming of CD8+ T cells. Mature

DCs can prime CD4+ T cells but can also receive help and then become ‘licensed’ to prime (helper-dependent) cytotoxic T lymphocyte (CTL) responses51. To account for all these observations, most researchers, including those who first proposed it, now accept a slightly modified form of the original model of DC maturation in which mature immunogenic DCs can induce TH1-cell dif-ferentiation, TH2-cell differentiation and/or CTL priming, depending on the nature of the maturation signal they received, as well as the constraints imposed by ontogeny and/or environmental modifiers (FIG. 3).

DC maturation and peripheral toleranceThere is now ample experimental evidence that DCs in the steady state — that is, in the absence of deliberate exposure to maturation signals — can tolerize peripheral CD4+ and CD8+ T cells by inducing deletion, anergy or regulation, depending on the model system studied2,52. It is thought that this represents an important physiological process designed to purge the peripheral T-cell repertoire of those autoreactive T cells that escaped thymic deletion and that might otherwise be activated by immunogenic mature DCs co-presenting self and foreign antigens during an infection2. However, it should also be noted that antigen presentation by steady-state DCs need not result in T-cell inactivation53 and, in some instances, can result in immunity54,55.

Given that many peripheral antigens are now known to promote central tolerance through promiscuous gene expression in thymic medullary epithelial cells, a process that is regulated by the autoimmune regulator (AIRE)56,57, one might question whether there is a need for peripheral tolerance. Indeed, AIRE deficiency results in an autoimmune syndrome58, albeit with variable penetrance59, indicating that peripheral tolerance mecha-nisms cannot entirely compensate for inef-ficiencies in central tolerance. Nevertheless, it is clear that negative selection in the thymus is not sufficient to eliminate all potentially pathogenic autoreactive T cells, because mice in which the development or the action of regulatory T cells is compromised often develop autoimmunity60. Peripheral tolerance induced by DCs in the steady state might, therefore, contribute to limiting autoimmunity.

Independent of its physiological sig-nificance, if antigen presentation by DCs in the steady state can induce tolerance, what should these tolerogenic DCs be called? According to the maturation model, only DCs that prime immune responses are

Figure 1 | The classical dendritic-cell-maturation paradigm. Initially derived from studies of Langerhans cells (LCs), this model states that dendritic cells (DCs) in the steady state are immature antigen-presenting cells (APCs) that can internalize exogenous antigens and process them for MHC-class-II-mediated presentation but cannot prime immune responses. Maturation induced by extrane-ous signals leads to an increase in immunogenicity and downregulation of antigen acquisition and antigen-processing ability. In the case of LCs, maturation can be accompanied by migration of the cells from the skin or mucosa to the draining lymph nodes. However, immature DCs are found both inside and outside secondary lymphoid tissues, and DC maturation need not be coupled to migration (BOX 1). For this reason, the localization of immature and mature DCs is deliberately omitted from this figure. Studies using truly immature DCs in vitro have shown that they possess little antigen acquisition and MHC-class-II presenting ability until they receive a maturation signal. As depicted here, this argues that, at least for some DC populations, antigen sampling might not be a constitutive process.




MHC class II

TCRNaiveT cell

NaiveT cell

TH1 cell

ImmatureTH1-cell-inducing DC

Mature TH1-cell-inducing DC


Maturation stimulusa

TH2 cell

TH1 cell

TH2 cell

TH2 cell

ImmatureTH2-cell-inducing DC


Maturation stimulus

b



NaiveT cell

TH1 cell

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c


termed mature. Therefore, tolerogenic DCs have been termed immature DCs2. This represents a revision of the original model (FIG. 1), in which antigen presentation by immature DCs had no role in determining T-cell fate. An updated version of the matura-tion model therefore proposes that DCs exist in the steady state in an immature form in which they can capture, process and present antigen to naive T cells, inducing deletion, anergy or differentiation into regulatory T cells2 (FIG. 3). Crucially, in this model, tolerance induction is a default pathway not requiring signals from the environment.

Re-phrasing the above model in ‘signal 1 and signal 2’ terminology: an immature DC delivers signal 1 but lacks the ability to deliver signal 2, resulting in tolerance;

and a mature DC delivers both signal 1 and signal 2 and induces immunity. Therefore, if tolerization by immature DCs is to solve the crucial issue of how to limit the priming of autoreactive T cells by DCs during infec-tion, immature DCs must be able to deliver signal 1 with the same efficiency as mature DCs. However, it remains unclear whether this is the case. Immature DCs express lower amounts of MHC and adhesion molecules, are not very motile and do not form con-jugates with T cells nearly as efficiently as mature DCs61. In addition, DCs matured by contact with a pathogen produce factors that make the responding T cells resistant to the suppressive action of regulatory T cells62. Therefore, autoreactive T cells would not only ‘see’ self-antigens as agonists more

efficiently when presented by mature DCs but would also potentially escape regula-tion. A related issue arises from the already mentioned fact that truly immature DCs, at least in vitro, do not efficiently capture or present antigens, so it is difficult to understand how they can present at the cell-surface peptide–MHC complexes at the levels necessary for delivery of a signal 1 comparable in strength to the signal 1 delivered by mature DCs.

A solution to these problems has been to invoke a specialized DC subset that is dedicated to tolerance63 or to suggest that, contrary to immature DCs in vitro, imma-ture DCs in secondary lymphoid tissues are unique in being able to process and present antigens efficiently without exposure to maturation stimuli2,36,64. However, DCs solely involved in tolerance remain to be identified and DCs in secondary lymphoid tissues still present exogenous antigens on MHC class II molecules more efficiently after receiving a maturation signal25,36,65. It therefore remains possible that immature DCs can tolerize high-affinity T cells (including those used in many experimental models) but spare low-affinity clones, which are the ones most likely to have escaped thymic deletion. If antigen presentation by immature DCs is the primary mechanism for peripheral-tolerance induction, such autoreactive low-affinity clones would per-sist in the repertoire and could potentially be activated during infection by mature DCs delivering higher levels of signal 1 together with signal 2.

Problems with terminologyA further problem with proposing that tolerogenic DCs are immature DCs arises when the characteristics of the former are examined. Unlike immature DCs, tolero-genic DCs can express substantial levels of co-stimulatory molecules and other matu-ration markers66,67. In fact, the expression of co-stimulatory molecules is often necessary for tolerance induction68,69 and, in some experimental systems, truly immature DCs that lack expression of ‘maturation markers’ are ignored by T cells rather than inducing tolerance66. This dichotomy between the expression of maturation markers and DC function is also apparent in the fact that high levels of expression of CD40, CD80, CD86 and MHC molecules are not predic-tive of an immunogenic DC. For example, co-stimulation-competent CD40-deficient DCs are unable to prime CD4+ or CD8+ T-cell responses70. Similarly, DCs exposed to endogenous levels of inflammatory

Figure 2 | Models of dendritic-cell maturation and the induction of different T-helper-cell fates. At least three models have been proposed to explain the ability of dendritic cells (DCs) to direct distinct T helper (TH)-cell responses. a | In the first model, distinct DC subsets are onto-genetically programmed to differentiate into mature TH1-cell- or TH2-cell-inducing DCs in response to environmental signals. b | In the second model, DCs mature sequentially: first into a mature TH1-cell-inducing DC and then into a mature TH2-cell-inducing DC. c | In the third model, a single DC subset can give rise to distinct types of mature DC that favour TH1-cell or TH2-cell differentia-tion. The quality of the maturational signal dictates which type of mature DC is generated. There is evidence in favour of all three models and many investigators believe that a combination of all three might regulate CD4+ T-cell immunity.




Immature DC:provides signal 1and only inducesT-cell tolerance

Time

Antigen captureand antigen-processing ability,tolerogenicity,CD4+ TH-cell-priming or CTLpriming

Maturationstimulus

?

Licensing by CD4+ TH cells

Mature DC:provides signal 1 andsignal 2, and primesCD4+ T-cell responses

Mature, licensed DC:induces CTL priming

Antigen capture andantigen processing ability Tolerogenicity CTL primingCD4+ TH-cell priming

mediators in vivo are unable to prime a CD4+ T-cell response but show conven-tional features of maturation, including the ability to stimulate naive T-cell prolifera-tion as efficiently as immunogenic DCs50. Notably, descendants of LCs found in lymph nodes in the steady state express maturation markers at levels comparable to those expressed under inflammatory condi-tions71. With the caveat that LCs could be constitutively immunogenic54,55, this would again indicate that the phenotype of a non-immunogenic DC can resemble that of an immunogenic DC.

In summary, using terminology that equates maturation with immunogenic-ity, we are forced to say that functionally immature DCs can express maturation markers and that DCs expressing maturation markers are not necessarily functionally mature. This is semantically unsatisfactory and necessitates that we either abandon the notion of maturation markers as identifiers of immunogenic DCs or modify the concept of DC maturation as the crucial switch between tolerance induction and immunity.

A terminology based on effector functionIs it possible to refine the concept of maturation markers so that they exclusively define immunogenic DCs? The two-signal paradigm predicts that there should be a qualitatively distinct ‘signal 2’ that switches DCs from tolerance to immunity (BOX 2). If this existed, the molecule(s) that deliv-ered signal 2 would be the true marker of DC maturation. Unfortunately, no such marker(s) has been found. Expression of CD80 and CD86 by APCs is crucial for delivering the co-stimulatory signals through CD28 that promote T-cell sur-vival, metabolic competence, cell-cycle progression and IL2 mRNA stabilization72. However, the same molecules also engage cytotoxic T-lymphocyte antigen 4 (CTLA4), a negative regulator of T-cell activation, and do not function as markers of immunogenic DCs. Similarly, OX40 ligand (also known as CD134), 4-1BB ligand and CD70 all contribute to immunity but can also have a dampening effect on T-cell activation73 and do not define an immunogenic DC. The truth is that we do not know of any set of molecules that is uniquely expressed by

immunogenic DCs and that functions as the qualitative basis of their immunogenicity.

Although such molecules might still be found, one should perhaps face up to the possibility that there might be no such thing as ‘signal 2’ in the original sense of the term (BOX 2). In adoptive transfer experiments, monoclonal T-cell populations have been observed to clonally expand and subse-quently contract under both tolerogenic and immunogenic conditions74. The crucial difference between deletional tolerance and immunity seems to be in the number of cells that survive the contraction phase, an event that might be decided by late and/or persistent exposure to antigen rather than by early exposure to a particular type of DC75,76. Furthermore, direct observations in mice have generated conflicting results regarding whether early interactions between T cells and DCs differ under immunogenic and tolerogenic conditions77,78. Ultimately, the distinction between an immunogenic and a tolerogenic DC might be in subtle quantita-tive differences in the expression of various known molecules, or even in DC longevity79, which add up over time to determine how many T cells survive the burst of clonal expansion and make it into the memory T-cell pool. If this is the case, it will be impos-sible to find markers that unambiguously define immunogenic DCs.

Does this mean that the expression ‘maturation markers’ ought to be abandoned altogether? Many researchers feel it is acceptable to perpetuate the tradition of using this expression in a phenotypic sense to refer to markers that are highly expressed by cultured LCs or in vitro cultured DCs exposed to maturation stimuli, irrespective of whether such cells are immunogenic. This corruption of the original meaning of ‘maturation’ might be irksome to those who have always been careful to use the term in its intended form but would be consistent with current usage by most of the immu-nological community13. Indeed, my own laboratory has been guilty of using the term in this sense35,50, as have others before us who introduced the concept of ‘semi-mature’ or ‘mature’ tolerogenic DCs66,67,80.

How then should one refer to DCs that induce immunity, tolerance or other responses? Throughout this article I have used the terms immunogenic DC or tolerogenic DC to refer to their properties. Others have used the term ‘licensed’ DC for immunogenic DCs13, and the terms DC1, DC2 and DCreg to refer to DCs that can induce TH1 cells, TH2 cells and regulatory T cells, respectively37. The exact terminology

Figure 3 | A dendritic-cell-maturation model and the induction of tolerance. This model of dendritic cell (DC) maturation is a refined version of that depicted in FIG. 1. It includes a step of DC ‘licensing’ by T helper (TH) cells, which allows for the induction of TH-cell-dependent cytotoxic T lym-phocyte (CTL) responses. It can also be easily coupled to any of the models depicted in FIG. 2 to provide a more comprehensive view of the role of mature DCs in the induction of distinct TH-cell responses and therefore distinct classes of adaptive immune response. However, the most important feature is that it ascribes a TH-cell-instructing function to immature DCs, namely inducing peripheral tolerance. This is difficult to reconcile with the earlier notion that efficient delivery of signal 1 by DCs necessitates a maturation signal that transiently increases antigen capture and processing. Therefore, the question mark denotes the possibility that some DC subsets in vivo might deliver signal 1 equally efficiently in the immature and mature state.




TH2 cell

Immature DC,a DC for inducingtolerance?

Mature DCfor inducingclonal expansionof naturalregulatory T cells

Mature DCfor inducing de novoregulatory T cells

Mature DCfor inducingdeletionaltolerance

Mature DCfor inducingCTL priming

MHC class ITCR

Mature DCfor inducingTH1-cellresponses

Mature DCfor inducingTH2-cell responses

NaiveCD8+

T cell

CTL

TH1 cell

NaiveT cell

Apoptotic T cell

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MHC class II Mature DCfor inducingIL-17-producingCD4+ T cells

IL-17-producingCD4+ T cells

NaiveT cell

is unimportant and it would be unwise to be overly prescriptive in a field that is domi-nated by personal preferences. The point is that, ultimately, all these terminologies define DCs empirically by what they do to T cells. In other words, there is a clear trend in the field to complement the use of the ‘maturation’ nomenclature with one that defines DCs by their ‘effector function’.

In the simplest view, DCs are ‘effector cells’ when they have the potential to inter-act with lymphocytes and regulate their function (although this does not exclude other possible effector roles for DCs, for

example, in innate immune responses81). However, ‘effector DCs’ can direct dis-tinct lymphocyte responses and should be further defined by reference to these responses. In this respect, it is illuminating to consider T cells, because so much of the effector activity of DCs seems to be focused on that leukocyte compartment. So, just as there are TH1 cells, TH2 cells and CTLs, there could be distinct DCs for TH1-cell, TH2-cell and CTL priming. Similarly, perhaps even DCs for peripheral tolerance or for the induction of regulatory T cells (FIG. 4). This open-ended view of DC

function could easily integrate future types of effector DC as they are discovered.

Is this just semantics? After all, what is the difference between a ‘mature DC for inducing TH1-cell priming’ and a ‘mature DC’? Depending on the context, the two descriptions could denote precisely the same cell. But terminology shapes thinking and the first nomenclature, although awkward, forces one to focus on DC function and thereby complements the second, which is all too often used merely as a phenotypic descrip-tion. A focus on effector activity also adds to the concept of ontogenetically distinct DC lineages. Therefore, one can allow for the fact that plasmacytoid DCs (pDCs) constitute a unique DC subset, but refer to them in different contexts: for example, as pDCs for CTL priming82,83 or as pDCs for regulating T-cell responses to inhaled antigens84. The advantage of complementing definitions of DC maturity (in terms of phenotype) with statements about effector function becomes clearest when the role of DCs in tolerance is considered. By accepting that phenotypically mature DCs can be tolerogenic, tolerogenic-ity does not need to be cited as a property of immature DCs. Therefore, the possibility that certain environmental signals can ‘mature’ DCs into a tolerogenic mode can be accepted. This would offer a solution to the issues raised earlier, by allowing for the exis-tence of tolerogenic maturation signals that allow DCs to selectively upregulate the ability to deliver signal 1 and thereby promote tolerization of the low-affinity repertoire of self-reactive T cells that is most likely to pose a problem during infection.

Is there any evidence that tolerance induc-tion by DCs in vivo requires a stimulus? It is notable that most protocols used to show tolerance induction by steady-state DCs involve delivery of potential signals, be they in the form of antibodies that crosslink cell-surface signalling molecules or Fc receptors, or as cell-associated antigens that can trigger receptors involved in the uptake of cellular material85–89. One apparent exception is the elegant model of Probst et al.69,90, in which antigen expression by steady-state DCs is acutely induced by tamoxifen administration and promotes peripheral tolerance of CD8+ T cells. Given that tamoxifen is unlikely to be acting as a signal for the generation of tolerogenic DCs, these data are generally seen as evidence for peripheral tolerance induction by DCs being a default pathway. Therefore, some populations of immature DCs might possess an intrinsic tolerogenic effector function, especially the blood-derived DCs that are present in secondary

Figure 4 | Dendritic-cell effector function. This view of dendritic cell (DC) function supposes that immature DCs can give rise to multiple types of ‘effector’ DC that instruct distinct T-cell fates, including immunity, tolerance and immune deviation. Maturation refers to the changes that accompany immature DC transition to a given effector state in response to environmental signals of exogenous (for example, microbial) or endogenous (for example, cytokines, hormones and dying cells) origin. The quality of these signals largely determines the choice of effector DC, although ontogeny can also have a role and certain DC subtypes might be predisposed towards particular effector states or have only a restricted repertoire of effector functions. DC activation might also occur spontaneously, that is, in the absence of environmental signals, automatically promoting DC transition to a given effector state after a fixed period of time. Note that this model differs from the conventional one by supposing that tolerogenic DCs can be a type of mature DC. In other words, some maturation signals can promote the generation of tolerogenic DCs. This does not negate the possibility that some immature DCs have an intrinsic tolerogenic function. CTL, cyto-toxic T lymphocyte; IL-17, interleukin-17; TCR, T-cell receptor; TH, T helper.




lymphoid tissues, and induce T-cell tolerance without the changes in phenotype indicative of maturation13. However, it is also possible that tolerogenicity in the steady state does not always indicate an intrinsic property of resting immature DCs but instead reflects the activity of an effector DC that has been rendered tolerogenic by signals from sur-rounding tissues. These signals might include immunosuppresive cytokines and apoptotic cells, all of which can give rise to tolerogenic DCs in vitro91, as well as signals that are gen-erated by certain pathogens or commensals37.

Future direction: defining the signalsIn the course of this Essay, I have tried to describe the evolution of our understand-ing of DC biology, starting from simple concepts of DC immaturity and maturation, and to show how, with time, new experi-mental evidence meant that these concepts had to be refined. I hope that I have been able to convey the notion that these modi-fications have not been wholly satisfactory and that there is currently no coherent and universal nomenclature that does full justice to the richness and diversity of DC biology. Until such a time arrives, I propose that we continue to follow the trend of defining DCs not only in terms of their phenotypic prop-erties but also in terms of their ontogeny and their effector properties. Describing DCs as ‘phenotypically immature conven-tional DCs with an effector function for tol-erance induction’, or ‘phenotypically mature pDCs with an effector function for tolerance induction’ might seem unduly cumbersome but is more informative and less confusing than referring to them simply as ‘immature’ or ‘mature’ DCs.

This proposal does not and could not represent a final resolution of the problems highlighted here. Refinement and evolution of DC nomenclature will probably come from a better definition of the many signals that are involved in generating different types of effector DC, both during infection and in the steady state (FIG. 4). This will help to determine whether there are unique patterns of gene expression that can unambiguously be used to define different effector DCs and that are predictive of their function, as postulated by ‘signal 3’ models49. Alternatively, it might lead to the conclusion that the ability of DCs to induce different T-cell fates is effectively a continuum in which tiny differences in the levels of a limited set of molecules can have a large and unpredictable impact92. Making sense of the exponential increase in information on DC heterogeneity, phenotype and function across different species and

experimental models will require better com-munication using a common, comprehensive and comprehensible language that is accept-able to the research community. The fully descriptive nomenclature, generated by defin-ing DCs not only in terms of their phenotypic properties but also in terms of their ontogeny and effector properties, discussed here, at best represents a staging post en route to a more effective terminology. Like the concept of maturation itself, it is clear that our views of DC biology will continue to mature over time.

Caetano Reis e Sousa is at the Immunobiology Laboratory, Cancer Research UK, London Research

Institute, Lincoln’s Inn Fields Laboratories, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK.

e-mail: [email protected]

doi:10.1038/nri1845Published online 8 May 2006

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AcknowledgementsI would like to thank K. Rowan for secretarial assistance. I am very grateful to M. Albert, S. Amigorena, W. Heath, M. Robertson, R. Steinman and members of my laboratory for discussions and critical review of earlier drafts of the manuscript. My expressions of gratitude should not be con-strued to mean that any of the named individuals concur with any of the views expressed in this Essay, which remains a personal and therefore biased opinion. I apologize to those colleagues whose work I have failed to cite, either through ignorance or space restrictions.

Competing interests statementThe author declares no competing financial interests.

DATABASESThe following terms in this article are linked online to:Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneCD40 | CD40L | CD80 | CD83 | CD86 | CTLA4 | IFNα | IL-12 | IL-17 | IL-18 | TNF

FURTHER INFORMATIONCaetano Reis e Sousa’s web page:http://science.cancerresearchuk.org/research/loc/london/lifch/sousac/?version=1Access to this links box is available online.




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Dendritic cells in a mature age