Pathways of development of human dendritic cells

ARTICLE

Dendritic cells (DC) are professional antigen-presenting cells necessary for the initiation of immune responses [1]. They are derived from the bone marrow and exist in trace amounts; they are characterised by a high expression of class II major histocompatibility complex (MHC) antigens and a dendritic morphology. They form a network present in: a) several zones of non-lymphoid tissues such as Langerhans cells of epithelia (skin, mucous membranes, lung) and interstitial DC of the heart, kidney and other organs, b) blood and lymph, including veiled cells of afferent lymphatics and blood DC, c) the T cell-rich zone of secondary lymphoid tissues, the so-called interdigitating cells (IDC), d) the medulla of the thymus, and e) B-follicles of secondary lymphoid tissue, the so-called germinal-centre DC (GCDC). Even though the relationship between these populations is not entirely understood, they are regarded as representing different steps of maturation, connected to each other by precise circulatory pathways. In peripheral tissues, DC such as Langerhans cells (LC) take up antigens (Ag); they subsequently migrate via lymphatic or blood vessels to the T cell-rich zone of secondary lymphoid tissues (Fig. 1) and present the processed Ag to naive T cells and initiate the immune response. This primary T immune response is followed by the primary extrafollicular B response, characterised by the generation of IgM-secreting plasma-cells. During their migration from the periphery to the draining lymph nodes, DC undergo phenotypic and functional changes, including loss of ability of Ag uptake and processing, and expression of accessory molecules.

Knowledge of the mechanisms of action of DC is important for the understanding of the activation pathways of T cells. This knowledge could allow an intervention in immune responses at the initiation step, i.e. negative regulation in the case of autoimmune reactions and in transplantation, or positive regulation in the case of infectious or neoplastic diseases. Furthermore, understanding DC physiology could have implications in the field of viral infections. DC are targets for viruses such as HIV, HTLV-1, measles, influenza and hepatitis, and could be involved in viral spread and general immunosuppression [2].

Whereas the bone marrow origin of DC was discovered twenty years ago, the conditions leading to their growth and differentiation were only recently defined. GM-CSF appears to be a key factor for both murine and human DC development. Since DC isolation from tissues is difficult, their generation in vitro is an important step for the understanding of DC physiology and their potential use in immunotherapy.

Generation of bone marrow-derived human DC in vitro

The expression of CD45 Ag by DC suggests their bone marrow origin; however a direct proof of this origin was obtained by reconstitution experiments following bone marrow irradiation and transplantation. Recipient DC originate from bone marrow precursors as shown by the expression of donor MHC Ag.

Recently, significant advances in generation of human and murine DC from precursor cells have been accomplished, allowing the in vitro production of large numbers of DC [3].

From peripheral blood monocytes

Ten years ago, Knight et al. [4] described monocytes having acquired, upon isolation, a dendritic and veiled aspect. It is now well known that monocytes cultured with GM-CSF plus IL-4 or IL-13 can differentiate into CD1a⁺ DC in the absence of proliferation [5, 6]. Monocyte-derived DC express a phenotype of immature DC, including weak expression of costimulatory molecules and expression of MHC Ag within intracytoplasmic compartments.

From haematopoietic CD34⁺ progenitors

In association with GM-CSF, TNF-alpha induces DC development. TNF-alpha strongly enhances the IL-3 or GM-CSF-induced proliferation of CD34⁺ haematopoietic progenitor cells (HPC) isolated from cord blood or from the bone marrow. The co-operation of TNF-alpha and GM-CSF or IL-3 is critical for the development of DC from CD34⁺ HPC in vitro. After eight days of culture in liquid medium, the addition of TNF-alpha to GM-CSF induces a 6 to 8-fold increase in DC numbers. After 12 days, 50-80% of cells express the CD1a Ag. Therefore, 10-30 x 10⁶ CD1a⁺ DC can be obtained from 10⁶ CD34⁺ HPC. Furthermore, SCF and FLT3-L increase from 3- to 6-fold the expansion of CD1a⁺. These CD1a⁺ cells are DC showing the following features: a) a typical dendritic morphology (Fig. 2), b) a strong expression of class II MHC Ag, CD4, CD40, CD54, CD58, CD80, CD83, CD86 and lack of CD64/Fc gammaRI and CD35/CR1, c) presence of Birbeck granules (BG), characteristic of LC, within 20% of cells, d) a potent capacity to stimulate proliferation of naive T cells and to present soluble Ag to clones of CD4⁺ T cells. CD1a⁺ cells are CD45RO⁺ and express the myeloid antigens CD13 and CD33. In this system, the effect of TNF-alpha on the development of DC seems to be mediated by TNFR1 [7].

In a semi-solid medium, DC form colonies with monocytes/macrophages, suggesting the existence of a common precursor [8-10].

Identification of two DC development pathways. Whereas after 12 days in culture most cells are CD1a⁺CD14^­, at an earlier stage two subsets of LC precursors characterised by the mutually exclusive expression of CD1a and CD14 develop independently [11, 12]. These two precursor types differentiate between days 12 and 14 into cells with morphological, phenotypic and functional features (stimulation of naive T cells) typical of DC. CD1a⁺ precursors differentiate into DC with features of LC (BG, Lag, E-cadherin). Conversely, CD14⁺ precursors undergo maturation into CD1a⁺ cells lacking these features but expressing CD2, CD9, CD11b, CD68 and the coagulation factor XIIIa of dermal DC. Interestingly, only the CD14⁺ precursor subset represents bipotent cells able to differentiate under the influence of M-CSF into macrophages lacking the ability to stimulate naive T cells. It is noteworthy that DC derived from CD14⁺ precursors ­ but not from CD1a⁺ ones ­ synthesise IL-10.

Recently it was shown that in cultures with serum-free media, TGFß is necessary for the development of DC with features of LC (BG, Lag, E-cadherin) [13]. Furthermore, TGF-ß induces expression of the molecule recognised by the antibody (Ab) DCGM4, specifically recognising LC in vivo (Valladeau, manuscript in preparation).

These two pathways of development have been confirmed and further studied by other investigators. The switch towards one or the other pathway would occur at the level of CD34⁺ HPC [14]. CD34⁺ cells of peripheral blood expressing the CLA molecule (cutaneous lymphocyte
antigen) differentiate in response to GM-CSF+TNF-alpha into CD1a⁺BG⁺Lag⁺ LC, whereas CLA^­ precursors differentiate into interstitial DC CD1a⁺BG^­Lag^­ (Fig. 3).

Whereas these subsets are both able to induce proliferation of naive T cells, they nevertheless show specific activities [11]. In particular, DC deriving from CD14 precursors show a strong and long-lasting capacity of Ag uptake via the mannose receptor (dextran-FITC or peroxidase). This capacity is regulated in parallel with the expression of non-specific esterases, markers of the lysosomal compartment. Conversely, this lysosomal activity is never present in the CD1a⁺ subset. From a functional point of view, the most remarkable difference between these two subsets is the possibility to induce differentiation of naive B-cells activated by the CD40 antigen, which is restricted to the CD14 subset (see below "DC and B cells").

DC derived from CD14⁺ precursors are closely linked to monocyte-derived DC both from a phenotypic (BG^­, Lag^­, E-cadherin^­, CD11b⁺, CD68⁺, fact. XIIIa⁺) and functional (efficient antigen uptake) point of view. Similarly, this cell population is close to blood CD11c⁺ DC, present also within tonsil B follicles.

It seems therefore that two pathways of development of DC could give rise to two DC populations: epithelial Langerhans cells, mainly involved in initiation of cellular immune responses, and monocyte-derived DC, close to blood or interstitial DC that would be preferentially involved in humoral immune responses.

Different maturation steps defined by specific functions

In vivo circulation of DC

Newly-generated DC migrate, probably via the bloodstream, from the bone marrow to non-lymphoid organs. In particular, in the lung, DC accumulate rapidly (within an hour) in bronchial epithelium after Ag inhalation (such as Moraxella catarrhalis) [15]. The precursor within the epithelium has not yet been characterised, but in vitro studies suggest that it could be a CD34⁺ precursor committed towards differentiation into LC and expressing the CLA Ag [14].

DC enter the spleen from the bloodstream. DC injected intravenously can migrate through the liver to the abdominal lymph nodes via afferent lymphatic vessels, but only a few enter lymph nodes directly from the bloodstream. Several experiments (subcutaneous Ag injection, allogeneic skin grafting, injection of labelled DC or of DC infected with Leishmania, etc.) have shown that DC enter the lymph nodes from the afferent lymphatic vessels. The local secretion of TNF-alpha in the dermis could be involved in the migration of LC from the epidermis to the lymph nodes [16]. Similarly, the injection of LPS induces migration of DC from non-lymphoid organs to the T zone of lymphoid tissues [17].

Antigen uptake

Although being professional antigen-presenting cells, the phagocytic capacity of DC is reduced compared with macrophages; they are nevertheless able to phagocytose relatively large particles, such as latex beads, apoptotic bodies, viruses, bacteria and intracellular parasites [18]. Furthermore, DC efficiently concentrate soluble extracellular substances within vacuoles through the process of macropinocytosis [19]. Ag uptake by DC may also occur via formation of immune complexes [20]. Finally, DC express membrane receptors bearing multiple lectin domains, such as the mannose receptor and the DEC-205 molecule [19, 21]. Through specific recognition of sugar moieties, these molecules take up the Ag and deliver it to intracytoplasmic compartments of class II MHC, leading to an optimal Ag presentation to CD4⁺ T cells. These surface lectins are likely to contribute to the uptake of bacterial Ag, showing a different pattern of glycosylation from that occurring in mammalian species, and could therefore act as a first-line mechanism for recognition of the "non-self" antigens.

Maturation

After Ag uptake, during their migration, DC undergo phenotypic and functional changes. Freshly-isolated LC are immature DC able to efficiently take up native proteins and to present processed peptides to memory T cells. Conversely, cultured LC and IDC of lymphoid organs are relatively inefficient at Ag uptake, although they have a noticeable capacity to activate naive T cells. In this context, epidermal LC express lower levels of class II MHC Ag and accessory molecules than cultured LC and IDC. Maturation of LC as it occurs in vitro is considered as a physiological event occurring during the in vivo migration of LC from the skin to the draining lymph nodes [22].

The mechanisms involved in this functional maturation have recently been studied by using DC generated in vitro. It has been shown that the factors known to induce migration of LC in vivo (i.e. LPS, TNF-alpha, IL-1, CD40L) also trigger their maturation [17, 19, 23]. DC activated in this way acquire a phenotype of mature cells, including typical morphology with long dendrites, loss of monocyte markers and expression of costimulatory molecules (CD58, CD80, CD86). During this maturation process, the capacity of Ag uptake is lost; furthermore, class II MHC molecules undergo a co-ordinated regulation (rapid and transitory increase in synthesis, increase of half-life, translocation to the plasma membrane) [24, 25]. These changes result in rapid accumulation of a large number of long-lasting peptide/MHC Ag complexes. These changes account for the functional differences between immature DC of non-lymphoid organs (i.e. Langerhans cells) and mature DC of lymphoid organs (i.e. interdigitating dendritic cells).

Regulation of DC migration

Although the factors triggering the in vivo migration of DC have been characterised (LPS, TNF-alpha), little is known concerning the regulation of DC migration from the bloodstream to the tissues and from there to lymphoid organs. In vitro-generated DC have recently been used to study the response of these cells to various chemokines.

In vitro-generated DC express the chemokine receptors C5aR, CCR1, CCR2, CCR5, CCR6, CXCR1, CXCR2 and CXCR4 [26]. They migrate in vitro in response to chemokines CCMCP3, MCP4, Rantes, MIP1alpha, MIP1ß, MIP5 and to the chemokine CXC SDF1 [26, 27]. Conversely, they do not respond to the following chemokines: CC eotaxin, CXC IL-8, IP-10, Groß, C lymphotactin. DC respond to C5a, formyl peptides and to PAF (platelet activated factor). DC migrate under the influence of a recently defined chemokine called MDC, produced by macrophages and DC [28]. Recently, a new receptor for the chemokines CC, CCR6 has been cloned and shown to be specifically expressed by DC derived from CD34⁺ HPC but not by monocyte-derived DC [29, 30]. A ligand of this receptor, MIP3-alpha, triggers the migration of DC derived from CD34⁺ HPC but not from monocytes C [30, personal data]. Similarly, a chemokine specifically expressed by thymic DC (TECK) has been reported to support migration of murine DC [31].

Concerning HIV infection, receptors to chemokines CCR5 and CXCR4 are involved in DC infection [32, 33].

It seems that several chemokines can trigger migration of DC in vitro. However, the migration of different DC populations (namely of immature DC from the bloodstream to a site of inflammation, or of activated DC from peripheral tissues to the draining lymph nodes) is probably regulated by different chemokines. Indeed, whereas the response to chemokines MIP1alpha, Rantes, MIP3ß is lost after maturation, recent data suggest that other chemokines are involved in the recruitment of mature DC [34].

It appears therefore that the definition of the maturation level of DC, their origin and of signals regulating secretion of various chemokines are a prerequisite to the understanding of the regulation of in vivo migration of DC.

Interaction between DC and T cells

Induction of primary T cell immune responses

By virtue of their unique ability to activate naive T cells, DC are necessary for the induction of immune responses. The in vivo ability of DC to activate naive T cells has been shown directly in animals by cell transfer experiments. Following reinjection within plantar pads or the blood, or following intrathecal injection, DC loaded in vitro with a proteinic Ag induce an antigen-specific, MHC-restricted immune response [35]. Furthermore, murine DC loaded with peptides induce in vivo a CD8 cytotoxic response leading to virus eradication or tumoural regression [36]. In vitro, DC are 100 to 300-fold more efficient in Ag presentation than other antigen-presenting cells (such as monocytes and B cells) [1, 37, 38].

DC generated in vitro from CD34⁺ HPC trigger a strong proliferation of allogeneic, naive CD4⁺ T cells and of syngeneic T cells in the presence of low amounts of superAg [39, 40]. The proliferation of allogeneic CD8⁺ T cells is weaker than that of CD4⁺ cells if cultured merely in the presence of DC, but reaches comparable levels upon addition of cytokines such as IL-2, IL-4 or IL-7. In parallel, allospecific lines of CD4⁺ or CD8⁺ cells can be generated by repeated cultures of T cells in the presence of DC, and the CD8⁺ lines are strongly cytotoxic in a MCH-restricted manner. DC generated from CD34⁺ HPC or from monocytes efficiently present soluble Ag to CD4⁺-specific T cell clones.

Significance of CD40-CD40L interaction

The CD40 Ag, which is critical for the development of T-dependant B cell responses, is also functional on other cell types [41]. In particular, the CD40 Ag is expressed by Langerhans cells, blood DC, IDC and DC generated in vitro. The CD40 Ag mediates the survival of DC and induces morphological, phenotypic and functional changes [42] (Fig. 4).

Since T cells activated by DC express CD40L, it seems likely that activation via CD40 of DC is an important physiologic interaction between DC and T cells. DC activated by CD40 express higher levels of accessory molecules (CD58, CD80, CD86) and produce cytokines (IL-10,
TNF-alpha) and chemokines. DC stimulated via this pathway activate in turn, T cells via the CD28 Ag with an optimal efficacy. Furthermore, the CD40 Ag appears as a unique trigger for IL-12 production by DC [43, 44], resulting in production of IFN-gamma by activated T cells [45]. On the other hand, some DC subsets induce T cells to synthesise IL-10 through a mechanism that is not yet entirely elucidated but that is dependent on CD40.

It is noteworthy that the engagement of CD40 induces the secretion of several cytokines. DC secrete namely MIP1a, MIP1b and IL-8, and express mRNA of several others (such as Rantes, I-309, MIP3a, MIP3b, MIP1g, MIG) [46, 47] (de Saint-Vis, manuscript in preparation). During the last year, several new chemokines specifically expressed by DC have been identified, including MDC (produced by macrophages and DC) [28], Tarc (produced mainly by DC) (de Saint-Vis, manuscript in preparation), DC-CK1 (strongly expressed by DC) [48] and Teck (produced by thymic DC of the mouse) [31]. Most of these DC-specific chemokines (Tarc, Teck, DC-CK1) trigger migration of T cells [31, 48, 49] and could be involved in the attraction of T cells in the paracortical zone of lymphoid organs during the induction of immune responses. The production of chemokines by DC upon CD40 engagement by T cells might also favour the encounter with B cells of appropriate antigenic specificity.

The engagement of CD40 Ag also induces maturation of DC as shown by induction of CD25 expression and loss of CD1a expression, mimicking the events occurring upon migration of Langerhans cells to lymphoid organs and differentiation into IDC. Furthermore, the engagement of CD40 inhibits antigen-uptake capacity, in line with the disappearance of endocytosis receptors (such as mannose receptors). The role of CD40 engagement has also been clearly shown using DC derived from monocytes [19, 23].

On the other hand, the CD40-mediated activation upregulates recently-discovered human molecules, the function of which is still unknown. In particular, CD40 engagement induces the expression of a new protease belonging to a family of enzymes cleaving TNF-alpha and cell-membrane FasL [50]. Similarly, a new lysosome-associated membrane protein homologous to CD68 and specifically expressed by DC is induced via CD40 activation and is detectable in vivo only on mature IDC of the T zone) (de Saint-Vis, manuscript in preparation). The expression of di-ubiquitin, encoded for by a gene located within the class I MHC locus, is induced via CD40 activation [51].

Conversely, in keeping with the loss of antigen-uptake capacity, CD40 activation induces the loss of recently-identified potential receptors of endocytosis (Garonne & Saeland, personal communication).

Interaction between DC and B cells

Although the primary T cell-dependant B cell response necessitates DC and occurs in the extrafollicular zone of secondary lymphoid organs, few data are so far available concerning the potential interactions between DC and B cells. In an in vitro system where T cells are mimicked by CD40L-transfected fibroblasts, DC directly regulate the growth and differentiation in B cells [52]. In particular, DC induce a 3- to 6-fold increase in B cell proliferation in the absence of exogenous cytokines. Furthermore, DC considerably increase (10- to 100-fold) the secretion of IgG, IgA and IgM by memory B cells activated by CD40, in the absence of exogenous cytokines. Interestingly, in the presence of DC, naive IgD⁺ B cells produce large amounts of IgM in response to IL-2. The latter effect is dependent on the production of soluble factors (namely IL-12) by DC, following engagement of CD40 [53]. Furthermore, DC strongly trigger proliferation of B cells of germinal centres as well as the isotypic switch of naive IgD⁺ B cells to IgA in the presence of IL-10 [54], or to IgG in the presence of IL-2 (Dubois, manuscript in preparation). These effects are restricted to the subset of DC derived from CD14⁺ precursors and from monocytes. Furthermore, GCDC isolated from tonsils, but not epidermal Langerhans cells, share these properties with in vitro generated DC. Therefore, DC derived from CD14⁺ precursors or from monocytes are phenotypically and functionally similar to GCDC. The location of GCDC within germinal centres suggests that the in vitro effects of DC on B cells could correspond to events occurring in the germinal centre reaction.

These data suggest that the subset deriving from monocytes (or from CD14⁺ precursors) close to GCDC could be involved in the regulation of humoral immune responses, whereas the subset derived from CD1a⁺ precursors would exert control on cellular immune responses.

DC and tolerance: pathway for the lymphoid development of DC

DC grown in vitro in the presence of GM-CSF are of bone marrow origin; they express the myeloid antigens CD13, CD33 and CD11c, and have a common origin with monocytes and granulocytes. In contrast, the existence of a population of DC of lymphoid origin has been suspected in the mouse.

Thymic DC of the mouse

In contrast to Langerhans cells, thymic T cells do not initiate a T immune response to a foreign antigen but rather induce death of incipient, potentially autoreactive T cells [55]. An endogenous precursor of DC has been identified in the thymus of adult mice [56]. In transfer experiments, this endogenous precursor generates DC and T cells in a ratio similar to that found in normal thymus, suggesting that it is the main, if not unique, source of thymic DC [57]. Rather than presenting foreign antigens collected at the periphery to T cells (as happens with Langerhans cells), such DC probably present self antigens produced locally.

Origin of thymic DC in the mouse

Thymic DC and a subset of DC of the spleen and lymph nodes of the mouse express markers (such as CD8a) usually associated with lymphoid cells. In experiments of in vivo transfer, the earliest T cell precursor CD4^low to be identified within the thymus of adult mice is not able to generate macrophages or granulocytes. This lymphoid precursor differentiates also into DC. The ability to differentiate into DC seems the last alternative to be lost when precursors commit themselves definitively toward the T cell lineage [58]. This suggests a close link between the development of T cells and DC in the thymus. In reconstitution experiments, this early thymic precursor generates only CD8a⁺ DC in the thymus and the spleen, whereas pluripotential bone marrow precursors produce both CD8a⁺ and CD8a^­ DC. This suggests that CD8a⁺ characterises DC linked to lymphoid lineage cells while CD8a^­ DC are those of myeloid origin.

This early thymic precursor can be induced to differentiate in culture toward DC with a cloning efficacy of 70%. Multiple cytokines are required for a maximal development of DC, including TNF-alpha, IL-1, IL-3, IL-7, SCF, FLT3-L and CD40L [59].

Possible function of thymic DC

The function of thymic DC seems different from that of bone marrow-derived DC. Lymphoid DC of the thymus are involved in the removal of potentially autoreactive T cells under development. Concerning mature T cells, lymphoid DC could be involved in the maintenance of
peripheral tolerance [60, 61]. Indeed, CD8a⁺ DC could kill CD4⁺ T cells via a mechanism of apoptosis involving Fas [62]. Furthermore, CD8a⁺ DC, contrary to CD8a^­ DC, could induce T cell proliferation without concomitant production of cytokines such as IL-2, IL-3, GM-CSF and IFN-gamma [63]. In this setting, lymphoid DC have been shown to express large amounts of class II MHC Ag/peptide complexes in the T zones of lymph nodes [64].

DC in mutant mice

Recent experiments performed in mice with inactivated Rel-B, TGFb1 or Ikaros genes highlight the different origins of DC subsets and upheld the concept of the lymphoid origin of murine DC. Rel-B^­/­ mice have epidermal Langerhans cells but have no DC in the spleen or the thymus [65, 66]; by contrast, TGFb^­/­ mice have no Langerhans cells but have CD11c⁺ DC in lymph nodes [67]. Ikaros^­/­ mice devoid of T, B and NK lymphocytes are deficient in CD11c⁺ DC in the spleen; however, epidermal Langerhans cells and all cells of myeloid lineage (granulocytes, monocytes) are unaffected [68, 69].

CD11c^­ DC precursors in the human

In human tonsils, a mucosa-associated lymphoid tissue, several DC subsets and DC precursors have been identified, including CD1a⁺ Langerhans cells of mucosal epithelium, CD40⁺CD80⁺CD83⁺CD86⁺ IDC, CD4⁺CD3^­CD11C⁺ plasmacytoid precursors of the T zone and CD4⁺CD3^­CD11c⁺ GCDC. A population of peripheral blood CD4⁺CD11c^­CD3^­ cells able to differentiate into DC has been identified [70]. These cells correspond to plasmacytoid T cells, an obscure cell type that had long been observed by pathologists in the secondary lymphoid organs. They express CD45RA but no specific markers of lymphoid or myeloid cells. In culture, they rapidly undergo apoptosis, which is readily prevented by IL-3. Addition of CD40L induces their differentiation into DC expressing very low amounts of the myeloid antigens CD13 and CD33. These cells could represent the precursors of lymphoid DC in man [71].

Possible physiological role (Fig. 5)

DC represent a family of heterogeneous cells with particular features. Their heterogeneity in terms of lineage and function has not yet been elucidated. At least three independent differentiation pathways leading to the development of various types of DC have been identified in man and mice. In man, two pathways of development leading to production of bone marrow-derived DC have been established in vitro. Langerhans cells can be generated from CD34⁺ HPC cultured with TNF-alpha and GM-CSF. Under the same culture conditions, another population of DC can be generated from CD34⁺ HPC; these cells do not show features of Langerhans cells and are probably related to monocyte-derived DC. Although these cells appear phenotypically similar to interstitial DC, blood CD11C⁺ DC and GCDC, their physiological counterpart has not yet been clearly established. Considering the effects of DC on the differentiation of naive B cells and the proliferation of germinal centre B cells, i.e. functions shared with CD14-derived DC, it is tempting to speculate that monocyte-derived DC are related to CD11c⁺ DC localised in the B follicles of the tonsil. Therefore these two pathways of DC development could also exist in vivo. The cell of the "Langerhans" type could be mainly involved in the induction of cellular immune responses, as shown by the involvement of these cells in delayed hypersensitivity reactions occurring after epicutaneous application of haptens; conversely, interstitial DC, probably derived from monocytes, could migrate, after antigen uptake, via the blood (or lymph) to the T cell-rich zone or B follicles where they could be more specifically involved in the regulation of the immune response.

In mice, the lymphoid origin of thymic DC and of a subset of DC of the spleen has been demonstrated by Shortman et al. Although this pathway of development has not been clearly shown in man, the CD4⁺CD11c^­CD13^­ DC precursors identified in the blood and tonsils could represent the equivalent of murine lymphoid DC. The results of Shortman et al. suggest that this lymphoid population could be involved in the negative regulation of T cell activation, thereby playing a role in the maintenance of peripheral tolerance.

The remarkable ability of DC to elicit an immune response and the availability of culture systems for DC allows their use in antitumour immunotherapy. Indeed, therapeutic antitumour responses based on protocols using DC have been successfully induced in mice models [for review see 36 and 72]. Different strategies have been used for loading DC with tumour-specific Ag in order to generate therapeutic antitumour responses, such as loading with tumoural peptides, fusion with tumour cells, transduction with recombinant viruses and transfection with tumour mRNA. All these protocols are based on the expansion and the handling of DC in vitro followed by their reinjection in animals. A better understanding of the system of DC could permit the handling of DC in vivo with the aim of obtaining an anti-tumour immunity, as highlighted by the use of FLT3-L in mice in vivo [73, 74].

Finally, the appropriate handling of the system of DC could permit the induction of tolerance rather than immunity, opening new possibilities of therapeutic intervention in autoimmune and allergic diseases, as well as in organ transplantation.

CONCLUSION

Acknowledgements

We would like to thank our many collaborators over the past three years who allowed us, through their work and discussions, to write this article. S. Ait-Yahia, C. Barthélémy, E. Bates, N. Bendriss, D. Blanchard, F. Brière, J.M. Bridon, L. Chalus, V. Clair, O. de Bouteiller, S. Denépoux, B. de Saint-Vis, M.C. Dieu, O. Djossou, B. Dubois, I. Durand, V. Duvert, J. Fayette, F. Fossiez, N. Fournier, C. Gaillard, E. Garcia, P. Garonne, G. Grouard, S. Ho, S. Lebecque, Y.J. Liu, C. Massacrier, C. Muller, C. Péronne, J.J. Pin, O. Ravel, M.C. Rissoan, S. Saeland, B. Salinas, V. Soumelis, J. Valladeau, B. Vanbervliet, S. Vandenabeele have greatly contributed to the most recent experiments. We thank O. Clear and L. Saïtta for wonderful daily organization, S. Bourdarel for outstanding help and D. Lepot and M. Vatan for invaluable assistance. We also thank J.F. Nicolas for his invitation to write this chapter and his collaborators for their help.

REFERENCES

1. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 1991; 9: 271-96.

2. Bhardwaj N. Interactions of viruses with dendritic cells: a double-edged sword. J Exp Med 1997; 186: 795-9.

3. Caux C, Dezutter-Dambuyant C, Liu YJ, Banchereau J. Isolation and propagation of human dendritic cells. In: Kabelitz D, Ziegler K, eds. Methods in microbiology: immunological methods. Academic Press Ltd., 1998; 505-38.

4. Knight SC, Farrant J, Bryan A. Non-adherent, low density cells from human peripheral blood contain dendritic cells and monocytes, both with veiled morphology. Immunology 1986; 57: 595-603.

5. Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 1994; 179: 1109-18.

6. Cella M, Sallusto F, Lanzavecchia A. Origin, maturation and antigen presenting function of dendritic cells. Cur Op Immunol 1997; 9: 10-6.

7. Lardon F, Snoeck HW, Berneman ZN, van Tendeloo VF, Nijs G, Lenjou M, Henckaerts E, Boeckxtaens CJ, Vandenabeele P, Kestens LL, van Bockstaele DR, Vanham GL. Generation of dendritic cells from bone marrow progenitors using GM-CSF, TNF-alpha, and additional cytokines: antagonistic effects of IL-4 and IFN-gamma and selective involvement of TNF-alpha receptor-1. Immunology 1997; 91: 553-9.

8. Reid CDL, Stackpoole A, Meager A, Tikerpae J. Interactions of tumor necrosis factor with granulocyte-macrophage colony-stimulating factor and other cytokines in the regulation of dendritic cell growth in vitro from early bipotent CD34⁺ progenitors in human bone marrow. J Immunol 1992; 149: 2681-8.

9. Santiago-Schwarz F, Belilos E, Diamond B, Carsons SE. TNF in combination with GM-CSF enhances the differentiation of neonatal cord blood stem cells into dendritic cells and macrophages. J Leukocyte Biol 1992; 52: 274-81.

10. Young JW, Szabolcs P, Moore MAS. Identification of dendritic cell colony-forming units among normal human CD34⁺ bone marrow progenitors that are expanded by c-kit-ligand and yield pure dendritic cell colonies in the presence of granulocyte/macrophage colony-stimulating factor and tumor necrosis factor alpha. J Exp Med 1995; 182: 1111-20.

11. Caux C, Massacrier C, Vanbervliet B, Dubois B, Durand I, Cella M, Lanzavecchia A, Banchereau J. CD34⁺ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNFalpha: II functional analysis. Blood 1997; 90: 1458-70.

12. Caux C, Vanbervliet B, Massacrier C, Dezutter-Dambuyant C, de Saint-Vis B, Jacquet C, Yoneda K, Imamura S, Schmitt D, Banchereau J. CD34⁺ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNFalpha. J Exp Med 1996; 184: 695-706.

13. Strobl H, Riedl E, Scheinecker C, Bello-Fernandez C, Pickl WF, Rappersberger K, Majdic O, Knapp W. TGF-B1 promotes in vitro development of dendritic cells from CD34⁺ hemopoietic progenitors. J Immunol 1996; 157: 1499-507.

14. Strunk D, Egger C, Leitner G, Hanau D, Stingl G. A skin homing molecule defines the Langerhans cell progenitor in human peripheral blood. J Exp Med 1997; 185: 1131-6.

15. McWilliam AS, Nelson D, Thomas JA, Holt PG. Rapid dendritic cell recruitment is a hallmark of the acute inflammatory response at mucosal surfaces. J Exp Med 1994; 179: 1331-6.

16. Cumberbatch M, Kimber I. Dermal tumour necrosis factor-alpha induces dendritic cell migration to draining lymph nodes, and possibly provides one stimulus for Langerhans' cell migration. Immunology 1992; 75: 257-63.

17. De Smedt T, Pajak B, Muraille E, Lespagnard L, Heinen E, De Baetselier P, Urbain J, Leo O, Moser M. Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo. J Exp Med 1996; 184: 1413-24.

18. Austyn JD. New insights into the mobilization and phagocytic activity of dendritic cells. J Exp Med 1996; 183: 1287-92.

19. Sallusto F, Cella M, Danieli C, Lanzavecchia A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: down-regulation by cytokines and bacterial products. J Exp Med 1995; 182: 389-400.

20. Harkiss GD, Hopkins J, McConnell I. Uptake of antigen by afferent lymph dendritic cells mediated by antibody. Eur J Immunol 1990; 20: 2367-73.

21. Jiang W, Swiggard WJ, Heufler C, Peng M, Mirza A, Steinman RM, Nussenzweig MC. The receptor DC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing. Nature 1995; 375: 151-5.

22. Larsen CP, Steinman RM, Witmer-Pack MD, Hankins DF, Morris PJ, Austyn JM. Migration and maturation of Langerhans cells in skin transplants and explants. J Exp Med 1990; 172: 1483-94.

23. Sallusto F, Nicolo C, de Maria R, Corinti S, Testi R. Ceramide inhibits antigen uptake and presentation by dendritic cells. J Exp Med 1996; 184: 2411-6.

24. Cella M, Engering A, Pinet V, Pieters J, Lanzavecchia A. Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells. Nature 1997; 388: 782-7.

25. Pierre P, Turley SJ, Gatti E, Hull M, Meltzer J, Mirza A, Inaba K, Steinman RM, Mellman I. Developmental regulation of MHC class II transport in mouse dendritic cells. Nature 1997; 388: 787-92.

26. Sozzani S, Luini W, Borsatti A, Polentarutti N, Zhou D, Piemonti L, D'Amico G, Power CA, Wells TN, Gobbi M, Allavena P, Mantovani A. Receptor expression and responsiveness of human dendritic cells to a defined set of CC and CXC chemokines. J Immunol 1997; 159: 1993-2000.

27. Sozzani S, Sallusto F, Luini W, Zhou D, Piemonti L, Allavena P, van Damme J, Valitutti S, Lanzavecchia A, Mantovani A. Migration of dendritic cells in response to formyl peptides, C5a, and a distinct set of chemokines. J Immunol 1995; 155: 3292-5.

28. Godiska R, Chantry D, Raport CJ, Sozzani S, Allavena P, Leviten D, Mantovani A, Gray PW. Human macrophage-derived chemokine (MDC), a novel chemoattractant for monocytes, monocyte-derived dendritic cells, and natural killer cells. J Exp Med 1997; 185: 1595-604.

29. Greaves DR, Wang W, Dairaghi DJ, Dieu MC, de Saint-Vis B, Franz-Bacon K, Rossi D, Caux C, McClanahan T, Gordon S, Zlotnik A, Schall TJ. CCR6, a CC chemokine receptor that interacts with macrophage inflammatory protein 3alpha and is highly expressed in human dendritic cells. J Exp Med 1997; 186: 837-44.

30. Power CA, Church DJ, Meyer A, Alouani S, Proudfoot AE, Clark-Lewis I, Sozzani S, Mantovani A, Wells TN. Cloning and characterization of a specific receptor for the novel CC chemokine MIP-3alpha from lung dendritic cells. J Exp Med 1997; 186: 825-35.

31. Vicari AP, Figueroa DJ, Hedrick JA, Foster JS, Singh KP, Menon S, Copeland NG, Gilbert DJ, Jenkins NA, Bacon KB, Zlotnik A. TECK: a novel CC chemokine specifically expressed by thymic dendritic cells and potentially involved in T cell development. Immunity 1997; 7: 291-301.

32. Ayehunie S, Garcia-Zepeda EA, Hoxie JA, Horuk R, Kupper TS, Luster AD, Ruprecht RM. Human immunodeficiency virus-1 entry into purified blood dendritic cells through CC and CXC chemokine coreceptors. Blood 1997; 90: 1379-86.

33. Granelli-Piperno A, Moser B, Pope M, Chen D, Wei Y, Isdell F, O'Doherty U, Paxton W, Koup R, Mojsov S, Bhardwaj N, Clark-Lewis I, Baggiolini M, Steinman RM. Efficient interaction of HIV-1 with purified dendritic cells via multiple chemokine coreceptors. J Exp Med 1996; 184: 2433-8.

34. Dieu MC, Vanbervliet B, Vicari A, Bridon JM, Oldham E, Aït-Yahia S, Brière F, Zlotnik A, Lebecque S, Caux C. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J Exp Med 1998; 188: 1-14.

35. Inaba K, Metlay JP, Crowley MT, Steinman RM. Dendritic cells pulsed with protein antigens in vitro can prime antigen-specific, MHC-restricted T cells in situ. J Exp Med 1990; 172: 631-40.

36. Mayordomo JI, Zorina T, Storkus WJ, Zitvogel L, Garcia-Prats MD, DeLeo AB, Lotze MT. Bone marrow-derived dendritic cells serve as potent adjuvants for peptide-based antitumor vaccines. Stem Cells 1997; 15: 94-103.

37. Croft M, Duncan DD, Swain SL. Response of naive antigen-specific CD4⁺ T cells in vitro: characteristics and antigen-presenting cell requirements. J Exp Med 1992; 176: 1431-7.

38. Macatonia SE, Hsieh CS, Murphy KM, O'Garra A. Dendritic cells and macrophages are required for Th1 development of CD4⁺ T cells from ab TCR transgenic mice: IL-12 substitution for macrophages to stimulate IFN-g production is IFN-g dependent. Int Immunol 1993; 5: 1119-28.

39. Caux C, Dezutter-Dambuyant C, Schmitt D, Banchereau J. GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells. Nature 1992; 360: 258-61.

40. Caux C, Massacrier C, Dezutter-Dambuyant C, Vanbervliet B, Jacquet C, Schmitt D, Banchereau J. Human dendritic Langerhans cells generated in vitro from CD34⁺ progenitors can prime naive CD4⁺ T cells and process soluble antigen. J Immunol 1995; 155: 5427-35.

41. Van Kooten C, Banchereau J. Functions of CD40 on B cells, dendritic cells and other cells. Curr Op Immunol 1997; 9: 330-7.

42. Caux C, Massacrier C, Vanbervliet B, Dubois B, van Kooten C, Durand I, Banchereau J. Activation of human dendritic cells through CD40 cross-linking. J Exp Med 1994; 180: 1263-72.

43. Cella M, Scheidegger D, Palmer-Lehmann K, Lane P, Lanzavecchia A, Alber G. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J Exp Med 1996; 184: 747-52.

44. Koch F, Stanzl U, Jennewein P, Janke K, Heufler C, Kämpgen E, Romani N, Schuler G. High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10. J Exp Med 1996; 184: 741-6.

45. Macatonia SE, Hosken NA, Litton M, Vieira P, Hsieh CS, Culpepper JA, Wysocka M, Trinchieri G, Murphy KM, O'Garra A. Dendritic cells produce interleukin-12 and direct the development of Th1 cells from naive CD4⁺ T cells. J Immunol 1995; 154: 5071-9.

46. Mohamadzadeh M, Poltorak AN, Bergstressor PR, Beutler B, Takashima A. Dendritic cells produce macrophage inflammatory protein-1 gamma, a new member of the CC chemokine family. J Immunol 1996; 156: 3102-6.

47. Zlotnik A. The expression by dendritic cells of a new CC or beta family chemokine. J Immunol 1996; 157: 2736.

48. Adema GJ, Hartgers F, Verstraten R, de Vries E, Marland G, Menon S, Foster J, Xu Y, Nooyen P, McClanahan T, Bacon KB, Figdor CG. A dendritic-cell-derived CC chemokine that preferentially attracts naive T cells. Nature 1997; 387: 713-7.

49. Imai T, Yoshida T, Baba M, Nishimura M, Kakisaki M, Yoshie O. Molecular cloning of a novel T cell-directed CC chemokine expressed in thymus by signal sequence trap using Epstein-Barr virus vector. J Biol Chem 1996; 271: 21514-21.

50. Mueller CGF, Rissoan MC, Salinas B, Ait-Yahia S, Ravel O, Bridon JM, Brière F, Lebecque S, Liu YJ. PCR selects a novel disintegrin-proteinase from CD40-activated germinal center dendritic cells. J Exp Med 1997; 186: 655-63.

51. Bates EEM, Ravel O, Dieu MC, Ho S, Guret C, Bridon JM, Ait-Yahia S, Brière F, Caux C, Banchereau J, Lebecque S. Identification and analysis of a novel member of the ubiquitin family expressed in dendritic cells and mature B cells. Eur J Immunol 1997; 27: 2471-7.

52. Dubois B, Vanbervliet B, Fayette J, Massacrier C, van Kooten C, Brière F, Banchereau J, Caux C. Dendritic cells enhance growth and differentiation of CD40-activated B lymphocytes. J Exp Med 1997; 185: 941-51.

53. Dubois B, Massacrier C, Vanbervlieet B, Fayette J, Brière F, Banchereau J, Caux C. Critical role of IL-12 in dendritic cell-induced differentiation of naive B lymphocytes. J. Immunol 1998 (in press).

54. Fayette J, Dubois B, Vandenabeele S, Bridon JM, Vanbervliet B, Durand I, Banchereau J, Caux C, Brière F. Human dendritic cells skew isotype switching of CD40-activated naive B cells towards IgA1 and IgA2. J Exp Med 1997; 185: 1909-18.

55. Brocker T, Riedinger M, Karjalainen K. Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo. J Exp Med 1997; 185: 541-50.

56. Ardavin C, Wu L, Li CL, Shortman K. Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Nature 1993; 362: 761-63.

57. Wu L, Vremec D, Ardavin C, Winkel K, Suss G, Georgiou H, Maraskovsky E, Cook W, Shortman K. Mouse thymus dendritic cells: kinetics of development and changes in surface markers during maturation. Eur J Immunol 1995; 25: 418-25.

58. Wu L, Li CL, Shortman K. Thymic dendritic cell precursors: relationship to the T-lymphocyte lineage and phenotype of the dendritic cell progeny. J Exp Med 1996; 184: 903-11.

59. Saunders D, Lucas K, Ismaili J, Wu L, Maraskovsky E, Dunn A, Shortman K. Dendritic cell development in culture from thymic precursor cells in the absence of granulocyte/macrophage colony-stimulating factor. J Exp Med 1996; 184: 2185-96.

60. Shortman K, Caux C. Dendritic cell development: multiple pathways to Nature's adjuvants. Stem Cells 1997; 15: 409-19.

61. Steinman RM, Pack M, Inaba K. Dendritic cells in the T-cell areas of lymphoid organs. Immunol Rev 1997; 156: 25-37.

62. Süss G, Shortman K. A subclass of dendritic cells kills CD4 T cells via Fas/Fas-Ligand-induced apoptosis. J Exp Med 1996; 183: 1789-96.

63. Kronin V, Winkel K, Süss G, Kelso A, Heath W, Kirberg J, von Boehmer H, Shortman K. A subclass of dendritic cells regulates the response of naive CD8 T cells by limiting their IL-2 production. J Immunol 1996; 157: 3819-27.

64. Inaba K, Pack M, Inaba M, Sakuta H, Isdell F, Steinman RM. High levels of a major histocompatibility complex II-self peptide complex on dendritic cells from the T cells areas of lymph nodes. J Exp Med 1997; 186: 665-72.

65. Burkly L, Hession C, Ogata L, Reilly C, Marconi LA, Olson D, Tizard R, Cate R, Lo D. Expression of rel/B is required for the development of thymic medulla and dendritic cells. Nature 1995; 373: 531-6.

66. Weih F, Carrasco D, Durham SK, Barton DS, Rizzo CA, Ryseck RP, Lira SA, Bravo R. Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-kB/Rel family. Cell 1995; 80: 331-40.

67. Borkowski TA, Letterio JJ, Farr AG, Udey MC. A role for endogenous transforming growth factor bB1 in Langerhans cell biology: the skin of transforming growth factor B1 null mice is devoid of epidermal Langerhans cells. J Exp Med 1996; 184: 2417-22.

68. Georgopoulos K, Bigby M, Wang JH, Molnar A, Wu P, Winandy S, Sharpe A. The Ikaros gene is required for the development of all lymphoid lineages. Cell 1994; 79: 143-56.

69. Wu L, Nichogiannopoulou A, Shortman K, Georgopoulos K. Cell-autonomous defects in dendritic cell populations of Ikaros mutant mice point to a developmental relationship with the lymphoid lineage. Immunity 1997; 7: 483-92.

70. O'Doherty U, Peng M, Gezelter S, Swiggard WJ, Betjes M, Bhardwaj N, Steinman RM. Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature. Immunology 1994; 82: 487-93.

71. Grouard G, Rissoan MC, Filgueira L, Durand I, Banchereau J, Liu YJ. The enigmatic plasmacytoid T cells develop into dendritic cells with IL-3 and CD40-ligand. J Exp Med 1997; 185: 1101-11.

72. Schuler G, Steinman RM. Dendritic cells as adjuvants for immune-mediated resistance to tumors. J Exp Med 1997; 186: 1183-7.

73. Chen K, Braun S, Lyman S, Fan Y, Traycoff CM, Wiebke EA, Gaddy J, Sledge G, Broxmeyer HE, Cornetta K. Antitumor activity and immunotherapeutic properties of Flt3-ligand in a murine breast cancer model. Cancer Res 1997; 57: 3511-6.

74. Lynch DH, Andreasen A, Maraskovsky E, Whitmore J, Miller RE, Schuh JC. Flt3 ligand induces tumor regression and antitumor immune responses in vivo. Nat Med 1997; 3: 625-31.