Traffic 2000 1: 312–317Munksgaard International Publishers

Review

Antigen Traffic Pathways in Dendritic Cells

Colin Wattsa* and Sebastian Amigorenab

a Department of Biochemistry, University of Dundee,Dundee, DD1 5EH, UKb Inserm U520, Institut Curie, 75005 Paris, France* Corresponding author: C. Watts, c.watts@dundee.ac.uk

Dendritic cells (DC) are now believed to be the principal

initiators of T cell-mediated immune responses. Their

location in body tissues, migratory behaviour in re-

sponse to inflammatory stimuli, endocytic properties,

expression of MHC molecules and key T cell stimulatory

molecules and many other attributes place these re-

markable cells in a unique and influential position in the

immune system. Progress in DC culture methods has

recently allowed in-depth studies on the cell biological

features that enable them to fulfil their crucial role in the

immune response.

Key words: Antigen presentation, dendritic cells, endocy-

tosis, MHC

Received 17 January 2000, revised and accepted for pub-

lication 18 January 2000

Dendritic cells (DC) were first described in 1973 as a low-abundance, novel cell type present in mouse lymphoid or-gans (1). Over the past few years, it has become clear thatthese cells are central to the induction of adaptive immuneresponses (2). DC are involved in inducing both CD8 and CD4T-cell responses to antigens presented on class I and class IIMHC molecules, respectively. Other cell types express MHCmolecules and can be recognised by previously activated Tcells, but DC possess unique attributes that enable them totrigger the expansion of naive T cells. Consequently, they arepivotal in the immune response and prime targets in at-tempts to manipulate the immune response for therapeuticeffect, for example, in vaccination.

A long standing observation is their striking ability to clusteravidly with T cells and to trigger their proliferation, particularlywhen the MHC molecules on the DC are different to thoseon the T cell population (and in the thymic environment inwhich those T cells developed). In this situation, the anti-genic stimulus is in-built, due to MHC differences and is thebasis of the so-called ‘mixed lymphocyte reaction’ which isan in vitro model for graft rejection. Surprisingly, early at-tempts to demonstrate that DC could also take up andpresent foreign antigens to T cells via MHC molecules metwith little success, due to the weak endocytic capacity of theisolated DC (3). It was paradoxical that a cell type that could

potently stimulate T cells, even when present in sub-stoichio-metric numbers, had such a poor capacity to capture environ-mental antigens. This puzzle was solved when it becameclear that isolated DC populations are frequently heteroge-neous and exhibit a continuum of developmental states orlife-stages. At the two extremes of this continuum are imma-ture DC, which are dedicated to antigen capture and matureDC which are dedicated to antigen presentation and T-cellengagement. The DC isolated in most early studies fell intothe latter group, their maturation being stimulated addition-ally by the manipulations of isolation. The true capacity ofthese cells to capture and process antigen remained hidden.As discussed below, immature DC are in fact the mostendocytically active cells known.

A key advance that has greatly facilitated our understandingof the DC system has been the development of cell culturesystems that allow immature DC to be propagated in vitroand maintained in the immature state (2). DC expanded inGM-CSF plus, according to different protocols, IL-4, TNFa orTGFb, can be induced to mature in vitro by the addition ofbacteria or bacterial products such as lipopolysaccharide(LPS), pro-inflammatory cytokines or indeed T cells them-selves, thus recapitulating, under controlled conditions, whathappens in the whole organism. These cells are remarkablypotent antigen-presenting cells able, in some cases, topresent antigens at picomolar concentrations. Many excel-lent reviews on various aspects of DC immunobiology haveappeared and the reader is referred to those for a morecomprehensive overview of their role in induction of B-cellresponses, in tolerance induction, their navigation throughdifferent tissues, their use in clinical immunology and theirpropagation in vitro (2,4–6). Here, we focus on some of theirproperties which allow them to perform their antigen cap-ture, processing and presentation function and which may beof particular interest to cell biologists.

Antigen Capture by Dendritic Cells

In vivo, immature DC are found scattered throughout periph-eral tissues such as the skin and airway epithelia where theyact as sentinels involved in capture and processing of envi-ronmental antigens. In response to immune stimuli, DC mi-grate out of damaged or infected tissue and move to thelymphoid organs. En route, so-called ‘co-stimulatory’molecules (e.g. CD80, CD86), adhesion molecules (e.g.CD50, CD54 (ICAMs) and signalling molecules such asCD40, appear on the cell surface. Upon arrival in thelymphoid organs, DC have partially matured, a process com-pleted upon interaction with T cells. At this stage, endocyto-sis has ceased to be of primary importance and is virtually

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shut down (Figure 1). The regulation of endocytosis in DC is,therefore, of some interest not least because successfulcapture of antigen by DC in vivo is a key step in vaccination.

Endocytosis in immature DC can involve receptor-mediatedendocytosis, macropinocytosis or phagocytosis. Differenttypes of antigen utilise different routes. For example, DCexpress a variety of receptors able to cluster in clathrin-coated pits including Fc receptors and members of the C-type lectin family. The members of this family featuremultiple carbohydrate or putative carbohydrate binding do-mains and include the macrophage mannose receptor, whichis also expressed on most DC and which drives very efficientuptake and presentation of mannosylated antigens (7,8). Theprecise specificity of other members of the family that areexpressed on DC, such as the DEC-205 molecule (9) is notknown, but, like other members of the family, it may recog-nise carbohydrate structures expressed on foreign patho-gens (10). Langerhans cells (LC) are immature DC that residein the skin and harbour a mysterious organelle known as theBirbeck granule, which has been suggested to be involved inantigen uptake and presentation. LC appear to lack mannosereceptors (11), but do express a novel member of the C-typelectin family called langerin. Interestingly, expression of lan-gerin in other cell types induces the dramatic appearance ofBirbeck granule structures ((12) and J. Valladeau et al., inpress). Hopefully, the function of this unusual organelle maysoon be known.

Non-specific antigen capture is particularly efficient in DCdue to a high level of constitutive macropinocytosis (13,14).In DC differentiated from blood monocytes, macropinocyto-sis rates are particularly high, such that the cell takes up its

own volume every 60 min (13). Little information is availableregarding the downstream processing of macropinosomes,although some evidence indicates that maturedmacropinosomes may act as distinct antigen storage com-partments (15). It is clear that antigen taken up via this routeis efficiently targeted to and presented on class II MHCmolecules (13).

DC are also able to phagocytose particulate material, al-though probably not as efficiently as macrophages. Nonethe-less, phagocytosis of bacteria, for example, leads not only topresentation of bacterial antigens on both class II and class IMHC molecules, but also drives DC maturation (16). DC canalso phagocytose apoptotic and necrotic cell fragments. Thiscan lead to presentation of fragment-derived peptides onclass II MHC molecules (17) and indeed on class I MHCmolecules (18), a very important route referred to as ‘cross-presentation’ (see below).

MHC Biosynthesis and Maturation in DC

DC maturation represents a co-ordinated set of cellular mod-ifications that contribute to efficient naive T-cell activation (2).One of the most significant of these modifications is amarked increase of MHC class II expression at the cellsurface. This increase is due to both the re-distribution ofMHC class II molecules stored in endosomes and lysosomesin immature DC and, later on, to a strong transient increasein the rate of MHC class II synthesis. A selective down-mod-ulation of MHC class II degradation also contributes to theincrease in its surface expression on mature DC (19,20).MHC class II delivery to endocytic compartments in DC, ascompared with other APC, presents some distinctive fea-

Figure 1: DC maturation and antigen presentation. Antigen presentation capacities of DCs are modified during their developmentinto fully mature DCs. The endocytic, processing and T-cell stimulating capacities of DCs at different maturation stages aresummmarised.

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tures. In human monocyte-derived DC, new Ii chain-associ-ated MHC class II molecules are transported to lysosomalcompartments after a short passage over the cell surface(21). This so-called indirect pathway, which is the major onein immature DC, only concerns a few percent of class IImolecules in B cells. DC also express a higher proportion ofp41 Ii chain isoform than B cells or macrophages. The highlevels of Iip41, however, do not seem to account for theunusual MHC class II transport routes in DC.

The pathways for MHC class II transport to the cell surfaceupon induction of maturation have been analysed in somedetail. In human monocyte-derived DC, class II MHC re-distri-bution is mainly due to a decrease in the rate of class IIinternalisation and degradation (19). In mouse DC, a selectivemechanism of control of Ii chain degradation determinesclass II MHC transport to the cell surface (20). In immatureDC, Ii chain degradation in lysosomes is incomplete. A 10-kDa fragment of Ii chain (Iip10), which includes CLIP (the Iiregion that occupies the class II peptide binding groove),remains associated to class II molecules and retains themintracellularly (22). This block in Ii degradation is due tocystatin C (23), an inhibitor of cathepsin S which is theproteolytic enzyme that normally degrades Iip10 (24). Uponinduction of maturation, cystatin C expression is down-regu-lated, which results in cathepsin S activation and Iip10 degra-dation, allowing peptide loading and MHC class II transport tothe cell surface. However, since DC from cathepsin S knock-out mice have normal levels of cell surface class II MHCmolecules, it is likely that other cystatin C sensitiveproteases can substitute for cathepsin S as far as Ii process-ing is concerned, albeit less efficiently (25,26).

The precise pathway used for class II transport from endo-cytic compartments to the cell surface is still unclear. Avesicular pathway, implicating the budding of class II trans-port vesicles from MHC class II -containing endosomes andthe fusion of these vesicles with the plasma membrane wasrecently described (27,28). Nevertheless, direct fusion ofendocytic compartments with the plasma membrane wasalso reported (29,30). This fusion event results in the deliveryof the MHC class II molecules present in the endosome’slimiting membrane to the plasma membrane, and in thesecretion of the endosome’s content, including internal en-dosomal vesicles, in the extracellular medium (see below). Itis, however, unclear to what extent these two pathwayscontribute to MHC class II delivery to the cell surface. Inmouse DC, MHC class II molecules were shown to re-dis-tribute from lysosomal to endosomal (class II vesicles, CIIV)compartments upon LPS-treatment (20). It is most likely,although not yet demonstrated, that CIIV directly fuse withthe plasma membrane, thus delivering peptide-loaded MHCclass II molecules to the cell surface.

Based on all these observations, a model for co-ordinatedMHC class II peptide loading and intracellular transport maybe proposed (Figure 1). In the absence of a maturation(danger) signal, DC internalise antigens, generate peptides in

endocytic compartments, but peptide loading is not veryefficient due to the association of Iip10 with class IImolecules. If MHC class II molecules are neverthelessloaded, the complexes are not expressed abundantly at thecell surface due to their high rate of internalisation and to theshort half-life of class II molecules in immature DC. When DCencounter a maturation signal, they enter an intermediateactivation state where the endocytic, macropinocytic andphagocytic activities are still high, thus allowing uptake ofpotential antigens. A marked increase of MHC class II syn-thesis, the release of the block of cathepsin S activity bycystatin C, which results in Iip10 degradation, and the re-dis-tribution of class II molecules to CIIV, all contribute to effi-cient peptide loading on MHC class II and transport of thecomplexes to the cell surface. During this intermediate matu-ration stage, DC thus optimise all the parameters for efficientgeneration of MHC class II-peptide complexes. In a thirdstage of DC maturation, the overall DC endocytic activity(macropinocytosis and phagocytosis) as well as MHC class IIsynthesis are reduced, thus preventing further antigen up-take and peptide loading. This reduction of the endocyticactivity also results in the stabilisation of surface class IImolecules, which prolongs the availability of presented epi-topes for T-cell activation after DC migration to the lymphnodes. MHC class II intracellular fate during DC developmentrepresents a remarkable example of regulated endocytictransport linked to a precise physiological function: the initia-tion of T-cell mediated immune responses.

‘Cross-Presentation’ of Exogenous Antigens onClass I MHC Molecules

In general, endocytosed antigens are presented on class IIMHC molecules but not on class I MHC molecules. This isbecause loading of class I MHC molecules takes place in theendoplasmic reticulum using peptides generated in the cyto-sol and not in endosomes (reviewed in (31)). This makesbiological sense, since cytotoxic T cells will recognise and killcells that are actually infected with pathogens and not by-stander cells that have passively taken them up. However,because presentation on DC alone seems necessary to ini-tiate CD8 T-cell responses, the question arises as to howCD8 T-cell responses are raised to viral antigens, which arenever expressed within DC. The same question can be askedof tumour antigens to which CD8 T-cell responses can some-times be induced. In vivo, this barrier is clearly overcome asrevealed by many experiments which demonstrate genera-tion of CD8 T-cell responses to antigens that are not actuallyexpressed in bone marrow-derived cells (i.e. DC ormacrophages) of the host, a phenomenon termed ‘cross-priming’ (32,33). One way around this problem would be toallow DC to translocate exogenous material into the cytosol.Clear evidence now exists that this occurs. The ability totranslocate material from endosomes to the cytosol appearsto be a constitutive feature of at least some DC endosomecompartments. Soluble antigen taken up by macropinocyto-sis and immune complexes taken up via Fc receptors canboth gain access to the cytosol. Not surprisingly, Fc receptor-

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mediated uptake leads to much more efficient presentation(34). Under these conditions, translocation is selective in thesense that there is no gross disruption of other lysosomecompartments and moreover it is size-selective: 50-kDa ma-terial is translocated more efficiently than 500 kDa material(35). A similar pathway has also been observed inmacrophages following phagocytosis (36), or stimulatedmacropinocytosis (37), but macrophages could not translo-cate material taken up via Fc receptors. Future work on theactual mechanism of translocation will undoubtedly focus onthe DC pathway because of the great potential this offers invaccination protocols aimed at inducing cytotoxic T-cell re-sponses. ‘Cross-presentation’ is emerging as important notonly for T-cell activation to viral and tumour antigens but alsomost likely to permit DC under certain non-inflammatoryconditions to induce tolerance to self-antigens not expressedin DC (6).

It is important also to note that several studies reviewed in(31,38) demonstrate loading of class I MHC molecules inendosomes, in other words without translocation to thecytosol. Typically, class I MHC presentation under theseconditions is Brefeldin A insensitive and independent of theproteasome and endoplasmic reticulum localised TAP trans-porters. The T-cell epitopes generated in endosomes maynot, however, be the same as those generated by cytosolicprocessing of the same antigen and any T cells elicited viathis route may not be useful for killing virally infected cells.

Exosomes

Exosomes are small membrane vesicles, of 60–90 nm di-ameter, secreted by cells in culture. Secretion is thought tooccur after fusion of multivesicular late endosomes with theplasma membrane, which results in the release of endo-some’s internal vesicles in the extracellular milieu. Exosomeswere initially described in erythrocytes, where they allow theelimination of several membrane receptors, including TfR, inthe final phases of erythrocyte development (39). Exosomesare also produced by hematopoietic cells, like B lymphocytesor DC (29,40). Exosomes resemble late endosome’s internalvesicles morphologically and concentrate a number of endo-somal markers present there. The most striking examplemay be the accumulation in both late endosomes and exo-somes of several members of the tetraspanin family, includ-ing CD9, CD37, CD63, CD82 and CD81 (40,41). Althoughsome of these proteins are also expressed at the plasmamembrane (like CD9), others mainly localise to multivesicularendosomes. In contrast, exosomes contain little or no LAMP-1 and -2, two abundant lysosomal glycoproteins.

A recent systematic analysis of exosomal proteins by peptidemapping and MALDI-TOF, revealed the presence of otherabundant exosomal components (42). Exosomes containCD11c/CD18 complexes, an integrin family member that alsoaccumulates in endocytic compartments in DC (our unpub-lished observation). The most abundant exosomal protein,however, is lactadherin, a secreted glycoprotein initially de-

scribed in milk fat globules. Lactadherin bears an EGF-like,RGD integrin-binding domain (which binds avb3 and avb5)and a phospholipid-binding domain, with high affinity forphosphatidyl serine (PS). Lactadherin may well interact withexosomes through it’s PS binding domain and target thevesicles to avb3 and avb5 expressing cells.

This proteomic analysis also revealed the presence in exo-somes of several cytosolic proteins, including annexin II,hsc70 and Gi2a. The role of these proteins in exosome’sbiogenesis and function is currently under study. Finally, andmost importantly from a functional point of view, exosomesalso contain high amounts of MHC class II and, to a lesserextent, MHC class I molecules, as well as CD86 (B7.2), a Tcell co-stimulatory molecule. In contrast, Ii chain or HLA-DM(H2M in mice), two other important players in the process ofMHC class II-restricted antigen presentation, which are veryabundant in endosomes and lysosomes, were not detectedin exosomes.

Therefore, a highly specific mechanism of protein segrega-tion within multivesicular endosomes, between the internalvesicles and the limiting external endosomal membrane re-sults in the selective enrichment of certain proteins in exo-somes. The question now is: why exosomes? Is there aspecific function for these vesicles or does their productiononly result from a cleaning pathway, whereby cells get rid ofpartially degraded lysosomal proteins and lipids? Recent re-sults demonstrate for the first time a biological effect ofexosomes in vivo.

Exosomes produced by mouse DC sensitised with tumour-derived peptides were injected into mice bearing the corre-sponding established tumours (40). A single exosomeinjection induced a strong immune response against thetumour, which resulted in a strong delay in tumour growthand complete tumour eradication in 60% of the mice. Exo-some-induced anti-tumour responses required T cells andspecific tumour peptides. Surprisingly, when compared withthe DC from which they were derived, exosomes demon-strated stronger anti-tumour effects. Therefore, although themechanisms of exosome action are still poorly understood,these results suggest that DC-derived exosomes may playsome role in the induction of immune responses in vivo.

Two possible mechanisms for exosome-mediated immunestimulation may be proposed. First, since exosomes bearMHC class I and II, as well as co-stimulatory molecules, theymay directly stimulate tumour specific T cells. Against thispossibility is the observation that direct T-cell stimulation bypeptide-bearing exosomes is extremely inefficient in vitro. Inaddition, it is unlikely that exosomes migrate on their ownfrom the sub-cutaneous site of injection to the lymph nodeswhere immune responses are initiated. Second, exosomesare targeted to other APCs, perhaps other DC, that somehowpresent them to specific T cells. Sensitisation of DC by MHCpeptide-bearing exosomes has been observed in vitro (L.Zitvogel and S. Amigorena, unpublished observations). In

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addition, the presence of lactadherin, which binds avb3 andavb5, two integrins expressed in DC and macrophages, fur-ther supports the possibility that the actual exosome targetcells are other APC and not directly T cells. This possibility isalso consistent with the observation that mature DC producelow levels of exosomes. If this is the case, DC-derivedexosomes could in fact represent an amplification step of theantigenic response: exosomes could transfer MHC- and/orhsc7O-bound peptides from a DC that has actually taken upand processed an antigen, to other neighbouring naive DC,which upon encountering a danger signal would also becomemature, migrate to the lymph nodes and contribute to theinitiation of immune responses.

Although these fascinating cells have been known for some30 years, their central role in immune responses has onlybeen appreciated in the last 10 years. Since then, because oftheir enormous clinical potential, many academic and indus-trial groups around the world have engaged in the effort tounderstand the biology of DC. Their transcriptional (genomic)and translational (proteomic) programs are being systemati-cally analysed. Their ontogeny and development – the cytoki-nes, the transcription and signalling pathways involved –have all been examined in detail. We hope that this reviewhas illustrated how DC have adapted their membrane trafficpathways to fulfil their unique immunological role. Their mi-gration fate, the adhesion molecules they express, thechemokines that the respond to and produce, are also understudy. Their role in the immune responses against viruses,bacteria and tumours as well as their implication in toleranceand autoimmunity have all been largely documented. Severalphase I clinical trials are being conducted in different infec-tious diseases and cancer. Because of this enormous effortto understand all the aspects of their biology, it is likely thatin the next few years, for the first time, we will be able tospecifically manipulate immune responses in patients.

References

1. Steinman RM, Cohn ZA. Identification of a novel cell type in periph-eral lymphoid organs of mice. I. Morphology, quantitation, tissuedistribution. J Exp Med 1973;137: 1142–1162.

2. Banchereau J, Steinman RM. Dendritic cells and the control ofimmunity. Nature 1998;392: 245–252.

3. Steinman RM, Swanson J. The endocytic activity of dendritic cells.J Exp Med 1995;182: 283–288.

4. Schuler G, Steinman RM. Dendritic cells as adjuvants for immune-mediated resistance to tumors. J Exp Med 1997;186: 1183–1187.

5. Cella M, Sallusto F, Lanzavecchia A. Origin, maturation and antigenpresenting function of dendritic cells. Curr Opin Immunol 1997;9:10–16.

6. Carbone FR, Kurts C, Bennett SR, Miller JF, Heath WR. Cross-pre-sentation: a general mechanism for CTL immunity and tolerance.Immunol Today 1998;19: 368–373.

7. Engering AJ, Cella M, Fluitsma D, Brockhaus M, Hoefsmit EC,Lanzavecchia A, et al. The mannose receptor functions as a highcapacity and broad specificity antigen receptor in human dendriticcells. Eur J Immunol 1997;27: 2417–2425.

8. Tan MC, Mommaas AM, Drijfhout JW, Jordens R, Onderwater JJ,Verwoerd D, et al. Mannose receptor-mediated uptake of antigensstrongly enhances HLA class II-restricted antigen presentation bycultured dendritic cells. Eur J Immunol 1997;27: 2426–2435.

9. Jiang W, Swiggard WJ, Heufler C, Peng M, Mirza A, Steinman RM,et al. The receptor DEC-205 expressed by dendritic cells and thymicepithelial cells is involved in antigen processing. Nature 1995;375:151–155.

10. Stahl PD, Ezekowitz RA. The mannose receptor is a pattern recogni-tion receptor involved in host defense. Curr Opin Immunol 1998;10:50–55.

11. Mommaas AM, Mulder AA, Jordens R, Out C, Tan MC, Cresswell P,et al. Human epidermal Langerhans cells lack functional mannosereceptors and a fully developed endosomal/lysosomal compartmentfor loading of HLA class II molecules. Eur J Immunol 1999;29:571–580.

12. Valladeau J, Duvert-Frances V, Pin JJ, Dezutter-Dambuyant C,Vincent C, Massacrier C, et al. The monoclonal antibody DCGM4recognizes Langerin, a protein specific of Langerhans cells, and israpidly internalized from the cell surface. Eur J Immunol 1999;29:2695–2704.

13. Sallusto F, Cella M, Danieli C, Lanzavecchia A. Dendritic cells usemacropinocytosis and the mannose receptor to concentrate macro-molecules in the major histocompatibility complex class II compart-ment: downregulation by cytokines and bacterial products. J ExpMed 1995;182: 389–400.

14. Norbury CC, Chambers BJ, Prescott AR, Ljunggren HG, Watts C.Constitutive macropinocytosis allows TAP-dependent major histo-compatibility complex class I presentation of exogenous solubleantigen by bone marrow-derived dendritic cells. Eur J Immunol1997;27: 280–288.

15. Lutz MB, Rovere P, Kleijmeer MJ, Rescigno M, Assmann CU,Oorschot VM, et al. Intracellular routes and selective retention ofantigens in mildly acidic cathepsin D/lysosome-associated mem-brane protein-1/MHC class II-positive vesicles in immature dendriticcells. J Immunol 1997;159: 3707–3716.

16. Rescigno M, Citterio S, Thery C, Rittig M, Medaglini D, Pozzi G, etal. Bacteria-induced neo-biosynthesis, stabilization, and surface ex-pression of functional class I molecules in mouse dendritic cells.Proc Natl Acad Sci USA 1998;95: 5229–5234.

17. Inaba K, Turley S, Yamaide F, Iyoda T, Mahnke K, Inaba M, et al.Efficient presentation of phagocytosed cellular fragments on themajor histocompatibility complex class II products of dendritic cells.J Exp Med 1998;188: 2163–2173.

18. Albert ML, Sauter B, Bhardwaj N. Dendritic cells acquire antigenfrom apoptotic cells and induce class I-restricted CTLs. Nature1998;392: 86–89.

19. Cella M, Engering A, Pinet V, Pieters J, Lanzavecchia A. Inflamma-tory stimuli induce accumulation of MHC class II complexes ondendritic cells. Nature 1997;388: 782–787.

20. Pierre P, Turley SJ, Gatti E, Hull M, Meltzer J, Mirza A, et al.Developmental regulation of MHC class II transport in mouse den-dritic cells. Nature 1997;388: 787–792.

21. Saudrais C, Spehner D, de la Salle H, Bohbot A, Cazenave JP,Goud B, et al. Intracellular pathway for the generation of functionalMHC class II peptide complexes in immature human dendritic cells.J Immunol 1998;160: 2597–2607.

22. Amigorena S, Webster P, Drake J, Newcomb J, Cresswell P, Mell-man I. Invariant chain cleavage and peptide loading in major histo-compatibility complex class II vesicles. J Exp Med 1995;181:1729–1741.

23. Pierre P, Mellman I. Developmental regulation of invariant chainproteolysis controls MHC class II trafficking in mouse dendritic cells.Cell 1998;93: 1135–1145.

24. Riese RJ, Wolf PR, Bromme D, Natkin LR, Villadangos JA, PloeghHL, et al. Essential role for cathepsin S in MHC class II-associatedinvariant chain processing and peptide loading. Immunity 1996;4:357–366.

25. Nakagawa TY, Brissette WH, Lira PD, Griffiths RJ, Petrushova N,Stock J, et al. Impaired invariant chain degradation and antigenpresentation and diminished collagen-induced arthritis in cathepsinS null mice. Immunity 1999;10: 207–217.

26. Driessen C, Bryant RA, Lennon-Dumenil AM, Villadangos JA, BryantPW, Shi GP, et al. Cathepsin S controls the trafficking and matura-tion of MHC class II molecules in dendritic cells. J Cell Biol1999;147: 775–790.

316 Traffic 2000: 1: 312–317

Antigen Traffic Pathways in Dendritic Cells

27. Pond L, Watts C. Characterization of transport of newly assembled,T cell-stimulatory MHC class II-peptide complexes from MHCclass II compartments to the cell surface. J Immunol 1997;159:543–553.

28. Ramm G, Pond L, Watts C, Stoorvogel W. Clathrin-coated latticesand buds on MHC class II compartments do not selectively recruitmature MHC-II. J Cell Sci 2000;113(Pt 2): 303–313.

29. Raposo G, Nijman HW, Stoorvogel W, Leijendekker R, Harding CV,Melief CJM, et al. B lymphocytes secrete antigen-presenting vesi-cles. J Exp Med 1996;183: 1161–1172.

30. Wubbolts R, Fernandez-Borja M, Oomen L, Verwoerd D, Janssen H,Calafat J, et al. Direct vesicular transport of MHC class II moleculesfrom lysosomal structures to the cell surface. J Cell Biol 1996;135:611–622.

31. Yewdell JW, Norbury CC, Bennink JR. Mechanisms of exogenousantigen presentation by MHC class I molecules in vitro and in vivo:implications for generating CD8+ T cell responses to infectiousagents, tumors, transplants, and vaccines. Adv Immunol 1999;73:1–77.

32. Bevan MJ. Antigen recognition. Class discrimination in the world ofimmunology. Nature 1987;325: 192–194.

33. Sigal LJ, Crotty S, Andino R, Rock KL. Cytotoxic T-cell immunity tovirus-infected non-hematopoietic cells requires presentation of ex-ogenous antigen. Nature 1999;398: 77–80.

34. Regnault A, Lankar D, Lacabanne V, Rodriguez A, Thery C,Rescigno M, et al. FcgR-mediated induction of dendritic cell matura-tion and MHC class I-restricted antigen presentation after immunecomplex internalization. J Exp Med 1999;189: 371–380.

35. Rodriguez A, Regnault A, Kleijmeer M, Ricciardi-Castagnoli P,Amigorena S. Selective transport of internalized antigens to thecytosol for MHC class I presentation in dendritic cells. Nat Cell Biol1999;1: 362–368.

36. Kovacsovics-Bankowski M, Rock KL. A phagosome-to-cytosolpathway for exogenous antigens presented on MHC class Imolecules. Science 1995;267: 243–246.

37. Norbury CC, Hewlett LJ, Prescott AR, Shastri N, Watts C. Class IMHC presentation of exogenous soluble antigen via macropinocyto-sis in bone marrow macrophages. Immunity 1995;3: 783–791.

38. Jondal M, Schirmbeck R, Reimann J. MHC class I-restricted CTLresponses to exogenous antigens. Immunity 1996;5: 295–302.

39. Johnstone RM, Adam M, Hammond JR, Orr L, Turbide C. Vesicleformation during reticulocyte maturation. Association of plasmamembrane activities with released vesicles (exosomes). J Biol Chem1987;262: 9412–9420.

40. Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza D, etal. Eradication of established murine tumors using a novel cell-freevaccine: dendritic cell-derived exosomes. Nat Med 1998;4: 594–600.

41. Escola JM, Kleijmeer MJ, Stoorvogel W, Griffith JM, Yoshie O,Geuze HJ. Selective enrichment of tetraspan proteins on the internalvesicles of multivesicular endosomes and on exosomes secreted byhuman B-lymphocytes. J Biol Chem 1998;273: 20121–20127.

42. Thery C, Regnault A, Garin J, Wolfers J, Zitvogel L, Ricciardi-Cas-tagnoli P, et al. Molecular characterization of dendritic cell-derivedexosomes. Selective accumulation of the heat shock protein hsc73.J Cell Biol 1999;147: 599–610.

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