- Email: [email protected]
Antigen processing and presentation: close encounters in the endocytic pathway
Class I and class lI molecules encoded by the major histocompatibility complex (MHC) bind peptides during their export to the cell surface and thereby display the peptide content of the cell to T lymphocytes (see Ref. 1 for a review). T cells are not activated when the peptides bound by MHC molecules are derived from self-proteins, but when a cell is infected by virus, cytotoxic T cells respond to the viral peptides that appear in conjunction with class I molecules. Likewise, when a class II-expressing cell endocytoses a foreign antigen, helper T cells are activated to stimulate the antibody response against that antigen. The association of class I and class II molecules with peptides generated in different intracellular compartments results in their ability to stimulate different populations of T cells. Class I molecules bind peptides generated from proteins produced within the same cell as the molecules themselves. The major source for class I peptides appears to be degraded cytoplasmic proteins, and considerable evidence suggests that there is a transport process* that allows peptides access to luminal compartments where class I molecules bind t h e m as part of their assembly process 2,3. Coassembly of the class I molecule with a peptide derived from an endogenously synthesized protein seems to be required for the stable expression of class I molecules at the cell surface 4. Differences in the biosynthetic assembly and stability of class II molecules compared with class I molecules account for the ability of class II molecules to bind peptides generated in the endocytic pathway from internalized exogenous proteins. Class II molecules avoid binding peptides during their initial assembly because their a and ~ subunits coassemble with a third polypeptide, the invariant chain, which prevents peptide binding (see Ref. 5 for a review). The invariant chain (I) then targets the class II molecules to intracellular compartments that are part of the endocytic pathway6,7, 8. It is believed that dissociation of the invariant chain in these compartments allows binding of antigenic peptides generated in the endocytic pathway, followed by release of class II molecules from the c o m p a r t m e n t and expression on the cell surface. Unlike class I molecules, class II molecules can be expressed stably on the cell surface even if they do not bind peptides9,10,11. Stable expression of ' e m p t y ' class II molecules on normal mouse spleen cells appears to result when invariant chain dissociates but there are insufficient levels of peptides available for binding in the endocytic pathway. Although empty class II molecules on normal cells have a shorter half-life than occupied class II molecules 11, they may represent a functional pool of class II molecules that could bind antigenic peptides at the cell surface or following endocytosis and recycling. The extent of internalization of class II molecules varies considerably between cell types 1, but this pathway may provide a second opportunity for class II molecules to encounter peptides generated in the endocytic pathway, after their initial export. The degree to which this second passage TRENDS IN CELL BIOLOGY VOL. 2 APRIL 1992
Antigen processing and presentation: close encounters in the endocytic pathway Stimulation of helper T cells by class II molecules occurs when the class I! molecules bind and display peptides derived from foreign antigens that have been endocytosed. The formation of peptide-class II complexes requires antigen degradation and exposure of the peptide-binding site of class II molecules, both of which depend on proteolysis and low pH in the endocytic pathway. This review discusses the role of specific compartments of the endocytic pathway in the generation of antigenic peptides, and in the binding of antigenic peptides to newly synthesized class II molecules and those that are internalized from the cell surface.
through the endocytic pathway may contribute to peptide binding by empty and occupied class II molecules is discussed in this review. For a class II molecule to interact with an antigenspecific T-cell receptor, native (intact) antigen must be degraded into peptides (antigen processing), and the class II molecule with an exposed peptidebinding site must encounter and bind the antigenic peptide and display it on the cell surface (antigen presentation). Experimental evidence points to a central role for the endocytic pathway in these events. Antigen processing and presentation are defective in cells that are unable to acidify their endosomes (although their lysosomes are properly acidified) 12, and in cells treated with agents that neutralize endosomal and lysosomal pH 13 or inhibit endosomal/lysosomal proteolysis 14. These findings reflect the fact that the endocytic pathway provides favourable conditions for both the degradation of antigen and the binding of antigenic fragments by class II molecules, and it is thus likely to be the site of interaction of class II molecules with antigenic peptides. Because class II molecules are stable without bound peptide, it is possible that they might meet and bind antigenic peptides in endocytic compartments other than those in © 1992 ElsevierScience PublishersLtd (UK) 0962-8924/92/$05.00
*See the last issue of TCBfor a review of this topic [TCB 2, 81-86 (1992)].
Frances M. Brodsky is at the Departments of Pharmacy, Pharmaceutical Chemistry, and Microbiology and Immunology, University of California, San Francicso, CA 94143-0446, USA. 1 09
BOX 1 - ANTIGEN-PRESENTINGCELLSAND CLASSII MOLECULEEXPRESSION The classical definition of antigen-presenting cells is those cells that constitutively express class II molecules and stimulate helper T cells. These include B cells, monocytes, macrophages and dendritic cells. • Tissue macrophages (e.g. Kupfer cells in the liver) and monocytes (their circulating precursors) are highly endocytic and phagocytic cells that internalize antigen at the site of infection in tissues and in lymph nodes. By presentation of antigenic peptides in association with their surface class II molecules they stimulate helper T cells in the vicinity, which in turn secrete lymphokines that stimulate local B cells to mature and secrete antibodys2. • B cells can also present antigen directly to helper T cells s3. In this case, antigen uptake occurs through specific surface immunoglobulin expressed on the B-cell surface Is. Thus, if the B cell encounters its antigen, it can receive antigen-specific help from the T cell. This positive feedback contributes to the development of the secondary immune response. • Dendritic cells are also important antigen-presenting cells. Their antigen uptake and processing pathways may be specialized for this function and are currently being characterizeds4. • Endothelial cells and fibroblasts express class II molecules during a sustained inflammatory response, due to the upregulation of class II molecule expression by interferon 7. Under such conditions, these cells can also present antigen to helper T cells, thereby expanding the repertoire of cells that stimulate helper T cells during infection ss. Class II molecules are also expressed on some nonlymphoid tumours, such as melanomas4s. Presentation of tissue-specific antigens by class II molecules expressed during chronic inflammation on tissues that are normally class llnegative has been hypothesized to be a stimulus of autoimmune diseases6.
which the peptides are generated. Current work in the field is attempting to define the specific roles of different compartments of the endocytic pathway in either antigen processing or presentation. In this review, evidence implicating particular endocytic compartments in antigen processing or in antigen presentation is first considered separately. Then, evidence for the intersection of these processes is discussed. Production of antigenic peptides in the endocytic pathway
Pathways of antigen uptake have been studied extensively for two types of cells that present antigen: B cells and macrophages (Box 1). B cells internalize antigen by receptor-mediated endocytosis using antigen-specific surface immunoglobulin as the receptor 15. Macrophages are actively endocytic and phagocytic 13. For the purposes of this article, the endocytic pathway of B cells and macrophages can be considered as three increasingly acidic and proteolytic compartments: early endosomes (pH 6.0-6.5), late endosomes (pH 5.0-6.0) and lysosomes (pH 4.5-5.0) 16. For reviews of transport through the endocytic pathway see Refs 17 and 18. In both h u m a n and mouse B-cell lines, specific delivery of native antigen to the endocytic pathway through binding to surface immunoglobulin, as compared to random pinocytosis, can result in considerably more efficient stimulation of T cells15,19. However, if antigen is targeted to the endocytic pathway of mouse B cells by coupling it to antibodies against different cell surface receptors, its efficiency of presentation varies 19. For example, internalization of cytochrome c into the early part 1 10
of the endocytic pathway after coupling to an antibody against the transferrin receptor does not result in efficient presentation, while the same antigen is efficiently presented if coupled to an antibody against immunoglobulin. These results suggest that this particular antigen must have access to the late compartments of the endocytic pathway for complete processing and/or presentation to occur. Targeting of antigens to different stages of the macrophage endocytic pathway, by using liposomes that disintegrate in the early endosome (liposomes sensitive to < pH 6.5) or that require degradation in lysosomes, has also suggested that later stages of the endocytic pathway are most efficient at generating antigenic peptides recognized by T cells2°, 21. Delivery of native antigen and antigenic peptides in liposomes that dissolve at early stages of the pathway results in some antigen presentation, but it is not clear whether this occurs in the early endosomal compartments or as a result of ultimate delivery of the antigens to later compartments. If lysosomal degradation results in highly efficient presentation of antigen, one interesting prediction is that there may be a pathway by which peptides generated in lysosomes have access to class II molecules, presumably in another endocytic compartment. Cleavage of antigens into peptides is mediated by acid-dependent proteases such as cathepsin D and neutral pH proteases such as cathepsin B14, as well as lysosomal proteases. Cathepsin B and D, formerly believed to be strictly lysosomal, have been localized in the endosomes of several cell types 22-26 and shown to be proteolytically active in endosomes of macrophages and 3T3 cells. The lysosomal proteases that bind to the mannose 6phosphate receptor are present in the late endosomes in most cell types, since this is the site at which they dissociate from the receptor and are sorted to lysosomes 27. Although direct measurem e n t of proteolytic activity in late endosomes has not been made, it is possible that these proteases are active to some extent, even if the pH is not optimal for efficient function. In two h u m a n B-cell lines, the protease content of endosomes was found to differ, but the samples were prepared under different experimental conditions. Cathepsin D was observed in the late, but not the early, endosomes of JY cells that were not exposed to any endocytic marker 28, while both cathepsin D and B were observed in the early and late endosomes of IM-9 cells that had internalized crosslinked surface immunoglobulin 26. Preliminary studies indicate that in IM-9 cells cathepsin B and D can be detected in endosomes accessed by internalized surface immunoglobulin, but not in endosomes accessed by the transferrin receptor (L. Guagliardi and F. Brodsky, unpublished). This suggests a possible difference in the endocytic fate of surface immunoglobulin, which perhaps enters a late endosomal compartment more rapidly. Alternatively, the uptake of surface immunoglobulin might stimulate increased delivery of proteases to the endocytic pathway, through a signalling mechanism. Rapid TRENDS IN CELL BIOLOGY VOL. 2 APRIL 1992
~~ /
~ ~
interseotionwith ~ Ii~ @ class II molecules
~
II
I
(a)
(b)
,~ ~
(c)
II
(d)
II
II
(e)
FIGURE 1
Representationof processingof antigen boundto surfaceimmunoglobulin(Ig) and classII molecules.(a) Nativeantigenis bound by surfaceIg that recognizesantigenicdeterminantA. RegionB representsa secondantigenicdeterminantthat would be boundand protectedby a surfaceIg of differentspecificity.(b) Initial degradationin the endocyticpathway.The Ig or a proteolyticfragmentgeneratedfrom it is still bound,protectingsiteAsT.(c) Furtherdegradationafterdissociationof the Ig; site A remainsintact longer. (d) Bindingof two differentclassII molecules(allelicforms specificfor the AI and B2 peptides)to the N-termini of peptidesgeneratedin the endocyticpathway.(e) Furthertrimming of the boundpeptides,showinghow the allelicform of classII moleculepresentcould influencethe peptidesgenerated.
encounter with proteases would explain the results of experiments demonstrating efficient processing of antigen internalized via surface immunoglobulin15, ]9. Thus, in antigen-presenting cells, such as B cells and macrophages, proteases may be present in early as well as late stages of the endocytic pathway. However, due to the higher pH, the proteases present in early compartments are likely to be less active than those in later endocytic compartments of lower pH. Consequently, while some antigenic peptides might be generated in early stages of the endocytic pathway, complete degradation of antigen and release of most antigenic peptides probably does not occur until the prelysosomal or lysosomal stage of the endocytic pathway, as suggested by the antigen-delivery experiments in both B cells and macrophages. This is illustrated by studies of the degradation of iodinated tetanus toxin (TT), internalized by binding to an immunoglobulin on the surface of h u m a n B cells that recognizes TT. Fragments of TT were present in an early endosomal fraction purified on Percoll density gradients and also in late endosomal and lysosoma] fractions 29. Although the kinetics of association of TI" fragments with class II molecules, as determined by coimmunoprecipitation, suggest that class IIbinding fragments are generated later in the pathway 30, initial degradation of TT antigen begins earlier in the pathway and for some antigens this could potentially release antigenic peptides. Antigen degradation in the endocytic pathway may not simply be a function of how easily the available proteases can cleave the antigenic molecule (Fig. 1). H u m a n B cells bearing various antibodies against TT on their surface generate different antigenic fragments, indicating that the receptor immunoglobulin bound to the antigen influences its pattern of degradation 31. In addition, different class II alleles (with polymorphic differences clustered in the peptide-binding site) may influence which fragments of antigen are presented32, 33. One interpretation of these results is that partially cleaved and unfolded antigen binds to class II molecules and is then 'trimmed' to fit the binding site. This idea has gained some credence
TRENDSIN CELLBIOLOGYVOL.2 APRIL1992
with the recent report that peptides bound by class II molecules differ from those bound by class I molecules. Peptides eluted from class I molecules are either octamers or nonamers34, 3s and the crystal structure of peptide bound to the HLA-B27 molecule indicates that both the C- and N-termini of the peptides are deeply imbedded in the binding cleft 36. By contrast, peptides eluted from class II molecules are of variable length (13-17 residues) and some have the same N-termini but different C-termini 37. This result implies that the N-terminus of the peptide is embedded in the peptide-binding site but the C-terminus is exposed to variable cleavage. Thus, it is possible that class II molecules bind partially processed antigenic fragments in a proteolytic environment and influence their subsequent processing. Maturation of class II molecules in the endocytic pathway
Proteases and the low pH of the endocytic pathway contribute to the binding of antigenic peptides by class II molecules. The endosomal proteases cathepsin B and D have been implicated in the dissociation of invariant chain from class II molecules38, 39, thereby exposing the peptide-binding site and releasing the c~ chains of class II molecules for expression on the cell surface. In addition, the binding of synthetic antigenic peptides by class II molecules is enhanced at pH < 6.0, within the range of all endocytic compartments40, 41. This enhancement of peptide binding at endosomal pH may affect only certain subsets of class II molecules: those that are empty and those that are not tightly associated with peptide (Fig. 2). Earlier studies of purified class II molecules bound to peptide suggested that most of these complexes are very stable. Even at lysosomal pH, the half-life of the complex is -30 min, and at endosomal pH it is > 2.5 h 42. It is, therefore, unlikely that exchange of bound peptides on class II molecules would occur spontaneously by recycling through endosomes 43. There is evidence (Fig. 2) that a chaperonin molecule of the 70 kDa heat shock protein family could play a role in the acquisition of antigenic peptides by class II molecules 19. Whether such a
11 1
reviews
methods to study class II molecule distribution in the h u m a n B-cell line JY, Peters e t al. 28 reported PM that the majority of class II molecules, but not invariant chain, were localized to late stages of the endocytic pathway in an endocytic compartment with some lysosomal characteristics 28. These compartments, dubbed MIIC, contain the lysosomal H+, peptide r:cycling t ~ P membrane protein lamp-1 and [~-hexosaminidase, endocytosis II d e gH+ r a dta t i o ~ ', (hsp 70) TGN --11------ PM but not cathepsin D, and could represent subregions of the late endosome or novel, specialized Y endocyticpathway compartments for the sequestration of class I/ molecules. MIIC compartments have also been FIGURE 2 observed in IM-9 cells (H. Ploegh, P. Peters and Intracellular processes influencing peptide binding to class II molecules. The c¢ J. Neefjes, pers. commun.). Quantitative immunoand [3 chains of class II molecules coassemble with the invariant chain to form electron microscopy of the h u m a n melanoma cell the c¢[31complex (containing three c~chains, three [3 chains and three invariant line Mel JuSo 45 revealed that both class II molecules chainsSS), which is targeted from the trans Golgi network (TGN) to the and invariant chain were present at equivalent endocytic pathway48 as a result of trafficking signals in the cytoplasmic domain levels in early endosomes and late endosomes and of the invariant chain s. Low pH and proteolysis in the endocytic pathway a few class II molecules were seen in lysosomes. remove the invariant chain, releasing the class II molecules for export to the Invariant chain expressed in CV1 monkey kidney plasma membrane (PM). The low pH in the endocytic pathway is favourable for cells was also observed in intracellular compartstable binding of peptide (P) present in the endocytic pathway. If insufficient ments with characteristics of both early and late peptide is present, empty class II molecules11 will be released to the cell surface. endosomes 7. Studies of murine macrophages reEndocytosis and recycling of class II molecules that remain empty or associate ported the presence of class II MHC molecules in weakly with peptides (Pw) at the cell surface would provide re-exposure to 'sac-like' endosomes and small endosomes conpeptides in early endosomes at pH 6.0, which may be sufficiently low to favour taining lamp1 and cathepsin D, and murine B cells exchange of weakly bound peptides, as well as to load empty molecules. were reported to have class II MHC molecules in Exchange of weakly bound peptides is suggested by the exchange of 'typical' endosomes 46. exogenously bound synthetic peptide for other exogenously added peptide in Together, the morphological data from many the early endosome59. Complexes formed from peptides derived from cell lines indicate that class II molecules and internalized antigen have long half-lifes on the cell surface, but there is some invariant chain are present in all compartments of evidence that stably bound peptides exported with class II molecules can be the endocytic pathway from early endosomes to exchanged in early endocytic compartments60. A potential role for a 70 kDa prelysosomal compartments. The differences obheat shock protein in the peptide-binding and exchange process should be served between the distribution of class II molconsidered. An antiserum specific for this hsp70 inhibits presentation of peptides ecules in various cell lines might be explained by derived from cytochrome c antigen19. The hsp70 protein binds antigenic cell-specific trafficking patterns of these molecules, peptides, and is localized to several compartments of the endocytic pathway6+. or there may be differences in the rates of transport or proteolytic processing of the molecules involved so that they accumulate in different commolecule functions by sequestering peptide in the partments in different cells. The morphological endocytic pathway or by loading peptides onto data suggest three possible export pathways for the class II molecules remains to be determined. c~I complex via the endocytic pathway (Fig. 3). All Given that the activity of endocytic compartthree are compatible with complete dissociation of ments is necessary for peptide binding to newly the cz[3Icomplex occurring primarily at late stages synthesized class II molecules, evidence for localizof the endocytic pathway, correlating with the ation of class II molecules and invariant chainfunctional data discussed below. class II complexes to endocytic compartments has Internalization of class II molecules after their been sought. Cell fractionation suggests that, in expression on the cell surface also contributes to several cell types, the class II and invariant chain their presence in the endocytic pathway° The level molecules are present in endosomal compartof internalization varies with the cell type, ranging ments 6,44. Immunoelectron microscopy studies26,28 from a few percent on h u m a n B-cell lines26,47,48,49 have characterized the nature of these endosomal to 35% on mouse B-cell lines 50. However, rapid compartments in more detail, and there is some uptake and recycling 49 could result in a substantial variation in endosomal distribution of class II proportion of the surface class II molecules on all molecules and invariant chain depending on the cells passing through early endosomal compartments cell type analysed and the microscopy approach with time. Antigenic peptides present in these used. In the h u m a n B-cell line IM-9, we have early compartments would have the opportunity to observed class II molecules and invariant chain in be bound by empty class II molecules internalized endocytic compartments accessed by surface imby this mechanism. munoglobulin or the transferrin receptor after two rain and 30 rain of uptake of these ligandsl, 26. Intersection of class II molecules and antigenic These findings suggest that c~I complexes are peptides in the endocytic pathway found throughout the pathway used for uptake of Functional and biochemical studies have sugforeign antigen 26. Using more quantitative gested that there is segregation of peptide binding
eo+
7 12
TRENDS [N CELL BIOLOGYVOL. 2 APRIL 1992
by newly synthesized ~3I complexes and class II molecules that have already been expressed at the cell surface (Fig. 4). Davidson et al. 3o found that the class II molecules that associate with TT fragments generated from internalized intact TT antigen came exclusively from the biosynthetic pathway and not from the cell surface. They also observed that both cell surface and internal class II molecules could bind antigen fragments introduced as synthetic peptides. These results suggest that peptides derived from complex antigens requiring extensive degradation are not generated until they reach the later stages of the endocytic pathway. At this later stage, the only class II molecules they encounter are molecules from the biosynthetic pathway that have not yet been expressed on the cell surface. However, if antigenic peptides are supplied to the early compartments of the endocytic pathway, they can bind to class II molecules from the cell surface and also from the intracellular pool. If the limited degradation that occurs in the early stages of the endocytic pathway generates antigenic peptides, they should be presented by those class II molecules in the early part of the pathway. In support of this mechanism, Kakiuchi et al. 51 found that ovalbumin introduced by pinocytosis, and presumably degraded in the early part of the endocytic pathway, can be presented by a pool of class II molecules that is insensitive to inhibitors of protein synthesis (i.e. recycling, empty class II molecules in early endosomes), whereas the same antigen bound to surface immunoglobulin and internalized by receptor-mediated endocytosis is presented by a pool of class II molecules that is depleted by inhibition of protein synthesis (i.e. newly synthesized molecules in late endosomes). On the basis of the properties of peptide binding by class II molecules (Fig. 2), it is likely that peptides in the early endosome are mostly bound by internalized empty molecules or molecules with weakly bound peptide, unless there is a specialized mechanism for peptide loading and exchange. Given that the morphological studies described above indicate that in some antigen-presenting cells cz[3I complexes and proteases colocalize early in the endocytic pathway, an unresolved question is whether the early colocalization simply reflects the pathway of transport by which these molecules accumulate in later endocytic compartments or whether they interact to form class II-peptide complexes in early as well as late endocytic compartments. There is little evidence that such complexes form in early endosomes but there is also no evidence that rules out this possibility. It is possible that antigen degradation and maturation of class II molecules are gradual processes, such that partially degraded antigen can associate with class II binding sites on complexes from which the portion of invariant chain that blocks peptide binding is removed. These complexes would then be exposed to further proteolysis in later compartments of the pathway, which would trim the bound peptide TRENDS IN CELL BIOLOGY VOL. 2 APRIL 1 992
(a)
(b)
(c)
FIGURE 3
Models for the maturation of class II MHC molecules in the endocytic pathway. After export from the TGN, the c~131 complex is localized to the endocytic pathway. There are three routes by which this localization could take place, which have been documented for other molecules targeted to the endocytic pathway. (a) ~131may be transiently expressed at the cell surface and rapidly internalized into the endocytic pathway, as observed for lysosomal acid phosphatase62, such that only low amounts are detected on the cell surface 63. (b) c~l~lmay be targeted from the TGN to the early endosome (EE), as observed for cathepsin D23. (c) c~131may be targeted from the TGN to a late endosomal compartment (LE), as observed for the mannose 6-phosphate receptor27. The class II-rich late endocytic compartment observed in human B-cell lines (MIIC) is shown as a possible subregion of the late endosome, but may be a separate organelle28. Once targeted to the endocytic pathway, the c~131complex will be retained until the invariant chain is degraded. The class II ~ and 13chains associated with peptide (c~13P)are then released to the plasma membrane (PM). On the basis of the distribution and activity of proteases in the endocytic pathway, it is assumed that the main site of release is from the later stages of the endocytic pathway. However, potential release of some class II-peptide complexes in the early stages might occur, following well-documented recycling pathways to the cell surface (dotted line in A and B). 113
(a)
fPw[
endocytosis
~6
PN endosomal i r a, targeting PEE fI! Ps
=13
(c)
e=,
retention
targeting
References
release II
(d)
targeting [
I l i '+
retention trimming
II
II
PM
flpE i
releas%,
0@ I" late endosome
c~61' early endosome
c
~13
PM
c~61 late endosome
c~131 TGN
Ill
. ?,1p, II II
early endosome
(b)
I
recycling
~6
PM
I
PEE
l P
flpE E 0@ PM
releas%,
Ip
1 i I
~,61 TGN
o~[~I' early endosome
late endosome
PM FIGURE 4
Segregation of antigens bound by endocytosed and exported class II molecules. (a) Functional experiments indicate that class II molecules [which may be empty or may weakly bind peptide (Pw)] internalized from the cell surface bind pinocytosed synthetic peptides (Ps) or peptides generated in the early endosome (PEE) but do not have access to peptides generated from internalized native antigen in the late stages of the endocytic pathway. (b) Class II molecules delivered to the late stages of the endocytic pathway from the biosynthetic pathway can, however, bind such peptides (PN), in addition to binding peptides delivered from the early stages of the pathway (Ps and PEE).(C) If biosynthetically derived class II molecules pass through earlier compartments, as suggested in Fig. 3a and 3b, peptides generated in early endosomes could also be bound in these compartments, provided that the invariant chain dissociates to expose the peptide-binding site. (d) The influence of class II molecules on antigen degradation suggests that partially degraded antigen (P+) could be bound before invariant chain is completely removed, and then trimmed (P+ to P) in a later compartment. Stepwise degradation of invariant chain (I to I') as shown in (c) and (d), for which there is biochemical evidence4s, would result in accumulation of c~131'molecules in the late stages of the endocytic pathway and correlate with kinetics indicating that processing and presentation of native antigen are completed in late endocytic compartments, even if they begin in early compartments.
and finally release the peptide--~[~ complex to the cell surface (Fig. 4d). Thus, many questions remain to be answered. What is the nature of the late endosomal compartment that provides most of the antigen processing and presentation functions? Is class II maturation spatially separated from the efficient degradation of antigen and, if so, where do these molecules meet and what is their pathway to the cell surface?
1 14
Finally, since redundancy is characteristic of the immune response, do several mechanisms of peptide binding by class II molecules (Fig. 4) occur in different locations in the endocytic pathway simultaneously? Given the nature of the problem, immunologists can be expected to be delving around in the endocytic pathway for some time to come.
1 BRODSKY,F. M. and GUAGLIARDI, L. E. (1991) Annu. Rev. ImmunoL 9, 707-744 2 SPIES,T. and DEMARS, R. (1991) Nature 351,323-324 3 POWlS, S. J., TOWNSEND, A. R. M., DEVERSON,E V., BASTIN, J., BUTCHER,G. W. and HOWARD, J. C. (1991) Nature 354, 528-531 4 LJUNGGREN,H-G. et al. (1990) Nature 346, 476-480 5 TEYTON, L. and PETERSON,P. A. Trends Cell BioL 2, 52-56 6 LOTTEAU,V. et aL (1990) Nature 348, 600-605 7 BAKKE,O. and DOBBERSTEIN,B. (1990) Cell 63, 707-716 8 LAMB, C. A., YEWDELL,J. W., BENNINK, J. R. and CRFSSWELL, P. (1991 ) Proc. Natl Acad. Sci. USA 88, 5998-6002 9 PETERSON,M. and MILLER, J. (1990) Nature 345, 172-174 10 MELLINS,E., SMITH, L, ARP, B., COTNER, T., CELLS,E and PIOUS, D. (1990) Nature 343, 71-74 11 GERMAIN, R. N. and HENDRIX, L. R. (1991) Nature353, 134-139 12 MCCOY, K. L., MILLER,J., JENKINS, M., RONCHESE,F., GFRMAIN, R. N. and SCHWARTZ, R. H. (1989) J. Immunol. 143, 29-38 13 UNANUE, E R. (1984)Annu. Rev. ImmunoL 2, 395428 14 BERZOFSKY,A., BRETi-,S. J., STREICHER,H. Z. and TAKAHASHI, H. (1988) ImmunoL Rev. 106, 5-31 15 LANZAVECCHIA,A. (1990) Annu. Rev. ImmunoL 8, 773-793 16 YAMASHIRO,D. J. and MAXFIELD, F. R. (1987) J. CellBiol. 105, 2723-2733 17 MURPHY,R. F. (1991 ) Trends Cell BioL 1, 77-82 18 GRIFFITHS,G. and GRUENBERG,J. (1991) Trends Cell BioL 1, 5-9 19 PIERCE,S. K. et al. (1988) ImmunoL Rev. 149-180 20 HARDING, C. V., COLLINS, D., SLOT, J. W., GEUZF, H. J. and UNANUE, E. R. (1991) Ce1164, 393-401 21 HARDING,C. V., COLLINS, D. S., KANAGAWA, O. and UNANUF, E. R. (1991 ) J. ImmunoL 147, 2860-2863 22 DIMENT, S. and STAHL, P. (1985) J. BioL Chem. 260, 15311-15317 23 LUDWIG, T., GRIFFITHS,G. and HOFLACK, B. (1991) J. Cell BioL 115, 1561-1572 24 WlLFY, H. S., VAN NOSTRAND, W., MCKINLEY, D. N. and CUNNINGHAM, D. D. (1985)J. BioL Chem. 260, 5290-5295 25 ROEDERER,M., BOWSER,R. and MURPHY,R. F. (1987) J. Cell. PhysioL 131,200-209 26 GUAGLIARDI,L. E., KOPPELMAN, B., BLUM, J. S., MARKS, M. S., CRESSWELL,P. and BRODSKY,F. M. (1990) Nature 343, 133-139 27 KORNFELD,S. and MELLMAN, I. (1989)Annu. Rev. CeifBiol. 5, 483-525 28 PETFRS,P. J., NEFFJES,J. J., OORSCHOT, V., PLOEGH, H. L. and GEUZE, H. J. (1991 ) Nature 349, 669-676 29 DAVIDSON, H. W., WEST, M. A. and WATi-S, C. (1990) J. ImmunoL 144, 4101-4109 30 DAVIDSON, H. W., REID, P. A., LANZAVECCHIA,A. and WATTS, C. (1991) Ce1167, 105-116 31 DAVIDSON, H. W. and WATi-S, C. (1989) J. Cell Biol. 85-92 32 DONERMEYER,D. L. and ALLEN, P. M. (1989) J. ImmunoL 142, TRENDS IN CELL BIOLOGY VOL. 2 APRIL 1992
1063-1068 33 AMETANI, A., APPLE,R., BHARDWAI,V., GAMMON, G., MILLER,A. and SERCARZ,E. (1989) Cold Spring Harbor Syrup. Quant. BioL 54, 505-511 34 ROTZSCHKE,O. and FALK,K. (1991 ) ImmunoL Today12, 447-455 35 JARDFTZKY,T. S., LANE,W. S., ROBINSON, R. A., MADDEN, D. R. and WILEY, D. C. (1991 ) Nature 353, 326-329 36 MADDEN, D. R., GORGA,J. C., STROMINGER,J. L. and WILEY, D. C. (1991) Nature 353, 321-325 37 RUDENSKY,A. Y., PRESTON-HURLBURT,P., I-lONG, S-C., BARLOW, A. and JANEWAY,C. A. Jr. (1991 ) Nature 353, 622-627 38 BLUM, J. S. and CRESSWELL,P. (1988) Proc. Natl Acad. Sci. USA 85, 3975-3979 39 NGUYEN,Q. V., KNAPP,W. and HUMPHREYS,R. E. (1989) Hum. Immunol. 24, 153-163 40 JENSEN,P. E. (1990) J. Exp. Med. 171, 1770-1784 41 WETTSTEIN,D. A., LIN, A. Y., MATIS, L. A. and DAVIS, M. M. (1991 )J. Exp. Med. 174, 219-228 42 BUUS,S., SEI-I-E,A., COLON, S. M., JENIS,D. M. and GREY, H. M. (1986) Ce1147,1071-1077 43 LEE,J. M. and WAI-FS, T. H. (1990) J. ImmunoL 144, 1829-1844 44 HARDING,C. V. and UNANUE, E. R. (1989)J. ImmunoL 142, 12-19 45 PIETERS,J., HORSTMANN, H., BAKKE,O., GRIFFITHS,G. and LiPP, J. (1991) J. CellBioL 115, 1213-1223 46 HARDING,C. V., UNANUE, E. R., SLOT,J. W., SCHWARTZ, A. L. and GEUZE, H. J. (1990) Proc. NatlAcad. Sci. USA87, 5553-5557
TRENDS IN CELL BIOLOGY VOL. 2 APRIL 1992
47 DAVIS,J. E. and CRESSWELL,P. (1990)J. ImmunoL 144, 990-997 48 NEEFJES,J. J., STOLLORZ,V., PETERS,P. J., GEUZE,H. J. and PLOEGH,H. L. (1990) Cell61, 171-183 49 REID,P. A. and WATI-S, C. (1990) Nature 346, 655-657 50 HARDING,C. V., ROOF, R. W. and UNANUE, E. R. (1989) Proc. Notl Acad. Sci. USA86, 4230-4234 51 KAKIUCHI,T., WATANABE, M., HOZUMI, N. and NARIUCHI,H. (1990) J. ImmunoL 145, 1653-1658 52 UNANUE, E. R. and ALLEN, P. M. (1987) Science236, 551-557 53 ASHWELL,J. D. (1988)J. ImmunoL 140, 3697-3700 54 KING, P. and KATZ, D. R. (1990) ImmunoL Today11,206-211 55 POBER,J. S. and COTRAN, R. S. (1990) PhysioLRev. 70, 427-451 56 DONIACH, D., SPENCER,K. M. and BOTAZZO, G. F. (1985) in The AutoimmuneDiseases(Rose, N. R. and Mackay, I. R., ed.), pp. 227-242, Academic Press 57 WAI-rS, C., WEST, M., REID, P. A. and DAVIDSON, H. W. (1989) Cold Spring Harbor Syrup. Quant. BioL 54, 345-352 58 ROCHE,P. A., MARKS,M. S. and CRESSWELL,P. (1991) Nature 354, 392-394 59 CEPPELLINI,R., FRUMENTO,G., FERRARA,G. B., TOSI, R., CHERSI,A. and PERRIS,B. (1989) Nature 339, 392-394 60 MARSH,E. W., DALKE,D. P. and PIERCE,S. K. (1992) J. Exp. Med. 175, 425-436 61 VANBUSKIRK,A. M., DENAGEL,D. C., GUAGLIARDI,L. E., BRODSKY,F. M. and PIERCE,S. K. (1991)J. ImmunoL 146, 500-506 62 BRAUN,M., WAHEED, A. and VON FIGURA,K. (1989) EMBOJ. 8, 3633-3640 63 WRAIGHT,C. J. (1990)J. BioL Chem. 265, 5787-5792
Acknowledgments I thank G. Griffiths and C. Watts for communication of data prior to publication and K. Mostov for comments on the manuscript. The work from my laboratory that is described in this review was supported by NIH grant AR20894 and by the Pew Charitable Trusts.
1 15
Antigen processing and presentation: close encounters in the endocytic pathway Stimulation of helper T cells by class II molecules occurs when the class I! molecules bind and display peptides derived from foreign antigens that have been endocytosed. The formation of peptide-class II complexes requires antigen degradation and exposure of the peptide-binding site of class II molecules, both of which depend on proteolysis and low pH in the endocytic pathway. This review discusses the role of specific compartments of the endocytic pathway in the generation of antigenic peptides, and in the binding of antigenic peptides to newly synthesized class II molecules and those that are internalized from the cell surface.
through the endocytic pathway may contribute to peptide binding by empty and occupied class II molecules is discussed in this review. For a class II molecule to interact with an antigenspecific T-cell receptor, native (intact) antigen must be degraded into peptides (antigen processing), and the class II molecule with an exposed peptidebinding site must encounter and bind the antigenic peptide and display it on the cell surface (antigen presentation). Experimental evidence points to a central role for the endocytic pathway in these events. Antigen processing and presentation are defective in cells that are unable to acidify their endosomes (although their lysosomes are properly acidified) 12, and in cells treated with agents that neutralize endosomal and lysosomal pH 13 or inhibit endosomal/lysosomal proteolysis 14. These findings reflect the fact that the endocytic pathway provides favourable conditions for both the degradation of antigen and the binding of antigenic fragments by class II molecules, and it is thus likely to be the site of interaction of class II molecules with antigenic peptides. Because class II molecules are stable without bound peptide, it is possible that they might meet and bind antigenic peptides in endocytic compartments other than those in © 1992 ElsevierScience PublishersLtd (UK) 0962-8924/92/$05.00
*See the last issue of TCBfor a review of this topic [TCB 2, 81-86 (1992)].
Frances M. Brodsky is at the Departments of Pharmacy, Pharmaceutical Chemistry, and Microbiology and Immunology, University of California, San Francicso, CA 94143-0446, USA. 1 09
BOX 1 - ANTIGEN-PRESENTINGCELLSAND CLASSII MOLECULEEXPRESSION The classical definition of antigen-presenting cells is those cells that constitutively express class II molecules and stimulate helper T cells. These include B cells, monocytes, macrophages and dendritic cells. • Tissue macrophages (e.g. Kupfer cells in the liver) and monocytes (their circulating precursors) are highly endocytic and phagocytic cells that internalize antigen at the site of infection in tissues and in lymph nodes. By presentation of antigenic peptides in association with their surface class II molecules they stimulate helper T cells in the vicinity, which in turn secrete lymphokines that stimulate local B cells to mature and secrete antibodys2. • B cells can also present antigen directly to helper T cells s3. In this case, antigen uptake occurs through specific surface immunoglobulin expressed on the B-cell surface Is. Thus, if the B cell encounters its antigen, it can receive antigen-specific help from the T cell. This positive feedback contributes to the development of the secondary immune response. • Dendritic cells are also important antigen-presenting cells. Their antigen uptake and processing pathways may be specialized for this function and are currently being characterizeds4. • Endothelial cells and fibroblasts express class II molecules during a sustained inflammatory response, due to the upregulation of class II molecule expression by interferon 7. Under such conditions, these cells can also present antigen to helper T cells, thereby expanding the repertoire of cells that stimulate helper T cells during infection ss. Class II molecules are also expressed on some nonlymphoid tumours, such as melanomas4s. Presentation of tissue-specific antigens by class II molecules expressed during chronic inflammation on tissues that are normally class llnegative has been hypothesized to be a stimulus of autoimmune diseases6.
which the peptides are generated. Current work in the field is attempting to define the specific roles of different compartments of the endocytic pathway in either antigen processing or presentation. In this review, evidence implicating particular endocytic compartments in antigen processing or in antigen presentation is first considered separately. Then, evidence for the intersection of these processes is discussed. Production of antigenic peptides in the endocytic pathway
Pathways of antigen uptake have been studied extensively for two types of cells that present antigen: B cells and macrophages (Box 1). B cells internalize antigen by receptor-mediated endocytosis using antigen-specific surface immunoglobulin as the receptor 15. Macrophages are actively endocytic and phagocytic 13. For the purposes of this article, the endocytic pathway of B cells and macrophages can be considered as three increasingly acidic and proteolytic compartments: early endosomes (pH 6.0-6.5), late endosomes (pH 5.0-6.0) and lysosomes (pH 4.5-5.0) 16. For reviews of transport through the endocytic pathway see Refs 17 and 18. In both h u m a n and mouse B-cell lines, specific delivery of native antigen to the endocytic pathway through binding to surface immunoglobulin, as compared to random pinocytosis, can result in considerably more efficient stimulation of T cells15,19. However, if antigen is targeted to the endocytic pathway of mouse B cells by coupling it to antibodies against different cell surface receptors, its efficiency of presentation varies 19. For example, internalization of cytochrome c into the early part 1 10
of the endocytic pathway after coupling to an antibody against the transferrin receptor does not result in efficient presentation, while the same antigen is efficiently presented if coupled to an antibody against immunoglobulin. These results suggest that this particular antigen must have access to the late compartments of the endocytic pathway for complete processing and/or presentation to occur. Targeting of antigens to different stages of the macrophage endocytic pathway, by using liposomes that disintegrate in the early endosome (liposomes sensitive to < pH 6.5) or that require degradation in lysosomes, has also suggested that later stages of the endocytic pathway are most efficient at generating antigenic peptides recognized by T cells2°, 21. Delivery of native antigen and antigenic peptides in liposomes that dissolve at early stages of the pathway results in some antigen presentation, but it is not clear whether this occurs in the early endosomal compartments or as a result of ultimate delivery of the antigens to later compartments. If lysosomal degradation results in highly efficient presentation of antigen, one interesting prediction is that there may be a pathway by which peptides generated in lysosomes have access to class II molecules, presumably in another endocytic compartment. Cleavage of antigens into peptides is mediated by acid-dependent proteases such as cathepsin D and neutral pH proteases such as cathepsin B14, as well as lysosomal proteases. Cathepsin B and D, formerly believed to be strictly lysosomal, have been localized in the endosomes of several cell types 22-26 and shown to be proteolytically active in endosomes of macrophages and 3T3 cells. The lysosomal proteases that bind to the mannose 6phosphate receptor are present in the late endosomes in most cell types, since this is the site at which they dissociate from the receptor and are sorted to lysosomes 27. Although direct measurem e n t of proteolytic activity in late endosomes has not been made, it is possible that these proteases are active to some extent, even if the pH is not optimal for efficient function. In two h u m a n B-cell lines, the protease content of endosomes was found to differ, but the samples were prepared under different experimental conditions. Cathepsin D was observed in the late, but not the early, endosomes of JY cells that were not exposed to any endocytic marker 28, while both cathepsin D and B were observed in the early and late endosomes of IM-9 cells that had internalized crosslinked surface immunoglobulin 26. Preliminary studies indicate that in IM-9 cells cathepsin B and D can be detected in endosomes accessed by internalized surface immunoglobulin, but not in endosomes accessed by the transferrin receptor (L. Guagliardi and F. Brodsky, unpublished). This suggests a possible difference in the endocytic fate of surface immunoglobulin, which perhaps enters a late endosomal compartment more rapidly. Alternatively, the uptake of surface immunoglobulin might stimulate increased delivery of proteases to the endocytic pathway, through a signalling mechanism. Rapid TRENDS IN CELL BIOLOGY VOL. 2 APRIL 1992
~~ /
~ ~
interseotionwith ~ Ii~ @ class II molecules
~
II
I
(a)
(b)
,~ ~
(c)
II
(d)
II
II
(e)
FIGURE 1
Representationof processingof antigen boundto surfaceimmunoglobulin(Ig) and classII molecules.(a) Nativeantigenis bound by surfaceIg that recognizesantigenicdeterminantA. RegionB representsa secondantigenicdeterminantthat would be boundand protectedby a surfaceIg of differentspecificity.(b) Initial degradationin the endocyticpathway.The Ig or a proteolyticfragmentgeneratedfrom it is still bound,protectingsiteAsT.(c) Furtherdegradationafterdissociationof the Ig; site A remainsintact longer. (d) Bindingof two differentclassII molecules(allelicforms specificfor the AI and B2 peptides)to the N-termini of peptidesgeneratedin the endocyticpathway.(e) Furthertrimming of the boundpeptides,showinghow the allelicform of classII moleculepresentcould influencethe peptidesgenerated.
encounter with proteases would explain the results of experiments demonstrating efficient processing of antigen internalized via surface immunoglobulin15, ]9. Thus, in antigen-presenting cells, such as B cells and macrophages, proteases may be present in early as well as late stages of the endocytic pathway. However, due to the higher pH, the proteases present in early compartments are likely to be less active than those in later endocytic compartments of lower pH. Consequently, while some antigenic peptides might be generated in early stages of the endocytic pathway, complete degradation of antigen and release of most antigenic peptides probably does not occur until the prelysosomal or lysosomal stage of the endocytic pathway, as suggested by the antigen-delivery experiments in both B cells and macrophages. This is illustrated by studies of the degradation of iodinated tetanus toxin (TT), internalized by binding to an immunoglobulin on the surface of h u m a n B cells that recognizes TT. Fragments of TT were present in an early endosomal fraction purified on Percoll density gradients and also in late endosomal and lysosoma] fractions 29. Although the kinetics of association of TI" fragments with class II molecules, as determined by coimmunoprecipitation, suggest that class IIbinding fragments are generated later in the pathway 30, initial degradation of TT antigen begins earlier in the pathway and for some antigens this could potentially release antigenic peptides. Antigen degradation in the endocytic pathway may not simply be a function of how easily the available proteases can cleave the antigenic molecule (Fig. 1). H u m a n B cells bearing various antibodies against TT on their surface generate different antigenic fragments, indicating that the receptor immunoglobulin bound to the antigen influences its pattern of degradation 31. In addition, different class II alleles (with polymorphic differences clustered in the peptide-binding site) may influence which fragments of antigen are presented32, 33. One interpretation of these results is that partially cleaved and unfolded antigen binds to class II molecules and is then 'trimmed' to fit the binding site. This idea has gained some credence
TRENDSIN CELLBIOLOGYVOL.2 APRIL1992
with the recent report that peptides bound by class II molecules differ from those bound by class I molecules. Peptides eluted from class I molecules are either octamers or nonamers34, 3s and the crystal structure of peptide bound to the HLA-B27 molecule indicates that both the C- and N-termini of the peptides are deeply imbedded in the binding cleft 36. By contrast, peptides eluted from class II molecules are of variable length (13-17 residues) and some have the same N-termini but different C-termini 37. This result implies that the N-terminus of the peptide is embedded in the peptide-binding site but the C-terminus is exposed to variable cleavage. Thus, it is possible that class II molecules bind partially processed antigenic fragments in a proteolytic environment and influence their subsequent processing. Maturation of class II molecules in the endocytic pathway
Proteases and the low pH of the endocytic pathway contribute to the binding of antigenic peptides by class II molecules. The endosomal proteases cathepsin B and D have been implicated in the dissociation of invariant chain from class II molecules38, 39, thereby exposing the peptide-binding site and releasing the c~ chains of class II molecules for expression on the cell surface. In addition, the binding of synthetic antigenic peptides by class II molecules is enhanced at pH < 6.0, within the range of all endocytic compartments40, 41. This enhancement of peptide binding at endosomal pH may affect only certain subsets of class II molecules: those that are empty and those that are not tightly associated with peptide (Fig. 2). Earlier studies of purified class II molecules bound to peptide suggested that most of these complexes are very stable. Even at lysosomal pH, the half-life of the complex is -30 min, and at endosomal pH it is > 2.5 h 42. It is, therefore, unlikely that exchange of bound peptides on class II molecules would occur spontaneously by recycling through endosomes 43. There is evidence (Fig. 2) that a chaperonin molecule of the 70 kDa heat shock protein family could play a role in the acquisition of antigenic peptides by class II molecules 19. Whether such a
11 1
reviews
methods to study class II molecule distribution in the h u m a n B-cell line JY, Peters e t al. 28 reported PM that the majority of class II molecules, but not invariant chain, were localized to late stages of the endocytic pathway in an endocytic compartment with some lysosomal characteristics 28. These compartments, dubbed MIIC, contain the lysosomal H+, peptide r:cycling t ~ P membrane protein lamp-1 and [~-hexosaminidase, endocytosis II d e gH+ r a dta t i o ~ ', (hsp 70) TGN --11------ PM but not cathepsin D, and could represent subregions of the late endosome or novel, specialized Y endocyticpathway compartments for the sequestration of class I/ molecules. MIIC compartments have also been FIGURE 2 observed in IM-9 cells (H. Ploegh, P. Peters and Intracellular processes influencing peptide binding to class II molecules. The c¢ J. Neefjes, pers. commun.). Quantitative immunoand [3 chains of class II molecules coassemble with the invariant chain to form electron microscopy of the h u m a n melanoma cell the c¢[31complex (containing three c~chains, three [3 chains and three invariant line Mel JuSo 45 revealed that both class II molecules chainsSS), which is targeted from the trans Golgi network (TGN) to the and invariant chain were present at equivalent endocytic pathway48 as a result of trafficking signals in the cytoplasmic domain levels in early endosomes and late endosomes and of the invariant chain s. Low pH and proteolysis in the endocytic pathway a few class II molecules were seen in lysosomes. remove the invariant chain, releasing the class II molecules for export to the Invariant chain expressed in CV1 monkey kidney plasma membrane (PM). The low pH in the endocytic pathway is favourable for cells was also observed in intracellular compartstable binding of peptide (P) present in the endocytic pathway. If insufficient ments with characteristics of both early and late peptide is present, empty class II molecules11 will be released to the cell surface. endosomes 7. Studies of murine macrophages reEndocytosis and recycling of class II molecules that remain empty or associate ported the presence of class II MHC molecules in weakly with peptides (Pw) at the cell surface would provide re-exposure to 'sac-like' endosomes and small endosomes conpeptides in early endosomes at pH 6.0, which may be sufficiently low to favour taining lamp1 and cathepsin D, and murine B cells exchange of weakly bound peptides, as well as to load empty molecules. were reported to have class II MHC molecules in Exchange of weakly bound peptides is suggested by the exchange of 'typical' endosomes 46. exogenously bound synthetic peptide for other exogenously added peptide in Together, the morphological data from many the early endosome59. Complexes formed from peptides derived from cell lines indicate that class II molecules and internalized antigen have long half-lifes on the cell surface, but there is some invariant chain are present in all compartments of evidence that stably bound peptides exported with class II molecules can be the endocytic pathway from early endosomes to exchanged in early endocytic compartments60. A potential role for a 70 kDa prelysosomal compartments. The differences obheat shock protein in the peptide-binding and exchange process should be served between the distribution of class II molconsidered. An antiserum specific for this hsp70 inhibits presentation of peptides ecules in various cell lines might be explained by derived from cytochrome c antigen19. The hsp70 protein binds antigenic cell-specific trafficking patterns of these molecules, peptides, and is localized to several compartments of the endocytic pathway6+. or there may be differences in the rates of transport or proteolytic processing of the molecules involved so that they accumulate in different commolecule functions by sequestering peptide in the partments in different cells. The morphological endocytic pathway or by loading peptides onto data suggest three possible export pathways for the class II molecules remains to be determined. c~I complex via the endocytic pathway (Fig. 3). All Given that the activity of endocytic compartthree are compatible with complete dissociation of ments is necessary for peptide binding to newly the cz[3Icomplex occurring primarily at late stages synthesized class II molecules, evidence for localizof the endocytic pathway, correlating with the ation of class II molecules and invariant chainfunctional data discussed below. class II complexes to endocytic compartments has Internalization of class II molecules after their been sought. Cell fractionation suggests that, in expression on the cell surface also contributes to several cell types, the class II and invariant chain their presence in the endocytic pathway° The level molecules are present in endosomal compartof internalization varies with the cell type, ranging ments 6,44. Immunoelectron microscopy studies26,28 from a few percent on h u m a n B-cell lines26,47,48,49 have characterized the nature of these endosomal to 35% on mouse B-cell lines 50. However, rapid compartments in more detail, and there is some uptake and recycling 49 could result in a substantial variation in endosomal distribution of class II proportion of the surface class II molecules on all molecules and invariant chain depending on the cells passing through early endosomal compartments cell type analysed and the microscopy approach with time. Antigenic peptides present in these used. In the h u m a n B-cell line IM-9, we have early compartments would have the opportunity to observed class II molecules and invariant chain in be bound by empty class II molecules internalized endocytic compartments accessed by surface imby this mechanism. munoglobulin or the transferrin receptor after two rain and 30 rain of uptake of these ligandsl, 26. Intersection of class II molecules and antigenic These findings suggest that c~I complexes are peptides in the endocytic pathway found throughout the pathway used for uptake of Functional and biochemical studies have sugforeign antigen 26. Using more quantitative gested that there is segregation of peptide binding
eo+
7 12
TRENDS [N CELL BIOLOGYVOL. 2 APRIL 1992
by newly synthesized ~3I complexes and class II molecules that have already been expressed at the cell surface (Fig. 4). Davidson et al. 3o found that the class II molecules that associate with TT fragments generated from internalized intact TT antigen came exclusively from the biosynthetic pathway and not from the cell surface. They also observed that both cell surface and internal class II molecules could bind antigen fragments introduced as synthetic peptides. These results suggest that peptides derived from complex antigens requiring extensive degradation are not generated until they reach the later stages of the endocytic pathway. At this later stage, the only class II molecules they encounter are molecules from the biosynthetic pathway that have not yet been expressed on the cell surface. However, if antigenic peptides are supplied to the early compartments of the endocytic pathway, they can bind to class II molecules from the cell surface and also from the intracellular pool. If the limited degradation that occurs in the early stages of the endocytic pathway generates antigenic peptides, they should be presented by those class II molecules in the early part of the pathway. In support of this mechanism, Kakiuchi et al. 51 found that ovalbumin introduced by pinocytosis, and presumably degraded in the early part of the endocytic pathway, can be presented by a pool of class II molecules that is insensitive to inhibitors of protein synthesis (i.e. recycling, empty class II molecules in early endosomes), whereas the same antigen bound to surface immunoglobulin and internalized by receptor-mediated endocytosis is presented by a pool of class II molecules that is depleted by inhibition of protein synthesis (i.e. newly synthesized molecules in late endosomes). On the basis of the properties of peptide binding by class II molecules (Fig. 2), it is likely that peptides in the early endosome are mostly bound by internalized empty molecules or molecules with weakly bound peptide, unless there is a specialized mechanism for peptide loading and exchange. Given that the morphological studies described above indicate that in some antigen-presenting cells cz[3I complexes and proteases colocalize early in the endocytic pathway, an unresolved question is whether the early colocalization simply reflects the pathway of transport by which these molecules accumulate in later endocytic compartments or whether they interact to form class II-peptide complexes in early as well as late endocytic compartments. There is little evidence that such complexes form in early endosomes but there is also no evidence that rules out this possibility. It is possible that antigen degradation and maturation of class II molecules are gradual processes, such that partially degraded antigen can associate with class II binding sites on complexes from which the portion of invariant chain that blocks peptide binding is removed. These complexes would then be exposed to further proteolysis in later compartments of the pathway, which would trim the bound peptide TRENDS IN CELL BIOLOGY VOL. 2 APRIL 1 992
(a)
(b)
(c)
FIGURE 3
Models for the maturation of class II MHC molecules in the endocytic pathway. After export from the TGN, the c~131 complex is localized to the endocytic pathway. There are three routes by which this localization could take place, which have been documented for other molecules targeted to the endocytic pathway. (a) ~131may be transiently expressed at the cell surface and rapidly internalized into the endocytic pathway, as observed for lysosomal acid phosphatase62, such that only low amounts are detected on the cell surface 63. (b) c~l~lmay be targeted from the TGN to the early endosome (EE), as observed for cathepsin D23. (c) c~131may be targeted from the TGN to a late endosomal compartment (LE), as observed for the mannose 6-phosphate receptor27. The class II-rich late endocytic compartment observed in human B-cell lines (MIIC) is shown as a possible subregion of the late endosome, but may be a separate organelle28. Once targeted to the endocytic pathway, the c~131complex will be retained until the invariant chain is degraded. The class II ~ and 13chains associated with peptide (c~13P)are then released to the plasma membrane (PM). On the basis of the distribution and activity of proteases in the endocytic pathway, it is assumed that the main site of release is from the later stages of the endocytic pathway. However, potential release of some class II-peptide complexes in the early stages might occur, following well-documented recycling pathways to the cell surface (dotted line in A and B). 113
(a)
fPw[
endocytosis
~6
PN endosomal i r a, targeting PEE fI! Ps
=13
(c)
e=,
retention
targeting
References
release II
(d)
targeting [
I l i '+
retention trimming
II
II
PM
flpE i
releas%,
0@ I" late endosome
c~61' early endosome
c
~13
PM
c~61 late endosome
c~131 TGN
Ill
. ?,1p, II II
early endosome
(b)
I
recycling
~6
PM
I
PEE
l P
flpE E 0@ PM
releas%,
Ip
1 i I
~,61 TGN
o~[~I' early endosome
late endosome
PM FIGURE 4
Segregation of antigens bound by endocytosed and exported class II molecules. (a) Functional experiments indicate that class II molecules [which may be empty or may weakly bind peptide (Pw)] internalized from the cell surface bind pinocytosed synthetic peptides (Ps) or peptides generated in the early endosome (PEE) but do not have access to peptides generated from internalized native antigen in the late stages of the endocytic pathway. (b) Class II molecules delivered to the late stages of the endocytic pathway from the biosynthetic pathway can, however, bind such peptides (PN), in addition to binding peptides delivered from the early stages of the pathway (Ps and PEE).(C) If biosynthetically derived class II molecules pass through earlier compartments, as suggested in Fig. 3a and 3b, peptides generated in early endosomes could also be bound in these compartments, provided that the invariant chain dissociates to expose the peptide-binding site. (d) The influence of class II molecules on antigen degradation suggests that partially degraded antigen (P+) could be bound before invariant chain is completely removed, and then trimmed (P+ to P) in a later compartment. Stepwise degradation of invariant chain (I to I') as shown in (c) and (d), for which there is biochemical evidence4s, would result in accumulation of c~131'molecules in the late stages of the endocytic pathway and correlate with kinetics indicating that processing and presentation of native antigen are completed in late endocytic compartments, even if they begin in early compartments.
and finally release the peptide--~[~ complex to the cell surface (Fig. 4d). Thus, many questions remain to be answered. What is the nature of the late endosomal compartment that provides most of the antigen processing and presentation functions? Is class II maturation spatially separated from the efficient degradation of antigen and, if so, where do these molecules meet and what is their pathway to the cell surface?
1 14
Finally, since redundancy is characteristic of the immune response, do several mechanisms of peptide binding by class II molecules (Fig. 4) occur in different locations in the endocytic pathway simultaneously? Given the nature of the problem, immunologists can be expected to be delving around in the endocytic pathway for some time to come.
1 BRODSKY,F. M. and GUAGLIARDI, L. E. (1991) Annu. Rev. ImmunoL 9, 707-744 2 SPIES,T. and DEMARS, R. (1991) Nature 351,323-324 3 POWlS, S. J., TOWNSEND, A. R. M., DEVERSON,E V., BASTIN, J., BUTCHER,G. W. and HOWARD, J. C. (1991) Nature 354, 528-531 4 LJUNGGREN,H-G. et al. (1990) Nature 346, 476-480 5 TEYTON, L. and PETERSON,P. A. Trends Cell BioL 2, 52-56 6 LOTTEAU,V. et aL (1990) Nature 348, 600-605 7 BAKKE,O. and DOBBERSTEIN,B. (1990) Cell 63, 707-716 8 LAMB, C. A., YEWDELL,J. W., BENNINK, J. R. and CRFSSWELL, P. (1991 ) Proc. Natl Acad. Sci. USA 88, 5998-6002 9 PETERSON,M. and MILLER, J. (1990) Nature 345, 172-174 10 MELLINS,E., SMITH, L, ARP, B., COTNER, T., CELLS,E and PIOUS, D. (1990) Nature 343, 71-74 11 GERMAIN, R. N. and HENDRIX, L. R. (1991) Nature353, 134-139 12 MCCOY, K. L., MILLER,J., JENKINS, M., RONCHESE,F., GFRMAIN, R. N. and SCHWARTZ, R. H. (1989) J. Immunol. 143, 29-38 13 UNANUE, E R. (1984)Annu. Rev. ImmunoL 2, 395428 14 BERZOFSKY,A., BRETi-,S. J., STREICHER,H. Z. and TAKAHASHI, H. (1988) ImmunoL Rev. 106, 5-31 15 LANZAVECCHIA,A. (1990) Annu. Rev. ImmunoL 8, 773-793 16 YAMASHIRO,D. J. and MAXFIELD, F. R. (1987) J. CellBiol. 105, 2723-2733 17 MURPHY,R. F. (1991 ) Trends Cell BioL 1, 77-82 18 GRIFFITHS,G. and GRUENBERG,J. (1991) Trends Cell BioL 1, 5-9 19 PIERCE,S. K. et al. (1988) ImmunoL Rev. 149-180 20 HARDING, C. V., COLLINS, D., SLOT, J. W., GEUZF, H. J. and UNANUE, E. R. (1991) Ce1164, 393-401 21 HARDING,C. V., COLLINS, D. S., KANAGAWA, O. and UNANUF, E. R. (1991 ) J. ImmunoL 147, 2860-2863 22 DIMENT, S. and STAHL, P. (1985) J. BioL Chem. 260, 15311-15317 23 LUDWIG, T., GRIFFITHS,G. and HOFLACK, B. (1991) J. Cell BioL 115, 1561-1572 24 WlLFY, H. S., VAN NOSTRAND, W., MCKINLEY, D. N. and CUNNINGHAM, D. D. (1985)J. BioL Chem. 260, 5290-5295 25 ROEDERER,M., BOWSER,R. and MURPHY,R. F. (1987) J. Cell. PhysioL 131,200-209 26 GUAGLIARDI,L. E., KOPPELMAN, B., BLUM, J. S., MARKS, M. S., CRESSWELL,P. and BRODSKY,F. M. (1990) Nature 343, 133-139 27 KORNFELD,S. and MELLMAN, I. (1989)Annu. Rev. CeifBiol. 5, 483-525 28 PETFRS,P. J., NEFFJES,J. J., OORSCHOT, V., PLOEGH, H. L. and GEUZE, H. J. (1991 ) Nature 349, 669-676 29 DAVIDSON, H. W., WEST, M. A. and WATi-S, C. (1990) J. ImmunoL 144, 4101-4109 30 DAVIDSON, H. W., REID, P. A., LANZAVECCHIA,A. and WATTS, C. (1991) Ce1167, 105-116 31 DAVIDSON, H. W. and WATi-S, C. (1989) J. Cell Biol. 85-92 32 DONERMEYER,D. L. and ALLEN, P. M. (1989) J. ImmunoL 142, TRENDS IN CELL BIOLOGY VOL. 2 APRIL 1992
1063-1068 33 AMETANI, A., APPLE,R., BHARDWAI,V., GAMMON, G., MILLER,A. and SERCARZ,E. (1989) Cold Spring Harbor Syrup. Quant. BioL 54, 505-511 34 ROTZSCHKE,O. and FALK,K. (1991 ) ImmunoL Today12, 447-455 35 JARDFTZKY,T. S., LANE,W. S., ROBINSON, R. A., MADDEN, D. R. and WILEY, D. C. (1991 ) Nature 353, 326-329 36 MADDEN, D. R., GORGA,J. C., STROMINGER,J. L. and WILEY, D. C. (1991) Nature 353, 321-325 37 RUDENSKY,A. Y., PRESTON-HURLBURT,P., I-lONG, S-C., BARLOW, A. and JANEWAY,C. A. Jr. (1991 ) Nature 353, 622-627 38 BLUM, J. S. and CRESSWELL,P. (1988) Proc. Natl Acad. Sci. USA 85, 3975-3979 39 NGUYEN,Q. V., KNAPP,W. and HUMPHREYS,R. E. (1989) Hum. Immunol. 24, 153-163 40 JENSEN,P. E. (1990) J. Exp. Med. 171, 1770-1784 41 WETTSTEIN,D. A., LIN, A. Y., MATIS, L. A. and DAVIS, M. M. (1991 )J. Exp. Med. 174, 219-228 42 BUUS,S., SEI-I-E,A., COLON, S. M., JENIS,D. M. and GREY, H. M. (1986) Ce1147,1071-1077 43 LEE,J. M. and WAI-FS, T. H. (1990) J. ImmunoL 144, 1829-1844 44 HARDING,C. V. and UNANUE, E. R. (1989)J. ImmunoL 142, 12-19 45 PIETERS,J., HORSTMANN, H., BAKKE,O., GRIFFITHS,G. and LiPP, J. (1991) J. CellBioL 115, 1213-1223 46 HARDING,C. V., UNANUE, E. R., SLOT,J. W., SCHWARTZ, A. L. and GEUZE, H. J. (1990) Proc. NatlAcad. Sci. USA87, 5553-5557
TRENDS IN CELL BIOLOGY VOL. 2 APRIL 1992
47 DAVIS,J. E. and CRESSWELL,P. (1990)J. ImmunoL 144, 990-997 48 NEEFJES,J. J., STOLLORZ,V., PETERS,P. J., GEUZE,H. J. and PLOEGH,H. L. (1990) Cell61, 171-183 49 REID,P. A. and WATI-S, C. (1990) Nature 346, 655-657 50 HARDING,C. V., ROOF, R. W. and UNANUE, E. R. (1989) Proc. Notl Acad. Sci. USA86, 4230-4234 51 KAKIUCHI,T., WATANABE, M., HOZUMI, N. and NARIUCHI,H. (1990) J. ImmunoL 145, 1653-1658 52 UNANUE, E. R. and ALLEN, P. M. (1987) Science236, 551-557 53 ASHWELL,J. D. (1988)J. ImmunoL 140, 3697-3700 54 KING, P. and KATZ, D. R. (1990) ImmunoL Today11,206-211 55 POBER,J. S. and COTRAN, R. S. (1990) PhysioLRev. 70, 427-451 56 DONIACH, D., SPENCER,K. M. and BOTAZZO, G. F. (1985) in The AutoimmuneDiseases(Rose, N. R. and Mackay, I. R., ed.), pp. 227-242, Academic Press 57 WAI-rS, C., WEST, M., REID, P. A. and DAVIDSON, H. W. (1989) Cold Spring Harbor Syrup. Quant. BioL 54, 345-352 58 ROCHE,P. A., MARKS,M. S. and CRESSWELL,P. (1991) Nature 354, 392-394 59 CEPPELLINI,R., FRUMENTO,G., FERRARA,G. B., TOSI, R., CHERSI,A. and PERRIS,B. (1989) Nature 339, 392-394 60 MARSH,E. W., DALKE,D. P. and PIERCE,S. K. (1992) J. Exp. Med. 175, 425-436 61 VANBUSKIRK,A. M., DENAGEL,D. C., GUAGLIARDI,L. E., BRODSKY,F. M. and PIERCE,S. K. (1991)J. ImmunoL 146, 500-506 62 BRAUN,M., WAHEED, A. and VON FIGURA,K. (1989) EMBOJ. 8, 3633-3640 63 WRAIGHT,C. J. (1990)J. BioL Chem. 265, 5787-5792
Acknowledgments I thank G. Griffiths and C. Watts for communication of data prior to publication and K. Mostov for comments on the manuscript. The work from my laboratory that is described in this review was supported by NIH grant AR20894 and by the Pew Charitable Trusts.
1 15