26

Proteolysis generates the peptides that bind to class II MHCmolecules and, by destruction of the invariant chain, preparesthe class II MHC molecule for capture of those peptides. Aclearer picture is emerging of the proteases, proteaseinhibitors and other factors that together control theenvironment for class II MHC peptide loading. However, thedetails of invariant-chain processing and antigen processingmay differ depending on the allele of class II and the antigensubstrate under consideration.

AddressesWellcome Trust Biocentre, University of Dundee, Dow Street, Dundee,DD1 5EH, UK; e-mail: c.watts@dundee.ac.uk

Current Opinion in Immunology 2001, 13:26–31

0952-7915/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.

AbbreviationsAEP asparaginyl endopeptidaseAPC antigen-presenting cellCLIP class-II-associated Ii peptideDC dendritic cellHEL hen egg lysozymeIi invariant chain

IntroductionThe capture of peptides by class II MHC molecules is animpressive salvage operation performed under seeminglyunfavourable circumstances. Class II MHC molecules,chaperoned by the invariant chain (Ii), are delivered to theendosome/lysosome system and exposed to an environ-ment rich in proteolytic enzymes. Somehow, processedantigenic material, usually present in very small amounts,survives the proteolytic onslaught and finds a safe refugein the binding groove of newly synthesised and — once Iiis removed — potentially unstable class II MHC mol-ecules. The DM protein plays a stabilising role here and,assisted by DO in some cells, steers peptide loading inimmunologically desirable directions. Here I review recentprogress on the proteolytic events that may create ordestroy antigenic epitopes in endocytic compartments andwhich are central to Ii removal; more participants are beingidentified, their roles elucidated and their potential, astargets for immune intervention, evaluated.

Proteases that are expressed in antigen-presenting cellsThe list of proteases found within the endosome/lysosomesystem is long and getting longer [1–3,4•]. At least a dozenenzymes related to the cysteine protease papain are nowknown to exist and, of these, cathepsins B, H, L, S, F, Z, V,O, C and possibly K are expressed in antigen-presentingcells (APCs) of different types. These are joined in APCsby the aspartic proteases cathepsins D and E and byasparaginyl endopeptidase (AEP). Other enzymes, such as

cathepsins J and W, are predominantly expressed in othercell/tissue types and probably do not play a role in normalantigen-presentation. Most of these proteases have acidicpH optima and rather broad substrate specificity; however,cathepsin S is an exception to the former rule and AEP aclear exception to the latter. Some, such as cathepsins H,C, B and Z, have structural features that limit access to theactive site and function as amino (cathepsins H and C) orcarboxyl (cathepsins B and Z) exopeptidases [1].

The ‘occluding loop’ of cathepsin B is such a structuralfeature but can, at lower pH values, become more flexible,allowing endoproteolytic cleavages [5]. Exopeptidases arelikely to account, at least in part, for the heterogeneoustermini of naturally processed T cell epitopes that bindclass II MHC [6,7]. Few of these enzymes are expressedexclusively in APCs and most have homologues in plantsor simple animals. Therefore, the mammalian immunesystem appears to have taken advantage of ancient prote-olytic systems to perform the essential steps in antigenpresentation. Expression in APCs of the most ‘useful’enzymes and regulation of expression of some of thesewith cytokines such as IFN-γ tailor the processing machineryto the needs of the APC.

The list of proteases implicated in class II MHC restrictedantigen presentation is unlikely to be complete.Radiolabelled, active-site-directed probes allow the levelsof active cysteine proteases in different APCs, and evendifferent compartments within cells, to be assessed. Someof these reagents label multiple targets, not all of whichhave been identified [8,9,10•]. Some may be cysteine pro-teases expressed in the endosome/lysosome system andmay play a role in class II MHC related events.

Antigen processingProteases involved in antigen processingSeveral studies have identified proteases that can liberateT cell epitopes from protein antigens in vitro but it hasbeen more difficult to establish which enzymes are actuallyinvolved in vivo [11]. Approaches that have been informa-tive here include the identification and mutagenesis ofprocessing sites in protein antigens, the use of specificinhibitors and the use of protease-gene-targeted mice.

Cathepsins D and B, both of which have been implicatedin antigen processing from in vitro digestion studies,appeared to be dispensable for both Ii processing and forthe generation of several T cell epitopes [12,13]. However,equivalent antigen-dose/T-cell-response titrations (as usedin these studies) can sometimes mask significant differ-ences in the kinetics of presentation [14], so it may bepremature to rule out any role for these abundant proteasesin antigen processing and they continue to be implicated

Antigen processing in the endocytic compartmentColin Watts

Antigen processing in the endocytic compartment Watts 27

in other studies [15]. A recent study [16] showed that pro-cessing of exogenous glutamate decarboxylase (GAD), atarget autoantigen in diabetes mellitus, was sensitive tocathepsin D inhibitors as well as cysteine proteaseinhibitors. In this study, presentation of endogenouscytosolic GAD on class #II MHC molecules was alsoobserved and was sensitive to inhibitors of the proteasomeand calpain, indicating an unexpected requirement forpre-processing by cytoplasmic proteases.

Cathepsin L is only expressed in certain APCs, notablycortical thymic epithelial cells where it is responsible forthe terminal stages of Ii processing [17]. Its role in gener-ating the peptide pool for positive-selection of CD4+

T cells is not known.

Cathepsin S is now known to be required for the latestages of Ii processing on some murine class II MHC alle-les, particularly I-Ab (see below), making it more difficultto identify a role for cathepsin S in specific antigen pro-cessing. However, other class II MHC alleles — such asI-Aq, I-Ak, I-Au and I-As — and certain cell types(macrophages) are less dependent on cathepsin S for Iiremoval [12,18••]. An I-Aq-restricted type II collagen epi-tope was not presented efficiently by cathepsin S nullmacrophages in vitro and may therefore requirecathepsin S for its processing [18••]. Although IgGresponses to ovalbumin were suppressed in cathepsinS null mice, IgE responses to nebulised ovalbumin werenormal, indicating that enzymes other than cathepsinS process this protein in lung APCs [10•,19••].

Relatively few protease-processing sites in native antigenshave been mapped and even fewer tested for their actualimportance. Recently, AEP (the mammalian form of plantlegumain) was identified as the enzyme that initiates pro-cessing of TTCF — a carboxy-terminal domain of thetetanus toxin antigen [20]. Mutagenesis (i.e. an Asn→Glnchange) of each of the three major AEP processing sites inTTCF revealed that one site is crucial for efficient pre-sentation of a range of T cell epitopes, some surprisinglydistant from the AEP processing site [21••]. A model forprocessing of this antigen was suggested whereby initialprocessing can only be efficiently performed by AEP. Asingle cleavage is proposed to ‘unlock’ the native struc-ture, leading to further, as-yet uncharacterised unfoldingand processing events.

The converse strategy — introduction of processing sitesinto antigens — is more difficult since there is no certaintythat recognition will actually occur. For example, AEP onlycleaves about 10% of asparaginyl bonds in TTCF and othernative proteins [20,22]. Nonetheless, a recent report [23]showed that a Phe→Arg mutation in hen egg lysozyme(HEL) that created a dibasic (Lys–Arg) motif resulted in~10-fold more efficient presentation of the adjacentHEL23–32 I-Ad-restricted determinant. The identity of theenzyme(s) that may cleave this dibasic site is not yet known.

Other factors that may be involved in antigen processingAre there other factors besides proteases involved in gen-eration of suitable substrates for loading onto class II MHCmolecules? Reduction of disulphide bonds was shown ear-lier to be important for presentation of certain T cellepitopes and to depend on a reducing environment inendosomes and lysosomes [24,25]. An interesting novelparticipant in this reaction is GILT, an IFN-γ-induciblelysosomal thiol reductase identified recently in endosomesthat were positive for class II MHC [26•]. GILT reducesprotein disulphide bonds optimally at acidic pH and maytherefore be involved in reduction and possibly unfoldingof protein antigens [27•].

The open ends of the class II MHC peptide-binding groovemay be well suited to capture of unfolded and extendedantigen domains and this may be essential to avoid over-digestion and destruction of T cell epitopes. Peptideseluted from bulk class II MHC populations generally have amaximum size of ~25 residues but earlier studies indicatedthat longer processing products might be captured by classII MHC molecules [28,29]. A recent study performed onHEL-pulsed splenocytes identified a class II MHC dimermade up of one I-Ek and one I-Ak class II MHC molecule.Interestingly, this association was only seen in HEL-pulsedcells and appeared to be due to a 7 kDa processed fragmentof HEL linking the two class II MHC molecules [30•].Intermediates of this type are very difficult to detect due totheir transient nature; the I-Ek–HEL–I-Ak complex wasdetected probably because of its unusual longevity and sta-bility. Nonetheless, early capture of unfolded, extendedsequences by class II is an attractive proposition in anaggressive proteolytic environment. On the basis of the datacurrently available, a speculative model for the processing ofprotein antigens is shown in Figure 1.

Processing of IiConversion of Iip10 to CLIPExamination of Ii processing in H-2b mice lacking eithercathepsin S or L demonstrated a key role for cathepsin S inmost cells that are positive for class II MHC [31](e.g. B cells and dendritic cells [DCs]) and for cathepsin Lin cortical thymic epithelial cells [17]. Inhibition or dele-tion of either enzyme results in the accumulation of a10 kDa amino-terminal Ii fragment (Iip10) that under nor-mal conditions is then converted by cathepsin S or L toCLIP (class-II-associated Ii peptide), the smallest Ii frag-ment that continues to stably associate with class II MHC.As detailed elsewhere (reviewed in [32,33]), this is thenexchanged for a foreign or self peptide in a reaction catalysedby the DM or in some cells the DM/DO proteins.

Surprisingly, macrophages from mice lacking both cathep-sins S and L showed virtually normal Ii processing andpeptide loading. Shi et al. [10•] examined the spectrum ofcysteine proteases expressed in different APC types andfound that two recently described cathepsins, F and Z (thelatter is also known as cathepsin X), were specifically

28 Antigen processing and recognition

expressed in macrophages. Cathepsin F, but not Z, was ableto process Iip10 at least as well in vitro as cathepsin S and islikely to be the enzyme responsible for the final stages of Iiprocessing in cathepsin S deficient macrophages. Becausecathepsin F has not so far been demonstrated to beexpressed in DCs or B cells, the results of Shi et al. [10•]also point to the importance of lung macrophages as APCs.

Conversion of intact Ii to Iip10Which enzyme(s) act upstream of cathepsin S to generatethe Iip10 fragment? This is currently unresolved. Althoughcathepsin S (and perhaps cathepsins L and F) can convertp31 (the major isoform of intact Ii) to CLIP in vitro it isclearly not essential in vivo. Leupeptin, a broad-spectrumcysteine protease inhibitor, arrests Ii processing at an earlierstage, inducing the accumulation of an Ii fragment ofaround 22/23 kDa, particularly in human cells [34,35].Thus one or more leupeptin-insensitive enzymes can acton the intact Ii substrate whereas a leupeptin-sensitiveenzyme(s) other than cathepsins S, L or F is likely to beinvolved in conversion of p22/p23 to p10. AEP, cathepsinD, cathepsin E or another leupeptin-insensitive enzymemight perform the first step. However, the precise cellularlocalisation of cathepsin E remains unclear and in spite ofreported effects of aspartic protease inhibitors on Ii pro-cessing [15,36], no defect in Ii processing was observed incathepsin D deficient mice, at least for the H-2b haplotype[12]. Conceivably, other proteases sensitive to theinhibitors used in these studies are at work. The p22, p10

and other less prominent intermediates can often beseen, albeit to a lesser extent, in non-inhibited cells, indi-cating that these are bone fide intermediates in normal Iiprocessing (reviewed in [2]).

The role of early endosomesPerturbations of the endocytic pathway can reveal the traf-ficking pathways taken by complexes of class II with Ii,and possible alternative pathways for Ii removal. Whenearly endosomes that are positive for transferrin-receptorwere selectively ablated, Ii processing was blocked,demonstrating that early endosomes are an obligatory stopon the itinerary leading to maturation of complexes of classII with Ii [37,38]. When such complexes were trapped inearly endosomes by concanamycin-B treatment, a propor-tion of complexes were able to eliminate Ii without theinvolvement of leupeptin-sensitive proteases or DM [39].The extent to which this pathway operates under normalconditions is not yet clear.

Protease inhibitors and regulation of antigenprocessing Cystatins The DC has provided interesting examples of how antigenpresentation can be tightly regulated, for example byinflammatory mediators, as immature DCs are driven tomaturation [40]. More recently, insight into some of themechanisms at work has been obtained. Differentialexpression of cystatin C in immature (compared with

Figure 1

A general pathway for processing in theendocytic compartment. Not all substrates willrequire all steps but it is suggested thatproteases will fall into three types. (a) The firsttype are those with preference for polar orcharged sidechains that can introducecleavages into native protein structures. Thesewill be relatively nonredundant and initiateprocessing. (b) These enzymes may continueto act later but may be joined by the secondtype of protease — those (such as cathepsin D)with preference for cleavage at hydrophobicsites. More redundancy may be found as theantigen substrate is made more accessible.GILT, and perhaps other chaperones, mayfacilitate the process of disulphide bondcleavage and unfolding. (c) Capture ofextended and unfolded regions by class IIMHC molecules recently divested of Ii mayoccur at any stage downstream of the initiatingcleavages and serves to protect the antigensubstrate from unrestricted proteolysis andperhaps to stabilise class II MHC moleculesthat are not DM-associated. The initialsequence captured may be suboptimal but theopen nature of the class II MHC groovepermits, in principle, ‘scanning’ of the extendedsequence for a better ligand. (d) This could befacilitated by the peptide editor function of DMoperating in ‘cis’ (i.e. on the same polypeptide

chain). (e) In the third type of protease action,further endo- and exo-peptidase action wouldgenerate the short but heterogenous ligandsthat can be eluted from cell surface class II

MHC molecules. Aminopeptidase action maybe arrested by proline, as suggested bysequencing of bulk peptides eluted fromhuman HLA-DR molecules [59].

S S

SH

S

SH SHHS

SH HS SH HS

HS

S SGILT S GILT

(a) (b)

(d)

(e)

(c)Initiating protease(AEP, cathepsins Sand B, others?)

Secondary protease(cathepsins Dand E, others?)

Exopeptidases(cathepsins H,Z, C, B, others)

MHC-class-II–DM

MHC class II

Peptide ligandCurrent Opinion in Immunology

Antigen processing in the endocytic compartment Watts 29

mature) DCs inhibited terminal processing of Ii andaccounted, at least in part, for the low cell-surface levels ofclass II MHC in immature H-2b DCs [41].

Using an antibody (C4H3) specific for a complex of anHEL peptide with I-Ak, Inaba et al. [42••] have now shownthat immature DCs do not generate this complex eventhough internalised HEL and I-Ak co-localise. Delivery ofa maturation stimulus, which could be given up to 48 hoursafter antigen pulsing, produced a dramatic appearance ofthe I-Ak–HEL complex and its transport to the cell surfacetogether with co-stimulatory molecules [43•]. In apparentcontrast to these results, an earlier study found that pep-tide-loaded (i.e. SDS-stable) class II MHC molecules wereformed in immature DCs but retained intracellularly [44].Possibly these were in fact I-Ab–Iip10 complexes, whichare unusually stable in SDS [45]. Do immature DCs fail togenerate the C4H3-reactive complex because of suppres-sion of cathepsin S activity by cystatin C and theconsequent failure of Ii and/or HEL processing?Additional mechanisms must be at work, since the I-Ak

molecule was able to mature normally in cathepsin S nullsplenocytes [12] and immature DCs also failed to formC4H3-reactive complexes even when pulsed with thepre-processed HEL peptide [42••].

Cystatin C inhibits several members of the papain-familyof cysteine proteases besides cathepsin S, via a conserved‘edge’ that binds in the active-site cleft [46]. Interestingly,cystatin C was recently shown to have a second inhibitorysite specific for asparaginyl endopeptidase [47•]. Thus,cystatin C is a ‘double-headed’ protease inhibitor capableof simultaneously inhibiting a wide range of cysteineproteases and not just cathepsin S.

Examples of pathogen interference in the class II MHCpathway are few in comparison with instances of interfer-ence with the class I MHC pathway. Nonetheless,proteases in the class II MHC pathway are possible targetsfor such interference and, interestingly, cystatin-like geneproducts have been found in several parasites [48,49].Recent studies show that Bm-CPI-2, a cysteine proteaseinhibitor secreted by the filarial parasite Brugia malayi, caninterfere with presentation of tetanus toxin epitopes byhuman APCs (B Manoury, W Gregory, RM Maizels,C Watts, unpublished data). Like cystatin C, Bm-CPI-2can inhibit both cathepsin S and AEP.

The p41/p31 isoforms of Ii Apart from the cystatins, the propeptides of cysteine pro-teases also act as natural inhibitors. Another intriguing‘natural’ inhibitor is a 64 amino acid peptide unique to thep41 isoform of Ii. This peptide unexpectedly turned upassociated with cathepsin L isolated from human kidney.This peptide, homologous to repeat domains in thyroglob-ulin, equistatin and other proteins [50], was shown to be apotent and specific (Ki = 1.7 pM) inhibitor of cathepsin Lbut not cathepsin S with a protein fold distinct to that of

cystatin C [51,52,53•]. The existence of this domain withinIi remains enigmatic since cathepsin L is not expressed inthose APCs (e.g. DCs) where p41 expression is mostprominent. Perhaps closer examination of the cells in thekidney, from which the complex of cathepsin L with p41was originally isolated, would shed more light on thefunctional significance of this striking association.

Evidence that Ii may exert a more general (i.e. indepen-dent of the association between the p41 fragment andcathepsin L) inhibitory effect on proteolysis in the class IIMHC pathway comes from a recent study on the stabilityof the DM protein in cells from Ii deficient mice.Unexpectedly, DM was found to be much less stable insplenocytes and mature DCs lacking Ii [54•]. Although thep41 form of Ii seemed preferentially to associate with DM,the p31 form was also effective. Exactly how Ii stabilisesDM remains to be established but Pierre et al. [54•] pointout that the defects in selection of CD4+ T cells observedearlier in Ii knockout mice may be due to compromisedDM function as well as aberrant class II MHC trafficking.

Cytokines and endosomal pH Cytokines and chemokines control many aspects of APCfunction but there is limited information on the extent towhich the cytokine/chemokine milieu influences pro-cessing and epitope capture in the class II MHC systembeyond the IFN-γ-mediated inducibility of several lysosomal enzymes [55].

A recent study found that the set of T cell epitopes fromHEL displayed by murine DCs differed markedlybetween IL-6-treated and -untreated cells. One dominantAk-restricted determinant (HEL46–61) was presented muchworse by IL-6-treated DCs whereas the Ed-restricteddeterminant HEL2–16, which is normally cryptic, was pre-sented much better both in vitro and in vivo [56•]. IL-6treatment rendered early endosomes more acidic, possiblyby interference with the electrogenic Na+/K+ ATPase thatis thought to limit early endosome acidification [57].Oubain, a classical inhibitor of this pump, was also able toboost presentation of the cryptic HEL2–16 epitope.Endosomal pH changes might in turn modulate proteaseactivity/specificity and/or affect DM function. However,DM activity, which should be boosted by endosomal acid-ification, was recently reported to disfavour presentation ofother cryptic epitopes in HEL [58•].

Conclusions and future directionsWe can look forward to further progress in understandingantigen processing in the endocytic compartment. Only afew of the proteolytic enzymes known to be present havebeen knocked out or specifically inhibited and even fewerantigens systematically analysed for their processingrequirements. A key issue concerns the level of redundan-cy that exists for both Ii and antigen processing.Surprisingly, the late stages of Ii processing, at least onsome class II alleles, and the initial stages of processing of

30 Antigen processing and recognition

at least one antigen (tetanus toxin) can only be performedby specific enzymes. No general rules can be establishedfrom one antigen but a reasonable guess might be that alimited but overlapping set of proteases will be requiredfor efficient processing of each antigen. It will be particu-larly important to identify the enzymes that participate inautoantigen and allergen processing. Are proteases, anti-gen substrates and chaperoned class II molecules the onlyplayers needed or is there a more concerted and controlledreaction that takes place? GILT may be one additionalparticipant but perhaps there are others.

AcknowledgementsWork in my laboratory is supported by the Wellcome Trust, the EuropeanUnion and the Association for International Cancer Research.

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42. Inaba K, Turley S, Iyoda T, Yamaide F, Shimoyama S, Reis e Sousa C,•• Germain RN, Mellman I, Steinman RM: The formation of

immunogenic major histocompatibility complex class II-peptideligands in lysosomal compartments of dendritic cells is regulatedby inflammatory stimuli. J Exp Med 2000, 191:927-936.

Together with [43•], this shows that the formation of a complex of peptidewith class II MHC is inhibited in immature dendritic cells (DCs). The use ofan antibody specific for the I-Ak–HEL46–61 peptide allows striking visuali-sation of the formation and subsequent transport of the complex uponinduction of DC maturation.

43. Turley SJ, Inaba K, Garrett WS, Ebersold M, Unternaehrer J,• Steinman RM, Mellman I: Transport of peptide-MHC class II

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45. Brachet V, Raposo G, Amigorena S, Mellman I: Ii chain controls thetransport of major histocompatibility complex class II moleculesto and from lysosomes. J Cell Biol 1997, 137:51-65.

46. Bode W, Engh R, Musil D, Thiele U, Huber R, Karshikov A, Brzin J,Kos J, Turk V: The 2.0 A X-ray crystal structure of chicken eggwhite cystatin and its possible mode of interaction with cysteineproteinases. EMBO J 1988, 7:2593-2599.

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Elegant demonstration that cystatin C has two distinct cysteine-proteaseinhibitor sites sufficiently distant that a single cystatin molecule can bind twodifferent protease molecules.

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49. Hartmann S, Kyewski B, Sonnenburg B, Lucius R: A filarial cysteineprotease inhibitor down-regulates T cell proliferation andenhances interleukin-10 production. Eur J Immunol 1997,27:2253-2260.

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51. Fineschi B, Sakaguchi K, Appella E, Miller J: The proteolyticenvironment involved in MHC class II-restricted antigenpresentation can be modulated by the p41 form of invariant chain.J Immunol 1996, 157:3211-3215.

52. Bevec T, Stoka V, Pungercic G, Dolenc I, Turk V: Majorhistocompatibility complex class II-associated p41 invariant chainfragment is a strong inhibitor of lysosomal cathespin L. J Exp Med1996, 183:1331-1338.

53. Guncar G, Pungercic G, Klemencic I, Turk V, Turk D: Crystal structure• of MHC class II-associated p41 Ii fragment bound to cathepsin L

reveals the structural basis for differentiation between cathepsinsL and S. EMBO J 1999, 18:793-803.

First of the thyrogloblin type 1 family of protease inhibitors, some of whichare still putative inhibitors, to be solved. It is superficially similar to cystatin C;however, the distinct fold explains the stricter specificity for cathepsin L.

54. Pierre P, Shachar I, Matza D, Gatti E, Flavell RA, Mellman I: • Invariant chain controls H2-M proteolysis in mouse

splenocytes and dendritic cells. J Exp Med 2000,191:1057-1062.

Intriguing evidence that the protease-inhibitory activity of the invariantchain may extend beyond the well-documented association of the p41specific domain with cathepsin L (see [53•]). This paper demonstrates anunexpected instability of the DM protein in cells lacking Ii. Both isoforms(p41 and p31) were effective although p41 seems to associate morestrongly with DM.

55. Chapman HA: Endosomal proteolysis and MHC class II function.Curr Opin Immunol 1998, 10:93-102.

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An example of how cytokine treatment can modulate the outcome of antigenpresentation. A cryptic epitope in hen egg lysozyme (HEL) is presented onlyby IL-6-treated dendritic cells. IL-6 is proposed to induce hyper-acidificationof early endosomes, which in turn could influence the antigen, proteases ,DM or other factors. Ouabain, an inhibitor of the endosomal Na+K+ ATPasemimicked the IL-6 effect.

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58. Nanda NK, Sant AJ: DM determines the cryptic and• immunodominant fate of T cell epitopes. J Exp Med 2000,

192:781-788.Clear evidence that expression of DM can extinguish presentation of crypticT cell epitopes and boost presentation of dominant epitopes. DiminishedDM expression leading to presentation of cryptic epitopes might have patho-logical consequences.

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