INTRODUCTION

Presentation of antigenic peptides to T cells in the context ofMHC class II molecules by antigen presenting cells, such asmacrophages, B lymphocytes, and dendritic cells, requires acomplex sequence of intracellular events. Antigens areinternalized, then degraded into peptides which associate withMHC class II molecules before reaching the cell surface tostimulate T cells (Watts, 1997). MHC class II α and β subunitsassemble in the endoplasmic reticulum with the invariant chain(Ii), then reach endocytic compartments (Cresswell, 1994). Inthese compartments, Ii is degraded, leaving MHC class IImolecules free to bind peptides derived from endocytosedantigens. Depending on the cell type analyzed, compartmentseither related to endosomes (Class II vesicles; CIIVs in murineB cells) or to lysosomes (MHC class II compartments; MIICsin human B cells, dendritic cells, macrophages) have beenidentified. CIIVs and MIICs have been proposed as the meetingpoint between class II molecules and processed antigenicpeptides (Amigorena et al., 1994; Tulp et al., 1994; West et al.,1994).

In bone marrow-derived mast cells (BMMCs) MHC class IImolecules accumulate in secretory granules with lysosomalcharacteristics, called secretory lysosomes (Raposo et al.,1997). Three types of secretory granules were identified on the

basis of their ultrastructural morphology, their protein contentand their accessibility to endocytic tracers. The majority ofMHC class II molecules accumulate in Type I granulesdisplaying intralumenal vesicles and in Type II granulescontaining an electron dense core, rich in serotonin, surroundedby a multivesicular cortex (Raposo et al., 1997). Two questionsarise from these observations: what are the molecularmechanisms responsible for the retention of MHC class IImolecules in secretory granules and what are the consequencesof this peculiar localization of MHC class II molecules onantigen presentation in mast cells?

To approach these issues, BMMCs are not the bestexperimental model. First, these cells need to be cultured in acocktail of lymphokines (IL3, IL4 and GM-CSF) which mayhave pleiotropic effects on MHC class II transport andfunctions. Second, BMMCs are an heterogenous cellpopulation since only 30% of cells express MHC class IImolecules. Therefore, we designed a new experimental modelto study MHC class II transport and functions in mast cells, byexpressing IA α and β chains, as well as murine Ii, in the MHCclass II negative rat basophilic cell line, RBL-2H3 (Barsumianet al., 1981). In the present study, we show that most MHCclass II molecules were retained in intracellular compartmentsdisplaying markers of lysosomes and secretory granules. Inthese compartments, MHC class II molecules were found in

323

Bone marrow-derived mast cells as well as dendriticcells, macrophages and B lymphocytes express majorhistocompatibility complex (MHC) class II molecules. Inmast cells, the majority of MHC class II molecules residein intracellular cell type-specific compartments, secretorygranules.

To understand the molecular basis for the localisation ofMHC class II molecules in secretory granules, MHC classII molecules were expressed, together with the invariantchain, in the mast cell line, RBL-2H3. Using electron andconfocal microscopy, we observed that in RBL-2H3 cells,mature and immature class II molecules accumulate insecretory granules. Two particular features of class IItransport accounted for this intracellular localization:first, a large fraction of newly synthesized MHC class IImolecules remained associated with invariant chain

fragments. This defect, resulting in a slower rate of MHCclass II maturation, was ascribed to a low cathepsin Sactivity. Second, although a small fraction of class II dimersmatured (i.e. became free of invariant chain), allowing theirassociation with antigenic peptides, they were retained insecretory granules. As a consequence of this intracellularlocalization, cell surface expression of class II moleculeswas strongly increased by cell activation stimuli whichinduced the release of the contents of secretory granules.Our results suggest that antigen presentation, and therebyantigen specific T cell stimulation, are regulated in mastcells by stimuli which induce mast cell activation.

Key words: MHC class II, Mast cell, Invariant chain, Secretarygranule, Antigen presentation

SUMMARY

Secretory granules of mast cells accumulate matureand immature MHC class II moleculesHélène Vincent-Schneider 1, Clotilde Théry 1, Daniela Mazzeo 2, Danièle Tenza 3, Graça Raposo 3

and Christian Bonnerot 1,*1INSERM, U. 520, Institut Curie,12, rue Lhomond, 75005 Paris, France2Department of Biochemistry, University of Dundee, MSI/WTB Complex, Dow Street, Dundee, DD1 5EH, UK3CNRS, UMR 144, Institut Curie,12, rue Lhomond, 75005 Paris, France*Author for correspondence (e-mail: bonnerot@curie.fr)

Accepted 1 November 2000Journal of Cell Science 114, 323-334 © The Company of Biologists Ltd

RESEARCH ARTICLE

324

two different forms: the majority of class II molecules wereimmature, i.e. associated to a partially digested form of Ii, thep10 fragment, which was due to a low cathepsin S activity inmast cells. However, a fraction of class II molecules retainedin the secretory granules matured into αβ dimers able to bindantigenic peptides. Consequently, we show that stimuli whichinduce mast cell activation, and therefore degranulation, alsoallow exposure of functional MHC class II-peptide complexesat the cell surface and thus T cell stimulation.

MATERIALS AND METHODS

Chemical reagents and antibodiesChemical reagents used in this study were all from Sigma (St Louis,MO, USA). RPMI 1640, fetal calf serum (FCS), phosphate bufferedsaline (PBS), penicillin – streptomycin, sodium pyruvate and L-glutamine were purchased from Gibco (Paisley, Scotland). RPMIdepleted for methionine and cysteine, and [35S]methionine/cysteinelabelling mix were obtained from ICN (Orsay, France). Protein G-Sepharose was obtained from Pharmacia (Uppsala, Sweden). Theantibodies used were the mouse anti-mouse IAd mAb MKD6(Kappler et al., 1981), the mouse anti-mouse IAb mAb Y3P (Janewayet al., 1984), the mouse mAb YAe anti-mouse IAb associated withpeptide 52-68 of IEd α (Murphy et al., 1992), the rat anti-IAb andIAd mAb M5/114 (Bhattacharya et al., 1981), a polyclonal rabbitserum against the conserved cytoplasmic tail of the α chain of themouse MHC class II molecules IAb, d (anti-IAα), the rat anti-mouseIi chain mAb IN1 (directed against Ii’s cytoplasmic tail) (Peterson andMiller, 1990) and a rabbit anti-serum specific for the cytoplasmic tailof Ii chain (IiNH2) (a gift from J. Davoust, CIML, Marseille, France).Other antibodies used were the mouse anti-rat CD63 mAb AD1 (a giftfrom Dr R. P. Siraganian, NIH, Bethesda, MD) (Kitani et al., 1991),the mouse anti-rat lamp1 mAb LY1C6 (a gift from Dr W. Hunziker,Epalinges, Switzerland) (Lewis et al., 1985), the mouse anti-rat p80(protein contained in the secretory granules of RBL-2H3; Bonifacinoet al., 1989) 5G10 (a gift from J. Bonifacino, NIH, Bethesda, MD), arabbit anti-serotonin anti-serum (Seralabo), a rabbit anti-mousecathepsin S anti-serum (a gift from Dr H. Chapman, HarvardUniversity, Boston, MA). To specifically detect mouse, rat or rabbitantibodies, horseradish peroxidase (HRP-), FITC- or Texas Red-coupled F(ab′)2 fragments of donkey antiserum were obtained fromJackson Immunoresearch (Jackson laboratory, West Grove, PE, USA).

CellsRBL-2H3 (Barsumian et al., 1981), B414, IIA1.6 and T cellhybridoma were grown in RPMI 1640, 10% FCS, 1% penicillin-streptomycin, 0,1% β-mercaptoethanol, 2% sodium pyruvate. The Blymphoma IIA1.6 is a FcγR-defective variant of A20 B cells(Bonnerot et al., 1992). The B lymphoma B414 is a A20 B lymphomacell line transfected with cDNAs encoding IAb α and β chains (kindlyprovided by A. Barlow and C. Janeway, Yale University, New Haven,CT). TH30 is a T cell hybridoma which recognizes Eα 52-68 peptideassociated with IAb (Barlow et al., 1998). Peptides have beensynthesised by Synt:em (Nice, France).

cDNAs were cloned downstream of an Srα promoter (Takebe et al.,1988) in expression vectors carrying resistance genes for hygromycinB (NTHygro), neomycin (NTNeo) or zeocin (NTZeo). The IAd andIAb cDNAs (respectively provided by Dr R. Germain, NationalInstitutes of Health, and Drs A. Barlow and C. Janeway, YaleUniversity, New Haven, CT) were subcloned in the NThygro vectorfor α chains and NTNeo vector for β chains. The cDNA encodingmurine Ii chain (provided by Dr R. Germain) was subcloned in theNTZeo vector. Expression vectors were co-transfected into the RBL-2H3 cell line as described (Bonnerot et al., 1992). Briefly, cells wereelectroporated at 260 V, 975 µF with 50 µg of linearized plasmids.

Two days after transfection, cells were switched into selectionmedium (containing 1 mg/ml of hygromycin, neomycin-G418- andzeocin, respectively). Growing cells were subcloned by limitingdilution in 96-well plates. Clones were then analyzed for surfaceMHC class II molecules expression by cytofluorimetry with aFACScan® flow cytometer (Becton Dickinson) after staining withM5/114 and FITC-coupled anti-rat antibodies.

Western blotCells were lysed in lysis buffer: 0.5% Triton X-100, 300 mM NaCl,50 mM Tris, pH 7.4, 10 µg/ml of leupeptin, chemostatin, aprotinin,pepstatin, 20 mM N-ethylmaleimide, 1 mM PMSF and centrifuged at20,000 g for 15 minutes. Cell lysates were diluted in Laemmli bufferand boiled at 95°C for 5 minutes before analysis on 12% SDS-polyacrylamide gels. Proteins were transferred to a polyvinylidenefluoride (PVDF) membrane (Millipore). After incubation withblocking solution, membranes were incubated with specific antibodiesfollowed by HRP-conjugated anti-rabbit IgG antibodies.Chemiluminescence was detected using a Boehringer kit.

Cell fractionation and immunoblottingPercoll gradients were performed as described (Bonnerot et al.,1998). Briefly, 3×107 RBL IAbIi cells were washed twice in PBS andresuspended in 1 ml of 10 mM triethanolamine, 10 mM aceticacid, 1 mM EDTA, 250 mM sucrose, pH 7.4 (TEA). They werehomogenized in a ball-bearing homogenizer (Balch and Rothman,1985) and centrifuged for 10 minutes at 300 g. Three hundredmicroliters of the resulting postnuclear supernatant (PNS) werediluted with TEA 250 mM sucrose and 90% Percoll to obtain a finalconcentration of 22% Percoll, loaded on top of a 1.5 M sucrosecushion and centrifuged for 30 minutes at 33,000 rpm in a TLA100.4rotor (Beckman Instruments, Inc., Palo Alto, CA). One milliterfractions were collected. β-Hexosaminidase activity in 1/100 of eachfraction was revealed with 6 mM 4-methyl-umbelliferyl-N-acetyl-β-D-glucosaminide (Sigma) in NaCitrate-PO4 buffer, pH 4.5(Green et al., 1987; Harms et al., 1980; Pool et al., 1983). Alkalinephosphodiesterase activity was revealed on 1/10 of each fractionsusing thymidine-5′-monophosphate-p-nitrophenyl ester (Sigma)(Green et al., 1987; Pool et al., 1983). Before being analysed by SDS-PAGE, fractions were lysed for 30 minutes at 4°C in 1/10th volumeof 10× lysis buffer (3 M NaCl, 5% Tx-100, 0.5 M Tris-HCl, pH 7.4)with 1 mM PMSF at 4°C for 30 minutes, adjusted to pH 12 with 2M NaOH and centrifuged at 20,000 g for 30 minutes at 4°C in orderto pellet Percoll (Marsh et al., 1987). Proteins were analysed bywestern blotting with the rabbit anti-IAα chain or anti-Ii-NH2antibodies.

Metabolic labelling and immunoprecipitationImmunoprecipitations were performed as described (Amigorena et al.,1994). After 45 minutes starving in methionine- and cysteine-freemedium, the cells were metabolically labelled with [35S]methionine/cysteine labelling mix for 30 minutes and chased for various times.Cells were lysed in 0.5% Nonidet P40, 300 mM NaCl, 50 mM Tris-HCl, pH 7.4, and 1 mM PMSF. Immunoprecipitations were performedon nucleus-free cell lysates with Y3P-, IN1- or M5/114-coated ProteinG-Sepharose beads. After washing, 30% of immunoprecipitates wereeluted in 20µl of Laemmli buffer containing 50 mM DTT for 30minutes at room temperature to release SDS-stable complexes fromthe beads. The remaining 70% were eluted in 50 mM DTT Laemmlibuffer, heated at 95°C for 5 minutes. Samples were analysed on 12%SDS-acrylamide gels.

Assays for antigen presentationFor antigen presentation assay by degranulated cells, RBL IAbIi cellswere incubated overnight with various concentrations of IEd α 52-68peptide. They were then incubated at 37°C for 30 minutes with orwithout 1µM ionomycin in DMEM (Gibco) then fixed at 4°C for 30

JOURNAL OF CELL SCIENCE 114 (2)

325Antigen presentation in mast cells

seconds with 0.05% glutaraldehyde. TH30 cells were added at 106

cells/ml. After 24 hours, IL-2 release by the T-cell hybridoma into theculture supernatants was determined as described before (Bonnerot etal., 1995), using a CTL.L2 proliferation assay: 50µl of supernatantswere removed, frozen at −80°C for 1 hour, thawed and addedto IL-2-dependent CTL.L2 cells for 16 hours. [3H]TdR (3H-labelled deoxythymidine) incorporation was measured after anadditional 6 hours incubation in the presence of 0.25µCi[3H]TdR/well. Each point represents the average of duplicate sampleswhich varied by less than 5%.

Immunofluorescence staining and confocal microscopyCells were washed in complete medium and allowed to adhere onglass coverslips for 2 hours. All solutions were completed with Ca2+

and Mg2+ in order to allow the adhesion of RBL-2H3 cells oncoverslips. Cells were fixed in 3% paraformaldehyde for 10 minutesat room temperature and then incubated with 100 mM glycine inPBS. For staining with rabbit anti-serotonin antibody, cells werefixed for 10 minutes at room temperature with 2% glutaraldehydeand then incubated with sodium borohydride (Sigma) at 1 mg/ml for15 minutes at room temperature. After permeabilization with 0.05%saponin in PBS 0.2% BSA, cells were incubated with specificantibodies. The mouse mAbs Y3P, MKD6, YAe, AD1, the rat mAbM5/114, the rabbit sera anti-serotonin, anti-IiNH2, anti-IA α chainwere all used at 7-10µg/ml. After three washes the mouse, rat orrabbit antibodies were, respectively, revealed by F(ab′)2 donkeyanti-mouse, anti-rat or anti-rabbit IgG coupled to FITC or TR.Coverslips were mounted in Mowiol. Confocal laser scanningmicroscopy was performed using a Leica TCS microscope based ona DM microscope interfaced with a mixed gas Argon/Krypton laser(Leica Laser Technik, Germany) as previously described (Bonnerotet al., 1995).

Immunoelectron microscopyRBL IAbIi cells were fixed with a mixture of 2% paraformaldehydein phosphate buffer 0.2 M, pH 7.4 (PB) and 0.125% glutaraldehydefor 2 hours at room temperature. Fixed cells were processed forultrathin sectioning and immunolabelling as described previously(Raposo et al., 1997). After washing with PB and PB-50 mM glycine,cells were embedded in 7.5% gelatin. Small blocks were infiltratedwith 2.3 M sucrose at 4°C for 2 hours and then frozen in liquidnitrogen. Ultrathin cryosections prepared with a Leica ultracut FCS(Wien, Austria) were retrieved with a mixture of 2% methylcelluloseand 2.3 M sucrose (vol/vol) and indirectly immunogold labelled withM5/114 followed by a rabbit anti-rat antibody (Dako, Danemark) anda rabbit polyclonal anti-serotonin antiserum. Rabbit antibodies weredetected with Protein A coupled to 10 and 15 nM gold particules(purchased from Dr J. W. Slot, Department of Cell Biology, UtrechtUniversity, The Netherlands). The sections were finally contrasted andembedded in a mixture of methylcellulose and uranyl acetate andviewed under a CM120 Twin Phillips electron microscope (Eindover,The Netherlands).

Fluorometric test for cathepsins activitiesPNS of RBL IAbIi and B414 cells were prepared as described above.Membranes were obtained by centifugation of PNS at 70,000 g for1 hour at 4°C. The same amounts of proteins (measured by Bradfordassay) diluted in 1 M NaAc, 0.2M EDTA, 0.5 M DTT, 5% CHAPS(pH 5.5 or pH 7.0) were incubated for 1 hour at 37°C in a 96-wellplate with the substrates of cathepsins: 200µM Z-Val-Val-Arg-NHMec (a gift from P. Morton, Chesterfield, MO, USA) for cathepsinS in the presence or not of 200 nM of the inhibitor N-morpholinurea-leucine-homophenylalanine vinylsulfone-phenyl (LHVS), 80µM Z-Phe-Arg-NHMec (Sigma) for cathepsins B/L. Reactions werequantified at λexc.=355 nM and λem.=460 nM with a fluorometer(1420 VICTOR2TM multilabel counter EG&G WALLAC) (Manouryet al., 1998). The same samples were tested for β hexosaminidase

activity as described before. LHVS was kindly provided by Dr H.Ploegh (Harvard University, Boston, MA).

Labelling of active site of cysteine proteasesRBL IAbIi and B414 were incubated in complete RPMI with orwithout 50 nM cathepsin S inhibitor LHVS. Detection of activecysteine proteases with Mu-125I-Tyr-Ala-CH2F was performed asdescribed elsewhere (Riese et al., 1996). The cysteine proteaseinhibitor Mu-Tyr-Ala-CH2F was iodinated as previously reported(Mason et al., 1989). RBL IAbIi and B414 cells (107 cells/sample)were incubated with LHVS (50 nM) at 37°C for 1 hour prior tolabelling. Cells were then incubated with 50 nM Mu-125I-Tyr-Ala-CH2F for 2 hours at 37°C, washed twice with cold PBS 1% FCS. Celllysates were boiled for 5 minutes before analysis in a 12% SDS-acrylamide gel.

RESULTS

RBL-2H3 cells accumulate MHC class II molecules insecretory lysosomesTo study MHC class II transport in mast cells, we haveexpressed recombinant murine class II molecules in the ratbasophilic cell line RBL-2H3. This mast cell line has afunctional phenotype of mucosal mast cell: i.e. expresses highaffinity receptors for IgE (FcεRI) and contains secretorygranules that accumulate biogenic amines, such as histamineand serotonin (Barsumian et al., 1981). These amines arereleased by the cells upon stimulation through IgE receptor ora calcium ionophore (Dvorak et al., 1983).

Fig. 1.Cell surface expression of functional MHC class II moleculeson RBL-2H3 cells. Cell surface expression of IAb and IAd MHCclass II molecules on transfected-RBL-2H3 cells was controlled byFACScan© analysis using the rat anti-IAb/d mAb, M5/114, followedby FITC-conjugated goat anti-rat antibody. In Grey, the same cellswere incubated with the secondary Ab alone. B414 cells, a Blymphoma cells expressing IAb and IAd class II molecules was usedas positive control.

RBL

RBL IAbIi RBL IAdIi

B414

326

RBL-2H3 cells have been transfected with cDNAs encodingthe α and the β chains of the b or the d haplotypes of IA MHCclass II molecules as well as murine Ii. Numerous clones were

isolated and characterized by immunofluorescence usingmonoclonal antibodies specific for the αβ dimers of IAd(MKD6), of IAb (Y3P) (data not shown) or of IAb/d (M5/114)

JOURNAL OF CELL SCIENCE 114 (2)

Fig. 2. MHC class II complexes are mainly retained in lysosomes and secretory granules. RBL-2H3 cells expressing class II molecules werefixed with 3% paraformaldehyde (A,B) or 1% glutaraldehyde (C) before permeabilization with 0.05% saponin and incubation with variousantibodies. Staining was detected with FITC-coupled or Texas red-coupled secondary antibodies, and was analyzed by confocal microscopy.(A) MHC class II molecules and Ii co-accumulate in the same vesicles. RBL IAbIi and IAdIi cells have been stained with rat mAb anti-β chain,M5/114 (green) and a rabbit antiserum specific for the cytoplasmic tail of murine Ii (red). (B) MHC class II molecules accumulate in lysosomalcompartments. RBL IAbIi were stained with the rabbit anti-IAα chain cytoplasmic tail and two antibodies directed against lysosomal proteins:AD1 specific for CD63 and LY1C6 specific for Lamp1 (red). (C) MHC class II molecules accumulate in secretory granules of RBL-2H3 cells.RBL IAbIi cells were labelled with the anti-CD63 (AD1) or the anti-class II (M5/114) antibodies (green) and a rabbit anti-serotonin antiserum(red). (D) MHC class II-peptide complexes accumulate intracellularly. RBL IAbIi cells incubated overnight with IEα 52-68 peptide were fixedand permeabilized with 0.05% saponin. IAb/IEα 52-68 peptide complexes were detected with the mouse mAb YAe (red) and MHC class IImolecules with the rat anti-IAb/d mAb, Μ5/114(green). Intracellular staining was analyzed by confocal microscopy. (B,C,D) Arrows indicatecolocalisation of the two labellings.

327Antigen presentation in mast cells

class II molecules. The RBL IAbIi cells as well as the RBLIAdIi cells, shown in Fig. 1, have been chosen because theyexpressed a high and homogenous level of MHC class IImolecules at their surface as analyzed by cytofluorimetry(Fig. 1) and similar levels of Ii as detected by western blotting(see Fig. 4D).

The intracellular localization of MHC class II wasdetermined by immunofluorescence. IAb and IAd moleculeswere detected with rabbit antiserum directed against thecytoplasmic domain of the IA α chain or with monoclonalantibodies specific for mature IAb (Y3P) or mature IAd(MKD6) αβ dimers, while the murine Ii was detected with arabbit antiserum specific for the cytosolic NH2 domain. Thelabelling was analyzed by confocal microscopy in both RBLIAbIi and RBL IAdIi cells. Class II molecules and Ii weremostly detected in intracellular vesicular compartments(Fig. 2A). Only a small fraction of class II molecules wasdetectable at the cell surface.

The intracellular site of MHC class II accumulation wascharacterized by colocalization with different markers ofendo/lysosomal compartments. The results obtained byimmunofluorescence and confocal analysis indicated that classII molecules, CD63 (Kitani et al., 1991; Nishikata et al., 1992)and Lamp 1 (Kleijmeer et al., 1996) were mostly detectedin the same compartments (Fig. 2B). In contrast, class IImolecules never colocalized with the transferrin receptor, amarker of early endosomes (Marsh et al., 1995) (data notshown). We then analyzed the intracellular localization of classII molecules into secretory granules containing serotonin. Thecells were fixed with 2% glutaraldehyde to preserve thecomposition of the secretory granules, then class II molecules,CD63 and serotonin were labelled with specific antibodies. Theresults shown in Fig. 2C illustrate a clear accumulation ofclass II molecules or CD63 with serotonin in vesicularcompartments sharing molecular components of typical andsecretory lysosomes.

The next question was: are intracellular MHC class IImolecules able to bind antigenic peptides? To answer thisquestion, we used the monoclonal antibody, YAe, specific forthe MHC class II-peptide complex formed by the associationof the 52-68 peptide derived from the IEd α chain with themurine IAb class II molecules (Farr et al., 1996; Murphy et al.,1992). RBL IAbIi cells were incubated overnight with theIEd peptide coupled to HEL, then the cells were fixed andpermeabilized to determine the localization of peptide-associated MHC class II molecules. The results shown in Fig.2D illustrate the labelling we obtained with the YAe antibodyin 20% of the cells. A faint labelling was detectable at thecell surface whereas most of the YAe labelling was found invesicular structures colabelled by the anti-class II, M5/114.These data suggested that antigens can be processed inantigenic peptides that associate with MHC class II moleculespresent in secretory granules.

To determine the ultrastructure of the compartments whereMHC class II molecules accumulated, ultrathin cryosections ofRBL IAbIi were immunogold-labelled with the rat monoclonalanti-class II M5/114, and the anti-serotonin rabbit antiserum.Fig. 3 shows that in these cells, MHC class II molecules mainlylocalized in compartments displaying internal membranevesicles. Class II molecules’ labelling was present on the smallinternal vesicles of 60 to 80 nM diameter and on the limiting

membrane of the granule (Fig. 3). Serotonin could also bedetected in the same compartments although it is difficult tostate if the labelling is present in the lumen or associated withthe cytosolic side of the internal vesicles. The MHC class IIrich compartments in RBL-2H3 cells are similar to thelysosomal Type I granules observed in BMMCs (Raposo et al.,1997).

These morphological approaches allow to conclude that inRBL-2H3, most MHC class II molecules are found in secretorycompartments related to lysosomes where they can be loadedwith antigenic peptide.

The maturation of MHC class II -invariant chaintrimers is slowed down in RBL-2H3 cellsTo analyze further the trafficking of mature (α β-peptide) andimmature (α β-Ii) class II molecules in mast cells, we followedtheir biosynthesis. RBL IAbIi cells and B cells weremetabolically labelled for 30 minutes with 35S-labelledcysteine and methionine, then chased for different times innon radioactive medium. IAb class II molecules wereimmunoprecipitated with Y3P, specific for IAb α,β dimers freeor associated with Ii fragments. The samples were heated ornot at 95°C and analyzed by 12% polyacrylamide SDS-PAGE.When Y3P-immunoprecipitates were boiled with SDS, weobserved that similar amounts of class II molecules maturedwith the same kinetics in RBL-2H3 cells and murine B cells.When the same samples were not heated, αβ SDS-stabledimers, which account for the full maturation of class IImolecules, were detected early (2 hours) in murine B cellswhereas, in RBL-2H3 cells, only a small fraction of class IImolecules were detected in SDS-stable conformation after20 hours of chase. Surprisingly, an additional SDS-stable 70kDa complex was observed in RBL-2H3 cells after 6 hours ofchase (Fig. 4A). The molecular mass of this complex couldcorrespond to immature class II dimers (60 kDa) associated tothe p10 fragment of Ii (10 kDa).

To define the composition of this 70 kDa complex inRBL-2H3 cells, immunoprecipitations were performed withantibodies recognizing non conformational epitopes on the βchain of class II molecules (i.e. M5/114) or the cytoplasmicdomain of Ii (i.e. IN1). In boiling conditions, we observed(Fig. 4B) that the rate of Ii biosynthesis was similar in B cellsand RBL-2H3 cells. In contrast, its half-life was clearlydifferent since most of the p31 was degraded in B cells after6 hours of chase whereas 50% of p31 Ii remained detectablein RBL-2H3 cells after 20 hours of chase in complete medium(Fig. 4B). In addition, a p10 fragment accumulated in RBL-2H3 cells from 2 hours to 21 hours of chase. When the samesamples were not boiled to preserve SDS-stableconformation, IN1 antibody immunoprecipitated a p90complex at time 0 which remained detectable after 2 hours ofchase whereas a p70 complex appeared at 2 hours to peakafter 6 hours of chase and remained detectable after 21 hours.In control B cells, IN1 did not immunoprecipitate the p70complex. To further identify the proteins contained in p90 andp70 complexes, class II molecules were immunoprecipitatedwith the anti-class II β chain, M5/114 (Fig. 4C). The samebands were obtained than with the anti-Ii antibody, IN1(Fig. 4B). Indeed, M5/114 and IN1 immunoprecipitated thesame p90 and p70 complexes with the same kinetics whenthe samples were run in unboiled conditions to dissociate, in

328 JOURNAL OF CELL SCIENCE 114 (2)

Fig. 3. Immuno-electronmicroscopy of RBL-2H3transfected cells.(A) Ultrathincryosections of RBLIAbIi cells wereimmunogold labelledwith the rat monoclonalanti-IA Ab M5/114, arabbit anti-rat bridgingantibody and Protein Acoupled to 10 nM goldparticles (PAG 10). MHCclass II molecules aredetected in the numerouscompartments filling thecytoplasm of RBL-2H3cells. (B) Highermagnification showingthe multivesicularappearance of thecompartmentsaccumulating MHC classII molecules. (B, inset)Ultrathin cryosectionswere also doubleimmunogold labelledwith the rat monoclonalAb M5/114 (PAG 15)and a rabbit polyclonalanti-serotonin (PAG 10).Serotonin was detectedto some extent in theMHC class II positivecompartments. Bars,200 nM.

329Antigen presentation in mast cells

the same ratio, into α, β chains of class II molecules and theentire or the p10 fragment of Ii. These data indicate that, inRBL-2H3 cells, MHC class II molecules slowly mature intoa compact αβ mature dimer because Ii is poorly degraded andremains associated with class II molecules in its entire formor p10 and p20 fragments.

Lysates of RBL IAbIi and RBL IAdIi cells were analyzedby western blotting with rabbit antiserum specific for thecytoplasmic tail of Ii. In RBL-2H3 cells, Ii mostly accumulatedas different forms corresponding to p20 and p10 fragmentsof the p31 Invariant chain (Fig. 4D) in agreement with the

previous data. In murine B cells only a small proportion of Iicorresponded to the p10 fragment.

In order to quantify the accumulation of MHC class IImolecules in secretory granules, RBL IAb cells werefractionated and lysosomes were purified by ultracentrifugationon Percoll gradient (Bonnerot et al., 1998). The differentfractions were characterized by their β hexosaminidase activity(found in secretory granules and lysosomal compartments) andtheir alkaline phosphodiesterase (APDE) activity (found at theplasma membrane) (Fig. 5A). Each fraction was analyzed bywestern blotting with the rabbit antiserum against MHC class

Fig. 4. Defect of MHC class IImaturation in RBL-2H3 cells isrelated to a slow degradation of Ii.RBL IAbIi cells and the Blymphoma cells B414, bothexpressing IAb molecules, weremetabolically labelled with[35S]methionine/cysteine thenchased for the indicated times. Ateach time, immunoprecipitationsof newly synthesized MHC classII molecules were performed withdifferent monoclonal antibodies.Before analysis by 12% SDS-PAGE, the samples wereincubated in reducing Laemmlibuffer at RT for 30 minutes (non-boiled conditions, NB, left panel)or at 95°C for 5 minutes (boiledconditions, B, right panel).(A) αβ dimers did not mature intoa compact SDS-stableconformation in RBL-2H3 cellsbut remained as p70 isoforms.Immunoprecipitations of MHCclass II molecules were carriedout with the Y3P antibodyrecognizing αβ IAb dimers freeor associated with Ii fragment.(B,C) Invariant chain degradationwas slowed down and it remainedassociated with class II dimers inRBL-2H3 cells. Ii wasimmunoprecipitated with IN1, anantibody directed against thecytoplasmic tail of murine Ii.(B) whereas class II moleculeswere immunoprecipitated with theM5/114 antibody recognizingthe IA β chain in mature orimmature αβ complexes (C).(D) Accumulation of p10 Iifragment in RBL-2H3 cellsexpressing class II molecules.Lysates of RBL IAbIi and IAdIicells as well as lysates fromIIA1.6 and untransfected RBL-2H3 cells were analyzed bywestern blotting with a rabbitmonoclonal antibody directedagainst the cytoplasmic tail of murine Ii. Arrowheads indicate the bands corresponding to α chain (α) or β chain (β) of MHC class II molecules,Ii and its degradation fragments (p10 and p20), SDS-stable compact form of class II molecules (αβ) or SDS-stable class II molecules associatedwith the entire Invariant chain (p90) or its p10 degradation fragment (p70).

330

II molecules. A minor fraction of MHC class II molecules wasfound in light fractions corresponding to the cell surfacewhereas 80% accumulated in dense fractions, correspondingto lysosomes-related compartments, together with p10 Iidegradation fragment (Fig. 5B).

These results were very similar to those obtained afterleupeptin (a protease inhibitor) treatment of B cells (Brachet etal., 1997) or in immature dendritic cells (Pierre and Mellman,1998) where αβ-p10 complexes accumulated in lysosomalcompartments due to a decreased activity of cathepsin S. Wetherefore measured Cathepsin S activity in RBL-2H3 cells bythree independent assays (Fig. 6). Using a fluorometric assay,low cathepsin S activity can be detected at pH 5.5 or pH 7 inmembrane preparations of RBL-2H3 cells, whereas normalcathepsin L, cathepsin B and β-hexosaminidase activities weremeasured in the same samples (Fig. 6A). Significant cathepsinS activity was detected in membrane preparations of B414 cells

in the same experiment. Since this fluorometric assay wasreported as not perfectly specific for cathepsin S activity, wethen labelled RBL-2H3 and B414 cells with a iodinated-peptide which specifically bound the active site of cathepsin S.A 26 kDa protein, corresponding to cathepsin S, is specificallylabelled in these conditions in B414 cells whereas nocorresponding protein was detected in RBL-2H3 cells(Fig. 6B). This protein was suspected to be cathepsin S becauseit has the right size and its labelling by the iodinated peptidewas inhibited in the presence of a cathepsin S inhibitor, theLHVS peptide (Xing and Mason, 1998). This result was

JOURNAL OF CELL SCIENCE 114 (2)

Fig. 5.MHC class II molecules and Ii were mostly accumulated inheavy fractions of Percoll gradient. Postnuclear supernatants ofRBL-2H3 cells were fractionated using a 22% Percoll densitygradient. (A) Membranes pelleted from each fraction were tested forβ hexosaminidase (enriched in heavy density lysosomes) and APDEactivities (low density fractions). (B) Aliquots of each fraction wererun on 12% polyacrylamide gels, transferred on nylon membranesthen blotted with rabbit antiserum specific for the cytoplasmic tails ofIA α chain or Ii (upper panel). The amount of MHC class IImolecules and p10 fragment of Ii was quantified in each fraction thenplotted using arbitrary units (lower panel).

Fig. 6. Default of cathepsin S activity in the mast cell line RBL-2H3.(A) PNS of RBL IAbIi and B414 cells were tested for β-hexosaminidase, cathepsin S and cathepsin B/L activities withfluorometric assays. Both cell types show the same activity ofcathepsin B/L. Cathepsin S activity was tested at two pH: 5,5 and 7,0since cathepsin S is functional at neutral pH. There is no differencebetween activities of cathepsin S in both membranes treated withLHVS, a cathepsin S inhibitor. RBL IAbIi show a low cathepsin Sactivity as compared to B414 cells. (B) Presence of cathepsin Sactivity was evidenced with 125I-Mu-Tyr-Ala-CH2F in B cells but notin RBL-2H3 cells. (C) Western blot analysis of RBL-2H3 and B414cells with rabbit anti-cathepsin S in 12% SDS-PAGE gel reveal thatcathepsin S was not present in RBL-2H3 cells.

331Antigen presentation in mast cells

supported by the lack of cathepsin S signal in RBL-2H3 lysatesby western blot using a rabbit anti-cathepsin S antiserum(Fig. 6C). Therefore, the absence of cathepsin S in mast cellssuch as RBL-2H3 cells is likely to contribute to the low rateof Ii degradation and the slow MHC class II maturation.

Therefore, mature MHC class II molecules loaded withpeptides or immature class II molecules associated with thep10 fragment accumulate in secretory granules. Since thecontent of secretory granules is known to be released upon acytosolic calcium increase (Stump et al., 1987), mast cellsactivation should induce cell surface arrival of MHC class IImolecules.

A cytoplasmic Ca2+ increase regulates cell surface MHCclass II molecules peptide complexes expression in RBL-2H3cells. We therefore checked if MHC class II molecules, loadedor not with antigenic peptides, reach the cell surface after ashort intracellular calcium increase induced by a calciumionophore, ionomycin. RBL IAb Ii cells were loaded for2 hours with the IEd α 52-68 peptide (10µM) then cell surfaceexpression of total class II molecules (Y3P) or peptide-class IIcomplexes (YAe) was measured by FACScan© analysis beforeand after addition of 1 µM ionomycin and 3% PAF. Thiscalcium signaling, which induced cell degranulation asmeasured by serotonin release (data not shown), uniformilyincreased the labelling detected by the Y3P monoclonalantibody (Fig. 7A, right panel), and it induced the detection ofthe YAe epitope at the cell surface of 40% of the cells (Fig. 7A,left panel). Concomitantly, ionomycin induced also cell surface

expression of other transmembrane proteins, known toaccumulate in secretory granules, such as lamp 1 (LY1C6mAb) or CD63 (AD1 mAb) and the protein recognized by5G10 mAb (Fig. 7A, right panel). To test if redistributedpeptide-class II complexes were recognized by specific T cells,RBL IAbIi cells were incubated with different concentrationsof ΙΕd α 52−68peptide and antigen presentation was detected,before or after ionomycin treatment (1 µM in DMEM), by theT hybridoma TH30, specific for this peptide presented by IAbMHC class II molecules. The results shown in Fig. 7B illustratethat a calcium signaling increased by a factor 15 the ability ofmast cells to stimulate antigen specific T cells. These dataconfirmed that class II molecules loaded with antigenicpeptides in secretory granules were delivered to the cell surfaceupon mast cell activation.

In conclusion, mast cells are able to process antigens intopeptides that bind to MHC class II molecules in secretorylysosomes. This feature of MHC class II transport in mast cellsallows therefore to coordinate, upon similar stimuli, theexocytosis of inflammatory mediators and the cell surfaceexpression of class II-peptide complexes.

DISCUSSION

We recently reported that BMMCs accumulate MHC class IImolecules in secretory granules (Raposo et al., 1997). Thesecells are currently used as a model for studying mast cell

YAe

+ ionomycin

- ionomycin

[IEdα 52-68] (µg/mL)

0 1 10 100

6

12

0

+ ionomycin

- ionomycin

B

H3

Thy

mid

ine

upta

ke (

cpm

x103 )

Y3P AD1

LY1C6 5G10

-+

--

-

++

+

A

Fig. 7. Increase in intracellularcalcium concentration induced cellsurface expression of MHC class IImolecules associated or not withantigenic peptide. (A) RBL IAbIicells have been incubated overnightwith IEd α 52-68 peptide, washed inDMEM, then incubated (+) or not(−) with 1µM ionomycin for 30minutes at 37°C. The cells were thenwashed in PBS FCS 3% NaN3 0,1%and stained with YAe (left panel)mAb or with Y3P (IAb MHC class IImolecules), LY1C6 (lamp 1), AD1(CD63) and 5G10 (right panel).Negative control (in grey): cells wereincubated with secondary Ab alone.(B) RBL IAbIi cells were incubatedwith various concentrations of IEd α52-68 peptide then they were fixedwith 0,05% glutaraldehyde afterinduced degranulation with 1µMionomycin. MHC class II-peptidecomplexes were detected usingspecific T cells (TH30). T cellstimulation was evaluated after24 hours by measuring Il2 secretionin the supernatant using classicalCTL.L2 proliferation assay.

332

function. However, the level of MHC class II molecules is toolow to allow biochemical approaches and MHC class IIexpression is under the control of various cytokines, such asGM-CSF and IL4, which could interfere with MHC class IItransport in BMMCs (Frandji et al., 1995). Expressing IAmolecules in RBL-2H3 cells allowed us to analyze theintracellular transport of MHC class II in an homogenous mastcells population expressing physiological levels of class IImolecules without the addition of any lymphokines in theculture media. We mostly found that secretory granules orsecretory lysosomes of RBL-2H3 cells, like in BMMCs(Raposo et al., 1997), are the intracellular site of accumulationof mature class II molecules, i.e. αβ dimers loaded withantigenic peptides, and of immature class II molecules, i.e. αβdimers associated with p10 fragment of Ii.

How do mast cells prevent MHC class II transport to the cellsurface? Our results suggest that several mechanisms may beused by these cells. First, a delay in the maturation of class IImolecules, which remained associated with a fragment of Ii,likely due to a low cathepsin S activity, blocking the access ofthe class II pocket to the peptides; second, a defect in thetransport of class II molecules-peptide complexes fromlysosomal compartments to the cell surface. Altogether thesemechanisms contribute to the intracellular accumulation ofclass II molecules.

One important observation is that the intracellularmaturation of MHC class II molecules in the rat mast cell lineRBL-2H3, as well as in BMMCs, is altered. Indeed, althougha small fraction of αβ dimers became SDS stable, the majorityof class II molecules remained associated with Ii as well as itsdegradation intermediates fragments, p10 and p22. Similarresults have been obtained in B cells treated with leupeptin,an inhibitor of cysteine-proteases (Amigorena et al., 1995;Brachet et al., 1997). In the presence of leupeptin, thedegradation of Ii was inhibited, which resulted in itsaccumulation as a p10 form. As a consequence, MHC classII/Ii complexes were retained in lysosomal compartments(Brachet et al., 1997). The hypothesis that Ii may indeedregulate the localization and transport of MHC class IImolecules along organelles of the endocytic pathway has beenrecently extended by Pierre and coll (Pierre and Mellman,1998). Immature dendritic cells express high levels of cystatinC, an inhibitor of cathepsin S, inducing the accumulation ofαβp10 complexes in lysosome-related compartments (MIICs).In LPS-induced mature dendritic cells, the down regulation ofcystatin C expression induced cathepsin S activity and thedegradation of p10 Ii form, thus allowing the redistribution ofmature αβ dimers at the cell surface of dendritic cells (Pierreand Mellman, 1998). The low cathepsin S activity we observedin RBL-2H3 cells therefore accounts for the accumulation ofp10 Ii fragment with class II dimers in secretory lysosomes ofmast cells.

It remains that, even in the absence of cathepsin S, a smallfraction of class II molecules became SDS stable and could beloaded by antigenic peptide in RBL-2H3 cells. This suggestthat cathepsin S is not absolutely necessary for MHC class IImaturation and antigen presentation. Indeed, mature orimmature dendritic cells of cathepsin S knock-out miceefficiently present antigens and express similar level of cellsurface class II molecules than wild-type mice (Villadangos etal., 1999). In addition, it has been reported that cathepsin L can

replace cathepsin S in some cell types such as thymic epithelialcells or macrophages (Nakagawa and Rudensky, 1999;Villadangos et al., 1999); mast cells or RBL-2H3 cells couldbe other examples where other enzymes such as cathepsin Ldegrade Iip10 or where Iip10 is slowly displaced from theMHC class II dimer. Thus, it seems that class II molecules inRBL-2H3 cells and in immature dendritic cells have verysimilar behaviors since αβIip10 complexes and αβ-peptidecomplexes mainly accumulate in compartments withlysosomal features (Pierre and Mellman, 1998) and they may,both, express high levels of class II molecules at the cellsurface and present antigen after cell activation.

What are the intracellular compartments of MHC class IIaccumulation in mast cells? Cell fractionation of RBL-2H3cells indicated that 80% of the total class II molecules werefound in dense fractions of Percoll gradient, enriched inconventional lysosomes and secretory lysosomes (Shiver et al.,1992). In addition, MHC class II and Ii were strictlycolocalized by immunofluorescence with CD63, a commonmarker of these two types of lysosomal compartments. Whenthe intracellular localization of class II molecules wascompared with that of a specific marker of secretory granules,a significant fraction of class II molecules was found inserotonin-positive compartments. In BMMCs, we previouslyobserved that the bulk of class II molecules is contained in twodistinct compartments (type I and type II) with features of bothlysosomal compartments and secretory granules as defined bytheir morphology, their protein content and their accessibilityto endocytic tracers. Type I granules are multivesicularcompartments where MHC class II molecules co-localize withmannose-6-phosphate receptors and lysosomal membraneproteins such as lamp1 and lamp2. Type II granules displayin addition to small membrane vesicles, an electron densesecretory domain rich in serotonin (Raposo et al., 1997). Themorphological features of the intracellular compartments ofRBL-2H3 cells expressing murine MHC class II molecules aresomehow distinct from those of BMMCs. In RBL-2H3 cellswe observed compartments morphologically similar to Type Igranules of BMMCs since they display intralumenalmembrane vesicles. Typical Type II granules with theirelectron dense core were not detected, instead compartmentswith internal membrane sheets morphologically similar tolysosomes were observed. Interestingly, serotonin could bedetected together with MHC class II molecules in themultivesicular compartments. Thus, it seems that in mast cells,the bulk of class II molecules and lysosomal proteins arelocalized in particular MIICs which are secretory granules.

What are the consequences of the accumulation of MHCclass II molecules in secretory granules? Antigen presentationis the result of numerous intracellular steps which requireaccessory molecules to obtain MHC class II molecules loadedwith antigenic peptides (Kropshofer et al., 1997). In mast cells,although the degradation of Ii is largely inhibited, a fraction ofαβ dimer matured and was able to bind antigenic peptides insecretory granules. This unique intracellular localization ofMHC class II molecules in mast cells has several consequenceson the transport of class II molecules towards the plasmamembrane. First, since the content of secretory granulesremains inside the cells in the absence of signals whichdetermine exocytosis, immature and mature MHC class IImolecules are mostly retained intracellularly in mast cells.

JOURNAL OF CELL SCIENCE 114 (2)

333Antigen presentation in mast cells

Second, cell surface expression of class II molecules, loadedor not with antigenic peptides occurs upon cytosolic calciumincrease, a process which induces mast cell degranulation.Thus the nature of the intracellular compartment where MHCclass II molecules accumulate in mast cells must haveimportant consequences on mast cell functions during antigenprocessing and antigen presentation. An hypothesis could bethat, during inflammatory or allergic reaction, mast cells mightbe able to determine a two-steps process: mast cells captureantigens in a primary body site, then process antigens intopeptides that are loaded on MHC class II molecules insecretory granules, the content of which could be released,together with inflammatory mediators, after cell migration intoa second body site upon IgE-mediated stimulation. Therefore,mast cells may have developed an original mechanism todeliver, into mucosal tissue, antigen specific signals to modifyT cell response.

The authors thank the people of the U520 INSERM, specially V.Briken, A. Regnault, S. Amigorena and P. Benaroch for excitingdiscussions as well as D. Lankar for her technical contribution duringthis work. We thank also R. Golsteyn and C. Watts for critical readingof the manuscript. This work was supported by grants from the InstitutNational de la Santé et de la Recherche Médicale, Institut Curie andthe Comité de Paris of the Ligue Nationale contre le Cancer. H.Vincent-Schneider is a fellow from the Ministère de l’EnseignementSupérieur et de la Recherche.

REFERENCES

Amigorena, S., Drake, J. R., Webster, P. and Mellman, I.(1994). Transientaccumulation of new class II MHC molecules in a novel endocyticcompartment in B lymphocytes. Nature369, 113-120.

Amigorena, S., Webster, P., Drake, J., Newcomb, J., Cresswell, P. andMellman, I. (1995). Invariant chain cleavage and peptide loading in majorhistocompatibility complex class II vesicles. J. Exp. Med.181, 1729-1741.

Balch, W. E. and Rothman, J. E.(1985). Characterization of protein transportbetween successive compartments of the Golgi apparatus: asymmetricproperties of donor and acceptor activities in a cell-free system. Arch.Biochem. Biophys.240, 413-425.

Barlow, A. K., He, X. and Janeway, C. Jr (1998). Exogenously providedpeptides of a self-antigen can be processed into forms that are recognizedby self-T cells. J. Exp. Med.187, 1403-1415.

Barsumian, E. L., Isersky, C., Petrino, M. G. and Siraganian, R. P. (1981).Ig-E induced histamine release from rat basophilic leukaemia cell lines:isolation of releasing and nonreleasing clones. Eur. J. Immunol.11, 317.

Bhattacharya, A., Dorf, M. E. and Springer, T. A. (1981). A sharedalloantigenic determinant on Ia antigens encoded by the I-A and I-Esubregions: evidence for I region gene duplication. J. Immunol. 127, 2488-2495.

Bonifacino, J., Yuan, L. and Sandoval, I. (1989). Internalization andrecycling to serotonin-containing granules of the 80K integral membraneprotein exposed on the surface of secreting rat basophilic leukaemia cells.J. Cell Sci.92, 701-712.

Bonnerot, C., Amigorena, S., Choquet, D., Pavlovich, R., Choukroun, V.and Fridman, W. H. (1992). Role of associated gamma-chain in tyrosinekinase activation via murine Fc gamma RIII. EMBO J.11, 2747-2757.

Bonnerot, C., Lankar, D., Hanau, D., Spehner, D., Davoust, J., Salamero,J. and Fridman, W. H. (1995). Role of B cell receptor Ig alpha and Ig betasubunits in MHC class II-restricted antigen presentation. Immunity3, 335-347.

Bonnerot, C., Briken, V., Brachet, V., Lankar, D., Cassard, S., Jabri, B.and Amigorena, S.(1998). syk protein tyrosine kinase regulates Fc receptorgamma-chain-mediated transport to lysosomes. EMBO J.17, 4606-4616.

Brachet, V., Raposo, G., Amigorena, S. and Mellman, I. (1997). Ii chaincontrols the transport of major histocompatibility complex class IImolecules to and from lysosomes. J. Cell Biol.137, 51-65.

Cresswell, P. (1994). Assembly, transport, and function of MHC class IImolecules. Annu. Rev. Immunol.12, 259-293.

Dvorak, A., Galli, S. J., Schulman, E. S., Lichtenstein, L. M. and Dvorak,H. F. (1983). Basophil and mast cell degranulation: ultrastructural analysisof mechanisms of mediator release. Fed. Proc.42, 2510-2515.

Farr, A., DeRoos, P. C., Eastman, S. and Rudensky, A. Y. (1996).Differential expression of CLIP:MHC class II and conventional endogenouspeptide:MHC class II complexes by thymic epithelial cells and peripheralantigen-presenting cells. Eur. J. Immunol.26, 3185-3193.

Frandji, P., C., T., C., O., J., L., R., P., B., D., JG., G. and S., M.(1995).Presentation of soluble antigens by mast cells: upregulation by interleukin-4 and granulocyte/macrophage colony-stimulating factor anddownregulation by interferon-gamma. Cell. Immunol.163, 37-46.

Green, S. A., Zimmer, K. P., Griffiths, G. and Mellman, I. (1987). Kineticsof intracellular transport and sorting of lysosomal membrane and plasmamembrane proteins. J. Cell Biol.105, 1227-1240.

Harms, E., Kern, H. and Schneider, J. A. (1980). Human lysosomes can bepurified from diploid skin fibroblasts by free-flow electrophoresis. Proc.Nat. Acad. Sci. USA 77, 6139-6143.

Janeway, C. A. Jr, Conrad, P. J., Lerner, E. A., Babich, J., Wettstein, P.and Murphy, D. B. (1984). Monoclonal antibodies specific for Iaglycoproteins raised by immunization with activated T cells: possible roleof T cellbound Ia antigens as targets of immunoregulatory T cells. J.Immunol.132, 662-667.

Kappler, J. W., Skidmore, B., White, J. and Marrack, P. (1981). Antigen-inducible, H-2-restricted, interleukin-2-producing T cell hybridomas.Lack of independent antigen and H-2 recognition. J. Exp. Med.153, 1198-1214.

Kitani, S., Berenstein, E., Mergenhagen, S., Tempst, P. and Siraganian, R.P. (1991). A cell surface glycoprotein of rat basophilic leukemia cells closeto the high affinity IgE receptor (Fc epsilon RI). Similarity to humanmelanoma differentiation antigen ME491. J. Biol. Chem.266, 1903-1909.

Kleijmeer, M. J., Raposo, G. and Geuze, H. J.(1996). Characterization ofMHC class II compartments by immunoelectron microscopy. Methods: aCompanion to Methods in Enzymology 10, 191-207.

Kropshofer, H., Hammerling, G. J. and Vogt, A. B. (1997). How HLA-DMedits the MHC class II peptide repertoire: survival of the fittest? Immunol.Today18, 77-82.

Lewis, V., Green, S. A., Marsh, M., Vihko, P., Helenius, A. and Mellman,I. (1985). Glycoproteins of the lysosomal membrane. J. Cell Biol. 100,1839-1847.

Manoury, B., Hewitt, E. W., Morrice, N., Dando, P. M., Barrett, A. J. andWatts, C. (1998). An asparaginyl endopeptidase processes a microbialantigen for class II MHC presentation [see comments]. Nature 396, 695-699.

Marsh, E. W., Leopold, P. L., Jones, N. L. and Maxfield, F. R. (1995).Oligomerized transferrin receptors are selectively retained by a lumenalsorting signal in a long-lived endocytic recycling compartment. J. Cell Biol.129, 1509-1522.

Marsh, M., Schmid, S., Kern, H., Harms, E., Male, P., Mellman, I. andHelenius, A. (1987). Rapid analytical and preparative isolation of functionalendosomes by free flow electrophoresis. J. Cell Biol. 104, 875-886.

Mason, R. W., Wilcox, D., Wikstrom, P. and Shaw, E. N.(1989). Theidentification of active forms of cysteine proteinases in Kirsten-virus–transformed mouse fibroblasts by use of specific radiolabelledinhibitor. Biochem. J. 257, 125-129.

Murphy, D. B., Rath, S., Pizzo, E., Rudensky, A. Y., George, A., Larson,J. K. and Janeway, C. A. Jr(1992). Monoclonal antibody detection of amajor self peptide. MHC class II complex. J. Immunol.148, 3483-3491.

Nakagawa, T. Y. and Rudensky, A. Y. (1999). The role of lysosomalproteinases in MHC class II-mediated antigen processing and presentation[In Process Citation]. Immunol. Rev.172, 121-129.

Nishikata, H., Oliver, C., Mergenhagen, S. and Siraganian, R. (1992). Therat mast cell antigen AD1 (homologue to human CD63 or melanomaantigen ME491) is expressed in other cells in culture. J. Immunol. 149, 862-870.

Peterson, M. and Miller, J. (1990). Invariant chain influences theimmunological recognition of MHC class II molecules. Nature345, 172-174.

Pierre, P. and Mellman, I. (1998). Developmental regulation of invariantchain proteolysis controls MHC class II trafficking in mouse dendritic cells.Cell 93, 1135-1145.

Pool, R. R. Jr, Maurey, K. M. and Storrie, B. (1983). Characterization ofpinocytic vesicles from CHO cells: resolution of pinosomes from lysosomesby analytical centrifugation. Cell Biol. Int. Rep.7, 361-367.

334

Raposo, G., Tenza, D., Mecheri, S., Perronet, R., Bonnerot, C. andDesaymard, C. (1997). Accumulation of MHC class II molecules in mastcell secretory granules and their release upon degranulation. Mol. Biol. Cell8, 2631-2645.

Riese, R. J., Wolf, P. R., Brömme, D., Natkin, L. R., Villadangos, J. A.,Ploegh, H. L. and Chapman, H. A. (1996). Essential role for cathepsin Sin MHC class II-associated invariant chain processing and peptide loading.Immunity4, 357-366.

Shiver, J. W., Su, L. and Henkart, P. A. (1992). Cytotoxicity with target DNAbreakdown by rat basophilic leukemia cells expressing both cytolysin andgranzyme A. Cell 71, 315-322.

Stump, R. F., Oliver, J. M., Cragoe, E. J. Jr. and Deanin, G. G. (1987). Thecontrol of mediator release from RBL-2H3 cells: roles for Ca2+, Na+, andprotein kinase C1. J. Immunol.139, 881-886.

Takebe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokota, K., Arai, M., Yoshida,M. and Arai, N. (1988). SRalpha promoter: an efficient and versatilemammalian cDNA expression system composed of the simian virus 40 early

promoter and the R-U5 segment of human T-cell leukemia virus type typeI long terminal repeat. Mol. Cell. Biol.8, 466.

Tulp, A., Verwoerd, D., Dobberstein, B., Ploegh, H. L. and Pieters, J.(1994). Isolation and characterization of the intracellular MHC class IIcompartment. Nature369, 120-126.

Villadangos, J. A., Bryant, R. A., Deussing, J., Driessen, C., Lennon-Dumenil, A. M., Riese, R. J., Roth, W., Saftig, P., Shi, G. P., Chapman,H. A. et al. (1999). Proteases involved in MHC class II antigen presentation[In Process Citation]. Immunol. Rev.172, 109-120.

Watts, C. (1997). Capture and processing of exogenous antigens for presentationon MHC molecules. [Review]. Annu. Rev. Immunol. 15, 821-850.

West, M. A., Lucocq, J. M. and Watts, C. (1994). Antigen processing andclass II MHC peptide-loading compartments in human B-lymphoblastoidcells. Nature369, 147-151.

Xing, R. and Mason, R. W. (1998). Design of a transferrin-proteinaseinhibitor conjugate to probe for active cysteine proteinases in endosomes.Biochem. J.336, 667-673.

JOURNAL OF CELL SCIENCE 114 (2)