invariant chain protects class 11 histocompatibility

The EMBO Journal vol.15 no.2 pp.418-428, 1996

Invariant chain protects class 11 histocompatibilityantigens from binding intact polypeptides in theendoplasmic reticulum

Robert Busch', Isabelle Cloutier2,Rafick-Pierre Sekaly2 andGiunter J.Hammerling3Division of Somatic Genetics, Tumor Immunology Program, GermanCancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg,Germany and 2Laboratory of Immunology, Clinical Research Instituteof Montreal, Montreal, Canada'Present address: The Joseph Stokes Jr Research Institute, Children'sHospital of Philadelphia, 34th Street and Civic Center Boulevard,Philadelphia, PA 19104, USA

3Corresponding author

Unlike class I histocompatibility (MHC) antigens, mostnewly synthesized MHC class II molecules fail to beloaded with peptides in the endoplasmic reticulum(ER), binding instead to the invariant chain glycopro-tein (Ii). Ii blocks the class II peptide binding grooveuntil the class II:Ii complexes are transported to endo-somes where Ii is removed by proteolysis, thus permit-ting loading with endosomal short peptides ('12-25amino acids). Ligands from which the groove is pro-tected by Ii have not yet been identified; theoreticallythey could be short peptides or longer polypeptides (orboth), because the class II groove is open at both ends.Here we show that in Ti-deficient cells, but not in cellsexpressing large amounts of Ii, a substantial fractionof class II acB dimers forms specific, SDS-resistant 1:1complexes with a variety of polypeptides. Different setsof polypeptides bound to H-2Ak, Ek, Ed and HLA-DR1class II molecules; for Ak, a major species of Mr50 kDa (p50) and further distinct 20 and 130 kDapolypeptides were detectable. Class II binding of p50was characterized in detail. Point mutations within theAk antigen binding groove destabilized the p50:classII complexes; a mutation outside the groove had noeffect. A short segment of p50 was sufficient forassociation with Ak. The p50 polypeptide was synthe-sized endogenously, bound to Ak in a pre-Golgi com-partment, and was transported to the cell surface inassociation with Ak. Thus, Ii protects the class IIgroove from binding endogenous, possibly misfoldedpolypeptides in the ER. The possibility is discussedthat polypeptide binding is an ancestral function ofthe MHC antigen binding domain.Keywords: antigen presentation/H-2 Ak/HLA-DR/intracel-lular transport/MHC

IntroductionClass I and class II major histocompatibility complex(MHC)-encoded glycoproteins present antigenic peptidesat the cell surface for recognition by CD8+ and CD4+ Tlymphocytes, respectively (Germain and Margulies, 1993).

MHC class I molecules are loaded in the endoplasmicreticulum (ER) with endogenously synthesized peptides,most of them imported from the cytosol by specific peptidepumps (Momburg et al., 1994). In contrast, the bulk ofMHC class II proteins in B cells appears not to bindantigenic peptides in the ER (Germain and Hendrix, 1991;Neefjes and Ploegh, 1992a; Newcomb and Cresswell,1993). Instead, class II molecules are selectively targetedto specialized endosomes and bind peptides there beforecell surface expression (Neefjes and Ploegh, 1992b;Schmid and Jackson, 1994). This permits exogenousantigens to be presented by MHC class II molecules.

These differences in intracellular maturation are at leastpartly due to the selective association of class II moleculeswith the invariant chain (Ii; for a review, see Cresswell,1994). In the ER, Ii homotrimers rapidly associate withnewly synthesized MHC class II c43 dimers. The resultingnonameric (01Mi)3 complexes are then exported from theER and targeted to endosomes, where a43 dimers arereleased by proteolysis of Ii. Endosomal targeting isfacilitated by sorting signals in the cytoplasmic tail of Ii.In cells expressing class II a and 3 chains without Ii, ax3dimers assemble and exit the ER inefficiently and are tovarying degrees defective in endosomal targeting. Thisresults in a decreased ability to present exogenous proteinantigens to T cells and, in B cells from Ii- knockout mice,in reduced cell surface expression of class II molecules(Stockinger et al., 1989; Nadimi et al., 1991; Schaiffet al., 1992; Bikoff et al., 1993; Simonsen et al., 1993;Viville et al., 1993; Elliott et al., 1994).

In vitro, human Ii prevents access of peptides to theantigen binding groove of class II molecules (Roche andCresswell, 1990; Teyton et al., 1990; Roche et al., 1992;Bijlmakers et al., 1994). After isolation from B-lympho-blastoid cells, cx3 dimers, but not a,BIi complexes, containendogenous peptides (Newcomb and Cresswell, 1993).Thus, Ii has been proposed to prevent loading withendogenous peptides in the ER, thereby keeping thedistinction between the class I and class II processingpathways.A corollary of this blocking hypothesis is that the

antigen binding site of class II molecules expressed in Ii-negative cells should be loaded with endogenously derivedpeptides in the ER. Functional evidence for this wasobtained by Long et al. (1994), who showed that classII-restricted presentation of an influenza nucleoproteindeterminant could be inhibited by expression of Ii; mostlikely the antigen was loaded onto class II molecules asa short cytosolically generated peptide after TAP-mediatedimport into the ER. For antigens expressed in the ER,some experiments have also shown li-mediated inhibitionof antigen presentation, but others have reported a potenti-ating effect of Ii (Bodmer et al., 1994). In several celltypes, such as rat-2 or CHO fibroblasts, studies of peptide

41) Oxford University Press418

Protection function of li in vivo

loading in the ER are complicated, either because class IIcan reach endosomes and be loaded with peptides thereeven when Ii is absent (Simonsen et al., 1993; Schildet al., 1994; Hitzel et al., 1995), or because of backgroundIi expression, as may be seen in L cells grown at highdensity (Bertolino et al., 1991).The importance of li-mediated blocking in the ER for

separating the class I and class II presentation pathwayswas also called into doubt by observations indicating thatmost class II molecules in Ii- B cells and some transfectantswere misfolded, exited the ER slowly, and were onlyinefficiently loaded with peptides (Schaiff et al., 1992;Bikoff et al., 1993; Simonsen et al., 1993; Viville et al.,1993; Bonnerot et al., 1994; Elliott et al., 1994).Using peptide extraction after metabolic labelling, we

have previously compared loading of conformationallyintact Ak molecules with endogenous short peptides in Ii+and Ii- transfected HeLa cells, a cell line in whichendosomal transport of class II molecules depends rathertightly upon co-expression of Ii (Busch et al., 1995).These experiments provided direct evidence that loadingwith endogenous peptides was quite inefficient when Iiwas lacking. Instead, we found the Ak molecules in 1i-cells to be associated with a variety of intact polypeptides.Here, we have analysed the specificity and intracellularsite of polypeptide binding. The results suggest that Ii canprotect the class II groove from binding intact polypeptidesin the ER.

ResultsSpecific SDS-stable polypeptide: MHC class 11complexes in invariant chain-negative HeLatransfectantsOur initial approach to comparing class II ligands in Ii+and Ii- cells was to analyse SDS-stable forms of class IImolecules. Binding of appropriate high-affinity peptidesto the antigen binding groove of class II ca, dimers isknown to stabilize the dimers against dissociation by SDSat room temperature; the resultant peptide:class II (xacomplexes migrate at -50-55 kDa apparent Mr on SDS-PAGE gels unless they are dissociated into their subunitsby boiling (for review, see Sadegh-Nasseri and Germain,1992). To maximize the sensitivity and resolution withwhich SDS-resistant complexes could be detected, weemployed non-boiled/boiled two-dimensional SDS-PAGEanalysis (NBB-PAGE). SDS-resistant populations of classII molecules were first separated from unstable populationson SDS-PAGE tube gels, then boiled to dissociate theSDS-resistant complexes into their subunits, and re-runon SDS-PAGE slab gels. SDS-unstable complexes andany non-specifically co-precipitating material migrateidentically whether boiled or not, and therefore locateultimately on a diagonal across the gel; SDS-stable com-plexes migrate off the diagonal, with the migration inthe first dimension indicating their size. The subunitscomprising each distinct complex are aligned verticallywith one another in the second dimension.

Ii effects on SDS-stable forms of class II moleculeswere examined by comparing HeLa cells expressingtransfected Ak cx and D chains (named HKK cells) withtransfectants that additionally expressed murine Ii insufficient amounts to saturate >95% of newly synthesized

class II molecules in the ER (HKKI cells). The cells weresurface-iodinated, Ak molecules were immunoprecipitatedand analysed by NBB-PAGE (Figure 1). Ak moleculesisolated from HKKI cells migrated in two populations: anunstable population migrating on the diagonal, and anSDS-stable population (i.e. peptide:class II complexes)migrating at -55 kDa in the unboiled dimension (shownas 'C55' in Figure IA).Ak molecules from Ii- HKK cells gave quite a different

NBB-PAGE pattern (Figure lB and C). Only few C55complexes were found in HKK cells. Instead, a sizeableoff-diagonal, SDS-resistant Ak population was found,which migrated in a smear of molecular weight >55 kDain the unboiled dimension and appeared to be associatedwith a variety of polypeptides arrayed along a seconddiagonal to the left of the diagonal defined by the SDS-unstable complexes. Among these stably bound polypep-tides, two were discernible as distinct spots: one was astrongly labelled 50 kDa polypeptide ('p50') that formeda 100 kDa apparent Mr complex ('C100') with Ak molec-ules; the other was a less abundant p20 polypeptideforming a C70 complex with Ak. On the assumption thatthe Ak (x chains themselves contribute -50-54 kDa to theapparent Mr of the complexes in the unboiled dimension (asin Ak:peptide complexes in HKKI cells), the apparentmolecular weights of the Ak :polypeptide complexes indi-cated that one polypeptide molecule was stably bound perAk heterodimer. In multiple experiments, C100 and C70were seen consistently, albeit in somewhat differentamounts depending on culture conditions; C55 was consist-ently low or absent. Polypeptide binding was specificbecause negative control and MHC class I immunoprecipi-tates from HKK cells did not contain p50 or p20 (Figure1D and E). Furthermore, stably bound polypeptides werenot detected in association with Ak from HKKI cells(Figure 1 A), showing that co-expression of excess Iiprevented polypeptide binding. In addition to the effectson SDS-resistant complexes, Ii also seemed to affect SDS-unstable association of class II molecules with iodinatedmaterial. Comparing Figure IA and B, a smear of radio-activity was seen along the diagonal in HKK-derivedAk immunoprecipitates but much less in those fromHKKI cells.A different class II molecule, HLA-DR 1 Dw 1, expressed

in HeLa cells without Ii, also formed SDS-resistant com-plexes with a variety of polypeptides (Figure IF). Strik-ingly, the pattern of stably bound polypeptides wasdifferent for different class II molecules: dominant p50 orp20 spots were not found in DRI precipitates, whereas60, 70 and 80 kDa polypeptides were detected reproduciblyas distinct spots. A large proportion of class II moleculeswas found in SDS-resistant C160 complexes that werenot found in Ak precipitates from HKK cells. A smallamount of C55 was also seen in DR1 precipitates fromtransfected HeLa cells. As for Ak, the arrangement of thebound polypeptides along a second diagonal was indicativeof a 1:1 stoichiometry (Figure 1F), and co-expression ofIi reduced the amount of bound polypeptides (not shown).In conclusion, two different MHC class II molecules fromli- cells formed specific, SDS-resistant, 1:1 complexeswith heterogeneous but specific sets of intact polypeptides(and possibly to a small extent with short peptides), in

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Fig. 1. Specific polypeptide binding to Ak class II molecules in Ii negative HeLa transfectants. NBB-PAGE analysis of Ak (A-C), control (D) orHLA class I (E) immunoprecipitates from surface iodinated HKKI (A) or HKK (B-E) cells. (F) DRI immunoprecipitate from HeLa-DRI cellsanalysed by NBB-PAGE. Sizes of Mr markers (kDa) are given, as well as the apparent molecular weights of SDS-resistant complexes ('ClOO', etc.)and the position of disassembled class II chains and dominant polypeptides ('p50', etc.). The Ak a chain, although labelled poorly bylactoperoxidase (Viville et al., 1993), was present in the SDS-resistant complexes, because the anti-Ak mAb used requires the a chain forrecognition. In (E), 'H' and 'P2m' represent the class I (HLA-ABC) heavy chain and f2-microglobulin subunits, respectively. Heavy chain dimers(labelled 'H-H' in panel E) have been described (Capps et al., 1993). Panel (C) is a schematic interpretation of the Ak pattern obtained from HKKcells in (B). It shows the direction of electrophoresis in both dimensions (from cathode to anode) and the migration of the Ak complexes in the firstdimension tube gels. The subunits comprising each SDS-resistant complex are aligned in the second dimension, as shown for C100 by the verticaldashed line. The intersection of the dashed line with the diagonal gives the migration distance of the complex in the first dimension and can be usedto estimate the Mr of the intact complex, as shown by the horizontal dashed line for C100.

contrast to the predominant loading with short peptidesseen in the presence of excess Ii.Many anti-class II mAbs fail to dissociate from MHC

class II molecules in SDS sample buffer, particularly wheneluted under non-reducing conditions, but the antibodiesused in this work could be dissociated completely fromSDS-stable C55 class II-peptide complexes isolated fromappropriate B cell lines (not shown). Therefore, failure ofantibodies to dissociate from the class II molecules shouldnot have interfered with our results.

Specificity of polypeptide binding analysed inL cell transfectantsTo compare the polypeptides bound to different class IImolecules further and to extend our results to a differentcell type, H-2Ak, HLA-DR1, H-2Ed and H-2Ek class IImolecules were isolated from transfected murine fibrobla-stoid L cells and analysed by NBB-PAGE (Figure 2A, B,C, D, respectively). In Figure 2A, Ak transfected L cellsare shown to contain significant amounts of both C55peptide:Ak complexes and Ak subpopulations that were

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Fig. 2. SDS-stable forms of different class II molecules at the surface of transfected L cells analysed by NBB-PAGE. L cells expressing H-2Ak[L-KK in (A)], HLA-DRI [R/RIH-L in (B)], Ed [CA36.2.1 in (C)] or Ek [L2B9 in (D)] were surface-iodinated and used for immunoprecipitation ofAk using H 116-32, of HLA-DRl using ISCR3, and of Ekd using 14-4-4S. SDS-resistant complexes and their subunits are designated as in Figure 1.Note that a and P chains are well resolved in HLA-DRI immunoprecipitates.

stably associated with long polypeptides. In L cells, SDS-resistant C55 complexes are probably found becausesubstantial amounts of class II can reach the endosomesand bind peptides there. This is at least partly due to lowand variable levels of Ii expression in these cells (datanot shown; cf. Bertolino et al., 1991, and Figure SD). Thepattern of polypeptides that formed SDS-stable complexeswith class II molecules resembled that found in HKKcells: again, p50 and p20 were discernible as distinct spotsamong a heterogeneous set of polypeptides arrayed alonga second diagonal. These findings suggested that thedominant Ak-bound polypeptides were similar in humansand rodents and expressed in fibroblastoid as well asepithelial cells.

For HLA-DR1 precipitated from L cell transfectants(Figure 2B), large amounts of C55 and high apparent MrSDS-stable complexes containing a variety of bound

polypeptides were also found, including the C160 com-plexes seen in the HeLa transfectants. As in HeLa-DR Icells, the bound polypeptides were a heterogeneous smeararrayed along a second diagonal, except that those polypep-tides found as distinct spots in HeLa-DR1 cells were notdominant in L cells. Thus, for DRI the precise pattern ofbound polypeptides depended both on the class II moleculeand on the cell type. Ed and Ek molecules expressed in Lcells also contained C55 as well as higher molecularweight SDS-resistant complexes with specific sets ofstoichiometrically bound polypeptides, such as Ed-boundp40 and p80, and Ek-bound p120, p60 and p45. Similarresults were obtained for rat-2 fibroblasts expressing Ak,and for Chinese hamster ovary fibroblasts expressing Ek.Supertransfection of Ii increased the amount of C55 atthe expense of polypeptide:class II complexes in Ed_transfected L cells and in Ek-transfected CHO cells (data

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not shown). In conclusion, stable binding of heterogeneouspolypeptides to class II molecules expressed in the absenceof saturating amounts of Ii appears to be a generalphenomenon. Moreover, as in HeLa cells, the pattern ofpolypeptides stably associated with class II molecules inL cells was specific for each particular class II molecule,indicating that MHC polymorphism and isotypic variationcontrol binding of at least those polypeptides that can bediscerned as defined spots. In addition, the precise patternof stably bound polypeptides and the relative proportionof class II molecules found within C55 complexesdepended on the cell type. HeLa cells, known to be almostcompletely unable to transport class II into endosomeswithout Ii (Simonsen et al., 1993), seemed to be uniqueamong the transfectants tested in possessing particularlyfew class II molecules complexed with short peptides.The subcellular origin of the different molecular complexesin different cells will be discussed further below (cf.Figure 5).

Two mutations within the antigen binding groovedestabilize polypeptide:class 11 complexesThe site on the class II molecule where the p5O polypeptidebound was characterized further using Ak variants con-taining point mutations to alanine along the ax chain helicalregion (Figure 3). Positions a52 and cx56 have beenmodelled to point into the antigen-binding cleft of H-2Akmolecules and are known to impair peptide presentation toT hybridomas and/or binding to cell surface Ak (Dellabonaet al., 1990; T.Schmidt and R.Busch, unpublished data).The mutation at x78 is predicted to be located outside thegroove and has no effect on peptide presentation/binding(Dellabona et al., 1990) (cf. Figure 3A). The mutant Akmolecules were immunoprecipitated from surface-iodin-ated L cells and analysed by SDS-PAGE (Figure 3B).Wild-type Ak migrated in three major populations whenanalysed without boiling: unstable molecules ('1'), as wellas C55 and C100 complexes that were destroyed uponboiling. After boiling, the amount of monomeric f3 chainwas increased and the p5O polypeptide was released. Akmutated at position a78 contained the same set of SDS-stable complexes as the wild-type molecule (albeit withlarger amounts of C55 and less C100 in this experiment).Ak molecules mutated at positions a52 and ax56 also wereassociated with co-precipitating p5O; however, interes-tingly these complexes were not stable in SDS. Themonomeric p5O band was detected in similar amountswith and without boiling, and SDS-resistant Cl00 com-plexes were not detectable.The specificity of these bands was confirmed by precipit-

ating Ak from the B cell line, LK35.2, which expresseslarge, saturating amounts of Ii in the ER and completelylacks p5O or Cl00 on two-dimensional NBB-PAGE gels(not shown). Neither the p5O nor Cl 00 band were detect-able in Ak immunoprecipitates from LK35.2 cells, whereaslarge amounts of C55 were found (Figure 3B).

In summary, two mutations in the Ak x chain helixpredicted to point into the antigen binding groove specific-ally rendered p5O:Ak complexes unstable in SDS, whereasa mutation outside the groove did not affect the SDS-stability of p5O:Ak complexes. There was considerablevariation in the relative amounts of C55, CIOO and p5Oexpressed, making it difficult to exclude the possibility

Fig. 3. Effect of Akct chain point mutations on SDS-stable polypeptidebinding. (A) Proposed location of the mutations used in relation to theantigen binding groove of Ak. (B) One-dimensional SDS-PAGE (10%acrylamide) analysis of control ('C') or Ak (HI 16-32)immunoprecipitates from LK35.2 B lymphoma cells or from L cellsexpressing wild-type or mutated Ak, as indicated. Samples wereanalysed with or without boiling ('b' and 'nb', respectively).Monomeric 0 chains, the p50 polypeptide, 55 kDa peptide:Ak (C55)and 100 kDa p50:Ak (C100) complexes are indicated.

that the ct78 mutation additionally had an effect on theamount of C100 complexes formed. However, a numberof other factors, including fluctuations in background p50and Ii expression due to culture conditions and/or clonalvariability between different transfectants, could also havecontributed to these quantitative differenfces. The importantpoint remains that the p5O:Ak complex remained SDS-stable when a mutation outside the groove was made, butwas destabilized by mutations of two residues believed tomake direct contact with bound antigenic peptides.

Limited proteolysis of polypeptide:class 11complexes yields SDS-resistant peptide:class 11complexesIf polypeptide binding resembled the binding of antigenicpeptides to Ak, then only a small portion of the polypeptideshould be involved in binding to Ak, the remainderprotruding from the groove and being unimportant for

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immunoprecipitates was much lower than the peptideconcentrations needed for saturating binding to A'. Inaddition, the duration of trypsin treatment (I min) wasvery short compared with binding rates at known highaffinity at neutral pH (see Materials and methods), andtrypsinization was done in the presence of NP40, adetergent that does not readily permit peptide exchange(Avva and Cresswell, 1994). In conclusion, the most likelyexplanation for our results was that trypsinization removedthe protruding termini of the bound polypeptides from theSDS-stable polypeptide:class II complexes, thus givingrise to SDS-stable C60 complexes.

Fig. 4. Trypsin sensitivity of protruding polypeptide ends. Akimmunoprecipitates from HKK or HKKI cells were briefly treatedwith the indicated amounts of trypsin and washed before elution insample buffer and SDS-PAGE analysis with or without boiling. TheSDS-stable complex between AK and truncated polypeptides isindicated as C60. and the position of monomeric f chain aftertrypsinization as 'Ptry: the other bands are assigned as before. C.negative control immmunoprecipitates using L243 (anti-DR).

binding and SDS stability. To test this, Ak immunoprecipi-tates from surface-iodinated Ii+ and li- cells were treatedbriefly (1 min) with trypsin before SDS-PAGE (Figure 4).After elution at room temperature, untrypsinized Ak fromHKKI cells was mostly present as a C55 oc3:peptidecomplex. Migration of C55 was hardly affected by trypsin,although the a chain was truncated by about 2 kDa. InHKK cells, the most abundant SDS-resistant form of Akwas the C100 complex, which dissociated into the p50polypeptide and AK subunits upon boiling. Some C70 wasalso seen in unboiled, and p20 in boiled, untrypsinizedprecipitates. After trypsinization, the polypeptide:AK com-plexes were truncated to an SDS-resistant non-covalentcomplex migrating at -60 kDa apparent Mr (C60), slightlylarger than the compact (C55) class II molecules isolatedfrom HKKI cells. Trypsinization resulted in the degrada-tion of p50 and p20 polypeptides to < 15 kDa fragments,as evident from the loss of the p50 and p20 bands fromboiled, trypsinized HKK immunoprecipitates.The class II bound tryptic fragments may have been

larger than the oligopeptides generated by natural pro-cessing, accounting for the Mr difference between C60 intrypsinized HKK and C55 in LK35.2 cells. Alternatively.the polypeptides could have given rise to 'floppy', ratherthan 'compact', peptide:class II complexes on trypsiniz-ation (Viville et al., 1993). Different cc chain glycosylationin HKK and HKKI cells might also contribute to themobility differences (Anderson and Miller, 1992;Simonsen et al., 1993). C60 most likely was not generatedby complete dissociation of class II bound polypeptidesduring fragmentation followed by re-binding of trypticfragments, because the polypeptide concentration in the

Pre-Golgi site of formation of polypeptide:AkcomplexesThe intracellular compartment where the polypeptidesbound were examined next, using pulse-chase analysistogether with brefeldin A (BfA) treatment. BfA is a fungalmetabolite that inhibits vesicular transport from the ER(Lippincott-Schwartz et al., 1989), which should permitthe localization of polypeptide binding to a pre-Golgi orpost-Golgi site.

In HKKI cells incubated with 35S-labelled amino acidsfor 30 min, class II molecules were unstable in SDS andassociated with large amounts of Ii (Figure 5A, lanes 'noBfA'). After an overnight chase, Ak immunoprecipitateslacked Ii and contained some C55 complexes; SDS-stableassociation with polypeptides was not found. Mobilityshifts in the oc and ( chain indicated export from theendoplasmic reticulum during the chase, which was con-firmed by BfA treatment. In BfA-treated HKKI cells. Akmolecules and associated Ii accumulated in the ER andwere aberrantly glycosylated after chase, as shown bymobility shifts on SDS gels (Figure 5A, lanes '+ BfA').The latter phenomenon is known to be due to redistributionof glycosylating enzymes from the cis-Golgi compartmentunder prolonged BfA block (Lippincott-Schwartz et al.,1989). Formation of C55 complexes during the chaseperiod was completely abrogated by BfA, confirming thatpeptide loading was a post-Golgi event. The experimentalso showed that transport to endosomes was blockedcompletely under these conditions, otherwise residual C55would have been present in BfA-treated cells.

Figure 5B shows the same analysis for li-negative HKKcells. Only small amounts of radiolabelled Ak moleculeswere detected after precipitation from pulse-labelled cellsusing the conformation-sensitive H1 16-32 mAb, all ofwhich was unstable in SDS. After an overnight chasewithout BfA, more AK was precipitated, with most of theincrease being in the SDS-resistant population. DistinctSDS-resistant complexes appeared at 100 and 200 kDa(C100 and C200, respectively) in the unboiled, chasedsample. After boiling, Cl00 and C200 vanished, the (xand ( chain bands were increased, and the p50 polypeptidewas released. Two-dimensional NBB-PAGE analysis ofbiosynthetically labelled, chased HKK cells (not shown)confirmed that the p50 band arose from the C100 complex.C200 was shown to consist of an Ak oc3 heterodimercomplexed with an endogenously synthesized -130 kDapolypeptide. although p130 was labelled too weakly to beseen above background in Figure 5B without BfA treat-ment. We have been unable to detect p20 or C70 bymetabolic labelling, probably due to their lower abundance.

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Fig. 5. Generation of SDS-resistant forms of Ak in Ii+ and Ii- cells followed by metabolic labelling in the presence of brefeldin A (BfA). (A and B)Effect of BfA on the formation of SDS-resistant complexes in HKKI and HKK cells, respectively. Cells were pulse-labelled for 30 min and chasedovernight, where indicated, in the presence or absence of 11 RIg/ml BfA. The symbols ab, lib, lb in (A) refer to class II subunits that are aberrantlyglycosylated in extensively BfA-treated cells (Lippincott-Schwartz et al., 1989); note that this results in mobility shifts of the subunits in both HKKand HKKI cells, showing that BfA treatment was effective. The non-specific band at 43 kDa is actin. (C) Ability of BfA to inhibit loading of Ekwith short peptides and to enhance binding of long polypeptides in CHO transfectants. CHO cells transfected with Ek (N.1A4) were pulse-labelledand chased overnight with or without BfA as before. Note that C55 complexes decreased to background under BfA treatment but higher molecularweight SDS-resistant forms, only faintly visible in the absence of BfA, were increased. Similar results were obtained for Ak in L-KK cells (notshown).

More detailed pulse-chase kinetics revealed that C100and C200 were faintly visible after a 1 h chase andaccumulated continually over the entire 17 h chase period,in parallel with the gradual accumulation of Ak precipitablewith the HI 16-32 mAb (data not shown). This mayindicate slow folding of 4xi dimers in cells lacking Iiand the requirement for a groove-bound polypeptide tofacilitate folding.

Note that even after an overnight chase, the majorityof Ak monomers in HKK cells did not undergo complexglycan maturation (which would have been visible asa decrease in electrophoretic mobility). This confirmedprevious findings showing that class II remained in theER for a long time in li-negative transfectants, despite thefact that class II ultimately accumulates at the cell surfacein similar amounts as in transfectants additionallyexpressing Ii (Schaiff et al., 1992; Simonsen et al., 1993).The result also suggested that polypeptide binding occurredin a pre-Golgi compartment, which was tested using BfAtreatment (Figure 5B, lanes '+BfA'). Strikingly, whereasC55 formation in HKKI cells was completely inhibitedby BfA, C100 and C200 complexes were readily foundin cells chased in the presence of BfA. The abundance ofC100 decreased somewhat upon BfA treatment, whereasthat of C200 increased; correspondingly the amount ofco-precipitated p50 was slightly reduced and that of p130increased to a level that permitted clear identification ona one-dimensional gel. Despite these subtle, quantitativeeffects, there was clear evidence that the polypeptide:Akcomplexes, unlike C55, could form under conditions whereexport through the Golgi apparatus was abrogated byBfA. The electrophoretic mobility of the complexes wassomewhat altered in the presence of BfA due to aberrant

glycosylation of x and , chains during the chase. Weconcluded that the polypeptides bound Ak in the ER (orpossibly the cis-Golgi compartment), in contrast to thepost-Golgi localization of loading with short peptides inthe presence of Ii. Furthermore, this experiment confirmedthat Ii physically inhibited polypeptide binding in the ER,because Ak:Ii complexes artificially retained in the ER ofBfA-treated HKKI cells did not form C100 or C200complexes (Figure 5A). In other words, Ii did not merelyprevent polypeptide binding by accelerating transportfrom the potential binding compartment, but physicallyprotected the antigen binding site from the polypeptides.

Finally, we investigated the origin of C55 complexesseen in L cell and CHO cell transfectants. Figure 5Cshows the effect of BfA treatment during pulse-labellingand chase on the SDS-resistant complexes formed by Ekmolecules in CHO transfectants, i.e. cells in which classII molecules can reach endosomes without Ii (Schild et al.,1994). In the absence of BfA, only small amounts of highmolecular weight polypeptide:class II complexes wereseen after an overnight chase, whereas a substantial amountof C55 complexes was observed. In the presence of BfA,formation of C55 was abrogated, whereas the amount ofpolypeptide:class II complexes was greatly increased.Similar results were obtained for L cells expressing Ak,although these cells expressed less class II and the bandswere correspondingly weaker (not shown). We concludedthat even in cells that simultaneously formed SDS-resistantcomplexes with short peptides and with polypeptides inthe absence of Ii, the vast majority of C55 complexeswas generated in post-Golgi compartments, whereas thepolypeptide:class II complexes were generated pre-Golgi.

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DiscussionIn this study we have compared the ligands bound to MHCclass II molecules expressed in Ii- and Ii+ transfectants. InHeLa cells, efficient loading with oligopeptides requiredthe presence of Ii (Figure 1; cf. Busch et al., 1995). Inthe absence of Ii, a large proportion of conformationallyintact, heterodimeric class II molecules formed complexeswith heterogeneous polypeptides in the ER, and at leastsome of these complexes were transported to the cellsurface. Polypeptide binding was SDS-stable, specific forthe particular class II molecule used, and occurred inseveral different cell types. Loading with short peptidesin the ER appeared to be a minor pathway for HLA-DRland virtually undetectable for Ak expressed in HeLa cellswithout Ii, consistent with our previous observations(Busch et al., 1995).

In the absence of Ii, class II molecules misfold, areretained in the ER, and/or rapidly form aggregates con-taining other proteins (Germain and Rinker, 1993; Bon-nerot et al., 1994; Elliott et al., 1994). The present studyconfirmed that class II molecules were retained in the ERfor many hours and were only slowly recruited intopopulations that could be precipitated with conformation-sensitive mAbs (Figure SB). Aggregation with other poly-peptides could also explain the SDS-labile association ofclass II molecules with polypeptides seen as a smear ofradioactivity on the diagonal in lilow or Ii- transfectants(e.g. Figure 1B).

However, the distinct SDS-resistant polypeptide:classII complexes such as C 100 could not be non-stoichiometricaggregates, because they contained exactly one polypep-tide per 4x3 dimer. The finding that the polypeptide:classII complexes in HKK cells could be precipitated withconformation-sensitive mAbs suggested that the classII molecules within these complexes were not grosslymalfolded, although the folding rate might have beenretarded in the absence of Ii. Importantly, aggregationwould be expected to trap similar proteins irrespective ofthe class II molecule used, whereas in our experimentsthe pattem of stably bound polypeptides was sensitive toMHC polymorphism. Thus, the polypeptides seemed tointeract with the class II antigen binding groove. TheSDS-stability of the complexes and the finding that bindingof p5O can be destabilized by mutations inside the groove(Figure 3) also pointed in this direction. That a shorttryptic fragment of p50 was sufficient to stabilize Akagainst SDS (Figure 4) also was consistent with the ideathat the polypeptides interact with class II like antigenicpeptides. In addition, polypeptide binding, like loadingwith antigenic peptides in vitro, was blocked by Ii in theER (Figures 1A and SA). In summary, by biochemicalcriteria the class II binding properties of these heterogen-eous polypeptides equalled those of short antigenicpeptides.We hypothesize, therefore, that in the ER, class II

molecules can bind polypeptides that possess exposed,extended polypeptide segments available for interactionwith the antigen binding groove unless protected by Ii.As an active protein folding compartment, the ER wouldbe expected to generate incompletely folded structurescontinuously (Helenius et al., 1992). The pre-Golgi siteof polypeptide loading seen in BfA inhibition experiments

(Figure SB) is consistent with this hypothesis. It also fitswith the ability of artificially unfolded protein antigens toassociate with class II molecules in vitro and to bepresented to T cells (Sette et al., 1989). We also consideredthat the dominant polypeptides might act as molecularchaperones (Schaiff et al., 1992) or superantigens(Winslow et al., 1992). Although difficult to rule outformally, this is unlikely because the stably bound polypep-tides were very heterogeneous in size and the dominantspots were specific for the class II molecules used-properties that would not be expected of chaperones orsuperantigens. The only known ER chaperone in theappropriate Mr range, hsp47, and the ERGIC-53 proteinmarking the cis-Golgi reticulum were clearly distinctfrom the p50 polypeptide by SDS-PAGE analysis (datanot shown).

In Ii+ cells, polypeptide binding to the class II grooveis physically blocked by Ii in the ER. Thus, it appearsthat in intact cells, the presence of Ii not only assists thecorrect assembly of newly synthesized class II moleculesin the ER, but also helps to prevent their antigen bindingsites from being occupied by endogenous polypeptides.Several endogenously synthesized antigens are known tobe presented by class II molecules in the absence of Iibut less well in its presence (Clements et al., 1992;Bodmer et al., 1994; Dodi et al., 1994). Our results raisethe possibility that such antigens may bind class II asintact polypeptides in the ER before surface export andmay or may not (depending on the cell type used) besecondarily degraded to peptide:class II complexes byserum proteases or during endosomal recycling (Nijenhuiset al., 1994). We do not wish to exclude the additionalpossibility that cytosolically derived peptides might alsobe prevented by Ii from associating with class II in theER (Bijlmakers et al., 1994; Long et al., 1994), but suchpeptides clearly comprise only a minor fraction of thematerial bound to the groove of Ak or DR 1 in Ii-HeLa cells.

Sequence comparison and molecular modelling havebeen used to identify tentative structural resemblancesbetween MHC molecules and the ligand binding domainsof stress proteins of the hsp70 family, which are knownto bind heterogeneous polypeptides in vivo (Flajnik et al.,1991; Rippmann et al., 1991). Our observation that classII molecules bind polypeptides in the ER unless protectedby Ii is consistent with the idea that polypeptide bindingmight have been the function of an ancestral MHCmolecule (Figure 6). Recruitment of an ancestral MHCmolecule for binding and presenting shorter peptides mayhave required the evolution of mechanisms to avoidpolypeptide binding in the ER. Class II molecules, whichhave retained an open antigen binding groove capable ofbinding long polypeptides (Brown et al., 1993), associatein the ER with the Ii molecule, which protects them frompolypeptide binding and facilitates transport to endosomes,highly degradative compartments that are particularly richin processed peptides (Germain and Margulies, 1993).Structural peculiarities of class I molecules also makesense when viewed in this context. Class I molecules needto discriminate within the ER between long polypeptidesand short peptides translocated into the ER lumen, andmay do so by virtue of their intrinsic preference for shorterpeptides due to their antigen binding groove being closed

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10 mM HEPES, pH 7.2, and 5% dialysed fetal bovine serum, labelledfor 30 min with 100 ,uCi [35S]methionine and 30 ,tCi [35S]cysteine (ICNFlow)/ml supplemented methionine/cysteine-free RPMI, and chased incomplete RPM11640 for various times. Washed cells were lysed at _107cells/ml for -l h at 4°C in 50 mM Tris-HCl, pH 8.0, 150 mM NaCI,containing 1% NP-40, 5 mM MgCl2, 0.1% aprotinin (Sigma), 100 lMN-ethylmaleimide, and 0.5 mM phenylmethylsulfonyl fluoride. Adherentcells were labelled in 10-cm Petri dishes and harvested with PBS, 5 mMEDTA before lysis.

Cell surface iodinationCells were harvested with PBS, 5 mM EDTA, washed extensively inGoding's PBS (Goding, 1980), labelled with 1 mCi Na125I (Amersham)per 107 cells using lactoperoxidase/H202 as described (Neefjes andPloegh, 1992a), and washed again in Goding's PBS before lysis.

Fig. 6. Hypothetical explanation for the distinct structural features ofthe class I and class II antigen binding grooves as distinct solutions tothe problem of avoiding polypeptide binding. The ancestral MHCmolecule is proposed to function as a polypeptide binding protein,similar to the behaviour of class II molecules not protected by Ii(centre). Class 1I proteins would evolve by inactivation of this grooveby the Ii chain in the ER, blocking polypeptide binding and permittingpeptide loading later during maturation in endosomes upon removal ofIi (right). Since peptide loading is relegated to a later compartmentwhere short peptides are abundant, there is no need for a preciselength requirement for the bound peptide. Class I molecules (left) mustselect for short, TAP-transported peptides in the face of potentialcompetition by polypeptides present in the ER, and have evolved thecapacity to discriminate against polypeptide binding by closing thegroove at both ends, thus imposing stricter length requirements fortightly bound peptides.

at both ends, making specific contacts with the N- and C-termini of bound peptides (Madden et al., 1991, 1992;Bouvier and Wiley, 1994).

Materials and methodsCell lines and hybridomasHeLa cells expressing Ak a,B dimers either alone (HKK cells; Simonsenet al., 1993) or together with an excess of murine Ii (HKKI cells; Buschet al., 1995), HeLa cells transfected with DRI (HLA-DRI; Thibodeauet al., 1994), L cells expressing wild-type or mutated Ak (Dellabonaet al., 1990), HLA-DRI (R/RIH-L; Altmann et al., 1990) and Ed(CA36.2. 1; Shastri et al., 1985), as well as CHO fibroblasts transfectedwith Ek (N.1A4; Wettstein et al., 1991) have been described. L cellstransfected with Ek were generated similarly (L2B9; D.Wettstein andH.Schild, unpublished data). Ii supertransfectants of HeLa-DRI(expressing human p33Ii; I.Cloutier, unpublished data). CA36.2.1 (C-A36.2.1Ii, expressing genomic murine Ii; Stockinger et al., 1989) andN.1A4 (2A.Bll, expressing murine p4lIi; Schild et al., 1994) werealso used. Cells were cultured in RPMI1640 medium (Gibco-BRL)supplemented with 10% fetal bovine serum, 2 mM glutamine, 4Xl0-5M 2-mercaptoethanol and antibiotics; they were passaged by trypsiniz-ation at least 48 h before experiments. Ii expression was sufficient tosaturate >95% of newly synthesized Ak molecules in HKKI cells asshown by immunodepletion after 30 min biosynthetic labelling, whereasvariable but small, subsaturating amounts of Ii were found in pulse-labelled L cell transfectants when grown to confluency (not shown). ThemAbs used were H116-32 (anti-Ak, a conformation-sensitive, a-chain-specific mAb; Lemke et al., 1979; Landais et al., 1986; Peccoud et al.,1990), ISCR3 (anti-HLA-DR; Watanabe et al., 1983), L243 (anti-HLA-DR; Lampson and Levy, 1980), and W6/32 (anti-HLA-ABC; Barnstableet al., 1978), K22-42 (anti-I-Ed; Koch et al., 1984) and 14-4-4S (anti-I-E; Sachs et al., 1981). P4H5, a hamster IgG mAb reactive with thelumenal domain (Mehringer et al., 1991), and In-1, a rat IgG reactivewith the cytoplasmic domain (Koch et al., 1982), were used to detectIi. A mouse anti-rat K chain mAb, MARI8.1 (Lanier et al., 1982), wasused to capture In- I on protein A-Sepharose.

Biosynthetic labellingCells were washed in Dulbecco's PBS, starved for 30-45 min in Met/Cys-free RPMI 1640 (ICN Flow) supplemented with 2 mM glutamine,

ImmunoprecipitationNP-40 lysates were centrifuged (13 000 r.p.m., 30 min, 4°C, in anEppendorf microcentrifuge) to remove debris and precleared 5-6 timeswith fixed Staphylococcus aureus, rabbit anti-mouse Ig and preimmunemouse serum. Protein A-Sepharose 4B (Pharmacia) was pre-loaded withmAbs (0.5-1 ml tissue culture supernatant per 20 ,ul beads) at 4°C for1 h, washed in lysis buffer, and mixed with the lysates. Lysates wereprecipitated sequentially with irrelevant control mAb, followed by anti-class II mAb, and finally anti-li and/or anti-class I mAbs. Immunoprecipi-tates were washed extensively (5-10 times) in lysis buffer (withoutinhibitors and MgCl2) containing 10 mM EDTA.

SDS-PAGE analysisImmunoprecipitates were eluted at room temperature in Laemmli SDS-PAGE sample buffer containing 1-2% SDS and 5-7% 2-mercaptoethanolfor - I h, aliquots were removed for boiling, and equal amounts ofboiled and unboiled samples were resolved on 10% or 12% SDS-PAGEgels (Germain and Hendrix, 1991). Alternatively, unboiled samples wereresolved on 8% or 10% SDS-PAGE tube gels, which were then soakedin several changes of sample buffer, heated to 95°C for 15 min, and re-run in the second dimension on SDS-PAGE slab gels (non-boiled/boiled2-D SDS-PAGE, or NBB-PAGE; for independently developed, similartechniques, see Lanzavecchia et al., 1992; Hitzel et al., 1995). Gelswere fluorographed/autoradiographed on Kodak X-OMAT AR film forvarious times depending on the cell line used.

Limited proteolysis of immunoprecipitateslodinated Ak immunoprecipitates were treated with the indicated amountsof trypsin (Sigma) in 100 mM Tris, pH 8.0, 0.1% NP-40, at 370C for Imin. Precipitates were washed in trypsinization buffer plus 1 mM PMSFto inhibit trypsin, and the digested class II and associated materialsubjected to SDS-PAGE. These conditions should minimize re-bindingof tryptic polypeptide fragments to the class II groove after dissociation:the concentration of class II bound polypeptides during trypsinizationwas at most of the order 10-9 M, which is low compared with the Kdfor good groove-binding peptides (_l0-7 to 106 M), and trypsinizationwas short compared with rates of stable peptide binding at neutral pH(half-life of the order of several hours) (Rothbard and Gefter, 1991).

AcknowledgementsWe thank Drs H.Ikeda, J.Trowsdale, J.Bodmer, D.Wettstein, H.Schild,N.Koch and C.Benoist for cell lines, Prof. B.Dobberstein for discussions,and Dr J.J.Neefjes for technical advice. This work was supported bygrant CII -CT92-0027 of the European Union, and the Sonderforschungs-bereich 352.

ReferencesAltmann,D.M., Heyes,J.M., Ikeda,H., Sadler,A.M., Wilkinson,D.,

Madrigal,J.A., Bodmer,J.G. and Trowsdale,J. (1990) Fine mapping ofHLA class II monoclonal antibody specificities using transfected Lcells. Immunogenetics, 32, 51-55.

Anderson,K.S. and Cresswell,P. (1994) A role for calnexin (IP90) in theassembly of class II MHC molecules. EMBO J., 13, 675-682.

Anderson,M.S. and Miller,J. (1992) Invariant chain can function as achaperone protein for class II major histocompatibility complexmolecules. Proc. Natl Acad. Sci. USA, 89, 2282-2286.

Avva,R.R. and Cresswell,P. (1994) In vivo and in vitro formation and

426


dissociation of HLA-DR complexes with invariant chain-derivedpeptides. Immunity, 1, 763-774.

Bamstable,C.J., Bodmer,W.F., Brown,G., Galfre,G., Milstein,C.,Williams,A.F. and Ziegler,A. (1978) Production of monoclonalantibodies to group A erythrocytes, HLA and other human cell surfaceantigens - new tools for genetic analysis. Cell, 14, 9-20.

Bertolino,P., Forquet,F., Pont,S., Koch,N., Gerlier,D. and Rabourdin-Combe,C. (1991) Correlation between invariant chain expression leveland capability to present antigen to MHC class II-restricted T cells.Int. Immunol., 3, 435-443.

Bijlmakers,M.-J.J.E., Benaroch,P. and Ploegh,H.L. (1994) Assembly ofHLA-DRI molecules translated in vitro: binding of peptide in theendoplasmic reticulum precludes association with invariant chain.EMBO J., 13, 2699-2707.

Bikoff,E.K., Huang,L.-Y., Episkopou,V., van Meerwijk,J., Germain,R.N.and Robertson,E.J. (1993) Defective major histocompatibility complexclass II assembly, transport, peptide acquisition, and CD4+ T cellselection in mice lacking invariant chain. J. Exp. Med., 177, 1699-1712.

Bodmer,H., Viville,S., Benoist,C. and Mathis,D. (1994) Diversity ofendogenous epitopes bound to MHC class II molecules limited byinvariant chain. Science, 263, 1284-1286.

Bonnerot,C., Marks,M.S., Cosson,P., Robertson,E.J., Bikoff,E.K.,Germain,R.N. and Bonifacino,J.S. (1994) Association with BiP andaggregation of class II MHC molecules synthesized in the absence ofinvariant chain. EMBO J., 13, 934-944.

Bouvier,M. and Wiley,D.C. (1994) Importance of peptide amino andcarboxyl termini to the stability of MHC class I molecules. Science,265, 398-402.

Brown,J.H., Jardetzky,T.S., Gorga,J.C., Stern,L.J., Urban,R.G.,Strominger,J.L. and Wiley,D.C. (1993) Three-dimensional structureof the human class 11 histocompatibility antigen HLA-DRI. Nature,364, 33-39.

Busch,R., Vturina,I.Y., Drexler,J., Momburg,F. and Hammerling,G.J.(1995) Poor loading of major histocompatibility complex class IImolecules with endogenously synthesized short peptides in the absenceof invariant chain. Eur J. Immunol., 25, 48-53.

Capps,G.G., Robinson,B.E., Lewis,K.D. and Zufiiga,M.C. (1993) In vivodimeric association of class I MHC heavy chains. Possible relationshipto class I MHC heavy chain-,B2-microglobulin dissociation. J.Immunol., 151, 159-169.

Clements,V.K., Baskar,S., Armstrong,T.D. and Ostrand-Rosenberg,S.(1992) Invariant chain alters the malignant phenotype of MHC classII tumor cells. J. Immunol., 149, 2391-2396.

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

Dellabona,P., Peccoud,J., Kappler,J., Marrack,P., Benoist,C. andMathis,D. (1990) Superantigens interact with MHC class II moleculesoutside of the antigen binding groove. Cell, 62, 1115-1121.

Dodi,A.I., Brett,S., Nordeng,T., Sidhu,S., Batchelor,R.J., Lombardi,G.,Bakke,O. and Lechler,R.I. (1994) The invariant chain inhibitspresentation of endogenous antigens by a human fibroblast cell line.Eur J. Immunol., 24, 1632-1639.

Elliott,E.A., Drake,J.R., Amigorena,S., Elsemore,J., Webster,P.,Mellman,I. and Flavell,R.A. (1994) The invariant chain is requiredfor intracellular transport and function of major histocompatibilitycomplex class II molecules. J. Exp. Med., 179, 681-694.

Flajnik,M.F., Canel,C., Kramer,J. and Kasahara,M. (1991) Which came

first, MHC class I or class II? Immunogenetics, 33, 295-300.Germain,R.N. and Hendrix,L.R. (1991) MHC class II structure,

occupancy and surface expression determined by post-endoplasmicreticulum antigen binding. Nature, 353, 134-139.

Germain,R.N. and Margulies,D.H. (1993) The biochemistry and cellbiology of antigen processing and presentation. Annu. Rev. Immunol.,11, 403-450.

Germain,R.N. and Rinker,A.G.,Jr (1993) Peptide binding inhibits proteinaggregation of invariant chain-free class II dimers and promotessurface expression of occupied molecules. Nature, 363, 725-728.

Goding,J.W. (1980) Structural studies of murine lymphocyte surfaceIgD. J. Immunol., 124, 2082-2088.

Helenius,A., Marquardt,T. and Braakman,l. (1992) The endoplasmicreticulum as a protein-folding compartment. Trends Cell Biol., 2,227-231.

Hitzel,C., van Endert,P. and Koch,N. (1995) Acquisition of peptides byMHC class 11 polypeptides in the absence of the invariant chain. J.Immunol., 154, 1048-1056.

Koch,N., Koch,S. and Hammerling,G.J. (1982) Ia invariant chain detected

on lymphocyte surfaces by monoclonal antibody. Nature, 299, 644-645.

Koch,N., Sandrin,M.S., Hammerling,G.J., Tada,N., Kimura,S. andHammerling,U. (1984) Monoclonal antibodies detect polymorphicdeterminants shared by I-A and I-E antigens. Mol. Immunol., 21,293-299.

Lampson,L.A. and Levy,R. (1980) Two populations of la-like moleculeson a human B cell line. J. Immunol., 125, 293-299.

Landais,D. et al., (1986) The assignment of chain specificities for anti-Ia monoclonal antibodies using L cell transfectants. J. Immunol., 137,3002-3005.

Lanier,L.L., Gutmann,G.A., Lewis,D.E., Griswald,S.T. and Warner,N.L.(1982) Monoclonal antibodies against rat immunoglobulin kappachain. Hybridoma, 1, 125-131.

Lanzavecchia,A., Reid,P.A. and Watts,C. (1992) Irreversible associationof peptides with class II MHC molecules in living cells. Nature, 357,249-252.

Lemke,H., Hammerling,G.J. and Hammerling,U. (1979) Fine specificityanalysis with monoclonal antibodies of antigens controlled by themajor histocompatibility complex and by the Qa/TL region in mice.Immunol. Rev., 47, 175-206.

Lippincott-Schwartz,J., Yuan,L.C., Bonifacino,J.S. and Klausner,R.D.(1989) Rapid redistribution of Golgi proteins into the ER in cellstreated with brefeldin A: evidence for membrane cycling from Golgito ER. Cell, 56, 801-813.

Long,E.O., LaVaute,T., Pinet,V. and Jaraquemada,D. (1994) Invariantchain prevents the HLA-DR-restricted presentation of a cytosolicpeptide. J. Immunol., 153, 1487-1494.

Madden,D.R., Gorga,J.C., Strominger,J.L. and Wiley,D.C. (1991) Thestructure of HLA-B27 reveals nonamer self-peptides bound in an

extended conformation. Nature, 353. 321-325.Madden,D.R., Gorga,J.C., Strominger,J.L. and Wiley,D.C. (1992) The

three-dimensional structure of HLA-B27 at 2.1 A resolution suggestsa general mechanism for tight peptide binding to MHC. Cell, 70,1035-1048.

Mehringer,J.H., Harris,M.R., Kindle,C.S., McCourt,D.W. and Cullen,S.E.(1991) Characterization of fragments of the murine Ia-associatedinvariant chain. J. Immunol., 146, 920-927.

Momburg,F., Neefjes,J.J. and Hammerling,G.J. (1994) Peptide selectionby MHC-encoded TAP transporters. Curr Opin. Immunol., 6, 32-37.

Nadimi,F., Moreno,J.A., Momburg,F., Heuser,A., Fuchs,S., Adorini,L.and Hammerling,G.J. (1991) Antigen presentation of hen egg-whitelysozyme but not of ribonuclease A is augmented by the majorhistocompatibility complex class II-associated invariant chain. Eur J.Immunol., 21, 1255-1263.

Neefjes,J.J. and Ploegh,H.L. (1992a) Inhibition of endosomal proteolyticactivity by leupeptin blocks surface expression of MHC class IImolecules and their conversion to SDS-resistant axp heterodimers inendosomes. EMBO J., 11, 411-416.

Neefjes,J.J. and Ploegh,H.L. (1992b) Intracellular transport of MHCclass II molecules. Immunol. Today, 13, 179-184.

Newcomb,J.R. and Cresswell,P. (1993) Characterization of endogenouspeptides bound to purified HLA-DR molecules and their absence frominvariant chain-associated ax1 dimers. J. Immunol., 150, 499-507.

Nijenhuis,M., Calafat,J., Kuijpers,K.C., Janssen,H., de Haas,M.,Nordeng,T., Bakke,O. and Neefjes,J.J. (1994) Targeting majorhistocompatibility complex class II molecules to the cell surface byinvariant chain allows antigen presentation upon recycling. Eur J.Immunol., 24, 873-883.

Peccoud,J., Dellabona,P., Allen,P., Benoist,C. and Mathis,D. (1990)Delineation of antigen contact residues on an MHC class II molecule.EMBO J., 9, 4215-4223.

Rippmann,F., Taylor,W.R., Rothbard,J.B. and Green,N.M. (1991) Ahypothetical model for the peptide binding domain of hsp70 based on

the peptide binding domain of HLA. EMBO J., 10, 1053-1059.Roche,P.A. and Cresswell,P. (1990) Invariant chain association withHLA-DR molecules inhibits immunogenic peptide binding. Nature,345, 615-618.

Roche,P.A., Teletski,C.L., Karp,D.R., Pinet,V., Bakke,O. and Long,E.O.(1992) Stable surface expression of invariant chain prevents peptidepresentation by HLA-DR. EMBO J., 11, 2841-2847.

Rothbard,J.B. and Gefter,M.L. (1991) Interactions between immunogenicpeptides and MHC proteins. Annu. Rei: Immunol., 9, 527-565.

Sachs,D.H., Mayer,N. and Ozato,K. (1981) Hybridoma antibodiesdirected toward murine H-2 and Ia antigens. In Hiimmerling,G.J.,Hammerling,U. and Kearney,J.F. (eds), Monoclonal Antibodies and T

427

R.Busch et al.

Cell Hybridomas. Perspectives and Technical Advances. ElsevierBiomedical Press, Amsterdam, pp. 95-101.

Sadegh-Nasseri,S. and Germain,R.N. (1992) How MHC class IImolecules work: peptide-dependent completion of protein folding.Immunol. Today, 13, 43-46.

Schaiff,W.T., Hruska,K.A.,Jr, McCourt,D.W., Green,M. andSchwartz,B.D. (1992) HLA-DR associates with specific stress proteinsand is retained in the endoplasmic reticulum in invariant chain-negative cells. J. Exp. Med., 176, 657-666.

Schild,H., Mavaddat,N., Litzenberger,C., Ehrich,E.W., Davis,M.M.,Bluestone,J.A., Matis,L., Draper,R.K. and Chien,Y.-H. (1994) Thenature of major histocompatibility complex recognition by y6 T cells.Cell, 76, 29-37.

Schmid,S.L. and Jackson,M.R. (1994) Making class II presentable.Nature, 369, 103-104.

Sette,A., Adorini,L., Colon,S.M., Buus,S. and Grey,H.M. (1989) Capacityof intact proteins to bind to MHC class II molecules. J. Immunol.,143, 1265-1267.

Shastri,N., Malissen,B. and Hood,L. (1985) la-transfected L-cellfibroblasts present a lysozyme peptide but not the native protein tolysozyme-specific T cells. Proc. Natl Acad. Sci. USA, 82, 5885-5889.

Simonsen,A., Momburg,F., Drexler,J., Hammerling,G.J. and Bakke,O.(1993) Intracellular distribution of the MHC class II molecules andthe associated invariant chain (Ii) in different cell lines. Int. Immunol.,5, 903-917.

Stockinger,B., Pessara,U., Lin,R.H., Habicht,J., Grez,M. and Koch,N.(1989) A role of Ia-associated invariant chains in antigen processingand presentation. Cell, 56, 683-689.

Teyton,L., O'Sullivan,D., Dickson,P.W., Lotteau,V., Sette,A., Fink,P.and Peterson,P.A. (1990) Invariant chain distinguishes between theexogenous and endogenous antigen presentation pathways. Nature,348, 39-44.

Thibodeau,J., Cloutier,I., Lavoie,P.M., Labrecque,N., Mourad,W.,Jardetzky,T. and Sekaly,R.-P. (1994) Subsets of HLA-DR1 moleculesdefined by SEB and TSST-1 binding. Science, 266, 1874-1878.

Viville,S., Neefjes,J.J., Lotteau,V., Dierich,A., Lemeur,M., Ploegh,H.L.,Benoist,C. and Mathis,D. (1993) Mice lacking the MHC class II-associated invariant chain. Cell, 72, 635-648.

Watanabe,M., Suzuki,T., Taniguchi,M. and Shinohara,N. (1983)Monoclonal anti-Ia murine alloantibodies cross-reactive with the Ia-homologues of other mammalian species including humans.Transplantation, 36, 712-718.

Wettstein,D.A., Boniface,J.J., Reay,P.A., Schild,H. and Davis,M.M.(1991) Expression of a class II major histocompatibility complex(MHC) heterodimer in a lipid-linked form with enhanced peptide/soluble MHC complex formation at low pH. J. Exp. Med., 174,219-228.

Winslow,G.M., Scherer,M.T., Kappler,J.W. and Marrack,P. (1992)Detection and biochemical characterization of the mouse mammarytumor virus 7 superantigen (Mis-la). Cell, 71, 719-730.

Received on June 26, 1995; revised on September 18, 1995

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