Proc. NatI. Acad. Sci. USAVol. 88, pp. 7928-7932, September 1991Immunology

Characterization of naturally processed antigen bound to majorhistocompatibility complex class II molecules

(antigen processing/processed peptide/peptide-la complexes)

MALLIKA SRINIVASAN, ERIC W. MARSH, AND SUSAN K. PIERCE*Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208-3500

Communicated by Laszlo Lorand, June 6, 1991 (received for review April 15, 1991)

ABSTRACT Helper T lymphocytes recognize peptide frag-ments of antigen bound to major histocompatibility complex(MHC) class H molecules presented on the surface of antigen-presenting cells (APCs). Previous studies showed that theMHCclass II, I-Ek molecules purified from APCs that had processedDrosophika melanogaster cytochrome c (DMc) contained func-tional, processed antigenI4-Ek complexes. This was demon-strated by the ability of purified I-Ek, incorporated intoliposomes, to stimulate DMc-specific T cells in the absence ofany additional antigen. Here we describe the isolation andcharacterization of the processed antigen bound to I-Ek. Thiswas accomplished using DMc radiolabeled- across its entirelength by reductive methylation of its lysine residues, allowingan analysis of the totality of processed antigen bound to MHCclass H molecules. After processing, only about 0.2% of theAPC I-Ek molecules contained processed DMc (4800 per cell),yet these were sufficient to stimulate specific T cells. The DMcpeptides isolated from the I-Ek molecules showed only twopredominant radioactive peaks as analyzed by reverse-phasechromatography. Less processedantigen was bound to purifiedI-Ak molecules, and these peptides were distinct from thosebound to I-E]k. The association of processed DMc with the I-Ekand I-Ak molecules appears highly specific in that no radiola-beled peptides were isolated from purified MHC class I mol-ecules, Kk and Dk, or from the B-cell differentiation antigenB220. The majority of processed antigen-I-Ek complexes mi-grated more slowly than the majority of the IEk protein asanalyzed by SDS/PAGE under nonreducing conditions with-out heating of the sample. This form of I-Ek may be analogousto the earlier described "floppy" form of MIC class IImolecules [Dormair, K., Rothenhausler, B. & McConnell,H. M. (1990) Cold Spring Harbor Symp. Quant. Biol. 54,409-416]. Since newly processed antigen binds nearly exclu-sively to this slow-migrating form, it may be of functionalsignificance.

Considerable evidence indicates that helper T lymphocytesrecognize antigenic peptides bound to major histocompati-bility complex (MHC) class II molecules (1). Such peptidesare derived from native proteins by antigen-presenting cells(APCs) in a poorly delineated series of intracellular stepsreferred to as antigen processing. The nature and number ofpeptides derived from native protein antigens that are assem-bled with the MHC class II molecules intracellularly duringprocessing are not known. Synthetic peptides of 7-14 aminoacids containing an antigenic determinant are sufficient forT-cell activation by APCs (2-5). However, longer peptidescan be presented to T cells by APCs, apparently withoutfurther processing (6). Synthetic peptides can bind to purifiedclass II molecules in vitro (4, 7, 8). The binding is unusual inthat the association rate is extremely slow and the dissocia-

tion rate is negligible, making the binding nearly irreversible.In addition, only a small fraction (0.1-10%o) of the total classII molecules purified from APCs bind peptide under condi-tions of excess peptide. Studying the binding of syntheticpeptides to class II molecules on APCs, Harding and Unanue(9) showed that -500 synthetic peptide-MHC complexes aresufficient to activate specific T cells (9). A lower estimate(=60) was reached by Demotz et al. (10) comparing T-cellresponses to I-Ak molecules purified from APCs that hadprocessed ovalbumin with T-cell responses to synthetic pep-tides presented by purified I-Ak. It is not known whether thebinding of synthetic peptides to mature MHC class II mole-cules accurately reflects the assembly of processed antigen-MHC class II complexes in APCs. Critical to an understand-ing of the mechanism of this process in APCs is a knowledgeof the nature of the processed antigen resulting from intra-cellular proteolysis that is bound to class II molecules.Our earlier results showed that I-Ek molecules purified

from APCs that had processed a globular -protein antigenDrosophila melanogaster cytochrome c (DMc), when incor-porated into lipid membranes, stimulated cytochrome c-spe-cific T cells in the absence ofany additional antigenic peptide(11). Moreover, the purified I-Ek maintained the fine speci-ficity of the T-cell response to cytochrome c. In mice, thepredominant T-cell response to cytochrome c shows a het-eroclitic crossreactivity, with the stimulation by DMc requir-ing 10- to 50-fold less antigen compared with pigeon cy-tochrome c (12). The I-Ek molecules purified from APCs thathad processed DMc stimulated T cells at 10- to 30-fold lowerconcentration compared with I-Ek purified from APCs thathad processed pigeon cytochrome c (11). This unequivocallyshowed that I-Ek molecules containing functional, processedantigenic peptides can be purified from APCs. Here wedescribe the isolation and characterization of the processedantigen from MHC class II molecules purified from APCs thathad processed radiolabeled DMc.

MATERIALS AND METHODSAntigens. DMc was purified as a recombinant protein from

the yeast strain GM-3C-2 transformed with the plasmidYEpDMc 01 (kindly provided by E. Margoliash, Northwest-ern University). DMc was radiolabeled by reductive meth-ylation of the lysine residues with sodium cyano-boro[3H]hydnde (13, 14). DMc was dissolved at 2 mg/ml in10 mM P., pH 7.0, with sodium cyanoboro[3H]hydride (Am-ersham). Formaldehyde (1 M in 10mM Pi, pH 7.0) was addedsuch that the molar ratio of the reactants was 1:64:64,respectively. The reaction was allowed to proceed for 4 hr atroom temperature. 3H-DMc was separated from excess re-actants by gel filtration on a Sephadex G-25 column. The

Abbreviations: APC, antigen-presenting cell; DMc, Drosophila mel-anogaster cytochrome c; IL-2, interleukin 2; MHC, major histocom-patibiity complex.*To whom reprint requests should be addressed.

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3H-DMc contained an average of six to seven dimethylatedlysine residues per molecule. To determine whether thelysine residues in the C-terminal antigenic peptide weremethylated, a C-terminal 23-residue peptide of 3H-DMc wassequenced and the radioactivity at each residue was mea-sured. 3H-DMc was cleaved with CNBr (15), and the cleav-age products were separated on a C18 reverse-phase HPLCcolumn with a 0-60% (vol/vol) gradient of acetonitrile in0.1% trifluoroacetic acid. The C-terminal peptide, residues81-103, was subjected to microsequencing using a proteinsequencer with an on-line phenylthiohydantoin analyzer (Ap-plied Biosystems, model 477A, model 120A). Significantradioactivity was detected as predicted at each lysine resi-due.

Purification of MHC Molecules from 3H-DMc-Pulsed CH27Cells. The MHC class II (I-Ek and I-Ak) and class I (Kk andDk) molecules were sequentially purified from the lysates ofCH27 mouse B-lymphoma cells. CH27 cells (109) were incu-bated in a spinner flask for 12 hr in 500 ml of completeDulbecco's modified Eagle's medium (16) containing 5% fetalbovine serum with 3-6 AM 3H-DMc at 370C in 5% C02/95%air. The cells were washed, and I-Ek was purified from celllysates by immunoaffinity chromatography on an I-Ek_specific monoclonal antibody (14.4.4S; ref. 17) column asdescribed (11). Rechromatography of the flow-throughyielded no additional I-Ek. The I-Ek was dialyzed againstphosphate-buffered saline (PBS: 0.14 M NaCl/0.01 M phos-phate, pH 7.2-7.4) containing 30 mM n-octyl f-D-glucosideand concentrated using Centricon microconcentrators (Am-icon) with a molecular weight cutoff of 10,000. The amountof purified I-Ek was determined by bicinchoninic acid (BCA)protein assay (Pierce), and the radioactivity associated withthe protein was determined by liquid scintillation counting. Afraction of the purified I-Ek was analyzed by nonreducingSDS/10% PAGE. The I-Ek in the SDS sample buffer wasloaded on the gel without boiling to retain the association ofthe a and ,B chains. Gels were stained with Coomassiebrilliant blue and subjected to autoradiography. I-Ak waspurified by immunoaffinity chromatography on a I-Ak_specific monoclonal antibody (10.2.16; ref. 18) column andthe class I molecules on an H-2Kk/Dk-specific monoclonalantibody (15.3.1S; ref. 17) column. The proteins were ana-lyzed by SDS/10% PAGE and stained with Coomassie bril-liant blue to verify purity. The amount of I-Ak and Kk/Dkpurified was determined by BCA protein assay (Pierce) andthe radioactivity associated was measured by scintillationcounting.APC Assay. Graded numbers of CH27 cells that had been

incubated with 3H-DMc as described above were coculturedfor 24 hr with 5 x 104 TPc9.1 cells; TPc9.1 is an I-Ek_restricted cytochrome c-specific T-cell hybrid (11). Culturesupernatants were assayed for their interleukin 2 (IL-2)content by the ability to maintain the growth of an IL-2-dependent T-cell line (CTLL) as measured by the incorpo-ration of [3H]thymidine (19).

Analysis of Processed 3H-DMc Bound to I-Ek and I-Ak. I-Ekand I-Ak were purified from CH27 cells incubated with3H-DMc as described above. The purified proteins at 0.5-1mg/ml in PBS containing 30 mM n-octyl 1-D-glucoside wereprecipitated at 50% acetonitrile at 0°C for 15 min. Theprecipitates were centrifuged at 2000-3000 X g for 20 min at0°C. The supernatants were precipitated at 66% acetonitrileat 0°C for 15 min and the precipitates were centrifuged at2000-3000 x g for 20 min at 0°C. Both precipitates wereredissolved in 100-300 ,ul of 15% (vol/vol) acetic acid andincubated at 370C for 45 min to release the bound processedantigen. The acid-treated material was applied to a C18reverse-phase HPLC column and eluted with a 0-60o gra-dient of acetonitrile in 0.1% trifluoroacetic acid. One ml

fractions were collected and monitored by absorbance at 214nm and by the amount of 3H in 100 gl of each fraction.

RESULTSDMc was radiolabeled by reductive methylation of the lysineresidues with [3H]cyanoborohydride (3H-DMc). Methylationwas chosen as a means of radiolabeling DMc because such amodification was predicted to have little effect on the anti-genicity of the DMc. Methylation does not change the chargeon the lysine residues and adds little to the size of eachresidue. The amino acid sequence of DMc is given (Fig. 1)showing the minimal length peptide sufficient for the high-affinity heteroclitic response as well as the minimal peptideofDMc yielding the low-affinity response. There are 15 lysineresidues in DMc spread relatively evenly over the entirepolypeptide chain so that most peptides greater than 10-15amino acids in length contain at least 1 lysine. Significantly,the minimal antigenic determinant contains 2 lysine residues.All lysine residues are on the surface of the protein andaccessible for methylation. After reductive methylation theDMc proteins contained on average 6-7 dimethyllysine res-idues, which appeared to be randomly distributed. Indeed,microsequencing of CNBr cleavage fragments of 3H-DMcverified that all lysine residues were modified and contained3H4.CH27 cells (4 x 108) were incubated overnight with 3H-

DMc (5 ,M), allowing the production of a sufficient numberof processed 3H-DMc-MHC class II complexes for analysis.The 3H-DMc-pulsed CH27 cells were active in stimulating aDMc-specific, I-Ek-restricted T-cell hybrid, TPc9.1, to se-

1-36 GOVE5GEILFVORGAQCHTVEAGGIHBVGPNLHGLF37-73 GRITGOAEGFSYTDANSNNGITWGEDTLFEYLENPKR

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01 10 100 1000

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FIG. 1. CH27 cells incubated with 3H-DMc activate cytochromec-specific T cells. (Upper) Amino acid sequence of DMc with lysine(K) residues highlighted. Indicated by underlining are (i) the minimal(10-amino acid) C-terminal peptide of DMc necessary for T-cellactivation but not exhibiting the high-affinity response ofnative DMcand (i0) a (23-amino acid) C-terminal DMc peptide exhibiting thehigh-affinity response. The T-cell hybrid TPc9.1 is half-maximallyactivated when provided with CH27 cells as APCs and DMc-(94-103)at 2.8 AM or DMc-(81-103) at 0.1-0.28 ,uM. (Lower) CH27 cells (4x 108) were incubated at 37°C with 3H-DMc (5 AM) for 14 hr incomplete medium containing 15% fetal bovine serum (11) and werewashed to remove excess antigen. Graded numbers of 3H-DMc-pulsed cells were cultured with 5 x 104 irradiated TPc9.1 cells for 24hr. IL-2 secreted in the culture supernatants was assayed by theability to maintain the growth of an IL-2-dependent cell line, CTLL,measured by incorporation of [3H]thymidine (19).

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crete IL-2 (Fig. 1). The number of3H-DMc-pulsed CH27 cellsrequired for TPc9.1 stimulation was the same as the numberofCH27 cells pulsed with unmodified DMc previously shownto be necessary to activate TPc9.1 cells in a high-affinityfashion (11). Thus, the 3H-DMc retains its antigenicity and isprocessed to yield stimulatory, high-affinity peptide-I-Ekcomplexes. The I-Ek molecules were affinity-purified fromthe 3H-DMc-pulsed CH27 cells in an identical manner to thatpreviously described (11). The purified I-Ek was dialyzed intodetergent-containing PBS through a dialysis membrane witha molecular weight cutoff of 3500 and concentrated in amicroconcentrator with a membrane having a molecularweight cutoff of 10,000. There was negligible radioactivityeither in the dialysis buffer or in the flow-through of theconcentrator, indicating that loosely associated peptideswere not copurifying with the I-Ek. The radioactivity in thepreparation was measured and the amount of I-Ek proteinwas determined. Approximately 0.1% of the total cell-associated radiolabeled antigen was purified bound to theI-Ek molecules. Assuming that any 10- to 20-amino acidpeptide fragment from 3H-DMc could bind to an I-Ek mole-cule, we calculated that -800 molecules of I-Ek perCH27 cellcontained processed antigen (Table 1). Approximately 3000cells that had assembled this number of complexes weresufficient to activate the TPc9.1 cells (Fig. 1). Indeed, thissmall number of processed antigen-I-Ek complexes per cellwas sufficient for T-cell activation as demonstrated both bythe ability of the CH27 cells from which the I-Ek was purifiedto stimulate TPc9.1 cells (Fig. 1) and by the ability of I-Ekprepared in an identical manner from nonradiolabeled DMc-pulsed CH27 cells to activate specific T cells (11).The MHC class II I-Ak molecules were purified from the

same CH27 cells that processed 3H-DMc. In mice, there is nodetectable T-cell response to DMc presented by I-Ak mole-cules. However, the purified I-Ak molecules contained pro-cessed 3H-DMc (Table 1). About two-thirds less processedantigen was bound to I-Ak compared with I-Ek. By deter-mining the amount of I-Ak protein purified and again assum-ing that any 10- to 20-amino acid peptide of3H-DMc could bebound, we calculated that only -=200 I-Ak molecules perCH27 cell had bound processed antigen (Table 1). Becausethere are no T cells that recognize such complexes, it is notpossible to know whether these would be sufficient foractivation.The association ofprocessed antigen with the I-Ek and I-Ak

molecules appears highly specific in that negligible radioac-tivity was detected associated with the MHC class I (Kk andDk) molecules purified from the CH27 cells that had pro-cessed 3H-DMc (Table 1). Moreover, in control experiments,no radioactivity was purified bound to the B-cell differenti-ation antigen B220. B220 was immunoprecipitated using theB220-specific monoclonal antibody 14.8 under conditionswhere processed 3H-DMc was immunoprecipitated with I-Ek(data not shown). The observation that not all proteins

Table 1. Peptide occupancy of MHC class II and class Imolecules purified from 3H-DMc-pulsed CH27 cells

Specific ProcessedAmount activity, DMc-MHC

H-2 purified, Radioactivity cpm/,ug of complexes,protein ,ug bound, cpm protein no. per cellI-Ek 102 48,000 471 800-2000I-Ak 83 12,000 146 200-600Kk/Dk 182 3,000 16 20-60

CH27 lymphoma cells were incubated with 3H-DMc for 15 hr. I-Ek,

isolated from 3H-DMc-pulsed cells contained radioactivityexcluded the possibility that the radioactivity associated withI-Ek and I-Ak was the result of nonspecific exchange and/orincorporation of [3H]dimethyllysine from 3H-DMc into cel-lular proteins.The purified I-Ek containing processed 3H-DMc was sub-

jected to SDS/PAGE under nonreducing conditions. TheI-Ek in the SDS sample buffer was not boiled before analysis,to preserve the processed antigen-MHC class II complexassociation. As shown by Coomassie blue staining, the I-Ekmigrated as predicted for a-f3 heterodimer, at =60 kDa (Fig.2). A small but detectable amount of I-Ek migrated in a wideband, more slowly than the majority of the protein. Most ofthe processed 3H-DMc was bound to the more slowly mi-grating I-Ek as visualized by autoradiography (Fig. 2). Asmall portion of processed 3H-DMc appeared to be bound tothe major fraction of the I-Ek molecules migrating at 60 kDa.No free processed 3H-DMc was detected in any other regionof the gel, particularly in the lower molecular weight regions,indicating that most of the processed antigen isolated withI-Ek remained bound during SDS/PAGE.To analyze the processed 3H-DMc bound to I-Ek and I-Ak,

the purified proteins were precipitated with acetonitrile. Theprecipitates were solubilized with 15% acetic acid to elutebound peptides, and the preparation was applied to a C18reverse-phase HPLC column (Fig. 3). The small amount ofradioactivity in the void volume most likely represents thatamount of processed 3H-DMc that remained bound to I-Ekafter acid treatment. The void volume also contained the onlydetectable A214, indicating the presence of the I-Ek. Themajority of the processed 3H-DMc was obtained in twopeaks. The first peak is sharp and may represent a singlepeptide. The second peak is broader, showing a shoulder thatmay represent peptide length heterogeneity. The smallamount of processed antigen bound to I-Ek precluded defin-itive sequence determination. However, amino acid se-quence analysis of fractions 38-41 suggested a mixture ofpeptides of various lengths. Indeed, a mixture of syntheticpeptides of increasing length from 12 to 23 residues repre-senting the C terminus ofa closely related tobacco hornwormmoth cytochrome c, THMc-(92-103) to THMc-(81-103), waseluted in this region of the gradient when analyzed underidentical conditions. The shortest peptide was eluted at 24 ml,whereas THMc-(93-103) through THMc-(81-103) wereeluted later, at 30-36 ml. An 80-amino acid N-terminal CNBr

466

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. 31

1 2 3

FIG. 2. I-Ek purified from CH27 cells incubated With 3H-DMccontains processed antigen, as shown by nonreducing SDS/PAGEfollowed by staining with Coomassie blue (lane 2) and autoradiog-raphy (lane 1). The I-Ek in the SDS sample buffer was loaded on the

gel without boiling. Molecular weight markers (M, x 10-3) are shown

(lane 3).

I-Ak, Kk/Dlk were purified sequentially on immunoaffinity columns.Proteins were analyzed by SDS/PAGE to verify purity. Protein wasdetermined by BCA protein assay (Pierce), and the radioactivityassociated was measured by liquid scintillation counting.

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150I

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FIG. 3. Reverse-phase chromatogram of processed 3H-DMceluted from I-Ek (Upper) and I-Ak (Lower) molecules. I-Ek and I-Akwere purified from CH27 cells incubated with 3H-DMc. The purifiedproteins were precipitated with 50%o and 66% acetonitrile at 0C, andthe precipitates were pooled and treated with 15% acetic acid for 45min at 37TC to elute the 3H-DMc peptides associated with the classII proteins. The acid-treated material was fractionated on a C18reverse-phase HPLC column with a 0-60%o gradient of acetonitrile in0.1% trifluoroacetic acid, at a flow rate of1% acetonitrile per 1 ml permin. Fractions (1 ml) were collected and their absorbance at 214 nmis shown. Radioactivity of 100 of each fraction was determined byliquid scintillation counting.

cleavage fragment, THMc-(1-80), was eluted still later, at37-40 ml. The methylation oflysine residues in peptides doesnot change their mobility on the reverse-phase column, asC-terminal peptides (residues 81-103) of DMc and 3H-DMcwere eluted from the column identically. There was nodetectable A214 in the region of the chromatogram where theprocessed antigen was eluted, indicating that a significantnumber of nonradioactive peptides were not copurified withthe I-Ek and/or were not generated during the acid treatmentofthe I-Ek. That the A214 was separated from the radioactivityon this column indicates that the radioactivity detected withthe purified I-Ek (Fig. 2, Table 1) was not due to artifactualexchange of 3H from 3H-DMc to cellular proteins.The I-Ak was treated and chromatographed on a C18

reverse-phase HPLC column in the same fashion as the I-Ek(Fig. 3). A small amount of the processed 3H-DMc was elutedin the void volume along with most of the A214. The majorityof the processed 3H-DMc was eluted in a broad peak that isclearly distinct from the pattern of the I-Ek-associated pro-cessed 3H-DMc. As for I-Ek, little A214 is seen in this regionof the chromatograph, again indicating that little peptidematerial other than the processed 3H-DMc was eluted fromthe I-Ek molecules and/or generated by the acid treatment.

DISCUSSION

We have shown that the globular protein antigen cytochromec is processed by APCs to a relatively restricted set ofpeptides that bind to the MHC class II molecules. Demotz et

al. (20) reported the isolation of peptides from APCs that hadprocessed ovalbumin. In those experiments the antigen wasnot radiolabeled and peptides were detected in a functionalassay by the ability to stimulate a specific T cell. Thefunctional antigenic peptides were heterogeneous as shownby molecular sieve and reverse-phase chromatography. Byradiolabeling the DMc antigen across its entire length, we areable to view all of the processed antigen fragments that arebound to the class II molecules, not only those that are T-cellantigens in a particular system. At the same time, because theI-Ek proteins from which the peptides are isolated have beenshown to stimulate a specific T cell (11), the processedantigen eluted from the I-Ek must contain functional antigenicpeptide. Given the small number ofpeptide-MHC complexesassembled by the APCs and the limited number of peptidesisolated from the MHC, the stimulatory antigenic peptidesmust be one or both of the two major peaks.Although we observe a somewhat restricted set ofpeptides

eluted from MHC class II molecules, these are far moreheterogeneous than the processed antigenic peptide isolatedfrom the MHC class I molecules by Van Bleek and Nathen-son (21). In that case, class I molecules purified from APCsinfected with vesicular stomatitis virus contained a single8-amino acid antigenic peptide of the nucleocapsid proteinand two smaller peaks that may have been virus-derived. Thedifferences in the patterns of peptide bound to the class IIversus class I molecules may be a reflection of the confor-mation of the two molecules and their specificity for pro-cessed antigen. The MHC class I crystal structure suggesteda binding site that would accommodate at most an 8- to20-amino acid peptide (22, 23). The class II peptide-bindingsite may be more flexible than the class I site and able toaccommodate larger peptides. The mechanisms by whichpeptides are generated and transported to the MHC mole-cules may also dictate the types of peptides to which theMHC molecules are exposed. Proteins encoded in the MHC,related to the ATP-binding-cassette family of membranetransporters, may play a role in transport ofpeptides from thecytoplasm to the endoplasmic reticulum for association withMHC class I molecules (24). Such pores could restrict thesize and heterogeneity of peptides entering the endoplasmicreticulum for binding to the MHC class I proteins. A greatervariety of peptides may be available to the MHC class IIproteins after pinocytosis or endocytosis and proteolysis inendosomal compartments.That the peptides bound to MHC class II molecules are not

very heterogeneous suggests that there are restrictions on thepeptides that stably associate with class II molecules. Arestriction might be imposed by the specificity of the class IImolecule for peptide, as certain binding studies suggest (25).The processing mechanism may also restrict the peptides thatare produced by proteolysis and transported to the class IImolecules. Thus, not all peptides may survive degradation.Those that do may require transport to the MHC class IImicroenvironment, where assembly occurs if these are notone and the same. Transport may be mediated by proteinssuch as the heat shock protein/chaperonin peptide-bindingprotein (PBP72/74) (26-28). The specificity of proteins suchas PBP72/74 for peptides may dictate which peptides can betransported to and bound by the MHC class II molecules.A somewhat surprising observation made here is that the

majority of I-Ek bound to processed DMc migrates moreslowly than the major fraction of I-Ek. The change in mobilitycould reflect the additional molecular weight of the boundpeptide. The high-affinity, heteroclitic T-cell response toprocessed DMc requires a peptide minimally of 20 residues(ref. 5 and unpublished observation). The peptide could belonger, although not all longer peptides of cytochrome c maybe antigenic (3). Alternatively, the binding of more than asingle peptide to a single I-Ek molecule may account for the

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difference in mobility. However, MHC class II protein hasbeen shown to associate in a 1:1 stoichiometry with antigenicpeptides (7, 8), making it unlikely that the shift in mobility isdue entirely to the additional molecular weight of the pep-tides. That only a very small fraction of I-Ek migrates slowlysuggests that peptides which might be bound to I-Ek as aresult of processing of self or serum proteins do not changethe mobility of I-E . Alternatively, the majority of I-Ek in Bcells is not bound to processed antigen. Processed antigenmay bind only to newly synthesized I-Ek that is in a differentconformation than the majority of mature I-Ek molecules.Earlier work indicated that purified MHC class II moleculesare not stable but may form intermediate structures. Indeed,Dornmair et al. (29) described a "compact" form and a"floppy," or slow-migrating, form ofMHC class II molecule,both of which are able to bind synthetic peptides in vitro. Itis possible that the I-Ek to which the processed antigen isbound represents the floppy form. If so, it is likely to befunctionally significant, as nearly all the newly processedantigen is bound to this form and the processed antigen-I-Ekcomplexes function to stimulate T cells.We show here that very few (-800) molecules of I-Ek per

APC bind processed antigen and that this number is sufficientfor T-cell activation. This number is similar to the number ofsynthetic peptide-MHC class II complexes reported to benecessary for T-cell activation (9) and is an indirect estimateofthe number ofprocessed antigen-MHC class II complexesneeded for T-cell activation (10). This small number ofpeptide-MHC class II complexes may be functionally am-plified if they are concentrated on the APC surface in amembrane domain appropriate for T-cell receptor binding.We do not know whether the number of complexes can beincreased by any manipulation ofthe APC or whether a largernumber ofcomplexes would be more effective in stimulatinga T cell. The small number of processed antigen-I-Ek com-plexes formed in the APC is consistent with the observationin vitro that only 0.1-10% ofthe total MHC class II moleculespurified from APCs are capable ofbinding peptides (4, 8). Theremainder of the MHC class II molecules are presumablyeither filled with peptide or in a conformation unable to bindto peptide. At present it is not possible to discriminatebetween these alternatives.

In summary, we have shown that the globular proteinantigen cytochrome c is processed to two predominant pep-tides that bind to 800 I-Ek molecules per APC, which issufficient for T-cell activation. Radiolabeling by reductivemethylation oflysine residues may be applied to other proteinantigens to determine the generality of our findings.

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