Cysteinylation of MHC class II ligands: peptide endocytosis and reduction within APC influences T...

Cysteinylation of MHC Class II Ligands: Peptide Endocytosisand Reduction Within APC Influences T Cell Recognition1

M. Azizul Haque,* John W. Hawes,† and Janice S. Blum2*‡

Peptides bind cell surface MHC class II proteins to yield complexes capable of activating CD41 T cells. By contrast, protein Agsrequire internalization and processing by APC before functional presentation. Here, T cell recognition of a short peptide in thecontext of class II proteins occurred only after delivery of this ligand to mature endosomal/lysosomal compartments within APC.Functional and biochemical studies revealed that a central cysteine within the peptide was cysteinylated, perturbing T cellrecognition of this epitope. Internalization and processing of the modified epitope by APC, was required to restore T cell recog-nition. Peptide cysteinylation and reduction could occur rapidly and reversibly before MHC binding. Cysteinylation did notdisrupt peptide binding to class II molecules, rather the modified peptide displayed an enhanced affinity for MHC at neutral pH.However, once the peptide was bound to class II proteins, oxidation or reduction of cysteine residues was severely limited.Cysteinylation has been shown to radically influence T cell responses to MHC class I ligands. The ability of professional APC toreductively cleave this peptide modification presumably evolved to circumvent a similar problem in MHC class II ligandrecognition. The Journal of Immunology,2001, 166: 4543–4551.

A ntigenic peptides complexed with MHC class II mole-cules are displayed on the surface of APC for recogni-tion by CD41 T cells. The formation and abundance of

these peptide:class II complexes is regulated by peptide source aswell as reactions within APC. Protein Ags must be internalizedinto acidic endosomal and lysosomal compartments for processingto yield the short peptides of 12–25 aa that optimally bind MHCclass II proteins (1–4). These peptides generated within APC canintersect and bind class II proteins throughout the endosomal path-way (5–9). However, peptide association with newly synthesizedclass II molecules may be most favored in the late endosomal/prelysosomal compartment termed MIIC (6, 7, 9–11). Here, theexchange factor HLA-DM catalyzes the release of invariant chainfragments from class II molecules and exposes the ligand bindinggroove (12–15). Peptides complexed with class II proteins withinthis compartment are then shuttled to the cell surface (1, 16).

By contrast, antigenic peptides can also be generated outsideAPC for example, upon lysis of tumor or virally infected cells (17,18), or as a result of Ag processing by extracellular proteases dur-ing inflammatory or autoimmune responses (19, 20). These pep-tides may be acquired and presented via MHC molecules on by-stander APC for T cell recognition (21, 22). Synthetic peptideshave also been used as vaccine reagents, following their incubationwith potent APC, such as dendritic cells (23–25). Studies usingsynthetic or chemically generated short peptides had suggestedthat these exogenous ligands bind directly to available cell surface

class II proteins yielding complexes for T cell recognition (26–29).Yet, several reports have also indicated that T cell responses toselect peptides required their presentation by viable APC (29–33),raising the question of whether ligand binding to surface MHCalone is sufficient for T cell activation. Among several of the pep-tides requiring presentation by viable APC, a shared common fea-ture that emerges is the presence of one or more cysteine residues(29, 30, 34). Here through studies of a cysteine-containing peptidefrom the Ag human Igk, we have demonstrated that cysteine mod-ification can regulate T cell responses to class II-restrictedepitopes. Although cysteinylation of ligands for MHC class I hasbeen reported previously (35–37), this marks the first demonstra-tion that such a modification can alter class II-restricted T cellresponses. Furthermore, these studies demonstrate that endocytosisand processing of the cysteinylated peptide by viable APC wasnecessary to restore antigenicity. Although the importance of re-duction in processing native Ags has been appreciated (30, 38, 39),here a role for reduction in the functional presentation of peptideligands by APC has also been clearly demonstrated.

Materials and MethodsCell lines

APC were cultured in Iscove’s complete DMEM with 10% heat-inacti-vated calf serum, 50 U/ml penicillin, and 50mg/ml streptomycin. TheB-lymphoblastoid cell Frev expresses endogenous class II DR4w4(DRB1*0401) and DR1 (DRB1*0101) alleles as well as Igl light chains.The human monocyte cell THP-1.DR4 and the murine dendritic cell FS-DC.DR4 were transduced using retroviral vectors for constitutive expres-sion of HLA-DR4 (DRB1*0401) with linked drug selection markers forhygromycin and G418 resistance (40). Expression of surface DR4 com-plexes on cells was confirmed by cytofluorography using the DR4-specificmAb, 359F10 (41). T cell hybridomas specific for Igk peptides presentedin the context of HLA-DR4, were generated by immunization of DR4w4-transgenic mice with human IgG. The hybridoma line 2.18 recognizes pep-tides encompassing Igk residues 188–203 while the cell 1.21 responds toIg k residues 145–159 (42). T cell hybridomas and HT-2 cells were cul-tured in RPMI 1640 with 10% FBS, 50 U/ml penicillin, 50mg/ml strep-tomycin, and 50mM 2-ME. For HT-2 cells, 20% Con A supernatant (T-STIM; Collaborative Biomedical Products, Bedford, MA) was also added.

Departments of *Microbiology and Immunology and†Biochemistry and MolecularBiology, and ‡Walther Oncology Center, Indiana University School of Medicine,Indianapolis, IN 46202

Received for publication October 5, 2000. Accepted for publication January 22, 2001.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported with funds from the National Institutes of Health-NationalInstitute of Allergy and Infectious Diseases and National Institute of Diabetes andDigestive Kidney Diseases (to J.S.B.). M.A.H. was supported by National Institutesof Health Training Grant T32 DK07519.2 Address correspondence and reprint requests to Dr. Janice S. Blum, 635 BarnhillDrive, MS 255, Indianapolis, IN 46202. E-mail address: [email protected]

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

Peptides

The human IgG immunodominant (kI) peptidek188–203 (sequence KHKVYACEVTHQGLSS) and subdominant (kII) peptide k145–159 (se-quence KVQWKVDNALQSGNS) were produced using Fmoc technologyand an Applied Biosystems synthesizer (Foster City, CA). Peptide purity(.99%) and sequence were analyzed by reverse-phase HPLC purificationand mass spectroscopy. Peptides were labeled as indicated at thea aminotermini by the sequential addition of two molecules of Fmoc-6-aminohex-anoic acid followed by a single biotin to yield the sequence biotin-amin-ohexanoic acid-aminohexanoic acid-peptide. Mass spectrometry confirmedthat the peptide was tagged with a single biotin molecule at the N terminus.Preparation of purified cysteinylatedkI was achieved by peptide incubation(3 h at 37°C in HBSS) with cystine (0.29 mM) followed by dialysis (1000Da membrane cutoff) to remove residual cystine/cysteine. Peptide cystei-nylated was maintained at neutral or acidic pH in the absence of reductants.Substituted forms of thekI peptide were also generated by Fmoc technol-ogy with Ala, Ser, or 2-aminobutyric acid (aba)3 replacing Cys194. Peptideswere dissolved at 1 mM in either DMSO (Sigma, St. Louis, MO) or PBS,and stored at220°C until use.

T cell proliferation assays

APC were incubated with synthetick peptides for 3–24 h at 37°C in culturemedium, washed, and cocultured with T cell hybridomas for 24 h. T cellcytokine production was monitored by measuring [3H]thymidine (1mCi/well) incorporation using the IL-2/IL-4-dependent cell line, HT-2. In somecases, APC were prefixed with 1% paraformaldehyde for 8 min on icefollowed by extensive washing and peptide addition, or postfixed beforecoculture with T cell hybridomas. When THP-1.DR4 cells were used asAPC, these cells were first stimulated with 50 U/ml IFN-g (R&D Systems,Minneapolis, MN) for 48 h before the addition of peptides. APC were alsoincubated withk peptides at 18°C for 24 h, fixed, and cocultured withk-specific T cell hybridomas for 24 h. Assays were also performed usingAPC treated with DTT (Sigma), L-cysteine, or L-cystine, andk peptidesbefore or after aldehyde fixation.

For inhibition studies, APC were pretreated with inhibitors such asNaN3/deoxyglucose (2 mg/ml, 50 mM, respectively), 60mM colchicine, or100mM primaquine (Sigma) in complete medium for 30 min followed bythe addition of synthetic peptides. Cells were subsequently washed twice inPBS and fixed with 1% paraformaldehyde before cultivation with T cells.All assays were repeated at least three to four times with the SE for trip-licate samples within a single experiment reported. Data were corrected forisotope counting efficiency and expressed as corrected cpm (ccpm).

Peptide binding assays

Paraformaldehyde-fixed Frev cells were incubated overnight with biotin-ylated k peptides (kI and kII) in 150 mM citrate-phosphate buffer, (pH5.5–7.4), HBSS, or Iscoves’s DMEM medium (pH 7.4) with heat-inacti-vated serum, washed with PBS, and lysed on ice for 20 min with 50 mMTris buffer (pH 8) containing 0.15 M NaCl and 0.5% IGEPAL-CA 630(Sigma) as described (43). The lysate was centrifuged to remove intactnuclei, and the supernatant was added to plates (Costar, Cambridge, MA)previously coated overnight with either the anti-human MHC II Ab 37.1(kindly provided by L. Wicker (Merck Research Laboratories, Rahway,NJ) or anti-DR4 359F10 (41). The captured class II-peptide complexeswere detected with europium-labeled streptavidin (Pharmacia, Piscataway,NJ) using a fluorescence plate reader (Delfia; Wallac, Turku, Finland).Peptide binding to MHC was consistently lower in medium plus heat-inactivated serum compared with buffered solutions. This is presumablydue to competing serum peptides, as no proteolysis of thek peptide wasdetected. The relative affinity of the Cys-substitutedk peptides and HA-flupeptide was also measured in a competitive binding assay as described(42). The number of total DR molecules within APC was quantitated usingbiotinylated L243 and the capture Ab 37.1 as described (42). In all exper-iments, drug treatment of cells did not diminish the total amount of cellularHLA-DR as detected using this assay.

Liquid chromatography-mass spectroscopy

PeptidekI was analyzed by capillary liquid chromatography using an Ap-plied Biosystems 140D solvent delivery system. Samples were applieddirectly to 300-mm diameter fused silica capillaries packed with VydacC18 resin and separated with gradients of buffer A (2% acetonitrile and98% H2O containing 0.2% isopropanol, 0.1% acetic acid, and 0.001% tri-

fluoroacetic acid) and buffer B (95% acetonitrile and 5% H2O containing0.2% isopropanol, 0.1% acetic acid, and 0.001% trifluoroacetic acid). Pep-tide was eluted at a flow rate of 7ml/min directly into the electrosprayionization source of a Finnigan LCQ mass spectrometer. Nitrogen was usedas the sheath gas with a pressure of 35 psi with no auxiliary gas. Electro-spray ionization was conducted with a spray voltage of 4.8 kV, a capillaryvoltage of 26 V, and a capillary temperature of 200°C. Spectra werescanned over am/zrange of 200-2000. Base peak ions were trapped usingthe quadruple ion trap and further analyzed with a high resolution scan(zoom-scan) using an isolation width of 3m/zand collision-induced dis-sociation scans with a collision energy of 40.0.

ResultsMetabolically active APC are required for the functionalpresentation of a synthetic peptide in the context of MHC classII proteins

Exogenous protein Ags are internalized and processed by viableAPC, yielding epitopes that bind intracellular class II histocom-patibility Ags before surface expression and T cell engagement. Incontrast, short synthetic peptides bind directly to MHC class IImolecules on the surface of APC, and typically trigger T cell ac-tivation without a requirement for APC metabolic activity (27,44–46). During studies of the processing of an autoantigen, humanIgG class II-restricted epitopes were identified in viable HLA-DR41 APC using functional and biochemical approaches (42).Yet, DR4-restricted T cell activation via a synthetic analog of animmunodominant peptide, IgkI (residues 188–203) was detectedonly using viable and not prefixed APC (Fig. 1). By contrast, T cellhybridomas specific for another IgkII epitope (residues 145–159)were able to recognize synthetic forms of this peptide displayed on

3 Abbreviations used in this paper: aba, 2-aminobutyric acid; ccpm, corrected cpm;GILT, IFN-g-inducible lysosomal thiol reductase;m/z, mass to charge ratio.

FIGURE 1. HLA class II-restricted presentation of synthetick peptidesby the B cell Frev (A andB), IFN-g-activated macrophage THP-1.DR4 (C),and dendritic cell FSDC.DR4 (D). Live (E) or chemically fixed (F) APCwere incubated in complete medium with eitherkI or kII synthetic peptidesfor 24 h at 37°C, washed, and cocultured with the appropriatek peptide-specific T cell hybridomas (2.18a cell specific for DR4:kI and 1.21 cellspecific for DR4:kII) for 24 h. B, Frev cells were incubated with thekIpeptide with or without subsequent aldehyde fixation followed by coculturewith T cells. Alternatively, these cells were prefixed and then incubatedwith kI (fxAPC 1 kI) before coculture with T cells without or with ex-ternal costimulation via cross-linked anti-CD28 (fxAPC1 kI 1 cost.). Tcell production of IL-2 was determined by measuring proliferation of HT-2cells using [3H]thymidine incorporation.

4544 CYSTEINYLATION OF MHC CLASS II LIGANDS

both aldehyde-fixed and live APC. Experiments with DR41 hu-man and murine B cell lines, macrophages, and dendritic cellsdemonstrated that regardless of cell lineage, functional T cell rec-ognition of thekI synthetic peptide was observed only with met-abolically active APC (Fig. 1,A, C, andD). Aldehyde-fixation hasbeen shown in some instances to perturb APC-T cell interactions,by disruption of costimulatory and adhesion molecules on APC(32). Yet, the T cell hybridomas used in this study have minimalrequirements for costimulation and can detect isolated complexesof peptide-loaded class II molecules (42). Two additional lines ofexperimental evidence prove that the failure of fixed cells to func-tionally display thekI peptide was not linked to a defect in co-stimulation or cell adhesion. First, even in the presence of a strongcostimulatory signal delivered via Ab cross-linking of CD28, Tcells failed to respond to fixed APC and thekI peptide (Fig. 1B).The effectiveness of this exogenous costimulation was confirmedusing THP-1.DR4 cells and thekII peptide. T cell responses to thispeptide plus APC could be enhanced in the presence of cross-linking of CD28 (data not shown). Also, preincubation of thekIpeptide with viable APC (for 24 h) before cell fixation, resulted inefficient presentation and T cell responses to this epitope (Fig. 1B).Together, these results indicate that formation of functional com-plexes of class II molecules with thekI peptide, was dependentupon the metabolic activity of APC. In this respect the 16 aakIepitope behaves more like native protein Ags, potentially requiringprocessing before efficient display via class II molecules.

Endocytosis is required for functional presentation ofkI peptideby B cells

To investigate the role of endocytic transport in class II-restrictedpresentation of thekI peptide, APC were incubated with metabolicinhibitors that block the internalization of molecules via coatedpits into the endosomal pathway. Treatment of APC with sodiumazide and 2-deoxyglucose, substantially reduced the ability ofthese cells to present the synthetickI peptide to T cells (Fig. 2A).Yet, presentation of thekII peptide was unaffected by these inhib-itors of cellular ATP production. As expected, control studies re-vealed that these metabolic inhibitors blocked the processing andpresentation of native Ags, such as IgG (data not shown). Theseresults suggest that endocytic transport is important in the func-tional presentation of thekI peptide, while thekII epitope directly

binds cell surface class II molecules and mediates T cell activation.To distinguish whether the requirement for endocytosis was linkeddirectly to thekI peptide and/or class II molecules themselves,APC were treated with the drug primiquine, which blocks endo-cytic recycling of transmembrane proteins such as class II mole-cules and the transferrin receptor (47, 48). Earlier, we reported thatthe drug primiquine could block the presentation of a peptide thatintersected class II molecules in recycling early endosomes (33).Functional presentation of thekI peptide was not perturbed usingprimiquine-treated APC, demonstrating that recycling class II mol-ecules are not required for display of thekI peptide (Fig. 2A).Rather, these results indicate that thekI peptide must be trans-ported into the endocytic pathway for DR4-restricted presentationand T cell activation. Further proof for peptide internalizationcame from studies in which endocytic transport was blocked be-tween early and late endosomal compartments. Incubation of APCat 18°C has been shown to halt the delivery of Ags from early tolate endosomes (49–51). Thus, presentation of thek peptides wasexamined in DR41 B cells incubated at 18°C and 37°C (Fig. 2B).T cell recognition of thekI peptide was completely ablated usingAPC incubated with this peptide at low temperature. In contrast,only a slight reduction in the efficiency ofkII peptide display wasobserved with DR41 APC at 18°C compared with 37°C. In eachexperiment, APC were incubated at the indicated temperatureswith peptides followed by aldehyde-fixation before coculture withT cells at 37°C. Endocytic vesicles associate with microtubuleswithin mammalian cells, and transport of molecules from earlyendosomes to late endosomes/lysosomes can be perturbed via mi-crotubule depolymerization using drugs such as colchicine (52).Treatment of APC with colchicine inhibited in part, functionalpresentation of thekI peptide to T cells (Fig. 2A). Class II pre-sentation of thekII peptide was not altered by this drug, confirm-ing that surface class II expression was not grossly altered by mi-crotubule depolymerization. In sum, these studies strongly suggesta requirement for peptide internalization and transport beyondearly endosomes before functional display in the context of MHCclass II molecules.

Synthetick peptides bind MHC class II proteins at both acidicand neutral pH

Epitopes released during Ag processing preferentially bind class IImolecules within acidic endosomal compartments with the aid ofchaperones such as HLA-DM. Although direct binding of syn-thetic peptides to MHC class II proteins can occur at neutral pH onthe cell surface, the efficiency of this reaction varies among pep-tides with low pH enhancing the binding of many epitopes to MHCclass II Ags (53–55). Thus, the requirement for endocytosis beforefunctional presentation of thekI peptide in the context of HLA-DR4, might reflect a failure of this peptide to bind class II mole-cules at neutral pH. Studies to assess direct binding ofkI and kIIpeptides to DR4 were conducted over a broad pH range usingaldehyde-fixed APC and Ab capture of the resulting class II-pep-tide complexes (Fig. 3). Although thekI peptide clearly showedpreferential binding to class II molecules at acidic pH values foundwithin late endosomes/prelysosomes (i.e., pH 5.5), measurable as-sociation of this peptide with DR4 was detected at neutral pH. Tcell responses were also enhanced following loading of the nativepeptide in low pH buffer solutions onto APC. At pH values be-tween 7.4 and 6.3, the level of class II DR4 binding for bothkI andkII peptides was comparable. For these experiments, biotin-taggedkI andkII peptides were used to directly monitor binding to classII proteins. Functional studies not shown confirmed that additionof an amino-terminal biotin did not alter the presentation of eitherk peptide. Thus, T cell responses to the biotinylatedkI peptide

FIGURE 2. Requirement for internalization and processing of thekIpeptide by APC. Frev were pretreated with either NaN3/deoxyglucose, col-chicine, primaquine, or PBS as a control (A) for 30 min at 37°C, followedby addition of synthetick peptides for 3 h plus or minus these inhibitors.APC were then washed three times, fixed, and cocultured with T cell hy-bridomas. Alternatively, inB, Frev (13 106/ml) were incubated with syn-thetic k peptides at 18°C or 37°C for 24 h, fixed with paraformaldehyde,and cocultured with T cell hybridomas. T cell IL-2 activity was measuredusing HT-2 cells.A, Data are expressed as relative percent peptide presen-tation based upon a maximal response of 149,117 ccpm for 2.18a and159,881 ccpm for 1.21 T cells, respectively.

4545The Journal of Immunology

were only detected in the context of viable APC. Binding of theunmodifiedk peptides to surface class II molecules over a broadrange of pH values, was also detected in competition assays withbiotin-tagged flu hemagglutinin 307–319 peptide (data not shown).Thus, the data indicate that each synthetick peptide was capableof directly binding to cell surface HLA-DR4. Still, T cell recog-nition of thekI peptide was dependent upon endocytic transport ofthis epitope and potentially, processing in viable APC.

Reduction of cysteine residues within a synthetic peptide isessential for T cell recognition

Although proteolytic cleavage is important in Ag processing, de-naturation of proteins by acidic pH and disulfide reduction alsoplay key roles in the class II-restricted presentation of select Ags(30, 31, 56). Experiments using a broad panel of protease andpeptidase inhibitors failed to block the ability of viable APC toconvert the synthetickI peptide to a functional epitope (data notshown), prompting a search for alternate modifications that mightinfluence T cell recognition of this peptide. Studies of class I-re-stricted viral and tumor epitopes have revealed modifications ofcysteine residues, which alter CD81 T cell responses (35, 36). ThekI peptide (KHKVYACEVTHQGLSS) contains a central cysteineresidue, potentially susceptible to disulfide linkage or oxidation.To investigate whether disulfide formation influenced peptide an-tigenicity, thekI peptide was incubated with APC in culture me-dium containing a reductant (DTT) followed by analysis of T cellactivation (Fig. 4). The addition of a reducing agent restored pre-sentation of thekI peptide by class II DR4 complexes on aldehyde-fixed APC, as well as enhancing peptide display by viable cells.Similar results were obtained by preincubation of purifiedkI withDTT in medium before removal of the reductant, and peptide ad-dition to APC resuspended in HBSS (data not shown). By contrastwith the 10- to 20-fold increase inkI peptide presentation observedwith fixed cells plus reductant (Fig. 4B), presentation of thekIIpeptide remained unchanged suggesting DTT treatment did notalter class II Ag function in general. To exclude the possibility thatDTT enhanced class II binding ofkI, studies of peptide bindingwere conducted in the absence and presence of reductant. Remark-ably, binding of the biotin-labeledkI peptide to class II molecules

was diminished upon exposure of cells to culture medium contain-ing DTT (Fig. 4C). Yet, no change inkII association with DR4could be detected in the presence of reductants (Fig. 4D). Peptidebinding in general is diminished in culture medium compared withbuffered salt solutions; however, inclusion of the reductant DTTaltered only the association of class II proteins with cysteine-con-taining peptides such askI. Levels of surface class II proteins onAPC were unchanged following exposure to DTT as determinedusing an ELISA (data not shown). Together, these results suggestthat reduction of thekI peptide is a key processing step essentialto functional epitope presentation and T cell recognition.

Role of cystine in the modification of synthetic peptides

Mass spectral analysis of the synthetickI peptide dissolved inHBSS, revealed a single monomeric species (molecular mass,1785.7) with residue Cys194existing as a free sulfhydryl (Fig. 5A).However, upon incubation of the peptide in tissue culture medium(with or without serum), greater than 80% of the peptide was mod-ified by a cysteine adduct yielding the 1905.6 molecular mass cys-teinylated species (Fig. 5B). Mass spectral analysis failed to revealany significant amounts of dimeric peptide as might be predictedbased upon disulfide formation. Trace amounts of am/z 601.3species were detected by mass analysis, potentially representingthe sulfenic acid form of the peptide. Peptide sequence and thepresence of the modified Cys was confirmed by collision-induceddissociation. Other modifications such as peptide-glutathione con-jugation were not detected upon incubation of thekI peptide withmedium.

Cysteinylation of class I epitopes has been documented (35),arising potentially from posttranslational epitope modification orvia reaction with cystine in serum or tissue culture medium. To test

FIGURE 3. Synthetick peptide binding to HLA-DR4 was not affectedby paraformaldehyde fixation of APC. Paraformaldehyde-fixed Frev wereincubated overnight with varying concentrations of biotinylatedk peptides(0–10mM) at different pH (5.5–7.4) values in citrate-phosphate buffer at37°C. APC were then washed and lysed, and the extent of binding ofbiotinylatedk peptides to surface DR4 was examined in a capture ELISAusing europium-labeled streptavidin.A, kI; B, kII. Binding of thekI pep-tide was optimal at pH 5.5, yet measurable peptide:MHC association couldbe detected even at neutral pH. Maximal binding of thekII peptide to DR4was detected at pH 7.4 and pH 5.5 in repeated experiments. Data are rep-resentative of mean fluorescence6 SEM for at least three separateexperiments.

FIGURE 4. Reduction of thekI peptide restores presentation of thisepitope by fixed APC. Metabolically active (A) or paraformaldehyde-fixed(B) Frev cells were incubated in culture medium withk peptides (5–20mM) 6 the reducing agent, DTT (200mM) for 3 h at37°C. APC were thenwashed and cocultured with the appropriate T cell hybridomas for 24 h.The level of T cell IL-2 production was measured as described above.Binding of thekI peptide (C) or kII peptide (D) to class II DR4 in culturemedium (IMDM plus heat-inactivated calf-serum)6 DTT. The associationof biotin-labeled peptides with class II molecules was detected via fluo-rescence of europium-tagged strepavidin as indicated inMaterials andMethods.


whether cystine was responsible for modification and inactivationof the kI peptide, fixed APC were incubated with this peptide inbuffer plus or minus 0.29 mM cystine, the concentration found inIMDM tissue culture medium. Remarkably in the absence of cys-tine or tissue culture medium, efficient presentation of thekI pep-tide was observed with fixed APC (Fig. 6A). Incubation of thekIpeptide in buffer plus cysteine minimally inactivated peptide pre-sentation by fixed APC. However, following APC incubation withthekI peptide in HBSS plus cystine peptide presentation was com-pletely blocked, confirming that cystine was responsible for pep-tide modification and loss of function. Incubation of live or fixedAPC with thekII peptide in buffer with cystine had no effect on Tcell responses to this epitope, which lacks a central cysteineresidue.

In direct contrast with functional assays,kI peptide binding toMHC class II proteins at neutral pH was enhanced by cysteinyla-tion (Fig. 7A). Binding of the biotin-labeledkI peptide to class IIDR4 in the presence of cystine at pH 7.4 was enhanced up to 5-foldcompared with the unmodified peptide. A similar increase in pep-tide binding was observed using cysteinylatedkI peptide (.95%modified) purified to remove residual cystine/cysteine before in-cubation with MHC Ags. Cystine did not alter the general peptidebinding properties of class II molecules as no change was detected

in the association of thekII peptide with DR4 molecules plus orminus cystine (Fig. 7B). Under conditions of low pH mimickingthe environment within mature endosomes, binding of thekI pep-tide to class II molecules was minimally effected by cysteinylation(Fig. 7C). Because cysteinylation is less efficient at low pH, forthese studies thek peptides were pretreated with cystine at neutralpH and dialyzed, and greater than 95% cysteinylation was con-firmed by mass spectroscopy before incubation with MHC underacidic pH conditions. Association of thekII peptide with MHC atlow pH was unaltered by cystine pretreatment (Fig. 7D), againconfirming an overall lack of change in class II structure underthese incubation conditions. Thus while the cysteinylatedkI pep-tide failed to activate T cells, modification of this peptide promotedinteractions with class II molecules under select conditions.

Studies were conducted to further investigate the mechanism ofcysteine modification and specifically, the role of MHC class IImolecules in this process. Although cysteinylation and inactivationof the freekI peptide was efficient in solution (Fig. 5), preformedcomplexes ofkI and class II DR were moderately resistant to mod-ification by cystine (Fig. 6B). In this experiment synthetickI pep-tide was bound to surface class II molecules on aldehyde-fixedAPC in HBSS, followed by a postincubation in the presence orabsence of cystine. T cell activation was only reduced by 20–30%,

FIGURE 5. Mass spectroscopic analy-sis of synthetickI peptide revealed cystei-nylation of this peptide in culture medium.The kI peptide incubated 3 h inHBSS (A)or culture medium (B) and spectra ana-lyzed following capillary liquid chroma-tography using an Applied Biosystems 140solvent delivery system as described inMaterials and Methods. Analysis of b ionsubfragmentation by collision-induced dis-sociation confirmed peptide cysteinylation.The kI peptide fragmentation profile inHBSS was consistent with the calculatedmolecular mass of 1785.7 and the presenceof reduced cysteine at position 194 (notshown). Fragmentation of the peptide afterincubation in tissue culture medium with-out or with serum, indicated a mass of1905.6 consistent with cysteinylation atresidue 194 (not shown).


suggesting that cysteinylation of the peptide bound to class II mol-ecules could occur but with markedly less efficiency. These resultssuggest that once bound to class II molecules, the peptide’s centralcysteine residue may be somewhat shielded from modification.This result also explains why peptide prebound to class II mole-cules on fixed APC in the absence of cystine was not inactivatedupon later coculture in complete medium with T cells. The inclu-sion of reductants during peptide incubation with fixed APC inculture medium, prevented or rapidly reversed peptide cysteinyla-tion (Fig. 4). Reduction of the free peptide before binding to classII molecules in HBSS also enhanced T cell recognition (Fig. 6C).This effect was reversed with nearly complete loss of peptide ac-tivity when the reduced peptide was added to APC in cystine-containing medium. Thus, demonstrating that the reduced peptidewas highly susceptible to cysteinylation unless a reducing agentsuch as DTT was continually present. Further studies revealed thatonce bound to class II DR, the cysteinylated peptide could not bereduced as measured by restoration of functional T cell recognition(Fig. 6D). Here, peptide binding to class II DR4 was conducted incystine-containing medium to promote cysteinylation, followed bya postincubation in medium with or without reductant. T cell rec-ognition of the peptide:class II molecules was minimal with onlya very slight enhancement upon treatment of these complexes withDTT. This result is important with potential relevance to reductivepeptide processing in vivo, as the findings suggest that reduction ofthe cysteinylatedkI peptide occurs before binding to class II mol-ecules. Thus for the freekI epitope, peptide cysteinylation andreduction can occur efficiently and reversibly. By contrast, peptidebinding to the groove of MHC class II molecules severely limitsthe accessibility of this central cysteine residue despite its potentialrole in T cell recognition.

Substitution of cysteine residues enhances peptide presentation

To further demonstrate that modification of cysteine residues leadsto the requirement forkI peptide processing by APC before func-

tional presentation, the properties of analogkI peptides with con-servative substitutions of serine, alanine, or aba for Cys194 weretested (Fig. 8). Incubation of live or fixed APC with thekI analogcontaining serine substituted for cysteine, failed to elicit any T cell

FIGURE 6. Preferential cysteinylation and reductionof kI before binding MHC class II Ags.A, Cysteinyla-tion of kI peptide was favored in the presence of cystinevs cysteine. Aldehyde-fixed Frev cells were incubatedwith kI in HBSS, HBSS1 cysteine (0.29 mM), orHBSS1 cystine (0.29 mM) for 3 h, followed by wash-ing and coculture with T cells for 24 h.B, Binding of kIpeptide to class II DR4 prevents cysteinylation. PrefixedAPC were incubated withkI in HBSS for 3 h, washedand further incubated in either HBSS or HBSS1 cys-tine (0.29 mM) for 3 h. APC were then washed andcocultured with T cells.C, Cysteinylation of reducedkIpeptide upon postincubation in IMDM. ThekI peptidewas preincubated in HBSS or HBSS1 DTT for 3 h.Aldehyde-fixed APC were incubated with the peptide ineither HBSS or IMDM culture medium for 3 h, washed,and cocultured with T cells.D, CysteinylatedkI peptidebound to class II DR was resistant to reduction. PrefixedAPC were incubated withkI in IMDM for 3 h, washed,and further incubated with 200mM DTT or IMDMalone for 3 h. Cells were then washed and coculturedwith T cells. In each group (A–D), T cell activation wasassessed by cytokine production as quantitated by HT-2cell proliferation. The range of peptide concentrationstested, is indicated in the figure.

FIGURE 7. Cysteinylation influenceskI binding to class II DR4 depen-dent upon the environmental pH.A and B, Paraformaldehyde-fixed Frevcells were incubated overnight with varying concentrations of biotinylatedk peptides (0–5mM) in HBSS (pH 7.4)6 100mM cystine.C andD, Cellswere also incubated in citrate phosphate buffer (CPB; pH 5.5) with biotin-labeledk peptides that had been pretreated with cystine (100mM) at neu-tral pH to promote modification as described inMaterials and Methods.APC were washed after incubation with peptides and lysed, and the extentof peptide binding was assessed using a capture ELISA and europium-labeled streptavidin.A andC, kI peptide:DR4 binding;B andD, kII peptidebinding to DR4. Data are representative of mean fluorescence6 SEM forat least three separate experiments.


response (Fig. 8,A andB). In contrast, functional presentation ofan alanine-substituted form of thekI peptide could be detectedusing fixed APC (Fig. 8B). T cell responses to this alanine analogwere reduced compared with the originalkI peptide in studies withlive APC (Fig. 8A). Substitution of aba, which more closely ap-proximates cysteine in size, led to nearly equivalent functionalpresentation of this analog by live or fixed APC (Fig. 8,A andB).Each of the substituted peptides was tested alongside the originalkI epitope in a competitive binding assay with class II DR4. Re-sults indicated that peptides substituted with serine or aba at po-sition 194 bound MHC comparable to thekI epitope (data notshown). Binding of the alanine-substituted peptide was reducedcompared with the originalkI peptide by;2-fold. To further ad-dress the requirement for processing using these analog peptides,APC were incubated at 18°C with the aba-substituted form ofkI.In contrast with the cysteine-containingkI peptide, which requiredendocytic transport and processing at temperatures above 18°C,the abakI peptide was presented equally well at low or high tem-peratures (Fig. 8C). These results demonstrate that intracellularreduction of the cysteinylatedkI peptide, was the key processingstep required for functional presentation of this epitope to T cells.

DiscussionThe presence of accessible cell surface class II molecules on APCfacilitates peptide loading, and offers an efficient means of deliv-ering peptide vaccines for immunoregulation (23, 24, 57, 58). Pro-tein Ags by contrast, typically require endocytosis and processingwithin APC before MHC binding and T cell activation. Here, arequirement for peptide endocytosis and reductive processing wasestablished, as a result of the spontaneous modification of an es-sential cysteine within an MHC class II peptide ligand. Binding ofthe cysteine-containing peptidekI to cell surface class II DR wasreadily demonstrated, yet the resulting complexes failed to activateT cells suggesting the need for peptide endocytosis and processing.Indeed, treatment of APC with inhibitors of endocytic transport ormetabolic activity, blocked functional presentation of thekIepitope but did not alter the class II-restricted display of anotherepitope,kII, which lacks cysteine. Therefore, thekII peptide fol-lows the conventional or established pathway, with functional dis-play of this epitope upon direct association with surface class IImolecules (59). Studies with primaquine demonstrated that endo-somal transit of thekI peptide was essential rather than endocyticrecycling of class II molecules. Incubation of APC at low temper-atures or in the presence of drugs that impaired transit to late ormature endo/lysosomal compartments also prevented the process-ing and functional presentation of thekI peptide. A requirementfor peptide reduction before T cell recognition was documented,while no evidence of proteolytic processing was observed. Thus Tcell recognition of the peptide presented by aldehyde-fixed APC,was detected following incubation of APC in medium containing

reducing agents. Mass spectroscopy revealed that cysteinylation ofthe peptide spontaneously occurred upon incubation in the pres-ence of physiological concentrations of cystine found in tissue cul-ture medium or serum. Experiments with peptide analogs furtherdemonstrated that the requirement for endocytosis and processingbefore functional class II-restricted presentation was linked to themodification of a reactive cysteine within the peptide.

Biochemical and functional studies of MHC class I-restrictedepitopes derived from tumors (60), viruses (35), and H-Y minorAgs (61) have established that cysteine residues within theseepitopes are modified in vivo and in vitro thus influencing T cellrecognition. Although dimerization and oxidation of these cys-teine-containing peptides was detected, by far the most commonmodification was peptide cysteinylation. In vivo cysteinylation ofpeptides is highly likely due to the high circulating levels of cys-tine in serum (0.1 mM) and the reactivity of free sulfhydryls (36,62). The importance of cysteinylation in regulating class II-re-stricted T cell activation, has not been investigated despite elegantstudies pointing to the role of disulfide reduction and cysteine res-idues in Ag unfolding, processing, and class II presentation (29,30, 39, 42, 63, 64). Previous studies had noted that several shortpeptides or small Ags containing cysteine residues were presentedonly by metabolically active APC, strongly suggesting a require-ment for processing (29–31, 65). Inactivation of these epitopeswas attributed to peptide oxidation or conjugation with unknownserum factors (29, 30, 63, 65), although direct analysis of epitopestructure was not performed. The present study offers biochemicalevidence that cysteinylation of class II epitopes can occur sponta-neously and with a high efficiency in medium or serum, thus al-tering T cell recognition of these ligands. Significant amounts ofoxidized, dimerized, or conjugated peptide were not detected,strongly suggesting cysteinylation was the predominant modifica-tion of kI similar to MHC class I peptide ligands. Mass spectralanalysis of an epitope from hen egg lysozyme (residues 74–88with Cys80 substituted to ala, NLCNIPASALLSSDI) also revealednearly complete cysteinylation at position 76 in culture medium(data not shown). Presentation of this peptide in the context ofI-Ab was observed only with viable and not fixed APC in line withprevious functional studies (29). Cysteinylation of thekI peptidealso ablated the functional presentation of this epitope to DR1-restricted T cells (data not shown). Whether cysteinylation influ-ences the processing and presentation of antigenic proteins re-mains unclear, yet an increasing number of cysteinylated proteinshave been detected with the advent of sequence analysis by massspectroscopy (66–69). Remarkably, cysteinylation of thekI pep-tide interfered with T cell recognition but did not diminish class IIbinding. In fact, at neutral pH the binding ofkI peptide to DR4 wasactually enhanced due to cysteinylation. These findings fit wellwith our previous prediction based on algorithms that the minimalkI binding epitope for DR4 encompassed residues 191–200 (42).

FIGURE 8. Presentation of cysteine-substi-tutedkI peptides by B cells. Live (A) or fixed(B) Frev cells were incubated 24 h at 37°C withvariants of thekI peptide substituted with Ala,Ser, or aba at position 194, followed by cocul-ture with thekI peptide-specific T cell hybrid-oma for 24 h.C, Frev cells were also incubatedat 18°C with eitherkI or aba-kI for 24 h,washed, fixed, and cocultured with thekI-spe-cific T cell hybridoma. T cell IL-2 activity wasmeasured using the HT-2 cell line. The range ofpeptide concentrations tested, is indicated in thefigures.


Published studies defining the ligand binding motif for HLA-DR4indicated a preference for hydrophobic primary anchor residues atP1 and P6 as well as secondary anchors potentially at P4, P7, andP9 (70). Based upon the predicted alignment for thekI epitope,both P1 and P6 would be valine residues with a weaker secondaryanchor at P4, the site of cysteinylation. Indeed our binding studieswith the unmodified, cysteinylated, and analog peptides suggestthat changes in P4 anchor can influencekI peptide binding to classII DR4. Thus we would predict that cysteine residue within thekIpeptide serves as a contact for MHC as well as TCR. Similarly,studies of class I ligands indicate that epitope cysteinylation canalter either or both TCR contact and binding to MHC proteins (35,36, 71). Depending upon the position and number of cysteineresidues, cysteinylation may influence epitope association withclass II molecules potentially explaining the lack of MHC bindingreported for cysteine-rich insulin (30) and hen egg lysozyme (29)epitopes. Studies of MHC class I ligands have also suggested thatserine or alanine residues can sometimes replace cysteine, avoid-ing cysteinylation and peptide inactivation (35, 71, 61). Serinereplacements of cysteine residues within class II peptide ligands,however, have proven less predictable in restoring functionalactivity (Fig. 6; Ref. 29). Rather, the amino-acid analog aba testedin this study may prove to be a more reliable substitute in epitopedesign.

Evidence provided here suggests that professional APC includ-ing B cells, macrophages, and dendritic cells could efficiently con-vert the cysteinylatedkI peptide to its active form for class II-restricted T cell presentation. This may prove to be a keydifference between class I and class II pathways for Ag presenta-tion, as to date reductive processing of cysteinylated ligands beforeclass I presentation has not been reported. Studies of nonprofes-sional APC including tumors have revealed minimal capacity forAg reduction (39, 72), potentially suggesting that cysteinylatedpeptides may be more abundantly displayed by class I and class IImolecules on these cells. At least one lysosomal reductase,IFN-g-inducible lysosomal thiol reductase (GILT) is lacking inmelanoma cells and may play an important role in class II-restricted epitope presentation (72). The localization of activeGILT in MIIC and lysosomes, would fit well with the requirementfor kI peptide transit to mature endo/lysosomal compartmentsbefore functional presentation (Fig. 2). Mechanistic studies withthekI peptide suggest that cysteinylation and reduction of the freepeptide occur efficiently, in contrast with peptide bound to MHCclass II molecules. Accessibility of the peptide’s reactive cysteineto reductants or oxidants, was dramatically limited followingepitope association with class II molecules. Thus, once bound toclass II molecules, the cysteinylated peptide was not readilyreduced, nor could the peptide be easily cysteinylated once in placewithin the MHC binding groove. Studies with proteases havepreviously demonstrated the protective power of the MHC bindinggroove, sparing epitopes from over-digestion and inactivation (73).Here, evidence suggests that peptide reduction may actually bemore favored before binding class II molecules. Whether GILT orother catalysts of reductive processing in vivo also preferentiallyreduce peptides or Ag before association with MHC class IIproteins remains to be demonstrated. The importance of reductiveprocessing for not only Ag unfolding, but epitope presentation andrecognition, is clearly established by this study. Furthermore,cysteinylation of peptide ligands for class II Ags therefore must beconsidered in the design of vaccine reagents as has been proposedfor MHC class I ligands (36, 74), along with the potential forepitope reduction within target presenting cells.

AcknowledgmentsWe thank Drs. Randy Brutkiewicz and Mark Kaplan for their commentsregarding the manuscript, and Dr. Linda Wicker (Merck Research Labo-ratories) for her support and provision of cell lines. We also thank Dr. S.Pathak, Dr. C. Dunn, J. Lich, and J. Beitz for their discussion.

References1. Watts, C. 1997. Capture and processing of exogenous antigens for presentation

on MHC molecules.Annu. Rev. Immunol. 15:821.2. Rudensky, A. Y., P. Preston-Hurlburt, S. C. Hong, A. Barlow, and C. A. Janeway,

Jr. 1991. Sequence analysis of peptides bound to MHC class II molecules. Nature353:622.

3. Hunt, D. F., H. Michel, T. A. Dickinson, J. Shabanowitz, A. L. Cox,K. Sakaguchi, E. Appella, H. M. Grey, and A. Sette. 1992. Peptides presented tothe immune system by the murine class II major histocompatibility complexmolecule I-Ad. Science 256:1817.

4. Buus, S., A. Sette, S. M. Colon, C. Miles, and H. M. Grey. 1987. The relationbetween major histocompatibility complex (MHC) restriction and the capacity ofIa to bind immunogenic peptides.Science 235:1353.

5. Germain, R. N., and L. R. Hendrix. 1991. MHC class II structure, occupancy andsurface expression determined by post-endoplasmic reticulum antigen binding.Nature 353:134.

6. Harding, C. V., and H. J. Geuze. 1993. Immunogenic peptides bind to class IIMHC molecules in an early lysosomal compartment.J. Immunol. 151:3998.

7. Rudensky, A. Y., M. Maric, S. Eastman, L. Shoemaker, P. C. DeRoos, andJ. S. Blum. Intracellular assembly and transport of endogenous peptide-MHCclass II complexes.Immunity1:585.

8. Amigorena, S., P. Webster, J. Drake, J. Newcomb, P. Cresswell, and I. Mellman.1995. Invariant chain cleavage and peptide loading in major histocompatibilitycomplex class II vesicles.J. Exp. Med. 181:1729.

9. Qiu, Y., X. Xu, A. Wandinger-Ness, D. P. Dalke, and S. K. Pierce. 1994. Sep-aration of subcellular compartments containing distinct functional forms of MHCclass II. J. Cell Biol. 125:595.

10. Tulp, A., D. Verwoerd, B. Dobberstein, H. L. Ploegh, and J. Pieters. 1994. Iso-lation and characterization of the intracellular MHC class II compartment.Nature369:120.

11. Castellino, F., and R. N. Germain. 1995. Extensive trafficking of MHC classII-invariant chain complexes in the endocytic pathway and appearance of peptide-loaded class II in multiple compartments.Immunity 2:73.

12. Denzin, L. K., and P. Cresswell. 1995. HLA-DM induces CLIP dissociation fromMHC class IIab dimers and facilitates peptide loading.Cell 82:155.

13. Sloan, V. S., P. Cameron, G. Porter, M. Gammon, M. Amaya, E. Mellins, andD. M. Zaller. 1995. Mediation by HLA-DM of dissociation of peptides fromHLA-DR. Nature 375:802.

14. Sherman, M. A., D. A. Weber, and P. E. Jensen. 1995. DM enhances peptidebinding to class II MHC by release of invariant chain-derived peptide.Immunity3:197.

15. Weber, D. A., B. D. Evavold, and P. E. Jensen. 1996. Enhanced dissociation ofHLA-DR-bound peptides in the presence of HLA-DM.Science 274:618.

16. Pond, L., and C. Watts. 1997. Characterization of transport of newly assembled,T cell-stimulatory MHC class II-peptide complexes from MHC class II compart-ments to the cell surface.J. Immunol. 159:543.

17. Carbone, F. R., and M. J. Bevan. 1989. Induction of ovalbumin-specific cytotoxicT cells by in vivo peptide immunization.J. Exp. Med. 169:603.

18. Brossart, P., A. W. Goldrath, E. A. Butz, S. Martin, and M. J. Bevan. 1997.Virus-mediated delivery of antigenic epitopes into dendritic cells as a means toinduce CTL.J. Immunol. 158:3270.

19. Casciola-Rosen, L., F. Andrade, D. Ulanet, W. B. Wong, and A. Rosen. 1999.Cleavage by granzyme B is strongly predictive of autoantigen status: implicationsfor initiation of autoimmunity.J. Exp. Med. 190:815.

20. Lipham, W. J., T. M. Redmond, H. Takahashi, J. A. Berzofsky, B. Wiggert,G. J. Chader, and I. Grey. 1991. Recognition of peptides that are immunopatho-genic but cryptic: mechanisms that allow lymphocytes sensitized against crypticpeptides to initiate pathogenic autoimmune processes.J. Immunol. 146:3757.

21. Butz, E. A., and M. J. Bevan. 1998. Massive expansion of antigen-specific CD81

T cells during an acute virus infection.Immunity 8:167.22. Brossart, P., and M. J. Bevan. 1997. Presentation of exogenous protein antigens

on major histocompatibility complex class I molecules by dendritic cells: path-way of presentation and regulation by cytokines.Blood 90:1594.

23. Subklewe, M., A. Chahroudi, A. Schmaljohn, M. G. Kurilla, N. Bhardwaj, andR. M. Steinman. 1999. Induction of Epstein-Barr virus-specific cytotoxic T-lym-phocyte responses using dendritic cells pulsed with EBNA-3A peptides or UV-inactivated, recombinant EBNA-3A vaccinia virus.Blood 94:1372.

24. Dhodapkar, M. V., R. M. Steinman, M. Sapp, H. Desai, C. Fossella, J. Krasovsky,S. M. Donahue, P. R. Dunbar, V. Cerundolo, and D. F. Nixon. 1999. Rapidgeneration of broad T-cell immunity in humans after a single injection of maturedendritic cells.J. Clin. Invest. 104:173.

25. Steinman, R. M., K. Inaba, S. Turley, P. Pierre, and I. Mellman. 1999. Antigencapture, processing, and presentation by dendritic cells: recent cell biologicalstudies.Hum. Immunol. 60:562.

26. Shimonkevitz, R., J. Kappler, P. Marrack, and H. Grey. 1983. Antigen recogni-tion by H-2-restricted T cells. I. Cell-free antigen processing.J. Exp. Med. 158:303.


27. Monji, T., and D. Pious. 1997. Exogenously provided peptides fail to complexwith intracellular class II molecules for presentation by antigen-presenting cells.J. Immunol. 158:3155.

28. Fox, B. S., F. R. Carbone, R. N. Germain, Y. Paterson, and R. H. Schwartz. 1988.Processing of a minimal antigenic peptide alters its interaction with MHC mol-ecules.Nature 331:538.

29. Kang, H. K., J. A. Mikszta, H. Deng, E. E. Sercarz, P. E. Jensen, and B. S. Kim.2000. Processing and reactivity of T cell epitopes containing two cysteine resi-dues from egg-white lysosome (HEL74–90). J. Immunol. 164:1775.

30. Jensen, P. E. 1991. Reduction of disulfide bonds during antigen processing: ev-idence from a thiol-dependent insulin determinant.J. Exp. Med. 174:1121.

31. Williams, D. B., J. Ferguson, J. Gariepy, D. Mckay, Y. T. Teng, S. Iwasaki, andN. Hozumi. 1993. Characterization of the insulin A-chain major immunogenicdeterminant presented by MHC class II I-Ad molecules.J. Immunol. 151:3627.

32. Barlow, A. K., X. He, and C. Janeway, Jr. 1998. Exogenously provided peptidesof a self-antigen can be processed into forms that are recognized by self-T cells.J. Exp. Med. 187:1403.

33. Pathak, S. S., and J. S. Blum. 2000. Endocytic recycling is required for thepresentation of an exogenous peptide via MHC class II molecules.Traffic 1:560.

34. Takahashi, K., H. Kropshofer, A. B. Vogt, E. Gleichmann, and P. Griem. 1998.Drug-induced inhibition of insulin recognition by T-cells: the antirheumatic drugaurothiomalate inhibits MHC binding of insulin peptide.Mol. Immunol. 35:1081.

35. Chen, W., J. W. Yewdell, R. L. Levine, and J. R. Bennink. 1999. Modification ofcysteine residues in vitro and in vivo affects the immunogenicity and antigenicityof major histocompatibility complex class I-restricted viral determinants.J. Exp.Med. 189:1757.

36. Meadows, L., W. Wang, J. M. M. den Hann, E. Blokland, C. Reinhardus,J. W. Drijfhout, J. Shabanowitz, R. Pierce, A. I. Agulnik, C. E. Bishop, et al.1997. The HLA-A*0201-restricted H-Y antigen contains a posttranslationallymodified cysteine that significantly affects T cell recognition.Immunity 6:273.

37. Larson, J. K., L. Otvos, Jr., and H. C. J. Ertl. 1992. Posttranslational side chainmodification of a viral epitope results in diminished recognition by specific Tcells.J. Virol. 66:3996.

38. Harding, C. V., and E. R. Unanue. 1990. Quantitation of antigen-presenting cellMHC class II/peptide complexes necessary for T-cell stimulation.Nature 346:574.

39. Merkel, B. J., R. Mandel, H. J. P. Ryser, and K. L. McCoy. 1995. Characteriza-tion of fibroblasts with a unique defect in processing antigens with disulfidebonds.J. Immunol. 154:128.

40. Kovats, S., S. Drover, W. H. Marshall, D. Freed, P. E. Whiteley, G. T. Nepom,and J. S. Blum. 1994. Coordinate defects in human histocompatibility leukocyteantigen class II expression and antigen presentation in bare lymphocyte syn-drome.J. Exp. Med. 96:217.

41. Hiraiwa, A., K. Yamanaka, W. W. Kwok, E. M. Mickelson, S. Masewicz,J. A. Hansen, S. F. Radka, and G. T. Nepom. 1990. Structural requirements forrecognition of the HLA-Dw14 class II epitope: a key HLA determinant associatedwith rheumatoid arthritis.Proc. Natl. Acad. Sci. USA 87:8051.

42. Ma, C., P. E. Whiteley, P. M. Cameron, D. C. Freed, A. Pressey, S. L. Chen,B. Garni-Wagner, C. Fang, D. M. Zaller, L. S. Wicker, and J. S. Blum. 1998. Roleof APC in the selection of immunodominant T cell epitopes.J. Immunol. 163:6413.

43. Hill, C. M., A. Liu, K. W. Marshall, J. Mayer, B. Jorgensen, B. Yuan,R. M. Cubbon, E. A. Nichols, L. S. Wicker, and J. B. Rothbard. 1994. Explo-ration of requirements for peptide binding to HLA DRB1 0101 and DRB1 0401.J. Immunol. 152:2890.

44. Ceppellini, R., G. Frumento, G. B. Ferraram, R. Tosi, A. Chersi, and B. Pernis.1989. Binding of labeled influenza matrix peptide to HLA-DR in living B lym-phoid cells. Nature 339:392.

45. Jensen, P. E. 1991. Enhanced binding of peptide antigen to purified class II majorhistocompatibility glycoproteins at acidic pH.J. Exp. Med. 174:1111.

46. Pinet, V., M. Vergelli, R. Martin, O. Bakke, and E. O. Long. 1995. Antigen presen-tation mediated by recycling of surface HLA-DR molecules.Nature 375:603.

47. Schwartz, A. L., A. Bolognesi, and S. E. Fridovich. 1984. Recycling of the asia-loglycoprotein receptor and the effect of lysosomotrophic amines in hepatomacells.J. Cell Biol. 98:732.

48. Stoorvogel, W., H. J. Geuze, and G. J. Strous. 1987. Sorting of endocytosedtransferrin and asialoglycoprotein occurs immediately after internalization inHepG2 cells. J. Cell Biol. 104:1261.

49. Wieigel, P. H., and J. A. Oka. 1982. Endocytosis and degradation mediated by theasialoglycoprotein receptor in isolated rat hepatocytes.J. Biol. Chem. 257:1201.

50. Dunn, W. A., T. P. Connolly, and A. L. Hubbard. 1986. Receptor-mediated en-docytosis of epidermal growth factor by rat hepatocytes: receptor pathway.J. CellBiol. 102:24.

51. Sullivan, P. C., A. L. Ferris, and B. Storrie. 1987. Effects of temperature, pHelevators, and energy production inhibitors on horseradish peroxidase transportthrough endocytic vesicles.J. Cell. Physiol. 131:58.

52. Liu, S. M., K. E. Magnusson, and T. Sundqvist. 1993. Microtubules are involvedin transport of macromolecules by vesicles in cultured bovine aortic endothelialcells.J. Cell. Physiol. 156:311.

53. Beeson, C., J. Rabinowitz, K. Tate, I. Gutgemann, Y. H. Chien, P. P. Jones,M. Davis, and H. N. McConnel. 1996. Early biochemical signals arise from lowaffinity TCR-ligand reactions at the cell-cell interface.J. Exp. Med. 184:777.

54. Mellman, I., R. Fuchs, and A. Helenius. 1986. Acidification of the endocytic andexocytic pathways.Annu. Rev. Biochem. 55:663.

55. Sherman, M. A., H. A. Runnels, J. C. Morre, L. J. Stern, and P. E. Jensen. 1994.Membrane interactions influence the peptide binding behavior of DR1.J. Exp.Med. 179:229.

56. Jensen, P. E. 1993. Acidification and disulfide reduction can be sufficient to allowintact proteins to bind class II MHC.J. Immunol. 150:3347.

57. Santambrogio, L., A. K. Sato, G. J. Carven, S. L. Belyanskaya, J. L. Strominger,and L. J. Stern. 1999. Extracellular antigen processing and presentation by im-mature dendritic cells.Proc. Natl. Acad. Sci. USA 96:15056.

58. Fremont, D. H., W. A. Hendrickson, P. Marrack, and J. Kappler. 1996. Structuresof an MHC class II molecule with covalently bound single peptides.Science272:1001.

59. Kappler, J. W., B. Skidmore, J. White, and P. Marrack. 1981. Antigen-inducible,H-2-restricted, interleukin-2-producing T cell hybridomas: lack of independentantigen and H-2 recognition.J. Exp. Med. 153:1198.

60. Fisk, B., T. L. Blevins, J. T. Wharton, and C. G. Ioannides. 1995. Identificationof an immunodominant peptide of Her-2/neo protooncogene recognized by ovar-ian tumor-specific cytotoxic T lymphocyte lines.J. Exp. Med. 181:2109.

61. Pierce, R. A., E. D. Field, J. M. M. den Haan, J. A. Caldwell, F. M. White,J. A. Marto, W. Wang, L. M. Frost, E. Blokland, C. Reinhardus, et al. 1999. TheHLA-A*0101-restricted HY minor histocompatibility antigen originates fromDFFRY and contains a cysteinylated cysteine residue as identified by a novelmass spectrometric technique.J. Immunol. 163:6360.

62. Modugno, F. D., C. Mammi, L. Rosano, O. Rubiu, P. Nistico, and A. Chersi.1997. MHC-peptide binding: dimers of cysteine-containing nonapeptides bindwith high affinity to HLA-A2.1 class I molecules.J. Immunother. 20:431.

63. Naquet, P., J. Ellis, B. Singh, R. S. Hodges, and T. L. Delovitch. 1987. Processingand presentation of insulin. I. Analysis of immunogenic peptides and processingrequirements for insulin A loop-specific T cells.J. Immunol. 139:3955.

64. Collins, D. S., E. R. Unanue, and C. V. Harding. 1991. Reduction of disulfide bondswithin lysosomes is a key step in antigen processing.J. Immunol. 147:4054.

65. Regnier-Vigouroux, A., M. el Ayeb, M. L. Defendini, C. Granier, and M. Pierres.1988. Processing by accessory cells for presentation to murine T cells of apamin,a disulfide-bonded 18 amino acid peptide.J. Immunol. 140:1069.

66. Watarai, H., R. Nozawa, A. Tokunaga, N. Yuyama, M. Tomas, A. Hinohara,K. Ishizaka, and Y. Ishii. 2000. Posttranslational modification of the glycosyla-tion inhibiting factor (GIF) gene product generates bioactive GIF.Proc. Natl.Acad. Sci. USA 97:13251.

67. Dormann, P., T. Borchers, U. Korf, P. Hojrup, P. Roepstoff, and F. Spener. 1993.Amino acid exchange and covalent modification by cysteine and glutathione ex-plain isoforms of fatty acid-binding protein occurring in bovine liver.J. Biol.Chem. 268:16286.

68. Suo, Z., C. T. Walsh, and D. A. Miller. 1999. Tandem heterocyclization activityof the multidomain 230 kDa HMWP2 subunit ofYersinia pestisyersiniabactinsynthetase: interaction of the 1–1382 and 1383–2035 fragments.Biochemistry38:14023.

69. Brennan, S. O., A. P. Fellowes, and P. M. George. 1999. Albumin banks pen-insula: a new termination variant characterized by electrospray mass spectrom-etry. Biochem. Biophys. Acta 1433:321.

70. Southwood, S., J. Sidney, A. Kondo, M. de Guercio, E. Appella, S. Hoffman,R. T. Kubo, R. W. Chesnut, H. M. Grey, and A. Sette. 1998. Several commonHLA-DR types share largely overlapping peptide binding repertoires.J. Immu-nol. 160:3363.

71. Kittlesen, D. J., L. W. Thompson, P. H. Gulden, J. C. A. Skipper, T. A. Colella,J. A. Shabanowitz, D. F. Hunt, V. H. Engelhard, and C. L. Slingluff, Jr. 1998.Human melanoma patients recognize an HLA-A1-restricted CTL epitope fromtyrosinase containing two cysteine residues: implications for tumor vaccine de-velopment.J. Immunol. 160:2099.

72. Arunachalam, B., U. T. Phan, H. J. Geuze, and P. Cresswell. 2000. Enzymaticreduction of disulfide bonds in lysosomes: characterization of ag-interferon-inducible lysosomal thiol reductase (GILT).Proc. Natl. Acad. Sci. USA 97:745.

73. Donermeyer, D. L., and P. M. Allen. 1989. Binding to Ia protects an immuno-genic peptide from proteolytic degradation.J. Immunol. 142:1063.

74. Kawakami, Y., P. F. Robbins, X. Wang, J. P. Tupesis, M. R. Parkhurst, X. Kang,K. Sakaguchi, E. Appella, and S. A. Rosenberg. 1998. Identification of newmelanoma epitopes on melanosomal proteins recognized by tumor infiltrating Tlymphocytes restricted by HLA-A1, -A2, and -A3 alleles.J. Immunol. 161:6985.


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