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The Journal of Immunology, 2001, 166: 4543-4551.
Copyright © 2001 by The American Association of Immunologists

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

M. Azizul Haque*, John W. Hawes{dagger} and Janice S. Blum2,*,{ddagger}

Departments of * Microbiology and Immunology and {dagger} Biochemistry and Molecular Biology, and {ddagger} Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peptides bind cell surface MHC class II proteins to yield complexes capable of activating CD4+ T cells. By contrast, protein Ags require internalization and processing by APC before functional presentation. Here, T cell recognition of a short peptide in the context 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 cell recognition of this epitope. Internalization and processing of the modified epitope by APC, was required to restore T cell recognition. Peptide cysteinylation and reduction could occur rapidly and reversibly before MHC binding. Cysteinylation did not disrupt 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 to reductively cleave this peptide modification presumably evolved to circumvent a similar problem in MHC class II ligand recognition.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antigenic peptides complexed with MHC class II molecules are displayed on the surface of APC for recognition by CD4+ T cells. The formation and abundance of these peptide:class II complexes is regulated by peptide source as well as reactions within APC. Protein Ags must be internalized into acidic endosomal and lysosomal compartments for processing to yield the short peptides of 12–25 aa that optimally bind MHC class II proteins (1, 2, 3, 4). These peptides generated within APC can intersect and bind class II proteins throughout the endosomal pathway (5, 6, 7, 8, 9). However, peptide association with newly synthesized class II molecules may be most favored in the late endosomal/prelysosomal compartment termed MIIC (6, 7, 9, 10, 11). Here, the exchange factor HLA-DM catalyzes the release of invariant chain fragments from class II molecules and exposes the ligand binding groove (12, 13, 14, 15). Peptides complexed with class II proteins within this compartment are then shuttled to the cell surface (1, 16).

By contrast, antigenic peptides can also be generated outside APC for example, upon lysis of tumor or virally infected cells (17, 18), or as a result of Ag processing by extracellular proteases during inflammatory or autoimmune responses (19, 20). These peptides may be acquired and presented via MHC molecules on bystander APC for T cell recognition (21, 22). Synthetic peptides have also been used as vaccine reagents, following their incubation with potent APC, such as dendritic cells (23, 24, 25). Studies using synthetic or chemically generated short peptides had suggested that these exogenous ligands bind directly to available cell surface class II proteins yielding complexes for T cell recognition (26, 27, 28, 29). Yet, several reports have also indicated that T cell responses to select peptides required their presentation by viable APC (29, 30, 31, 32, 33), raising the question of whether ligand binding to surface MHC alone is sufficient for T cell activation. Among several of the peptides requiring presentation by viable APC, a shared common feature that emerges is the presence of one or more cysteine residues (29, 30, 34). Here through studies of a cysteine-containing peptide from the Ag human Ig {kappa}, we have demonstrated that cysteine modification can regulate T cell responses to class II-restricted epitopes. Although cysteinylation of ligands for MHC class I has been reported previously (35, 36, 37), this marks the first demonstration that such a modification can alter class II-restricted T cell responses. Furthermore, these studies demonstrate that endocytosis and processing of the cysteinylated peptide by viable APC was necessary to restore antigenicity. Although the importance of reduction in processing native Ags has been appreciated (30, 38, 39), here a role for reduction in the functional presentation of peptide ligands by APC has also been clearly demonstrated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

APC were cultured in Iscove’s complete DMEM with 10% heat-inactivated calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. The B-lymphoblastoid cell Frev expresses endogenous class II DR4w4 (DRB1*0401) and DR1 (DRB1*0101) alleles as well as Ig {lambda} light chains. The human monocyte cell THP-1.DR4 and the murine dendritic cell FSDC.DR4 were transduced using retroviral vectors for constitutive expression of HLA-DR4 (DRB1*0401) with linked drug selection markers for hygromycin and G418 resistance (40). Expression of surface DR4 complexes on cells was confirmed by cytofluorography using the DR4-specific mAb, 359F10 (41). T cell hybridomas specific for Ig {kappa} peptides presented in the context of HLA-DR4, were generated by immunization of DR4w4-transgenic mice with human IgG. The hybridoma line 2.18 recognizes peptides encompassing Ig {kappa} residues 188–203 while the cell 1.21 responds to Ig {kappa} residues 145–159 (42). T cell hybridomas and HT-2 cells were cultured in RPMI 1640 with 10% FBS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 50 µM 2-ME. For HT-2 cells, 20% Con A supernatant (T-STIM; Collaborative Biomedical Products, Bedford, MA) was also added.

Peptides

The human IgG immunodominant ({kappa}I) peptide {kappa}188–203 (sequence KHKVYACEVTHQGLSS) and subdominant ({kappa}II) peptide {kappa}145–159 (sequence KVQWKVDNALQSGNS) were produced using Fmoc technology and an Applied Biosystems synthesizer (Foster City, CA). Peptide purity (>99%) and sequence were analyzed by reverse-phase HPLC purification and mass spectroscopy. Peptides were labeled as indicated at the {alpha} amino termini by the sequential addition of two molecules of Fmoc-6-aminohexanoic acid followed by a single biotin to yield the sequence biotin-aminohexanoic acid-aminohexanoic acid-peptide. Mass spectrometry confirmed that the peptide was tagged with a single biotin molecule at the N terminus. Preparation of purified cysteinylated {kappa}I was achieved by peptide incubation (3 h at 37°C in HBSS) with cystine (0.29 mM) followed by dialysis (1000 Da membrane cutoff) to remove residual cystine/cysteine. Peptide cysteinylated was maintained at neutral or acidic pH in the absence of reductants. Substituted forms of the {kappa}I peptide were also generated by Fmoc technology with Ala, Ser, or 2-aminobutyric acid (aba)3 replacing Cys194. Peptides were dissolved at 1 mM in either DMSO (Sigma, St. Louis, MO) or PBS, and stored at -20°C until use.

T cell proliferation assays

APC were incubated with synthetic {kappa} peptides for 3–24 h at 37°C in culture medium, washed, and cocultured with T cell hybridomas for 24 h. T cell cytokine production was monitored by measuring [3H]thymidine (1µCi/well) incorporation using the IL-2/IL-4-dependent cell line, HT-2. In some cases, APC were prefixed with 1% paraformaldehyde for 8 min on ice followed by extensive washing and peptide addition, or postfixed before coculture with T cell hybridomas. When THP-1.DR4 cells were used as APC, these cells were first stimulated with 50 U/ml IFN-{gamma} (R&D Systems, Minneapolis, MN) for 48 h before the addition of peptides. APC were also incubated with {kappa} peptides at 18°C for 24 h, fixed, and cocultured with {kappa}-specific T cell hybridomas for 24 h. Assays were also performed using APC treated with DTT (Sigma), L-cysteine, or L-cystine, and {kappa} peptides before or after aldehyde fixation.

For inhibition studies, APC were pretreated with inhibitors such as NaN3/deoxyglucose (2 mg/ml, 50 mM, respectively), 60 µM colchicine, or 100 µM primaquine (Sigma) in complete medium for 30 min followed by the addition of synthetic peptides. Cells were subsequently washed twice in PBS and fixed with 1% paraformaldehyde before cultivation with T cells. All assays were repeated at least three to four times with the SE for triplicate samples within a single experiment reported. Data were corrected for isotope counting efficiency and expressed as corrected cpm (ccpm).

Peptide binding assays

Paraformaldehyde-fixed Frev cells were incubated overnight with biotinylated {kappa} peptides ({kappa}I and {kappa}II) in 150 mM citrate-phosphate buffer, (pH 5.5–7.4), HBSS, or Iscoves’s DMEM medium (pH 7.4) with heat-inactivated serum, washed with PBS, and lysed on ice for 20 min with 50 mM Tris buffer (pH 8) containing 0.15 M NaCl and 0.5% IGEPAL-CA 630 (Sigma) as described (43). The lysate was centrifuged to remove intact nuclei, 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 complexes were 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 presumably due to competing serum peptides, as no proteolysis of the {kappa} peptide was detected. The relative affinity of the Cys-substituted {kappa} peptides and HA-flu peptide was also measured in a competitive binding assay as described (42). The number of total DR molecules within APC was quantitated using biotinylated L243 and the capture Ab 37.1 as described (42). In all experiments, drug treatment of cells did not diminish the total amount of cellular HLA-DR as detected using this assay.

Liquid chromatography-mass spectroscopy

Peptide {kappa}I was analyzed by capillary liquid chromatography using an Applied Biosystems 140D solvent delivery system. Samples were applied directly to 300-µm diameter fused silica capillaries packed with Vydac C18 resin and separated with gradients of buffer A (2% acetonitrile and 98% H2O containing 0.2% isopropanol, 0.1% acetic acid, and 0.001% trifluoroacetic acid) and buffer B (95% acetonitrile and 5% H2O containing 0.2% isopropanol, 0.1% acetic acid, and 0.001% trifluoroacetic acid). Peptide was eluted at a flow rate of 7 µl/min directly into the electrospray ionization source of a Finnigan LCQ mass spectrometer. Nitrogen was used as the sheath gas with a pressure of 35 psi with no auxiliary gas. Electrospray ionization was conducted with a spray voltage of 4.8 kV, a capillary voltage of 26 V, and a capillary temperature of 200°C. Spectra were scanned over a m/z range of 200-2000. Base peak ions were trapped using the quadruple ion trap and further analyzed with a high resolution scan (zoom-scan) using an isolation width of 3 m/z and collision-induced dissociation scans with a collision energy of 40.0.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Metabolically active APC are required for the functional presentation of a synthetic peptide in the context of MHC class II proteins

Exogenous protein Ags are internalized and processed by viable APC, yielding epitopes that bind intracellular class II histocompatibility Ags before surface expression and T cell engagement. In contrast, short synthetic peptides bind directly to MHC class II molecules on the surface of APC, and typically trigger T cell activation without a requirement for APC metabolic activity (27, 44, 45, 46). During studies of the processing of an autoantigen, human IgG class II-restricted epitopes were identified in viable HLA-DR4+ APC using functional and biochemical approaches (42). Yet, DR4-restricted T cell activation via a synthetic analog of an immunodominant peptide, Ig {kappa}I (residues 188–203) was detected only using viable and not prefixed APC (Fig. 1Go). By contrast, T cell hybridomas specific for another Ig {kappa}II epitope (residues 145–159) were able to recognize synthetic forms of this peptide displayed on both aldehyde-fixed and live APC. Experiments with DR4+ human and murine B cell lines, macrophages, and dendritic cells demonstrated that regardless of cell lineage, functional T cell recognition of the {kappa}I synthetic peptide was observed only with metabolically active APC (Fig. 1Go, A, C, and D). Aldehyde-fixation has been 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 minimal requirements for costimulation and can detect isolated complexes of peptide-loaded class II molecules (42). Two additional lines of experimental evidence prove that the failure of fixed cells to functionally display the {kappa}I peptide was not linked to a defect in costimulation or cell adhesion. First, even in the presence of a strong costimulatory signal delivered via Ab cross-linking of CD28, T cells failed to respond to fixed APC and the {kappa}I peptide (Fig. 1GoB). The effectiveness of this exogenous costimulation was confirmed using THP-1.DR4 cells and the {kappa}II peptide. T cell responses to this peptide plus APC could be enhanced in the presence of cross-linking of CD28 (data not shown). Also, preincubation of the {kappa}I peptide with viable APC (for 24 h) before cell fixation, resulted in efficient presentation and T cell responses to this epitope (Fig. 1GoB). Together, these results indicate that formation of functional complexes of class II molecules with the {kappa}I peptide, was dependent upon the metabolic activity of APC. In this respect the 16 aa {kappa}I epitope behaves more like native protein Ags, potentially requiring processing before efficient display via class II molecules.



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  		FIGURE 1. HLA class II-restricted presentation of synthetic {kappa} peptides by the B cell Frev (A and B), IFN-{gamma}-activated macrophage THP-1.DR4 (C), and dendritic cell FSDC.DR4 (D). Live ({circ}) or chemically fixed (•) APC were incubated in complete medium with either {kappa}I or {kappa}II synthetic peptides for 24 h at 37°C, washed, and cocultured with the appropriate {kappa} peptide-specific T cell hybridomas (2.18a cell specific for DR4:{kappa}I and 1.21 cell specific for DR4:{kappa}II) for 24 h. B, Frev cells were incubated with the {kappa}I peptide with or without subsequent aldehyde fixation followed by coculture with T cells. Alternatively, these cells were prefixed and then incubated with {kappa}I (fxAPC + {kappa}I) before coculture with T cells without or with external costimulation via cross-linked anti-CD28 (fxAPC + {kappa}I + cost.). T cell production of IL-2 was determined by measuring proliferation of HT-2 cells using [3H]thymidine incorporation.

 
Endocytosis is required for functional presentation of {kappa}I peptide by B cells

To investigate the role of endocytic transport in class II-restricted presentation of the {kappa}I peptide, APC were incubated with metabolic inhibitors that block the internalization of molecules via coated pits into the endosomal pathway. Treatment of APC with sodium azide and 2-deoxyglucose, substantially reduced the ability of these cells to present the synthetic {kappa}I peptide to T cells (Fig. 2GoA). Yet, presentation of the {kappa}II peptide was unaffected by these inhibitors of cellular ATP production. As expected, control studies revealed that these metabolic inhibitors blocked the processing and presentation of native Ags, such as IgG (data not shown). These results suggest that endocytic transport is important in the functional presentation of the {kappa}I peptide, while the {kappa}II epitope directly binds cell surface class II molecules and mediates T cell activation. To distinguish whether the requirement for endocytosis was linked directly to the {kappa}I peptide and/or class II molecules themselves, APC were treated with the drug primiquine, which blocks endocytic recycling of transmembrane proteins such as class II molecules and the transferrin receptor (47, 48). Earlier, we reported that the drug primiquine could block the presentation of a peptide that intersected class II molecules in recycling early endosomes (33). Functional presentation of the {kappa}I peptide was not perturbed using primiquine-treated APC, demonstrating that recycling class II molecules are not required for display of the {kappa}I peptide (Fig. 2GoA). Rather, these results indicate that the {kappa}I peptide must be transported into the endocytic pathway for DR4-restricted presentation and T cell activation. Further proof for peptide internalization came from studies in which endocytic transport was blocked between early and late endosomal compartments. Incubation of APC at 18°C has been shown to halt the delivery of Ags from early to late endosomes (49, 50, 51). Thus, presentation of the {kappa} peptides was examined in DR4+ B cells incubated at 18°C and 37°C (Fig. 2GoB). T cell recognition of the {kappa}I peptide was completely ablated using APC incubated with this peptide at low temperature. In contrast, only a slight reduction in the efficiency of {kappa}II peptide display was observed with DR4+ APC at 18°C compared with 37°C. In each experiment, APC were incubated at the indicated temperatures with peptides followed by aldehyde-fixation before coculture with T cells at 37°C. Endocytic vesicles associate with microtubules within mammalian cells, and transport of molecules from early endosomes to late endosomes/lysosomes can be perturbed via microtubule depolymerization using drugs such as colchicine (52). Treatment of APC with colchicine inhibited in part, functional presentation of the {kappa}I peptide to T cells (Fig. 2GoA). Class II presentation of the {kappa}II peptide was not altered by this drug, confirming that surface class II expression was not grossly altered by microtubule depolymerization. In sum, these studies strongly suggest a requirement for peptide internalization and transport beyond early endosomes before functional display in the context of MHC class II molecules.



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  		FIGURE 2. Requirement for internalization and processing of the {kappa}I peptide by APC. Frev were pretreated with either NaN3/deoxyglucose, colchicine, primaquine, or PBS as a control (A) for 30 min at 37°C, followed by addition of synthetic {kappa} peptides for 3 h plus or minus these inhibitors. APC were then washed three times, fixed, and cocultured with T cell hybridomas. Alternatively, in B, Frev (1 x 106/ml) were incubated with synthetic {kappa} 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 measured using HT-2 cells. A, Data are expressed as relative percent peptide presentation based upon a maximal response of 149,117 ccpm for 2.18a and 159,881 ccpm for 1.21 T cells, respectively.

 
Synthetic {kappa} peptides bind MHC class II proteins at both acidic and neutral pH

Epitopes released during Ag processing preferentially bind class II molecules within acidic endosomal compartments with the aid of chaperones such as HLA-DM. Although direct binding of synthetic peptides to MHC class II proteins can occur at neutral pH on the cell surface, the efficiency of this reaction varies among peptides with low pH enhancing the binding of many epitopes to MHC class II Ags (53, 54, 55). Thus, the requirement for endocytosis before functional presentation of the {kappa}I peptide in the context of HLA-DR4, might reflect a failure of this peptide to bind class II molecules at neutral pH. Studies to assess direct binding of {kappa}I and {kappa}II peptides to DR4 were conducted over a broad pH range using aldehyde-fixed APC and Ab capture of the resulting class II-peptide complexes (Fig. 3Go). Although the {kappa}I peptide clearly showed preferential binding to class II molecules at acidic pH values found within late endosomes/prelysosomes (i.e., pH 5.5), measurable association of this peptide with DR4 was detected at neutral pH. T cell responses were also enhanced following loading of the native peptide in low pH buffer solutions onto APC. At pH values between 7.4 and 6.3, the level of class II DR4 binding for both {kappa}I and {kappa}II peptides was comparable. For these experiments, biotin-tagged {kappa}I and {kappa}II peptides were used to directly monitor binding to class II proteins. Functional studies not shown confirmed that addition of an amino-terminal biotin did not alter the presentation of either {kappa} peptide. Thus, T cell responses to the biotinylated {kappa}I peptide were only detected in the context of viable APC. Binding of the unmodified {kappa} peptides to surface class II molecules over a broad range of pH values, was also detected in competition assays with biotin-tagged flu hemagglutinin 307–319 peptide (data not shown). Thus, the data indicate that each synthetic {kappa} peptide was capable of directly binding to cell surface HLA-DR4. Still, T cell recognition of the {kappa}I peptide was dependent upon endocytic transport of this epitope and potentially, processing in viable APC.



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  		FIGURE 3. Synthetic {kappa} peptide binding to HLA-DR4 was not affected by paraformaldehyde fixation of APC. Paraformaldehyde-fixed Frev were incubated overnight with varying concentrations of biotinylated {kappa} peptides (0–10 µM) at different pH (5.5–7.4) values in citrate-phosphate buffer at 37°C. APC were then washed and lysed, and the extent of binding of biotinylated {kappa} peptides to surface DR4 was examined in a capture ELISA using europium-labeled streptavidin. A, {kappa}I; B, {kappa}II. Binding of the {kappa}I peptide was optimal at pH 5.5, yet measurable peptide:MHC association could be detected even at neutral pH. Maximal binding of the {kappa}II peptide to DR4 was detected at pH 7.4 and pH 5.5 in repeated experiments. Data are representative of mean fluorescence ± SEM for at least three separate experiments.

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

Although proteolytic cleavage is important in Ag processing, denaturation of proteins by acidic pH and disulfide reduction also play key roles in the class II-restricted presentation of select Ags (30, 31, 56). Experiments using a broad panel of protease and peptidase inhibitors failed to block the ability of viable APC to convert the synthetic {kappa}I peptide to a functional epitope (data not shown), prompting a search for alternate modifications that might influence T cell recognition of this peptide. Studies of class I-restricted viral and tumor epitopes have revealed modifications of cysteine residues, which alter CD8+ T cell responses (35, 36). The {kappa}I peptide (KHKVYACEVTHQGLSS) contains a central cysteine residue, potentially susceptible to disulfide linkage or oxidation. To investigate whether disulfide formation influenced peptide antigenicity, the {kappa}I peptide was incubated with APC in culture medium containing a reductant (DTT) followed by analysis of T cell activation (Fig. 4Go). The addition of a reducing agent restored presentation of the {kappa}I 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 purified {kappa}I with DTT in medium before removal of the reductant, and peptide addition to APC resuspended in HBSS (data not shown). By contrast with the 10- to 20-fold increase in {kappa}I peptide presentation observed with fixed cells plus reductant (Fig. 4GoB), presentation of the {kappa}II peptide remained unchanged suggesting DTT treatment did not alter class II Ag function in general. To exclude the possibility that DTT enhanced class II binding of {kappa}I, studies of peptide binding were conducted in the absence and presence of reductant. Remarkably, binding of the biotin-labeled {kappa}I peptide to class II molecules was diminished upon exposure of cells to culture medium containing DTT (Fig. 4GoC). Yet, no change in {kappa}II association with DR4 could be detected in the presence of reductants (Fig. 4GoD). Peptide binding in general is diminished in culture medium compared with buffered salt solutions; however, inclusion of the reductant DTT altered only the association of class II proteins with cysteine-containing peptides such as {kappa}I. Levels of surface class II proteins on APC were unchanged following exposure to DTT as determined using an ELISA (data not shown). Together, these results suggest that reduction of the {kappa}I peptide is a key processing step essential to functional epitope presentation and T cell recognition.



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  		FIGURE 4. Reduction of the {kappa}I peptide restores presentation of this epitope by fixed APC. Metabolically active (A) or paraformaldehyde-fixed (B) Frev cells were incubated in culture medium with {kappa} peptides (5–20 µM) ± the reducing agent, DTT (200 µM) for 3 h at 37°C. APC were then washed 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 the {kappa}I peptide (C) or {kappa}II peptide (D) to class II DR4 in culture medium (IMDM plus heat-inactivated calf-serum) ± DTT. The association of biotin-labeled peptides with class II molecules was detected via fluorescence of europium-tagged strepavidin as indicated in Materials and Methods.

 
Role of cystine in the modification of synthetic peptides

Mass spectral analysis of the synthetic {kappa}I peptide dissolved in HBSS, revealed a single monomeric species (molecular mass, 1785.7) with residue Cys194 existing as a free sulfhydryl (Fig. 5GoA). However, upon incubation of the peptide in tissue culture medium (with or without serum), greater than 80% of the peptide was modified by a cysteine adduct yielding the 1905.6 molecular mass cysteinylated species (Fig. 5GoB). Mass spectral analysis failed to reveal any significant amounts of dimeric peptide as might be predicted based upon disulfide formation. Trace amounts of a m/z 601.3 species were detected by mass analysis, potentially representing the sulfenic acid form of the peptide. Peptide sequence and the presence of the modified Cys was confirmed by collision-induced dissociation. Other modifications such as peptide-glutathione conjugation were not detected upon incubation of the {kappa}I peptide with medium.



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  		FIGURE 5. Mass spectroscopic analysis of synthetic {kappa}I peptide revealed cysteinylation of this peptide in culture medium. The {kappa}I peptide incubated 3 h in HBSS (A) or culture medium (B) and spectra analyzed following capillary liquid chromatography using an Applied Biosystems 140 solvent delivery system as described in Materials and Methods. Analysis of b ion subfragmentation by collision-induced dissociation confirmed peptide cysteinylation. The {kappa}I peptide fragmentation profile in HBSS was consistent with the calculated molecular mass of 1785.7 and the presence of reduced cysteine at position 194 (not shown). Fragmentation of the peptide after incubation in tissue culture medium without or with serum, indicated a mass of 1905.6 consistent with cysteinylation at residue 194 (not shown).

 
Cysteinylation of class I epitopes has been documented (35), arising potentially from posttranslational epitope modification or via reaction with cystine in serum or tissue culture medium. To test whether cystine was responsible for modification and inactivation of the {kappa}I peptide, fixed APC were incubated with this peptide in buffer plus or minus 0.29 mM cystine, the concentration found in IMDM tissue culture medium. Remarkably in the absence of cystine or tissue culture medium, efficient presentation of the {kappa}I peptide was observed with fixed APC (Fig. 6GoA). Incubation of the {kappa}I peptide in buffer plus cysteine minimally inactivated peptide presentation by fixed APC. However, following APC incubation with the {kappa}I peptide in HBSS plus cystine peptide presentation was completely blocked, confirming that cystine was responsible for peptide modification and loss of function. Incubation of live or fixed APC with the {kappa}II peptide in buffer with cystine had no effect on T cell responses to this epitope, which lacks a central cysteine residue.



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  		FIGURE 6. Preferential cysteinylation and reduction of {kappa}I before binding MHC class II Ags. A, Cysteinylation of {kappa}I peptide was favored in the presence of cystine vs cysteine. Aldehyde-fixed Frev cells were incubated with {kappa}I in HBSS, HBSS + cysteine (0.29 mM), or HBSS + cystine (0.29 mM) for 3 h, followed by washing and coculture with T cells for 24 h. B, Binding of {kappa}I peptide to class II DR4 prevents cysteinylation. Prefixed APC were incubated with {kappa}I in HBSS for 3 h, washed and further incubated in either HBSS or HBSS + cystine (0.29 mM) for 3 h. APC were then washed and cocultured with T cells. C, Cysteinylation of reduced {kappa}I peptide upon postincubation in IMDM. The {kappa}I peptide was preincubated in HBSS or HBSS + DTT for 3 h. Aldehyde-fixed APC were incubated with the peptide in either HBSS or IMDM culture medium for 3 h, washed, and cocultured with T cells. D, Cysteinylated {kappa}I peptide bound to class II DR was resistant to reduction. Prefixed APC were incubated with {kappa}I in IMDM for 3 h, washed, and further incubated with 200 µM DTT or IMDM alone for 3 h. Cells were then washed and cocultured with T cells. In each group (A–D), T cell activation was assessed by cytokine production as quantitated by HT-2 cell proliferation. The range of peptide concentrations tested, is indicated in the figure.

 
In direct contrast with functional assays, {kappa}I peptide binding to MHC class II proteins at neutral pH was enhanced by cysteinylation (Fig. 7GoA). Binding of the biotin-labeled {kappa}I peptide to class II DR4 in the presence of cystine at pH 7.4 was enhanced up to 5-fold compared with the unmodified peptide. A similar increase in peptide binding was observed using cysteinylated {kappa}I peptide (>95% modified) purified to remove residual cystine/cysteine before incubation with MHC Ags. Cystine did not alter the general peptide binding properties of class II molecules as no change was detected in the association of the {kappa}II peptide with DR4 molecules plus or minus cystine (Fig. 7GoB). Under conditions of low pH mimicking the environment within mature endosomes, binding of the {kappa}I peptide to class II molecules was minimally effected by cysteinylation (Fig. 7GoC). Because cysteinylation is less efficient at low pH, for these studies the {kappa} peptides were pretreated with cystine at neutral pH and dialyzed, and greater than 95% cysteinylation was confirmed by mass spectroscopy before incubation with MHC under acidic pH conditions. Association of the {kappa}II peptide with MHC at low pH was unaltered by cystine pretreatment (Fig. 7GoD), again confirming an overall lack of change in class II structure under these incubation conditions. Thus while the cysteinylated {kappa}I peptide failed to activate T cells, modification of this peptide promoted interactions with class II molecules under select conditions.



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  		FIGURE 7. Cysteinylation influences {kappa}I binding to class II DR4 dependent upon the environmental pH. A and B, Paraformaldehyde-fixed Frev cells were incubated overnight with varying concentrations of biotinylated {kappa} peptides (0–5 µM) in HBSS (pH 7.4) ± 100 µM cystine. C and D, Cells were also incubated in citrate phosphate buffer (CPB; pH 5.5) with biotin-labeled {kappa} peptides that had been pretreated with cystine (100 µM) at neutral pH to promote modification as described in Materials and Methods. APC were washed after incubation with peptides and lysed, and the extent of peptide binding was assessed using a capture ELISA and europium-labeled streptavidin. A and C, {kappa}I peptide:DR4 binding; B and D, {kappa}II peptide binding to DR4. Data are representative of mean fluorescence ± SEM for at least three separate experiments.

 
Studies were conducted to further investigate the mechanism of cysteine modification and specifically, the role of MHC class II molecules in this process. Although cysteinylation and inactivation of the free {kappa}I peptide was efficient in solution (Fig. 5Go), preformed complexes of {kappa}I and class II DR were moderately resistant to modification by cystine (Fig. 6GoB). In this experiment synthetic {kappa}I peptide was bound to surface class II molecules on aldehyde-fixed APC in HBSS, followed by a postincubation in the presence or absence of cystine. T cell activation was only reduced by 20–30%, suggesting that cysteinylation of the peptide bound to class II molecules could occur but with markedly less efficiency. These results suggest that once bound to class II molecules, the peptide’s central cysteine residue may be somewhat shielded from modification. This result also explains why peptide prebound to class II molecules on fixed APC in the absence of cystine was not inactivated upon later coculture in complete medium with T cells. The inclusion of reductants during peptide incubation with fixed APC in culture medium, prevented or rapidly reversed peptide cysteinylation (Fig. 4Go). Reduction of the free peptide before binding to class II molecules in HBSS also enhanced T cell recognition (Fig. 6GoC). This effect was reversed with nearly complete loss of peptide activity when the reduced peptide was added to APC in cystine-containing medium. Thus, demonstrating that the reduced peptide was highly susceptible to cysteinylation unless a reducing agent such as DTT was continually present. Further studies revealed that once bound to class II DR, the cysteinylated peptide could not be reduced as measured by restoration of functional T cell recognition (Fig. 6GoD). Here, peptide binding to class II DR4 was conducted in cystine-containing medium to promote cysteinylation, followed by a postincubation in medium with or without reductant. T cell recognition of the peptide:class II molecules was minimal with only a very slight enhancement upon treatment of these complexes with DTT. This result is important with potential relevance to reductive peptide processing in vivo, as the findings suggest that reduction of the cysteinylated {kappa}I peptide occurs before binding to class II molecules. Thus for the free {kappa}I epitope, peptide cysteinylation and reduction can occur efficiently and reversibly. By contrast, peptide binding to the groove of MHC class II molecules severely limits the accessibility of this central cysteine residue despite its potential role in T cell recognition.

Substitution of cysteine residues enhances peptide presentation

To further demonstrate that modification of cysteine residues leads to the requirement for {kappa}I peptide processing by APC before functional presentation, the properties of analog {kappa}I peptides with conservative substitutions of serine, alanine, or aba for Cys194 were tested (Fig. 8Go). Incubation of live or fixed APC with the {kappa}I analog containing serine substituted for cysteine, failed to elicit any T cell response (Fig. 8Go, A and B). In contrast, functional presentation of an alanine-substituted form of the {kappa}I peptide could be detected using fixed APC (Fig. 8GoB). T cell responses to this alanine analog were reduced compared with the original {kappa}I peptide in studies with live APC (Fig. 8GoA). Substitution of aba, which more closely approximates cysteine in size, led to nearly equivalent functional presentation of this analog by live or fixed APC (Fig. 8Go, A and B). Each of the substituted peptides was tested alongside the original {kappa}I epitope in a competitive binding assay with class II DR4. Results indicated that peptides substituted with serine or aba at position 194 bound MHC comparable to the {kappa}I epitope (data not shown). Binding of the alanine-substituted peptide was reduced compared with the original {kappa}I peptide by ~2-fold. To further address the requirement for processing using these analog peptides, APC were incubated at 18°C with the aba-substituted form of {kappa}I. In contrast with the cysteine-containing {kappa}I peptide, which required endocytic transport and processing at temperatures above 18°C, the aba {kappa}I peptide was presented equally well at low or high temperatures (Fig. 8GoC). These results demonstrate that intracellular reduction of the cysteinylated {kappa}I peptide, was the key processing step required for functional presentation of this epitope to T cells.



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  		FIGURE 8. Presentation of cysteine-substituted {kappa}I peptides by B cells. Live (A) or fixed (B) Frev cells were incubated 24 h at 37°C with variants of the {kappa}I peptide substituted with Ala, Ser, or aba at position 194, followed by coculture with the {kappa}I peptide-specific T cell hybridoma for 24 h. C, Frev cells were also incubated at 18°C with either {kappa}I or aba-{kappa}I for 24 h, washed, fixed, and cocultured with the {kappa}I-specific T cell hybridoma. T cell IL-2 activity was measured using the HT-2 cell line. The range of peptide concentrations tested, is indicated in the figures.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The presence of accessible cell surface class II molecules on APC facilitates peptide loading, and offers an efficient means of delivering peptide vaccines for immunoregulation (23, 24, 57, 58). Protein Ags by contrast, typically require endocytosis and processing within APC before MHC binding and T cell activation. Here, a requirement for peptide endocytosis and reductive processing was established, as a result of the spontaneous modification of an essential cysteine within an MHC class II peptide ligand. Binding of the cysteine-containing peptide {kappa}I to cell surface class II DR was readily demonstrated, yet the resulting complexes failed to activate T cells suggesting the need for peptide endocytosis and processing. Indeed, treatment of APC with inhibitors of endocytic transport or metabolic activity, blocked functional presentation of the {kappa}I epitope but did not alter the class II-restricted display of another epitope, {kappa}II, which lacks cysteine. Therefore, the {kappa}II peptide follows the conventional or established pathway, with functional display of this epitope upon direct association with surface class II molecules (59). Studies with primaquine demonstrated that endosomal transit of the {kappa}I peptide was essential rather than endocytic recycling of class II molecules. Incubation of APC at low temperatures or in the presence of drugs that impaired transit to late or mature endo/lysosomal compartments also prevented the processing and functional presentation of the {kappa}I peptide. A requirement for peptide reduction before T cell recognition was documented, while no evidence of proteolytic processing was observed. Thus T cell 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 of the peptide spontaneously occurred upon incubation in the presence of physiological concentrations of cystine found in tissue culture medium or serum. Experiments with peptide analogs further demonstrated that the requirement for endocytosis and processing before functional class II-restricted presentation was linked to the modification of a reactive cysteine within the peptide.

Biochemical and functional studies of MHC class I-restricted epitopes derived from tumors (60), viruses (35), and H-Y minor Ags (61) have established that cysteine residues within these epitopes are modified in vivo and in vitro thus influencing T cell recognition. Although dimerization and oxidation of these cysteine-containing peptides was detected, by far the most common modification was peptide cysteinylation. In vivo cysteinylation of peptides is highly likely due to the high circulating levels of cystine in serum (0.1 mM) and the reactivity of free sulfhydryls (36, 62). The importance of cysteinylation in regulating class II-restricted T cell activation, has not been investigated despite elegant studies pointing to the role of disulfide reduction and cysteine residues in Ag unfolding, processing, and class II presentation (29, 30, 39, 42, 63, 64). Previous studies had noted that several short peptides or small Ags containing cysteine residues were presented only by metabolically active APC, strongly suggesting a requirement for processing (29, 30, 31, 65). Inactivation of these epitopes was attributed to peptide oxidation or conjugation with unknown serum factors (29, 30, 63, 65), although direct analysis of epitope structure was not performed. The present study offers biochemical evidence that cysteinylation of class II epitopes can occur spontaneously and with a high efficiency in medium or serum, thus altering T cell recognition of these ligands. Significant amounts of oxidized, dimerized, or conjugated peptide were not detected, strongly suggesting cysteinylation was the predominant modification of {kappa}I similar to MHC class I peptide ligands. Mass spectral analysis of an epitope from hen egg lysozyme (residues 74–88 with Cys80 substituted to ala, NLCNIPASALLSSDI) also revealed nearly 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 with previous functional studies (29). Cysteinylation of the {kappa}I peptide also ablated the functional presentation of this epitope to DR1-restricted T cells (data not shown). Whether cysteinylation influences the processing and presentation of antigenic proteins remains unclear, yet an increasing number of cysteinylated proteins have been detected with the advent of sequence analysis by mass spectroscopy (66, 67, 68, 69). Remarkably, cysteinylation of the {kappa}I peptide interfered with T cell recognition but did not diminish class II binding. In fact, at neutral pH the binding of {kappa}I peptide to DR4 was actually enhanced due to cysteinylation. These findings fit well with our previous prediction based on algorithms that the minimal {kappa}I binding epitope for DR4 encompassed residues 191–200 (42). Published studies defining the ligand binding motif for HLA-DR4 indicated a preference for hydrophobic primary anchor residues at P1 and P6 as well as secondary anchors potentially at P4, P7, and P9 (70). Based upon the predicted alignment for the {kappa}I epitope, both P1 and P6 would be valine residues with a weaker secondary anchor at P4, the site of cysteinylation. Indeed our binding studies with the unmodified, cysteinylated, and analog peptides suggest that changes in P4 anchor can influence {kappa}I peptide binding to class II DR4. Thus we would predict that cysteine residue within the {kappa}I peptide serves as a contact for MHC as well as TCR. Similarly, studies of class I ligands indicate that epitope cysteinylation can alter either or both TCR contact and binding to MHC proteins (35, 36, 71). Depending upon the position and number of cysteine residues, cysteinylation may influence epitope association with class II molecules potentially explaining the lack of MHC binding reported for cysteine-rich insulin (30) and hen egg lysozyme (29) epitopes. Studies of MHC class I ligands have also suggested that serine or alanine residues can sometimes replace cysteine, avoiding cysteinylation and peptide inactivation (35, 71, 61). Serine replacements of cysteine residues within class II peptide ligands, however, have proven less predictable in restoring functional activity (Fig. 6Go; Ref. 29). Rather, the amino-acid analog aba tested in this study may prove to be a more reliable substitute in epitope design.

Evidence provided here suggests that professional APC including B cells, macrophages, and dendritic cells could efficiently convert the cysteinylated {kappa}I peptide to its active form for class II-restricted T cell presentation. This may prove to be a key difference between class I and class II pathways for Ag presentation, as to date reductive processing of cysteinylated ligands before class I presentation has not been reported. Studies of nonprofessional APC including tumors have revealed minimal capacity for Ag reduction (39, 72), potentially suggesting that cysteinylated peptides may be more abundantly displayed by class I and class II molecules on these cells. At least one lysosomal reductase,IFN-{gamma}-inducible lysosomal thiol reductase (GILT) is lacking in melanoma cells and may play an important role in class II-restricted epitope presentation (72). The localization of active GILT in MIIC and lysosomes, would fit well with the requirement for {kappa}I peptide transit to mature endo/lysosomal compartments before functional presentation (Fig. 2Go). Mechanistic studies with the {kappa}I peptide suggest that cysteinylation and reduction of the free peptide occur efficiently, in contrast with peptide bound to MHC class II molecules. Accessibility of the peptide’s reactive cysteine to reductants or oxidants, was dramatically limited following epitope association with class II molecules. Thus, once bound to class II molecules, the cysteinylated peptide was not readily reduced, nor could the peptide be easily cysteinylated once in place within the MHC binding groove. Studies with proteases have previously demonstrated the protective power of the MHC binding groove, sparing epitopes from over-digestion and inactivation (73). Here, evidence suggests that peptide reduction may actually be more favored before binding class II molecules. Whether GILT or other catalysts of reductive processing in vivo also preferentially reduce peptides or Ag before association with MHC class II proteins remains to be demonstrated. The importance of reductive processing for not only Ag unfolding, but epitope presentation and recognition, is clearly established by this study. Furthermore, cysteinylation of peptide ligands for class II Ags therefore must be considered in the design of vaccine reagents as has been proposed for MHC class I ligands (36, 74), along with the potential for epitope reduction within target presenting cells.


   Acknowledgments
 
We thank Drs. Randy Brutkiewicz and Mark Kaplan for their comments regarding the manuscript, and Dr. Linda Wicker (Merck Research Laboratories) 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.


   Footnotes
 
1 This work was supported with funds from the National Institutes of Health-National Institute of Allergy and Infectious Diseases and National Institute of Diabetes and Digestive Kidney Diseases (to J.S.B.). M.A.H. was supported by National Institutes of Health Training Grant T32 DK07519. Back

2 Address correspondence and reprint requests to Dr. Janice S. Blum, 635 Barnhill Drive, MS 255, Indianapolis, IN 46202. Back

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

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


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