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Role of Disulfide Bonds in Regulating Antigen Processing and Epitope Selection¹

Department of Microbiology and Immunology and Walther Oncology Center, Indiana University School of Medicine, and Walther Cancer Institute, Indianapolis, IN 46202

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Knowledge of the events governing Ag processing and epitope selection within APC is key to the development of novel immunotherapeutic strategies for infectious diseases, cancer, and autoimmunity. The influence of disulfides and Ag reduction on the hierarchy of epitope presentation via MHC class II molecules was investigated through studies of a self Ag, IgG . HLA-DR4⁺ B cells preferentially present an immunodominant IgG-derived epitope, I, relative to a subdominant II peptide. I contains a cysteine masked within the native Ag via an intrachain disulfide, the latter of which is reduced during Ag processing. Mutagenesis of this cysteine as well as others within minimally perturbed the abundance and overall conformation of IgG. Yet, disruptions in disulfide bonding within this Ag influenced the selective display of class II-restricted dominant and subdominant T cell epitopes. Presentation of the I epitope from both native and variant IgG was dependent upon cellular expression of IFN--inducible lysosomal thiol reductase. These studies indicate that disulfide bonds regulate Ag processing both locally and at distant sites, thus influencing epitope selection within the class II pathway.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
During Ag processing, a large pool of peptides is generated, with only a subset of these abundantly displayed in the context of MHC molecules on the surface of APC. This selective presentation of epitopes by MHC proteins modulates T cell responses to foreign Ags and guides the induction of self tolerance. The hierarchy of T cell responses observed in vivo, namely dominant, subdominant, and cryptic, reflects in part this selective presentation of epitopes by MHC molecules, as well as the influences of T cell responsiveness and repertoire. Biochemical and functional studies of MHC class II molecules have revealed the preferential display of immunodominant epitopes, with conversely lower levels of MHC-restricted presentation of subdominant or cryptic peptides derived from the same Ag (1, 2, 3). The specific reactions within APC which influence epitope selection remain poorly defined, with both Ag processing and MHC binding potentially playing key roles (1, 4, 5, 6, 7). In the latter case, both peptide affinity and stable binding to MHC may be important. Thus, expression of the MHC-encoded editor DM has been shown to disrupt the presentation of some cryptic epitopes (7). Ag processing regulates class II-restricted presentation at an earlier step, and may influence both the abundance and structure of peptides generated in APC. During processing, a series of events including proteolysis and reduction take place and may influence epitope selection. Studies using protease inhibitors and knockout animals suggest that multiple enzymes participate in Ag proteolysis, with considerable overlap in the specificity and function of these enzymes (8, 9, 10, 11). By contrast, the requirement for Ag reduction appears more conserved and has been consistently demonstrated for multiple Ags containing cysteine residues and disulfides (12, 13, 14).

Ag reduction occurs within distinct endosomal and lysosomal compartments of APC, with some controversy regarding the specific location and steps involved in this process (12, 15). A lysosomal reductase, IFN--inducible lysosomal thiol reductase (GILT),³ has been identified which can catalyze disulfide reduction and enhance Ag presentation (14, 16, 17). In this study, to examine the importance of Ag reduction and how disulfide bonding influences epitope selection, studies were conducted using a model self protein, human IgG (Ig). Functional studies using HLA-DR4 transgenic mice, as well as human and murine B cell lines, have demonstrated a conserved hierarchy of epitope presentation following the processing of native IgG (1). For exogenous Ag, the immunodominant I epitope identified in these studies (residues 188–203) was presented 15- to 20-fold more efficiently than the subdominant II peptide (residues 145–159). Yet, T cell responses to the synthetic forms of these peptides were similar, demonstrating that Ag processing reactions may control this hierarchy of epitope presentation. The dominant and subdominant epitopes bind to class II proteins with a similar high affinity and stability, such that editing of these complexes by HLA-DM is not observed in vitro (1). The hierarchy of epitope presentation was also confirmed biochemically in B cells expressing endogenous Ag via quantitative sequence analysis (1). The I epitope contains a central cysteine (Cys¹⁹⁴) which forms an intrachain disulfide linkage within native Ag, yet which exists in a reduced form following processing and binding to MHC class II molecules as determined by mass spectroscopy. Studies have demonstrated that reduction of this cysteine residue is critical for T cell recognition of I complexed with MHC (18). By contrast, the subdominant II epitope does not contain cysteine but is located downstream from Cys¹³⁴ which pairs with Cys¹⁹⁴. In vitro reduction of the multiple disulfide bonds within IgG altered the hierarchy of peptides displayed by class II DR4 with the nearly complete loss of I epitope presentation. These results lead to the suggestion that disulfide reduction during Ag processing may regulate the hierarchy of peptide display (1). Immunodominant epitopes from several Ags, including hen egg-white lysozyme (HEL) and insulin, contain or are located near cysteine residues (1, 19, 20), further raising questions as to the role of reductive processing in epitope selection. In this study, mutagenesis of select cysteine residues within Ig was used to examine the importance of disulfide bonding and Ag reduction in epitope selection. Remarkably, gross changes in Ig conformation were not detected following disruption of discrete disulfide linkages within this Ag. Yet, in B lymphocytes expressing endogenous variants, mutations in distinct cysteine residues altered both the hierarchy and efficiency of epitope presentation in the context of class II DR4. In contrast with in vitro chemical reduction of Ag, the systematic disruption of disulfides within this endogenous IgG did not ablate I epitope presentation but rather enhanced II epitope presentation. In some cases the increase in II epitope display was sufficient to actually shift the hierarchy of epitopes presented by class II complexes. These results suggest that the accessibility of epitopes for proteolytic processing or determinant capture by class II molecules may be influenced by disulfide reduction and Ag unfolding.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell lines

43.2.1DR4, a mouse B cell line (I-A^f/d and I-E^d) transfected with chimeric human and mouse DR4w4 and (1), was maintained in RPMI 1640 with 10% FBS. The human B lymphoblastoid cell Frev (DR4w4, DR1), melanoma cells J3.DR4 and J3.GILT.DR4 were cultured in Iscove’s complete DMEM with 10% heat inactivated calf serum, 50 U/ml penicillin, and 50 µg/ml streptomycin. The T cell hybridomas I/I2.18a and I/I1.21 are specific for epitopes I (188–203) and II (145–159), respectively, presented in the context of DR4 molecules. These T cells were maintained in RPMI 1640 supplemented with 10% FBS, 0.1% 2-ME, 50 U/ml penicillin, and 50 µg/ml streptomycin. Previous studies have demonstrated that while these two T cell hybridomas recognize distinct peptides coupled to MHC, the relative number of class II:peptide complexes necessary to trigger the activation of each of these cells is nearly identical (1). Thus, activation of these T cells can be used to quantitate and compare the relative levels of class II:peptide complexes expressed by APC.

Peptides

The human IgG immunodominant peptide I (188–203, KHKVYACEVTHQGLSS), subdominant peptide II (145–159, KVQWKVDNALQSGNS), and substituted forms of the I epitope (Cys¹⁹⁴ replaced with Ala in I-A, or Ile in I-I) were produced using Fmoc technology and an Applied Biosystems synthesizer (Foster City, CA). Peptide purity (95%) and sequence were analyzed and confirmed by reverse-phase HPLC purification and mass spectroscopy.

Site-directed mutagenesis of Ag

A monoclonal human IgG (Ig -wt) which specifically recognizes the ligand HEL was used as a model Ag in this study. Vectors aLys27 and aLys38 contain cDNA encoding the L and H chains, respectively, for this human Ab (21), and were kindly provided by Dr. J. Foote (Fred Hutchinson Cancer Center, Seattle, WA).

To examine the role of discrete cysteines in the processing of this Ag, site-directed mutagenesis of the vector encoding the Ig -chain was conducted. A 500-bp DNA fragment derived from the human C region gene spanning the I and II epitopes was excised from aLys27 with SacI, and then inserted into the corresponding site of pALTER-1. Cys¹⁹⁴ and Cys¹³⁴ contained within this inserted fragment were independently mutated to Ala or Ile using the Altered Sites II In Vitro Mutagenesis System (Promega, Madison, WI). Mutated inserts were screened by restriction analysis and sequencing. The mutated SacI fragments were reinserted in frame in the excised vector aLys27 to yield the vectors aLys27-134A, aLys27-194A, or aLys27-194I, respectively. The orientation of vector inserts was established by DNA sequencing. To generate Ig with Ala-substituted for Cys at positions 134 and 214, the vector aLys27-134A was linearized with BamHI. This linearized plasmid was introduced into the pALTER-1 which was previously cut with BamHI. Mutagenesis of Cys²¹⁴ to Ala was performed in a similar fashion. Excision of the mutated insert yielded the vector aLys27-134A214A. Oligonucleotides used to generate specific mutated forms of Ig are shown in Table I.

Vectors encoding wild-type or mutant L chain were cotransfected with the aLys38 H chain coding vector into 43.2.1DR4 cells. In this study, 1 x 10⁷ B cells were electroporated with 20 µg of the appropriate -chain coding vector, 14 µg of linearized aLys38/PvuI, and 10 µg/ml of DEAE-dextran at 200 V, 900 µF. Transfected cells were selected and cloned based upon vector-encoded drug resistance. To select for Ig transfectants, cells were exposed to 300 µg/ml hygromycin (Calbiochem, La Jolla, CA), 20 µg/ml mycophenolic acid (Life Technologies, Grand Island, NY), and 250 µg/ml xanthine (Sigma-Aldrich, St. Louis, MO). The melanoma J3.DR4 and J3.GILT.DR4 were also cotransfected with Ig H chain and L chain genes by electroporation, and the resulting cell lines were selected using antibiotics followed by subcloning and screening for functional-assembled IgG. Prior studies of the J3 tumor reveal no detectable GILT expression via Western blotting (14, 17). The absence of GILT in these melanomas following transfection with Ig as well as reductase overexpression in J3.GILT.DR4 cells expressing Ig, was confirmed by Western blotting with a GILT-specific polyclonal antisera (data not shown). Following transfection and selection, melanoma cells with and without GILT were also analyzed by FACS using an Ab specific for HLA-DR4. The studies indicated that each of the cell lines expressed similar levels of surface class II DR4.

ELISAs were devised to facilitate screening of IgG -transfected B cells and melanomas. These rely on the specific detection of -chains bound to either a ligand, HEL, or in a parallel assay, plate-bound anti- antisera. Control studies demonstrated that binding to HEL was not detectable using free , thus the former assay measures only assembled IgG H and L chain complexes. Serial dilutions of cell culture medium or cell lysates containing anti-HEL Ig, were incubated on an ELISA plate coated with HEL (8 µg/ml; Sigma-Aldrich) or rabbit anti-human -chain polyclonal Ab (0.8 µg/ml; Sigma-Aldrich), respectively. Biotin-labeled goat F(ab')₂ anti-human (0.1 µg/ml; Southern Biotechnology Associates, Birmingham, AL) and HRP-streptavidin (0.5 µg/ml; Pierce, Rockford, IL) were used to detect the captured proteins and HRP activity measured using ABTS (BioFX, Randallstown, MD).

Western blot

For immunoblots, cells (10⁷) expressing wild-type or mutant Ig -chains were lysed in buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 100 µM tosyl-Lys-chloromethyl ketone, and 200 µM PMSF) on ice followed by centrifugation at 1000 rpm, 5 min to remove nuclei. The protein concentration within cell lysates was determined using the Bio-Rad reagent (Hercules, CA), and equal amounts of protein from each sample were boiled in reducing buffer followed by separation on 10% SDS-PAGE. These proteins were transferred to a nitrocellulose membrane (Osmonics, Westborough, MA) and probed with the rabbit anti-human -chain polyclonal Ab (1:2000; Sigma-Aldrich). This Ab recognizes free -chains, which could be visualized using goat anti-rabbit HRP (1:4000; Jackson ImmunoResearch Laboratories, West Grove, PA) followed by epichemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). GAPDH was detected using an Ab from Chemicon International (Temecula, CA) as a control for protein loading among samples. Benchmark (Invitrogen, Carlsbad, CA) protein standards were used to estimate molecular mass on SDS-PAGE.

Ag presentation assays

For exogenous epitope presentation studies, 43.2.1DR4 was incubated with increasing concentrations of synthetic peptides or Ag for 4–24 h, followed by washing and cultivation with T cells. To monitor endogenous processing and presentation, APCs (43.2.1DR4 Ig) were titrated (100–5000 cells/well) and cocultured with T cells (1 x 10⁴/well) for 24 h. Using variable ratios of APC-T cells ensured that T cell responses to endogenous Ag accurately reflected epitope abundance. For J3.DR4 and J3.GILT.DR4 Ig transfectants, 5000 cells/well were cocultured with T cells (2 x 10⁴/well) for 24 h. Again, variable ratios of tumor cells to T cells were tested to determine assay conditions to ensure the linearity of T cell responses. In all experiments, a range of synthetic I or II peptide concentrations were tested with APC or tumor cells lacking endogenous Ag to ensure the specificity of T cells as well as the relative responsiveness of these cells to peptide:class II complexes. T cell IL-2 production was quantitated using HT-2, an IL-2 dependent T cell line. HT-2 cells were incubated with dilutions of cell culture supernatants from T cell hybridomas for 16 h, followed by the addition of [³H]thymidine (1 µCi/well) for 8 h and harvesting on filters. The levels of [³H]thymidine incorporation were determined by liquid scintillation counting (Microbeta; Wallac, Turku, Finland). Data are expressed as cpm with all assays performed in triplicate and the mean and SD calculated. Each figure is representative of a minimum of three independent experiments.

Peptide competitive binding assay

Aldehyde-fixed Frev cells were incubated overnight with 1 µM biotinylated I peptide and serial increasing concentrations of I, I-A, and I-I peptides in 150 mM citrate-phosphate buffer (pH 5.5), washed with HBSS, and lysed on ice for 20 min in 50 mM Tris buffer (pH 8) containing 0.15 M NaCl and 0.5% IGEPAL CA 630 (Sigma-Aldrich) as described by Pathak et al. (22). The lysates were centrifuged to remove intact nuclei, and the supernatant was added to 96-well polystyrene plates that had been previously coated overnight with the anti-human MHC class II Ab 37.1 and then blocked with FCS. The supernatants were neutralized using 0.02% N-dodecyl -D-maltoside in 50 mM Tris buffer (pH 8) and incubated at 4°C for 4 h. After washing with PBST (0.5% Tween 20 in PBS), DELFIA Eu-labeled streptavidin (PerkinElmer Life Sciences, Gaithersburg, MD) was added for 1 h. After washing, MHC complexes with biotin-tagged peptides were detected by the addition of 0.1 M acetate/phthalate buffer, pH 3.2, containing 0.1% Triton X-100, 15 µM 1-naphthoyltrifluoroacetone, and 50 µM tri-N-octylphosphine oxide, which released the chelated europium from streptavidin to form a fluorescent micellar solution. The resulting fluorescence was measured using a fluorescence plate reader (DELFIA; Wallac).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ig structure is minimally altered following mutation of specific cysteine residues

Disulfide bonds play a central role in maintaining Ag tertiary conformation and disruption of these linkages may influence epitope processing and presentation. Human IgG consists of two disulfide-bonded H chains, each linked via a sulfhydral to Cys²¹⁴ of the subunits. Studies using HLA-DR4 transgenic mice immunized with human IgG revealed several class II-restricted T cell epitopes located within the C region including the immunodominant I (188–203) and subdominant II (145–159) peptides (1). The constant domain forms a globular structure with two anti-parallel sheets held together by a conserved disulfide bond (Cys¹³⁴-Cys¹⁹⁴) (Fig. 1). Sequence analysis of peptides bound to class II DR4 suggests that reduction of this disulfide occurs during Ag processing as Cys¹⁹⁴ within the I epitope is found in a reduced state (1). Additionally, T cell responsiveness is dependent upon reduction of Cys¹⁹⁴ within the peptide (18). To test the hypothesis that disulfide bonds play a key role in guiding epitope selection and the hierarchy of Ig peptides displayed by APC, specific cysteine residues (positions 134, 194, and 214) within this Ag were mutated followed by structural and functional analyses (Table I). To disrupt the intrachain disulfide bond between residues 134 and 194, these cysteines were replaced conservatively by either Ala or Ile. An additional double mutant, with Cys¹³⁴ and Cys²¹⁴ substituted by Ala was also generated to block intrachain disulfide pairing with Cys¹⁹⁴. Despite single or double mutations at these discrete cysteine residues, Ig conformation was largely maintained with comparable expression and stable assembly of functional H and L chains in murine B cells endogenously producing either wild-type or mutant Ags (Fig. 2). Each mutated -chain paired with Ig H chains, forming a functional Ab complex comparable to wild-type Ig in terms of reactivity with the ligand, HEL. Control studies using cells transfected with free -chains alone confirmed that recognition of HEL required H and L chain assembly (data not shown). ELISA results also indicated that the amount of total protein produced within transfected B cell lines was nearly identical regardless of the mutation tested (Fig. 2). To confirm that the anti- Abs used in the ELISA were similarly reactive with equimolar amounts of protein, wild-type and mutant Ig were purified from culture medium and tested using this assay. All of the cells analyzed produced only soluble Ig complexes detectable in both cell lysates and medium. As the Ig H chain tested lacks a transmembrane domain, cell surface expression of these Abs was not predicted or observed.

View larger version (41K): [in this window] [in a new window]   FIGURE 1. The structure of Ig -chain constant domain. The position of three cysteine residues, 134, 194, and 214, is indicated with an intrachain disulfide linkage between Cys134 and Cys194. The location of dominant I 188–203 () and subdominant II 145–159 () epitopes are marked along with the primary sequence of each peptide.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. The stable expression and functional assembly of Ig was not altered by site-directed mutagenesis of conserved cysteine residues. The level of intracellular -chains, as well as IgG assembly and ligand binding, was found to be comparable in APC cotransfected with Ig H chains and either wild-type or mutated -chains. Lysates from 43.2.1DR4 cells transfected with Ig H chains and wild-type or Ig variants were used to quantitate the total amount of cellular protein () or total cellular Ig binding to its ligand HEL (). Detergent lysates of cells were incubated on ELISA plates coated with either an anti-human polyclonal antisera or the ligand HEL. Ig captured in these assays was quantitated using biotin-labeled goat F(ab')2 anti-human and HRP-streptavidin with the substrate ABTS. Results are representative of three independent experiments with the SE for triplicated samples indicated.

View larger version (49K): [in this window] [in a new window]   FIGURE 3. Structural differences in -chain variants were detectable after denaturation and reduction. Western immunoblot of unfolded wild-type or mutant -chains from transfected APC revealed differential reactivity of these proteins using a polyclonal antisera specific for denatured L chains. 43.2.1DR4 cells cotransfected with Ig H chains and wild-type or mutated proteins were lysed in detergent and samples (50 µg/lane) prepared by boiling and reduction for SDS-PAGE, followed by detection with a polyclonal antisera recognizing denatured human . This blot was reprobed with an Ab to GAPDH to quantitate protein loading.

Impact of disulfide bonding on the hierarchy of epitope presentation

To address the question of whether disulfide bonds influence the selection of immunodominant T cell epitopes, functional studies of Ag presentation were conducted using DR4⁺ B cell lines expressing endogenous wild-type and mutant human Ig proteins as APC. Murine DR4⁺ B cells expressing endogenous Ig preferentially displayed the immunodominant I epitope relative to the subdominant II peptide as assessed by T cell activation (Fig. 4 and Table II). Comparison of the relative amounts of epitope presentation is possible as the T cell hybridomas used in this study require similar numbers of peptide-MHC complexes to trigger activation (1, 18). The specificity and similar responsiveness of these T cells were confirmed using synthetic peptides and the B cell 43.2.1.DR4 which lacks endogenous (Fig. 4). In contrast, with results from cells producing endogenous wild-type IgG, distinct patterns of T cell responses to the epitopes were observed dependent upon whether Cys¹⁹⁴ or Cys¹³⁴ of the Ag was mutated to Ala (Fig. 4 and Table II). T cells recognizing the I epitope were unresponsive to B cells expressing Ig -194A, while T cell responses to the II epitope from this Ag were nearly doubled (Table II). By contrast, functional assays using the Ag Ig -134A, revealed an even greater increase in II epitope presentation, with only a slight increase in T cell responses to the I epitope when compared with wild-type Ag. Thus, disruption of a single disulfide bond within Ag radically altered T cell responses. In B cells expressing the double-mutant Ig -134A214A, T cell responses to the I epitope were nearly equivalent to that observed with wild-type Ag, yet there was a significant increase in the presentation of the II epitope (Fig. 4). Thus, mutation of Cys¹³⁴ within Ig led to an alteration in the hierarchy of T cell responses with nearly equivalent presentation of the I and II epitopes (Table II). These results suggest that changes in the disulfide bonding pattern of an Ag may influence T cell responses to distinct epitopes and the hierarchy of peptides displayed by APC.

View larger version (40K): [in this window] [in a new window]   FIGURE 4. Disruption of disulfide bonds within Ig altered T cell responses to distinct class II-restricted epitopes. The APC, 43.2.1DR4 transfected with either wild-type Ig or the mutant proteins Ig -194A, Ig -134A, Ig -134A214, or Ig -194I was cocultured with I- and II-specific T cells for 24 h. Data represent results from an assay in which 500 APC were incubated with 1 x 104 T cells, however, variable ratios of APC-T cells were tested showing a similar hierarchy of T cell responses to endogenous Ag. T cell responses to dominant I () and subdominant II () epitopes were assessed via cytokine production as detected by HT-2 cell proliferation. Results are also shown for 43.2.1DR4 cells incubated with 5 µM of synthetic I or II peptides, respectively, confirming the relative responsiveness of T cells.

View this table: [in this window] [in a new window]   Table II. Comparative analysis of T cell recognition of I and II epitope presentationa

T cell recognition of the I immunodominant epitope

The failure of T cells to detect the I epitope following processing of the mutant Ag Ig -194A contrasted with results using Ig -134A, yet each mutation disrupted the same disulfide linkage within IgG. Studies were conducted to dissect whether substitutions at position 194 within the Ag directly influence TCR or MHC contacts, rather than Ag processing. Prior studies of synthetic variants of the I peptide demonstrated that bulky, hydrophobic replacements at position 194 such as 2-aminobutyric acid were superior to weakly polar residues such as Ser in terms of triggering T cell recognition (18). With this in mind, the ability of synthetic peptides including the original I with Cys¹⁹⁴, I-A with Ala at 194, and I-I with Ile at position 194 were tested for binding to class II proteins as well as T cell activation. Competitive binding studies revealed little difference in the relative affinity of each of these peptides for class II DR4 molecules (Fig. 5A). By contrast, T cell recognition of the I-A peptide was reduced by approximately one-half compared with the original I and I-I peptides (Fig. 5B). This result suggested that position 194 of the I peptide is a TCR contact site, potentially explaining differential T cell responses to the mutated Ags Ig I-194A and Ig I-134A (Fig. 4 and Table II). However, T cell responses to the synthetic I-I peptide were comparable to the wild-type I peptide, suggesting that mutagenesis of position 194 to Ile within would be informative in terms of Ag processing. Endogenous presentation of Ig -194I resulted once more in the preferential activation of T cells responsive to the I-I epitope relative to II (Fig. 4). Yet T cell responses to both epitopes were enhanced with this mutant Ag compared with wild type, with the II epitope display approaching that observed with Ags lacking Cys¹³⁴ (Table II). The results of these functional assays again suggest that intrachain disulfide bonding can influence epitope selection, and the hierarchy of peptides displayed by class II proteins.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. Mutations of Cys194 within the I epitope influenced TCR recognition. A, Synthetic I, I-A, and I-I peptides bound with a similar affinity to class II DR4 molecules. Aldehyde-fixed B cells were incubated with 1 µM biotinylated I peptide and serial increasing concentrations of either I (), I-A (), or I-I () peptides overnight for competitive binding analysis. These cells were lysed and the extent of biotinylated-peptide binding to DR4 was quantitated via a capture ELISA using europium-labeled streptavidin. The percentage binding for each variant I peptide was nearly equivalent to that of the original I epitope. Results are representative of three separate studies. B, T cell response to the peptide I-A were significantly decreased. 43.2.1DR4 cells were incubated with either I, I-A, or I-I peptides at 37°C for 16 h, respectively, and were followed by addition of the T cell hybridoma I/I2.18a. IL-2 production was measured as described in Materials and Methods. These results are representative of three separate experiments.

Requirement for GILT in reducing mutated Ig Ag

A thiol reductase GILT has been demonstrated to play a role in Ag reduction, thus facilitating processing and presentation of some epitopes in the context of class II molecules (14, 17). Disruption of disulfide bonds within an Ag that are proximal to immunodominant and subdominant epitopes via mutagenesis might diminish the requirement for GILT during processing and thus providing further insights into the role of protein disulfides in epitope selection. Although B cells constitutively express GILT (16), this enzyme is lacking or greatly diminished in nonprofessional APC such as melanomas (16, 17). Efficient processing and presentation of the I epitope from endogenous, wild-type Ig was dependent upon GILT as demonstrated using J3.DR4 melanoma cells lacking or expressing this reductase (Fig. 6). Thus, in melanomas lacking GILT, T cell responses to the I epitope were greatly diminished compared with cells expressing GILT. Unexpectedly, T cell recognition of the I epitope following processing of endogenous Ig -134A214A, although improved relative to wild-type Ig, remained largely dependent upon GILT even with disruption of the disulfide linkage Cys¹³⁴-Cys¹⁹⁴ within this Ag. In both wild-type Ig and Ig -134A214A, presentation of the subdominant II epitope was found to be conserved and independent of cellular GILT expression. In these studies of endogenous Ag presentation, melanomas with or without GILT were cotransfected with Ig H chain and the appropriate -chain genes, followed by subcloning and analysis to ensure cell lines expressed similar levels of HLA-DR4 and human IgG. The transfected melanomas produced only soluble Ig proteins detectable in the cell lysates and culture medium. Control studies using the synthetic II peptide and tumor cells lacking endogenous Ag, demonstrated no requirement for GILT for presentation of this peptide to T cells. By contrast, the synthetic I peptide which is readily cysteinylated in cell culture medium, requires intracellular processing by APC such that T cell activation is only detectable using presenting cells expressing GILT (17, 18). These results suggest that processing of peptides within the Ag Ig was dependent upon disruption of disulfides both proximate and distant to the relevant epitopes. Interestingly, the hierarchy of epitope presentation in these melanoma cells differed from that observed in B lymphocytes even following GILT overexpression. These results suggest that reduction is one of several processing steps which may differ between tumors and professional APC, thus potentially influencing epitope selection and display.

View larger version (33K): [in this window] [in a new window]   FIGURE 6. Processing and display of the I eptiope from Ag variants lacking an intrachain disulfide was dependent upon cellular GILT expression. Melanomas J3.DR4 or J3.GILT.DR4 transfected with Ig or Ig 134A214A (5 x 103 cells/well) were incubated with T cells (2 x 104/well) specific for either the I () or II () epitopes, respectively, for 18–20 h. T cell activation was measured as indicated in Materials and Methods. To demonstrate that this requirement for GILT is linked to epitope processing, I and II peptides (10 µM) were incubated with J3.DR4 or J3.GILT.DR4 lacking endogenous Ag, followed by cell culture with the appropriate T hybridoma lines. Efficient presentation of the II peptide was observed independent of cellular GILT expression, in contrast with the I peptide which is cysteinylated and requires GILT for functional recognition by T cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Indeed, functional studies analyzing the processing and presentation of discrete T cell epitopes within wild-type and mutated Ig Ags revealed very specific changes in epitope selection with disruption of disulfide bonds. Although chemical reduction of all disulfides within IgG resulted in a loss of presentation of the dominant I epitope (1), in this study, selective mutations to disrupt specific thiol linkages had a very different effect. I-specific T cell responses to one mutant protein, Ig -194A were diminished, however, this may largely relate to reduced TCR recognition of Ala¹⁹⁴ within this peptide. In most cases, the MHC-restricted display of the dominant I epitope was preserved regardless of the presence or absence of a disulfide linkage within this peptide. These findings suggest that this epitope may be exposed or readily reduced early during Ag processing. Class II presentation of the I epitope could be slightly enhanced using variant Ags (Ig -194I; Ig -134A) with mutations disrupting the disulfide bond between Cys¹³⁴ and Cys¹⁹⁴. These results would be consistent with localized unfolding within this domain, facilitating I presentation.

Remarkably as shown in B cells, presentation of a subdominant II epitope (residues 145–159) was most dramatically influenced by select mutations of cysteine residues within Ig. This epitope lacks cysteine yet substitutions at either Cys¹³⁴ or Cys¹⁹⁴ within Ig consistently resulted in enhanced epitope presentation. These findings suggest that localized changes in the structure of Ig, due to disulfide bond disruption at Cys^134–194 can significantly enhance the processing and presentation of this epitope to shift the hierarchy of epitope display. Because an increase in II epitope presentation was observed with mutation of either Cys, it seems unlikely that the observed effects are linked to alterations in the primary structure of the Ag. Also, for the majority of the mutant Ags tested, T cell responses to the I epitope were only minimally enhanced or unchanged suggesting that there is not a competition between these two peptides for binding to MHC class II molecules. Functional presentation of the II epitope was observed in melanomas lacking GILT, suggesting that disulfide reduction is not absolutely required for formation of this peptide in these cells. It was somewhat surprising that the expression of GILT did not significantly alter T cell responses to the II epitope following endogenous Ag processing. Studies of exogenous IgG presentation in melanomas did indicate that II epitope display could be enhanced 3- to 4-fold with cellular GILT expression (17). This may indicate that there are alternate pathways for forming this epitope dependent upon the sources of Ag as well as the type of presenting cell, with only some of these routes relying on intracellular reduction.

Our studies demonstrate that for native IgG, efficient presentation of the I epitope required cellular expression of the endo/lysosomal reductase, GILT. Furthermore, GILT appears to be required for reduction of disulfides beyond the intrachain linkage at Cys¹³⁴-Cys¹⁹⁴. Efficient presentation of the I epitope from the mutant Ag Ig -134A214A, which lacks an intrachain disulfide, remained largely dependent upon intracellular GILT expression. Ag reduction by GILT may occur in a stepwise fashion coupled with other sequential processing events such as proteolysis or determinant capture. As indicated using IgG denatured and chemically reduced to disrupt all disulfides in vitro, a complete loss of I epitope presentation was observed (1). Yet, in this study, by selective disruption of disulfide linkages using mutated Ig, presentation of the I peptide was retained. These results suggest that in vivo, Ag reduction and processing very likely occur in discrete stages with selective disulfide bond cleavage influencing and guiding epitope selection.

For both MHC class I and class II presentation pathways, studies indicate that changes in Ag structure both proximal and distant to specific epitopes can alter the efficiency and hierarchy of epitope presentation (27, 28). These changes induced by site-directed mutagenesis of Ag, have been presumed to primarily influence protease susceptibility. Indeed, changes in predicted protease cleavage sites can alter epitope presentation (29, 30, 31). In this study, mutagenesis of specific disulfide bonds within an Ag was also shown to influence the efficiency of epitope presentation by MHC class II molecules. These studies demonstrate that cleavage of disulfide bonds within an Ag, both proximate and distant from a specific epitope can also regulate presentation and epitope selection.

	Acknowledgments

 
We thank Drs. Mark H. Kaplan, Randy R. Brutkiewicz, and Michael J. Klemsz for their comments regarding the manuscript, and Dr. Peter Cresswell for provision of J3 melanoma cells and reagents for GILT expression and detection. We also thank Dr. Q. Chen, D. Zhou, and P. O’Donnell for their helpful discussions, and S. K. Jackson for technical assistance.

	Footnotes

 
¹ This study was supported by National Institutes of Health Grant No. AI33418 and a Phi Beta Psi award (to J.S.B.). M.A.H. was supported by the Arthritis Foundation, Indiana Chapter.

² Address correspondence and reprint requests to Dr. Janice S. Blum, Medical Science Building, Room 255, 635 Barnhill Drive, Indianapolis, IN 46202-5120. E-mail address: jblum{at}iupui.edu

³ Abbreviations used in this paper: GILT, IFN--inducible lysosomal thiol reductase; HEL, hen egg-white lysozyme.

Received for publication April 23, 2002. Accepted for publication July 1, 2002.

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