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The Journal of Immunology, 2000, 164: 1775-1782.
Copyright © 2000 by The American Association of Immunologists

Processing and Reactivity of T Cell Epitopes Containing Two Cysteine Residues from Hen Egg-White Lysozyme (HEL74–90)1

Hee-Kap Kang2,*, John A. Mikszta23*, Hongkui Deng4,{dagger}, Eli E. Sercarz5,{dagger}, Peter E. Jensen{ddagger} and Byung S. Kim6,*

* Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, IL 60611; {dagger} Department of Microbiology and Molecular Genetics, University of California, Los Angeles, CA 90024; and {ddagger} Department of Pathology, Emory University, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Ag processing and structural requirements involved in the generation of a major T cell epitope from the hen egg-white lysozyme protein (HEL74–88), containing two cysteine residues at positions 76 and 80, were investigated. Several T cell hybridomas derived from both low responder (I-Ab) and high responder (I-Ak) mice recognize this region. These hybridomas are strongly responsive to native HEL, but unresponsive to the reduced and carboxymethylated protein. Air-oxidized HEL74–88 peptide was unable to bind I-Ak molecules and failed to stimulate T cells in the absence of intracellular Ag processing. Further functional competition assays showed that alkylation of cysteine residues with bulky methyl groups interferes with the contacts for the MHC class II molecules (I-Ak) of high responder mice and the I-Ab-restricted TCR of low responder mice. Serine substitutions of the cysteine residues of HEL74–88 either enhanced or abrogated T cell stimulation by the peptides without significant alterations in the class II binding. These results suggest that the cysteine residues of peptides must be free from disulfide bonding for efficient stimulation of T cells and yet frequently used modifications of cysteine residues may not be suitable for peptide-based vaccine development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The generation of both humoral and cell-mediated immune responses against exogenous foreign Ags requires the stimulation of Ag-specific Th cells. The general paradigm for the recognition of such Ag by Th cells is that, after internalization by an APC, the Ag must first be enzymatically processed to a sufficient degree to allow for MHC class II binding. These complexes of antigenic peptides and class II molecules are then transported to the cell surface where a trimolecular interaction with an Ag-specific TCR leads to activation of the Th cell and initiation of the immune response (reviewed in Refs. 1 and 2).

Through elution studies, it has been shown that peptides associated with class II molecules generally range in length from about 10–30 aa residues (3, 4, 5, 6). The recent determination of the crystal structures of human and mouse class II molecules complexed with peptides show that in contrast to class I, the peptide binding groove of class II is open at both ends, thus allowing for the binding of longer polypeptide chains (7, 8). T cell clones and hybridomas have been described that respond strongly to reduced and denatured protein Ag, even in the presence of enzymatic processing inhibitors or chemically fixed APC (9, 10, 11, 12). These studies provided the initial suggestion that unfolding may in many cases be sufficient for the binding of a protein sequence to class II molecules. Sette et al. (13) directly examined this possibility and determined that the reduced, but not the native, form of several proteins could bind to selective class II molecules. Jensen (14) has more recently described the capacity of various protein Ag to bind to class II molecules in the presence of a reducing agent at low pH. Thus, partial unfolding of a protein Ag, through reduction of disulfide bonds at low pH, is often sufficient for class II binding and subsequent T cell stimulation.

Interestingly, several investigators using a number of different Ag systems, have described T cells that appear to require native protein Ag or certain aspects of tertiary structure for stimulation (15, 16, 17, 18, 19). With the exception of fibrinogen (17), however, such studies were neither conducted in the presence of Ag processing inhibitors nor used chemically fixed APC. It is therefore uncertain whether the T cells used in these studies were, in fact, recognizing conformation-dependent regions of the proteins or peptide fragments. The possibility that intact, tightly folded, compact proteins can bind to class II molecules seems rather unlikely due to the structural constraints involved in MHC binding (7, 8), unless the particular epitope in question is localized to a conformationally flexible region of the protein (17, 20). However, the influence of protein or peptide folding via disulfide bonds on Ag processing and presentation to T cells has not fully been explored.

Previous studies using hen egg-white lysozyme (HEL)7 as a model Ag have determined that two sets of overlapping T cell epitopes are present within amino acid residues 46–61 (21, 22) as well as 74–96 (23, 24) for both high responder (I-Ak, I-Ek) and low responder (I-Ab) mice. Interestingly, three cysteine residues are located at positions 76, 80, and 94 within 74–96. These residues are involved in forming the tertiary structure of HEL by disulfide bonding with other residues (76–94 and 64–80) in HEL (25). C57BL/6 mice immunized with native HEL in CFA generate strong T cell responses against HEL74–90, but fail to respond to the overlapping HEL81–96 region (24). However, this strain immunized with the partially unfolded peptide fragment encompassing residues 13–105 responds strongly to HEL81–96, but not to HEL74–90 (24). Additionally, T cell hybridoma clones specific for the HEL81–96 region respond poorly to native HEL, but very strongly to the protein with reduced and carboxymethylated cysteines (CM-HEL), as well as to HEL covalently modified by a diazonium salt (12). These results suggest that complete or partial unfolding of HEL is capable of enhancing the presentation of this epitope.

To further understand the nature of T cell recognition for such a modification, T cell reactivity to the epitopes at the site of modification has been investigated. In this study we demonstrate that T hybridoma clones specific for the HEL74–90 epitope from low responder C57BL/6 (H-2b) and high responder C3H/HeJ (H-2k) mouse strains are completely unresponsive to covalently cysteine-modified CM-HEL, in sharp contrast to the T cells specific for the overlapping HEL81–96 region. Subsequently, the potential role of the HEL tertiary structure conferred by double-cysteine residues within the T cell epitopes was further investigated using native HEL, CM-HEL, as well as the peptides with/without cysteine modifications. The ability of the epitope(s) to stimulate T cells is highly dependent on the reduced cysteine form. The failure of air-oxidized HEL74–90 to bind class II or to stimulate T cell hybridomas supports the necessity of the reduction of disulfide bonds for presentation of these epitopes to T cells. The failure to respond to CM-HEL or CM-HEL74–90 appears to be due to the modification of the cysteine residues that are critically involved in contacting MHC class II and/or the TCR. These results suggest that unfolding and reduction of this epitope are important prerequisites for T cell recognition of the HEL74–90 epitope and provide a significant implication for peptide-based vaccine development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Inbred female C57BL/6 and C3H/HeJ mice (4–6 wk old) were purchased from Charles River Laboratories (Wilmington, MA) via the National Cancer Institute (Bethesda, MD). B10.A mice were purchased from The Jackson Laboratory (Bar Harbor, ME).

Antigens

Three times crystallized HEL was purchased from the Sigma (St. Louis, MO). Phosphorylcholine (PC) was covalently conjugated to HEL by diazonium linkage, as described previously (12, 21, 26). Synthetic peptides were prepared by the F-moc method (27) using the RaMPS system (DuPont, Wilmington, DE). Peptides used in this study included: HEL74–88, NLCNIPCSALLSSDI; HEL47–61, TDGSTDYGILQINSR; HEL81–93, SALLSSDITASVN; and a non-HEL-derived viral (A1Bb) peptide, PADVTDQLIGYTPSL (28). Cysteine-methylated HEL (CM-HEL) and HEL74–88 (CM-HEL74–88) were prepared according to the method of Lee and Atassi (29). Briefly, HEL or peptide (7 µmol) was dissolved in 5 ml of 0.25 M triethylamine-acetate buffer containing 8 M urea and subsequently reduced with a final concentration of 1.3 M 2-ME. The reduced and unfolded HEL was precipitated, washed with ethanol/HCl (98/2, v/v), and then carboxymethylated by addition of iodoacetic acid (0.1 M) for 2 h. CM-HEL was recovered by precipitation in ethanol/HCl followed by extensive washing and dialysis against H2O to remove excess 2-ME, iodoacetic acid, and urea. These low m.w. compounds were removed from the CM-HEL74–88 solution by ultrafiltration through Amicon-1000 membranes (Amicon, Beverly, MA), and the carboxymethylated peptide was then recovered by lyophilization. Oxidation of the peptide was accomplished by incubation of the sterile peptide solution at 37°C for 2–3 h followed by further storage at 4°C.

T cell hybridoma clones

T cell hybridoma clones used in this study included A2.5 specific for HEL83–93/I-Ab (12), A69.5 specific for HEL76–85/I-Ab (Y. S. Jang and B. S. Kim, unpublished observations), BO4H specific for HEL74–90/I-Ab (24), PCH4.1 specific for HEL51–60/I-Ab (26), AOIT2.11 (abbreviated AOIT) specific for HEL74–82/I-Ak (30), and AO4H.H.9.1 (abbreviated AO4H) specific for HEL73–82/I-Ak (31). Culture medium for maintenance and stimulation of the cell lines consisted of RPMI 1640 (Sigma) supplemented with 5% FCS and 5 x 10-5 M 2-ME.

Ag presentation assays

T hybridoma cells (1 x 105) were cultured in triplicate in flat-bottom 96-well microtiter plates (Costar, Cambridge, MA) for 24 h with various concentrations of Ag or PBS in the presence of 5 x 105 irradiated (3000 rad), syngeneic splenocytes as APC. T cell hybridoma stimulation was based on IL-2 production measured by the ability of the culture supernatants to support proliferation of the IL-2-dependent cell line, CTLL2 (32, 33). Briefly, 100 µl of supernatants were added to 7.5 x 103 CTLL2 cells in 100 µl of culture medium. After 24 h, wells were pulsed with [3H]TdR (1 µCi/well) and incubated for an additional 14–18 h before harvesting. Levels of [3H]TdR uptake were determined by liquid scintillation counting. Data represent the maximum counts per minute, in which the background level of [3H]TdR uptake in cultures with PBS alone was subtracted from the level of proliferation to Ag (the mean counts per minute of triplicate cultures ± SE). For assays using the reducing agent, DTT, paraformaldehyde-fixed APC were incubated for 24 h at 37°C with Ag in the presence or the absence of 2 mM DTT (14). Preloaded APC were then washed and cultured with T hybridoma cells as described above.

Inhibition of Ag processing

Inhibition of Ag processing was conducted as previously described (12, 26) using leupeptin or NH4Cl (Sigma). Briefly, irradiated splenocytes (5 x 105/well) were incubated at 37°C with 0–1 mM leupeptin or 0–15 mM NH4Cl for 15 min before Ag exposure and then further incubated with T hybridoma cells (1 x 105) for an additional 24 h. Alternatively, APC (1 x 107 cells/ml) were fixed with 0.1% paraformaldehyde (Sigma) according to previously described methods (26). The fixation was terminated by addition of an excess of cold 0.5% glycyl glycine in PBS. The cells were then washed three times with PBS containing 10% FCS, and further incubated for 1 h at 37°C in RPMI 1640 culture medium. The fixed splenocytes (1 x 106 cells/well) were used as APC.

MHC class II binding assay

A functional competition assay was used to evaluate the relative class II binding ability of the CM-HEL74–88 peptide (34). T hybridoma cells were cultured with live APC, as described above, in the presence of a suboptimal dose of stimulatory peptide (HEL74–88) and various concentrations of competitor test peptides or PBS. Test peptides capable of binding to class II molecules can compete with the stimulatory peptide for binding and result in inhibition of the T cell response. Data are expressed as the percent inhibition of the response induced by HEL74–88 as calculated by the following formula: [(cpm of PBS-treated control) - (cpm with inhibitor)/(cpm of PBS-treated control)] x 100.

Direct binding of MHC class II molecules with various peptides was assessed by the levels of competition of binding between isolated class II molecules and biotin-labeled, appropriate haplotype-reactive peptides using a capture ELISA with europium-labeled streptavidin as described previously (35). HEL46–61 was used as a probe peptide for I-Ak-binding. Data points represent mean fluorescent counts per second.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
T cells specific for the HEL74–90 region are unresponsive to CM-HEL

To examine the structural requirements involved in generation of the overlapping HEL74–90 and HEL81–96 regions, we initially compared the ability of T cell hybridomas specific for these regions to respond to native HEL and chemically modified HEL. Both I-Ab-restricted (A69.5 and BO4H) and I-Ak-restricted (AOIT and AO4H) T hybridomas specific for HEL74–90 were completely unresponsive to unfolded CM-HEL (Fig. 1Go). In addition, these hybridomas did not show an enhanced reactivity to HEL following diazonium-linked hapten conjugation (PC-HEL; Fig. 1Go). Such a modification has resulted in more efficient generation of HEL46–61 and HEL81–96 T cell epitopes (12, 26). For example, a HEL81–96-specific T hybridoma (A2.5) was stimulated very poorly by native HEL, but responded strongly to CM-HEL and PC-HEL (Fig. 1Go). Collectively, these results initially suggested that unfolding enhances the generation of HEL81–96, while the overlapping HEL74–90 epitopes (recognized by A69.5, BO4H, AOIT, and AO4H) are inhibited by this process. Thus, the failure of HEL74–90-specific T cells to respond to CM-HEL may reflect the T cell recognition of a conformational epitope that is destroyed in the unfolded derivative.



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  		FIGURE 1. Stimulation of I-Ab-restricted (A69.5 and BO4H) and I-Ak-restricted (AOIT and AO4H) T cell hybridoma clones specific for the HEL74–90 region as well as representative I-Ab-restricted hybridomas specific for HEL46–60 (PC-H4.1) and HEL81–96 (A2.5) with HEL, PC-HEL, and CM-HEL. Cultures were incubated with 30 µM of the indicated protein Ag for 24 h in the presence of irradiated, sygeneic splenocytes as APC. Levels of IL-2 in culture supernatants were determined using the IL-2-dependent cell line, CTLL2.

 
Generation of the HEL74–90 region requires Ag processing

To directly address the possibility that the conformation of Ag is critical for T cell stimulation, the requirement for Ag processing was examined. Conventional Ag processing inhibitors were used to determine the processing requirements for the generation of HEL74–90 from native HEL. Leupeptin was used to inhibit serine/cysteine proteases such as cathepsins, while ammonium chloride was used as a general inhibitor of acidic proteases by raising the intracellular pH of endosomal/lysosomal compartments (36, 37, 38). Interestingly, the stimulation of HEL74–90-specific T cell hybridomas from both MHC haplotypes was relatively resistant to leupeptin compared with the stimulation of T hybridomas recognizing other regions of HEL, such as PCH4.1 (21, 26), which is specific for HEL51–60 (Fig. 2Go and data not shown). However, the generation of HEL74–90 was less affected by NH4Cl treatment compared with that of HEL51–60 (Fig. 2Go). Collectively, these results suggest that generation of this HEL74–90 epitope region requires relatively low level of enzymatic processing.



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  		FIGURE 2. Effect of Ag processing inhibitors on the stimulation of HEL74–90-reactive T cell hybridoma clones. Cultures were incubated with 30 µM native HEL or PBS in the presence of irradiated, syngeneic splenocytes as APC with or without the processing inhibitors. Control wells were treated with PBS instead of the inhibitors. The PCH4.1 T cell hybridoma clone, specific for HEL51–60, was used as a control to verify that the concentrations of inhibitors were sufficient to block the stimulation of other HEL epitopes. The reduced stimulation of PCH4.1 was observed using native HEL, but not a minimal epitope peptide that does not require additional processing.

 
To conclusively determine whether intracellular processing is required to generate the HEL74–90 region from native HEL, APC were chemically fixed with paraformaldehyde and used to present HEL or freshly prepared peptide to representative T cell hybridomas (Fig. 3Go). HEL was nonstimulatory in the presence of fixed APC, while stimulation by the HEL74–88 peptide remained high. Thus, HEL appears to require Ag processing to reveal the HEL74–90 region, although the degree of such processing appears to be relatively low (Fig. 2Go) compared with that required to generate other T cell epitopes from the protein.



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  		FIGURE 3. Requirement for Ag processing to generate the HEL74–90 region. Representative HEL74–90-specific T cell hybridoma clones, A69.5 and AOIT2.11, were cultured with HEL (30 µM) or HEL74–88 peptide (10 µM) in the presence of paraformaldehyde-fixed or control (PBS)-treated syngeneic splenocyte APC.

 
Fixed APC can present unfolded HEL by reduction alone without carboxymethylation

The failure of HEL74–90-specific T cells to respond to CM-HEL may actually be due to the direct modification of cysteine residues at positions 76 and 80 rather than the inability to respond to unfolded protein. To determine whether protein unfolding without cysteine alkylation is able to stimulate the HEL74–90-specific T cells, paraformaldehyde-fixed APC were pulsed with HEL or peptide in the presence or the absence of the reducing agent, DTT, and subsequently cultured with the T hybridomas. Interestingly, all the hybridomas reactive to this region were responsive to only unfolded HEL, and representative results are shown in Fig. 4Go. However, such stimulation was rather inefficient, as even 2- to 10-fold higher molar concentrations of native HEL induced a much lower stimulation compared with DTT-reduced peptide presented by the fixed APC or HEL presented by intact APC (Fig. 4Go and data not shown). In addition, these hybridomas poorly recognized air-oxidized HEL74–88, and the stimulation was markedly increased in the presence of DTT, suggesting that the cystine(s) should be reduced even for the peptides to stimulate the T cells (Fig. 4Go). Unfixed APC have previously been shown to reduce disulfide bonds (39), which is consistent with the ability of unfixed APC to present oxidized peptide in the absence of added reducing agent. A T cell hybridoma specific for a different HEL epitope lacking cysteine was stimulated equally well by the peptide and fixed APC in the presence or absence of DTT (data not shown). Thus, this reducing reagent at the concentration used is not likely to confer a nonspecific enhancement of T cell stimulation. Together, the cysteine residues at positions 76 and/or 80 must be maintained in an unmodified and reduced form to be recognized by MHC class II and/or the epitope-specific TCR.



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  		FIGURE 4. Presentation of HEL and oxidized HEL74–88 peptide by fixed APC in the presence or the absence of DTT. Paraformaldehyde-fixed I-Ab (C57BL/6) splenocytes were pulsed with HEL (70 µM) or oxidized HEL74–88 peptide (30 µM) for 18 h in 0.15 M citrate/phosphate buffer (pH 5.0), plus 2 mM DTT or PBS as a control. Similarly fixed I-Ak (C3H/HeJ) splenocytes were pulsed with HEL (30 µM) or oxidized HEL74–88 peptide (30 µM). After extensive washing, T hybridoma cells were added and further incubated for 18–24 h with the prepulsed APC.

 
Internalization of Ag/peptide into APC is necessary for cystine reduction

To determine whether internalization of Ag is necessary for cystine reduction, APC were incubated for 6 h in the presence or the absence of 0.1% sodium azide at pH 7.0 before incubation with T cell hybridomas (Fig. 5Go). To prevent continuous internalization of Ag, aliquots of APC were fixed with paraformaldehyde immediately after the 6-h incubation. Internalization of oxidized HEL74–88 and HEL appears to be necessary for Ag presentation to T cells in the absence of DTT, strongly suggesting that the reduction occurs intracellularly. Similar results were observed at pH 5.0, although the levels of T cell stimulation were lower, and this may be due to the inhibition of Ag internalization at this pH (data not shown). In the presence of DTT, a relatively low level of T cell stimulation was obtained by HEL, and a high level was obtained by the oxidized peptide. In addition, membrane-associated proteases may not be involved in the trimming of HEL to facilitate the interaction between the reduced HEL and class II molecules, because the level did not increase during the 6-h incubation in the presence of sodium azide.



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  		FIGURE 5. Requirement of Ag internalization to present HEL and oxidized HEL74–88. Irradiated syngeneic splenocyte APC were incubated with native HEL (140 µM) or HEL74–88 (60 µM) in the presence or the absence of 0.1% NaN3 for 6 h at pH 7.0. After the 6-h incubation at 37°C, APC were washed and then fixed with paraformaldehyde. Unfixed APC were used as a control. T hybridoma cells (A69.5) were incubated with the APC for 18–24 h, and then the level of IL-2 production was measured as described above.

 
To determine the time required for the disulfide bond reduction of cystines on HEL and oxidized HEL74–88 peptide, a time-course experiment was performed (Fig. 6Go). Native HEL and oxidized peptide were processed for various time periods, fixed with paraformaldehyde, and then examined for their ability to stimulate specific T cells. The results strongly suggest that the epitopes efficiently presentable to T cells, including the reduction of cystine residues, are generated 2–4 h following internalization of the peptide or HEL. This relatively delayed generation suggests that reduction of the determinants may occur in a late stage of HEL processing.



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  		FIGURE 6. Determination of the time course required to present HEL and oxidized HEL74–88. Splenic APC were pulsed with either intact HEL or air-oxidized HEL74–88 for varying periods and then fixed with paraformaldehyde as described in Materials and Methods. T hybridoma cells (A69.5) were incubated for 24 h with the Ag-pulsed fixed APC as well as similarly treated unfixed control APC. The generation of the epitope was assessed by the stimulation of epitope-specific T cell hybridomas based on the level of IL-2 production.

 
Carboxymethylation of HEL74–88 peptide abrogates T cell stimulation

To directly investigate the status of cysteine residues for TCR recognition, the HEL74–88 peptide was carboxymethylated and tested for the ability to stimulate I-Ak-restricted AOIT and I-Ab-restricted A69.5 hybridomas (Fig. 7GoA). Interestingly, carboxymethylation of the peptide (CM-HEL74–88) completely abrogated the ability of the peptide to stimulate either of the T cell hybridomas. These results provide direct evidence that methylation of the cysteine residues destroys the epitope function. Thus, the unmodified cysteine residues are probably required for class II binding and/or TCR contact in both the I-Ak- and I-Ab-restricted responses to this region. The CM-HEL74–88 peptide was unable to inhibit the HEL74–88-induced response of AOIT, suggesting poor binding of the peptide to I-Ak (Fig. 7GoB). In contrast, this modified peptide appeared to interact with I-Ab, based on the significant inhibition of the HEL74–88-induced response of A69.5 similar to the level by a known I-Ab binding peptide, HEL81–93 (Fig. 7GoB). This result suggests that the lack of response by A69.5 to CM-HEL74–88 is due to the inability to interact with the TCR. Therefore, the failure of HEL74–88-specific T cells to respond to CM-HEL is probably due to the direct modification of the cysteine residues, rather than to alteration of the tertiary structure of HEL.



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  		FIGURE 7. Reactivity of T cell hybridomas with carboxymethylated HEL74–88. A, Failure of CM-HEL74–88 to induce T cell stimulation. AOIT and A69.5 were used as representative HEL74–90-reactive I-Ak- and I-Ab-restricted T cells, respectively. T hybridoma cells were cultured with various concentrations of the unmodified (HEL74–88) or carboxymethylated (CM-HEL74–88) peptide in the presence of irradiated, syngeneic splenocytes as APC. B, Competitive inhibition of the native, HEL74–88-induced response of AOIT and A69.5 T cell hybridomas. In both cases, cultures consisted of a suboptimal dose of native, unmodified HEL74–88 plus PBS or competitor peptides. A1Bb represents an unrelated, non-MHC binding negative control peptide. HEL47–61 and HEL81–93 represent positive control peptides for I-Ak and I-Ab binding, respectively. Data are expressed as the percent inhibition of the response induced by HEL74–88 in the absence of competitor peptides.

 
Serine-substituted peptide for both cysteine residues enhances stimulation of I-Ab-restricted T cell hybridomas but fails to stimulate I-Ak-restricted hybridomas

To further correlate the binding and oxidation of cysteine residues, the cysteine residues were conservatively substituted with serine residues, and the levels of T cell activation by these peptides were compared with that by oxidized peptide (Fig. 8GoA). Substitutions of both cysteine residues at position 76 and 80 drastically improved the stimulation of I-Ab-restricted T cell hybridoma, while such modifications completely abrogated stimulation of I-Ak-restricted T cell hybridoma. The I-Ak-dependent T cell stimulation with singly substituted peptides indicates that the substitution of cysteine at position 76, but not that at position 80 is responsible for the failure of stimulation. Such substitutions also completely abolished the I-Ak-restricted T cell activation by fixed APC in the presence of DTT, but not that in I-Ab-restricted T cell hybridomas (data not shown). These results are consistent with the above data (Fig. 7Go) indicating that modification of the cysteine residue(s) abrogates interaction with TCR of the I-Ak-restricted T cell hybridomas.



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  		FIGURE 8. T cell stimulation by HEL74–88 peptides containing serine substitutions of cysteine residues. HEL74–88-reactive AOIT (I-Ak-restricted) and BO4H (I-Ab-restricted) T cell hybridoma clones were cultured with serine-substituted HEL74–88 peptides (30 µM) in the presence of irradiated or paraformaldehyde-fixed syngeneic splenocytes. A, T cell hybridomas were cultured with HEL74–88 peptides substituted at positions 76 (76S), 80 (80S), or both 76 and 80 (76&80S) in the presence of syngeneic APC. The substituted or oxidized peptides were pretreated with or without 2.5 mM DTT for 30 min and then diluted to a final concentration of 100 µM in culture medium. B, Direct binding of the oxidized or substituted peptides to purified I-Ak molecules was examined based on the inhibition of HEL46–61 binding using a modified capture ELISA. Inhibition by HEL46–61 was also determined as a positive control. Data points represent mean fluorescent counts per second.

 
To correlate the functional assessment of T cell activation with their potential interactions with class II molecules, the ability of the oxidized peptide to bind class II molecules was further examined by direct binding assays (Fig. 8GoB). The oxidized HEL74–88 was clearly unable to interact with I-Ak molecules, while freshly prepared HEL74–88 displayed significant binding to the class II molecules. Substitution of the cysteine residues increased the binding 2- to 3-fold, suggesting that there is a significant level of cross-linked peptides in the freshly prepared solution. Despite the increase in the binding to I-Ak, T cell activation was abrogated by even conservative substitution with serine residues (Fig. 8GoA), suggesting the potential problems associated with substitutions or modifications of these cysteine residues maintaining the intact T cell reactivity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The selection of T cell epitopes used from a multideterminant protein Ag has recently been shown to influence the type of immune response induced (40). Thus, an understanding of the mechanisms involved in the generation of distinct T cell epitopes from a single protein Ag is likely to be of critical importance in the fields of vaccine development as well as autoimmunity. In the present study we have further defined the Ag processing and structural requirements involved in generation of the HEL74–90 region, which is recognized by T cells from both high responder (I-Ak) as well as low responder (I-Ab) mice. This region represents one of two overlapping epitopes (HEL74–90 and HEL81–96) within the 23-aa sequence of HEL74–96 for both strains. This region is particularly interesting because three cysteine residues are located within this sequence, and these form disulfide bridges with other cysteine residues to maintain the intact molecular structure of HEL.

Cysteine residues have either been substituted with similar serine residues or carboxymethylated to avoid cross-linking of cysteine residues via disulfide bonds. The failure of T cells to respond to reduced and alkylated protein or peptide derivatives has previously been taken as evidence for conformation-dependent epitopes (15, 16, 17, 18, 19). However, many of the previous studies were not conducted in the presence of processing inhibitors or fixed APC (15, 16, 18, 19). In contrast to HEL81–96-specific T cells (12, 24), those specific for the HEL74–90 region are strongly responsive to native HEL, but are unresponsive to the unfolded CM-HEL (Fig. 1Go). Thus, this result initially suggested that the T cell recognition may be affected by protein conformation or modified processing by carboxymethylation. Our additional studies with HEL74–90-specific T cell hybridomas revealed a failure to respond to native HEL in the presence of fixed APC (Fig. 3Go), demonstrating that a certain degree of processing is required to generate the epitope. In addition, Ametoni and Sercarz (unpublished observations) have recently observed that HEL74–96 coupled by a disulfide bond at residue 80 with residue 64 of HEL62–68 was unable to stimulate T cells specific for HEL85–96/I-Ek. This result confirms the possibility that complete reduction of disulfide bonds is necessary for the recognition of a closely located epitope by TCR. Furthermore, the failure of the T cells to respond to CM-HEL (Fig. 1Go) appears to be due to the methylation of cysteine residues, disrupting the interactions with class II and/or the TCR (Fig. 7Go). Consequently, modification of these residues can influence T cell recognition due to alteration of the contacting residues rather than the conformational changes as previously thought in some cases. This is strongly supported by the fact that serine substitution of the cysteine residue(s) also inhibits the stimulation of certain (I-Ak-restricted) hybridomas. Jensen (39) has likewise described the importance of reduced cysteine residues in class II contact using an insulin determinant that had previously been suggested to be conformation dependent (15, 16).

The idea that the reduction of disulfide bonds at positions 76 and 80 is required to present the HEL74–90 epitope was further supported by the observation that fixed APC poorly present oxidized peptide to the T cell hybridomas in the absence of added reducing agent (Fig. 4Go). Unfolding of HEL in the absence of additional processing (fixed APC) was sufficient to stimulate low, but significant, levels of both the I-Ab- and I-Ak-restricted T cell hybridomas (Fig. 4Go). Others have also described the ability of unfolded Ag to bind to class II molecules and stimulate T cells without additional processing (13, 14), suggesting that unfolding alone is sufficient to bind class II molecules and stimulate specific T cells. Through direct binding studies, the reduced, but not native, forms of proteins, including HEL, are able to bind to selected class II molecules (13). Polypeptides capable of binding to class II molecules may be very long (7, 8, 41), and additional trimming of the polypeptides after class II binding may be required for efficient stimulation of these T cell hybridomas (42). Although such trimming could potentially occur through the actions of proteases at the plasma membrane (43, 44), internalization of the Ag appears to be necessary for additional processing (Fig. 5Go) to generate the particular epitopes within this region.

The failure of fixed APC to optimally present reduced HEL to these T cell hybridomas suggests that supplemental processing is required in addition to unfolding to efficiently generate the HEL74–90 epitope. However, only minimal processing may be sufficient for generation of the determinant from this region, because T cell hybridomas can be stimulated even in the presence of intracellular processing inhibitors. (Fig. 2Go). The insensitivity to leupeptin suggests the lack of involvement of serine/cysteine proteases in the generation of HEL74–90 (Fig. 2Go). However, the sensitivity to ammonium chloride is only marginal (Fig. 2Go). Various compartments have been suggested to contain reducing activity, including lysosomes (45), endosomes (46), and Golgi vesicles (47), indicating that such reducing activity may not always require low pH. Furthermore, treatment with leupeptin or ammonium chloride does not inhibit intracellular reduction of disulfide bridges of a protein Ag (46). Also, the generation of class II binding peptides upon unfolding has been shown to require low pH for some, but not all, proteins (14, 39). Interestingly, Jensen (14) demonstrated that I-Ed can present HEL106–120 after unfolding of HEL at low pH, and others have shown that generation of the HEL46–61 region requires transport of the protein to the low pH environment of the lysosomes (45, 48).

The processing and structural requirements involved in the generation of HEL74–90 appear to be similar for both I-Ab- and I-Ak-restricted T cell hybridomas (Figs. 1Go and 2Go). Although this region has previously been suggested to represent a predominant T cell determinant region from native HEL in low responder C57BL/6 mice (24, 49), it does not represent a predominant epitope in I-Ak-bearing high responder mouse strains (49, 50). Nevertheless, the region of HEL74–96 serves as a major source of epitopes for both low responder as well as high responder mice. The immunodominance in T cell epitope selection is probably determined not only by class II binding affinities of individual peptides but also by availability of the epitopes (51, 52, 53). Although reduction itself is not sufficient for an optimal presentation of the epitopes derived from native HEL (Fig. 4Go), the generation of such epitopes may be relatively simple and less constrained, as the reduction of the molecules alone can provide a significant level of T cell stimulation. Despite the simple requirement for Ag processing to generate epitopes from this region, the kinetic study indicated that a prolonged time was required for their generation, suggesting the reduction may occur in a late endosome or lysosome (Fig. 6Go). Interestingly, the reduction/presentation of oxidized peptide was even slower than that of the intact HEL molecule. This difference may reflect the differential efficiency of the transport of the molecules, as a recent report suggested a deficient transport of peptide by APC (54). Taken together, our results indicate that the reduction of cysteine residues of either intact protein or peptide is essential for efficient T cell stimulation. Furthermore, modification of such cysteine residues with either chemical modification or substitution with other structurally similar residues may also alter the immunological properties of the proteins and/or epitopes. Thus, particular considerations should be given for cysteine residues in peptide-based vaccine development.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Research Grants RO1AI15446 (to B.S.K.) and AI11183 (to E.E.S.). Back

2 H.-K.K. and J.A.M. made equal contributions to this study. Back

3 Current address: Drug Delivery, Becton Dickinson Technologies, 21 Davis Drive, Research Triangle Park, NC 27709. Back

4 Current address: Ag Express, Inc., 1 Innovation Drive, Worcester, MA 01605-4306. Back

5 Current address: La Jolla Institute for Allergy and Immunology, San Diego, CA 92121. Back

6 Address correspondence and reprint requests to Dr. Byung S. Kim, Department of Microbiology-Immunology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611. E-mail address: Back

7 Abbreviations used in this paper: HEL, hen egg-white lysozyme; CM-, carboxymethylated; PC, phosphorylcholine. Back

Received for publication August 31, 1999. Accepted for publication December 3, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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