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The Journal of Immunology, 2001, 166: 6907-6913.
Copyright © 2001 by The American Association of Immunologists

Thiopalmitoylation of Myelin Proteolipid Protein Epitopes Enhances Immunogenicity and Encephalitogenicity1

Judith M. Greer2,*, Bérangère Denis3,{dagger}, Raymond A. Sobel{ddagger} and Elisabeth Trifilieff{dagger}

* Department of Medicine, University of Queensland, Royal Brisbane Hospital, Herston, Queensland, Australia; {dagger} Laboratoire de Chimie Organique des Substances Naturelles, Unité Mixte de Recherche 7509, Centre National de la Recherche Scientifique, Université Louis Pasteur, Strasbourg, France; and {ddagger} Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage. Two of the acylation sites are within epitopes of PLP that are encephalitogenic in SJL/J mice (PLP104–117 and PLP139–151) and against which increased immune responses have been detected in some multiple sclerosis patients. It is known that attachment of certain types of lipid side chains to peptides can result in their enhanced immunogenicity. The aim of this study was to determine whether thioacylated PLP peptides, as occur in the native protein, are more immunogenic than their nonacylated counterparts, and whether thioacylation influences the development of autoreactivity and experimental autoimmune encephalomyelitis. The results show that in comparison with nonacylated peptides, thioacylated PLP lipopeptides can induce greater T cell and Ab responses to both the acylated and nonacylated peptides. They also enhanced the development and chronicity of experimental autoimmune encephalomyelitis. Synthetic peptides in which the fatty acid was attached via an amide linkage at the N terminus were not encephalitogenic, and they induced greater proportions of CD8+ cells in initial in vitro stimulation. Therefore, the lability and the site of the linkage between the peptide and fatty acid may be important for induction of encephalitogenic CD4+ T cells. These results suggest that immune responses induced by endogenous thioacylated lipopeptides may contribute to the immunopathogenesis of chronic experimental demyelinating diseases and multiple sclerosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Myelin proteolipid protein (PLP)4 is the most abundant protein of CNS myelin, and is a potent immunogen and encephalitogen (1, 2, 3, 4). PLP is posttranslationally acylated by covalent attachment of long chain fatty acids to cysteine residues in the polypeptide backbone via thioester linkages. PLP acylation is highly conserved throughout evolution and during brain development, and is thought to play an important role in the normal functioning of PLP and in myelin stability (5, 6). Cys108 is the major acylation site (7), but a total of six acylation sites have been reported (8). The acylation sites Cys108 and Cys140 are within the encephalitogenic PLP epitopes PLP104–117 and PLP139–151, respectively (1, 4, 9, 10). Reactivity to these epitopes is also found in some patients with multiple sclerosis (MS) (11, 12, 13, 14, 15). However, the contribution that the thioacyl side chains make to the immunogenicity and encephalitogenicity of PLP has not been studied.

It has been shown that lipopeptides formed by the attachment of acyl side chains to peptides either via stable amide bonds (16, 17, 18, 19) or via the more labile thioester linkage (20), as is found in PLP, can act as natural adjuvants for the induction of Ab and CTL responses. We postulated that if thioacylated PLP lipopeptides show similar immune-enhancing properties, their release from the native protein during demyelination in MS or experimental autoimmune encephalomyelitis (EAE) might lead to enhanced autoimmune activation directed against PLP.

The aim of the present study was to determine whether thioacylated PLP lipopeptides affect the development of autoreactivity differently from their nonacylated counterparts. PLP peptides PLP104–117 and PLP139–151 were synthesized with a palmitic acid side chain attached via a thioester linkage (thiopalmitoylated; designated S-palm-PLP104–117 and S-palm-PLP139–151), as occurs in the native protein. Mice immunized with these peptides showed significant increases in T cell proliferative responses, and the incidence and duration of clinical EAE were enhanced. In contrast, peptides synthesized with a palmitic acid side chain attached to the N terminus via the amide group (N-palmitoylated (N-palm)-PLP104–117 and N-palm-PLP139–151) were only weakly immunogenic and not encephalitogenic, suggesting that the type of linkage between the peptide and the fatty acid may be important for the induction of CD4+ T cells. These results imply that immune responses induced by physiologically thioacylated PLP lipopeptides that are released with myelin breakdown may play a role in prolongation of autoimmune inflammatory demyelinating diseases in vivo.


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

Female SJL/J mice were purchased from the Animal Resources Center (Murdoch, Western Australia). Mice were age matched for each experiment, and were immunized at 7–10 wk of age.

Antigens

Peptides PLP104–117 and PLP139–151 were synthesized by solid-phase synthesis using a Fmoc/tBu strategy. The thiopalmitoylation of residues Cys108 and Cys140 was performed on the resin-bound peptide after selective deprotection of the Cys side chain (21). The N-palm peptides were obtained by coupling activated palmitic acid on the N terminus residue. After cleavage from the resin, the crude peptides were lyophilized and purified by C18 RP-HPLC. The purity of the peptides was >=95%, and their identities were confirmed by electrospray mass spectrometry. The sequences of the peptides are shown in Table IGo. In some experiments, peptides PLP40–59 and PLP178–191, or guinea pig myelin basic protein (MBP), prepared as previously described (22), were used as control Ags.


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  		Table I. Sequences of the peptides used in this study

 
Active induction of EAE

Mice were injected s.c. in the flank with 50 µg of peptide and 400 µg of Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI) in an emulsion consisting of equal volumes of water and CFA (Difco). Peptides were dissolved at a concentration of 5 mg/ml in 0.2 M acetic acid, and diluted to the appropriate concentration with distilled water. Each mouse was also injected i.v. on days 0 and 3 with 300 ng Bordetella pertussis toxin (List Biological Laboratories, Campbell, CA).

Clinical and histological evaluation

Mice were assessed clinically, as previously described (4, 23), according to the following criteria: 0, no disease; 1, decreased tail tone or slightly clumsy gait; 2, tail atony and/or moderately clumsy gait and/or poor righting ability; 3, limb weakness; 4, limb paralysis; 5, moribund state. Animals were sacrificed within 7 days of the initial appearance of clinical signs of disease or within 7 days of a relapse. Some mice that showed no clinical signs were also sacrificed for histological analysis. Brains and spinal cords were removed and fixed in 10% phosphate-buffered Formalin, and paraffin-embedded sections were stained with luxol fast blue hematoxylin and eosin for light microscopy. Histological disease was quantitated by counting the inflammatory foci in meninges and parenchyma, as previously described (23).

Proliferation assays

Pooled lymph node cells (LNC) were prepared from inguinal and axillary lymph nodes from two to five mice injected s.c. 9–10 days earlier with 50 µg peptide in CFA. The in vitro responses of LNC were assayed in triplicate in 96-well flat-bottom microtiter plates. Three hundred thousand LNC were added to each well, together with tissue culture medium as a control or various Ags. Cells were incubated for 72 h at 37°C in 5% CO2. [3H]Thymidine (1 µCi) was added during the final 18–20 h of culture. The plates were harvested onto filter mats and counted in a LKB beta plate counter. The data are expressed as stimulation indices (SI), which were determined by the formula SI = (Mean cpm of Ag-containing triplicate well)/(Mean cpm of control triplicate wells). All SD were <15% of the mean cpm.

ELISA

Blood was collected by heart puncture from mice at the time they were perfused for histology. Sera were stored at -20°C until being tested in ELISA. Plates (Immulon 4; Dynatech Laboratories, Chantilly, VA) were coated with 5 µg/ml peptide overnight at 4°C. Another peptide, PLP40–59, was used to coat control wells. Plates were then washed and blocked with 200 µl/well of PBS containing 0.05% Tween 20 and 2% skim milk powder (PBS-T-SM). After washing, 100 µl antiserum (diluted in PBS-T-SM) was added to each well, and the plates were incubated for 2 h at 37°C. The plates were washed four times with PBS containing 0.05% Tween 20 (PBS-T), and 100 µl of 1/1000 dilution of anti-mouse polyvalent Igs (Sigma, St. Louis, MO) was added to each well and incubated 2 h at 37°C. After extensive washing with PBS-T, 100 µl p-nitrophenylphosphate substrate (Sigma) was added to each well, and the plates were incubated 1 h in the dark at room temperature. The absorbance was read at 405 nm in a BioLumin 960 plate reader (Molecular Dynamics, Sunnyvale, CA). Data are expressed as the mean specific absorbance, which is the mean absorbance of test peptide-coated wells minus the mean absorbance of wells coated with the control peptide, ± SEM.

Flow cytometry

LNC or T cell lines were centrifuged through a Ficoll gradient and washed with PBS containing 1% FCS and 0.01% sodium azide (wash buffer). Aliquots of 1 million cells were incubated with Abs specific for CD4 (clone RM4-5; rat IgG2a) or CD8a (clone 53-6.7; rat IgG2a) together with Ab specific for the TCR {beta}-chain (H57-597; hamster IgG) for 30 min at 4°C in the dark, followed by FITC-conjugate anti-rat {kappa}-chain or PE-conjugated anti-hamster IgG for 30 min at 4°C in the dark. Isotype-matched primary Abs were used as controls. All Abs were purchased from PharMingen (San Diego, CA) and were used at 1 µg/ml dilution in wash buffer. After washing, cells were resuspended in wash buffer and analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of lipopeptides on induction of proliferative responses

Mice were immunized with nonacylated, S-palm, or N-palm PLP104–117 or PLP139–151 in CFA. After 9 days, lymph nodes were removed, and proliferation of the LNC in response to the immunizing peptide and other Ags was tested. LNC from mice immunized with nonacylated PLP104–117, a subdominant epitope of PLP (10), responded with a mean SI of >=3 to concentrations ranging from 4.5 to 36 nmol/ml of the immunizing peptide and to concentrations of S-palm-PLP104–117 greater than 18 nmol/ml, but made no significant response to the other concentrations of S-palm-PLP104–117 or to the N-palm-PLP104–117 peptides or unrelated Ags (guinea pig MBP or PLP178–191; data not shown) (Fig. 1GoA). The mean SI of LNC from mice immunized with S-palm-PLP104–117 in response to PLP104–117 was at least 4-fold greater than the response of LNC from mice immunized with PLP104–117 (Fig. 1GoB). In addition, these LNC also responded with SI >=3 to the S-palm-PLP104–117 peptide, but not to N-palm-PLP104–117. They did not cross-react nonspecifically with palmitoylated PLP139–151 peptides (data not shown), indicating that the T cell response is not directed against the palmitic acid side chain. The response of LNC from mice immunized with N-palm-PLP104–117 to all Ags was minimal (Fig. 1GoC). Thus, maximal proliferative responses were observed in LNC from S-palm-PLP104–117-immunized mice.



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  		FIGURE 1. Proliferative responses to nonacylated and acylated PLP peptides. Mice were immunized with the peptide shown above each graph, and then LNC were tested for their proliferative responses to the nonacylated peptide (•), S-palm peptide ({blacksquare}), or N-palm peptide ({blacktriangleup}). Each point on the graph represents the SI (mean ± SD of three to five repetitions of each experiment) at a particular peptide concentration.

 
PLP139–151 is an immunodominant epitope of PLP (1, 4, 23), and LNC responses of PLP139–151-immunized mice to the nonacylated peptide were substantially greater than those seen in response to PLP104–117, with mean SI ranging from 12 to 40 over the 1–35 nmol/ml peptide concentrations tested (Fig. 1GoD). The response to S-palm-PLP139–151 was comparable with the response to the nonacylated peptide at the highest peptide concentrations, although at lower peptide concentrations, the response to S-palm-PLP139–151 was greater than the response to the nonacylated peptide (p = 0.03 for peptide concentrations of 4 and 8 nmol/ml and p = 0.06 for lower concentrations). The response to the N-palm-PLP139–151 peptide was significantly less than to either of the other two peptides. The same pattern of reactivity was seen in LNC from mice immunized with S-palm-PLP139–151 (Fig. 1GoE), except that the responses to all three peptides were stronger than those in mice immunized with nonacylated PLP139–151. The responses to S-palm-PLP139–151 were significantly greater than the response to nonacylated peptide for concentrations <8 nmol/ml. For LNC from mice immunized with N-palm-PLP139–151, the magnitude of the responses was much less than responses of LNC from mice immunized with nonacylated PLP139–151 or S-palm-PLP139–151 (Fig. 1GoF).

Phenotype of T cells from lipopeptide-immunized mice

Lipopeptides have been widely used for induction of CD8+ CTL responses; however, encephalitogenic T cells have invariably been found to be CD4+ Th cells. Therefore, we investigated whether the T cells from lipopeptide-immunized mice were predominantly of the CD4+ or CD8+ phenotype. The CD4/CD8 ratio was measured for T cells taken from mice injected with nonacylated, S-palm, or N-palm peptide 9 days previously. As expected from the proliferative data, mice injected with S-palm peptides showed an increased CD4/CD8 ratio compared with cells from mice injected with nonacylated peptide, suggesting a stronger CD4+ T cell response. In contrast, T cells taken from mice immunized with either N-palm-PLP104–117 or N-palm-PLP139–151 showed a reduction in the CD4/CD8 ratio compared with mice immunized with the corresponding nonacylated peptide or S-palm peptide, suggesting that the N-palm peptides either do not induce as strong a CD4+ response or skew the response in favor of a CD8+ T cell response (Table IIGo). After one in vitro stimulation, the percentages of activated CD4+ T cells specific for nonacylated PLP139–151 and S-palm-PLP139–151 were 92% and 95%, respectively. In addition, the percentage of activated T cells of the CD8 phenotype responding to N-palm-PLP139–151 was substantially higher than the percentage of CD8+ T cells responding to PLP139–151 (16.1% vs 5.5%); however, <10% of {alpha}{beta}TCR+ cells responded to the N-palm peptide, compared with nearly 50% of {alpha}{beta}TCR+ cells stimulated with the nonacylated peptide. In contrast, after four in vitro stimulations, almost all of the responding cells were CD4+ for T cells specific for both the nonacylated and N-palm-PLP139–151 peptides. T cell lines specific for N-palm-PLP104–117 could not be generated.


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  		Table II. CD4/CD8 ratios of activated LNC from mice (4 per group) immunized with nonacylated or palmitoylated peptides

 
Effect of lipopeptides on induction of Ab responses

The ability of the thioacylated PLP lipopeptides to affect the production of Ab was investigated. Sera from mice immunized with nonacylated PLP104–117 make a moderate response to that peptide, but recognize the immobilized S-palm-PLP104–117 or N-palm-PLP104–117 peptides very poorly (Fig. 2GoA). In contrast, sera from mice immunized with S-palm-PLP104–117 showed a much stronger Ab response to the nonacylated peptide and to the S-palm-PLP104–117, but these Abs did not interact with the N-palm peptide (Fig. 2GoB). Sera from mice immunized with N-palm-PLP104–117 did not contain Abs specific for any of the peptides (Fig. 2GoC). The lack of cross-reactivity between any of the sera and N-palm-PLP104–117 suggests that the Abs against PLP104–117 may be directed against an N-terminal region of the peptide, and that the attachment of the acyl side chain to this part of the peptide may interfere spatially with Ab/Ag interaction. None of the sera reacted with PLP40–59, which was used as a control peptide in each assay (data not shown).



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  		FIGURE 2. Ab responses to palmitoylated and nonacylated peptides. Sera were collected from mice immunized with the peptide shown above each graph, and then tested for their ability to interact with nonacylated peptide (•), S-palm peptide ({blacksquare}), or N-palm peptide ({blacktriangleup}) immobilized on ELISA plates. Each point is the mean specific absorbance ± SEM of sera (n = 8 for A–C and F; n = 10 for D; n = 6 for E).

 
Nonacylated PLP139–151 induces a strong Ab response to both the nonacylated and S-palm peptides (Fig. 2GoD). In comparison, S-palm-PLP139–151 induced Abs with higher titers for the nonacylated and S-palm peptides (Fig. 2GoE). Abs induced by both the nonacylated and S-palm peptides bound very well to immobilized N-palm-PLP139–151, suggesting either that the palmitic acid attached at the N terminus enhances binding of this peptide to the plate, or that it may act as a spacer between the plate and the peptide, and allow improved binding of the Ab to the peptide. N-palm-PLP139–151 induced a moderate Ab response against itself, but these Abs did not cross-react with nonacylated or S-palm peptide (Fig. 2GoF). None of the Abs bound nonspecifically to the unrelated control peptide PLP40–59 (data not shown).

EAE induction with lipopeptides

The nonacylated and acylated peptides were tested for their ability to induce EAE in vivo (Table IIIGo). PLP104–117 is only weakly encephalitogenic in SJL/J mice, and during the 95 days that mice immunized with this peptide were followed, none developed EAE. Histologically, two of these mice had small numbers of inflammatory cells in the meninges, but none in the parenchyma. In contrast, four of four SJL/J mice immunized with S-palm-PLP104–117 developed EAE with an average day of onset of the first attack of EAE of 40.5 days. All of the S-palm-PLP104–117-immunized mice subsequently recovered and developed one or more relapses before they were perfused for histology. Histologically, these mice showed evidence of demyelinated plaque-like lesions in the spinal cord, with some Wallerian degeneration.


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  		Table III. Comparison of features of the first episode of EAE induced by nonacylated or palmitoylated PLP peptides

 
PLP139–151 is highly encephalitogenic in SJL/J mice, and four of four mice immunized with PLP139–151 developed EAE (Table IIIGo). Mice immunized with the S-palm-PLP139–151 peptide induced EAE with the same incidence and mean day of onset of EAE. The mean clinical score of the mice immunized with S-palm-PLP139–151 was slightly higher than that of mice immunized with the nonacylated peptide, but this difference was not statistically significant (p = 0.06). The most striking feature of EAE induced by S-palm-PLP139–151 peptide was that the duration of the first attack of EAE was significantly longer in the mice immunized with S-palm-PLP139–151 (p = 0.007). In addition, mice immunized with S-palm-PLP139–151 developed a more chronic disease (i.e., more severe disease with more frequent relapses) than did the mice immunized with the nonacylated peptide (Fig. 3Go). The mean number of CNS-inflammatory lesions was slightly higher in the mice immunized with S-palm-PLP139–151 compared with mice immunized with PLP139–151 (140 ± 48 vs 116 ± 78), reflecting their higher clinical scores. Histologically, no differences in the composition or distribution of the lesions induced by the S-palm-PLP139–151 peptide compared with those induced by the nonacylated PLP139–151 peptide could be detected. A representative lesion from the spinal cord of a mouse immunized with S-palm-PLP139–151 is shown in Fig. 4Go. As expected from the in vitro proliferation assays, none of the mice immunized with N-palm peptides developed EAE (Table IIIGo).



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  		FIGURE 3. Pattern of disease in mice immunized with PLP139–151 (A) or S-palm-PLP139–151 (B). Each panel represents the clinical course of disease for an individual animal. The duration of the first episode of EAE was significantly increased in mice immunized with S-palm-PLP139–151 compared with those immunized with PLP139–151 (p = 0.007). Mice were followed for up to 95 days, or perfused for histology at an earlier time point (indicated by {dagger}) if their clinical signs were severe.

 


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  		FIGURE 4. Leptomeningeal mononuclear cell infiltrate (arrow) and a large demyelinated plaque-like lesion in the spinal cord of an SJL/J mouse immunized with S-palm-PLP139–151. Intact white matter (white asterisk) is on the right side of the field. Luxol fast blue hematoxylin and eosin. Magnification, x428.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Posttranslational modifications of myelin proteins have been largely overlooked in studies of the development of autoreactivity to those proteins in demyelinating diseases such as MS; however, recent evidence suggests that they may be important. For example, it has been shown that MS patients accumulate over time a posttranslationally modified form of MBP, citrullinated MBP (24, 25), which appears to induce a stronger immune response than noncitrullinated MBP (26). We now show that another posttranslational modification, namely palmitoylation of peptides of PLP via a thioester linkage, as occurs in the native protein in myelin, can enhance autoimmune responses to PLP in an animal model of MS.

PLP palmitoylation is a dynamic and reversible event, and, although the dynamics of PLP palmitoylation change during development (5), the total percentage of acyl side chains covalently linked to PLP remains remarkably constant at ~3 mol of fatty acid/mol of protein throughout the life of the animal (6). The physiological role(s) of the acyl groups bound to PLP has not been fully elucidated. Several lines of evidence support the concept that the number of acyl side chains, the conformation of PLP in the myelin membrane, and the stability of the myelin sheath are interrelated, and that changes in one may lead to changes in the others. For example, in adrenoleukodystrophy, another human disease in which there is an inflammatory infiltrate with demyelination in the CNS, there is an increase in the proportion of very long chain fatty acids in PLP that may contribute to the myelin instability characteristic of this disorder (27). In addition, it has been shown that during spontaneous demyelination in transgenic mice carrying multiple copies of cDNA for DM20, the alternatively spliced isoform of PLP, the amount of palmitic acid linked to PLP increases 3-fold (28).

Regardless of whether the demyelination in these cases was a causal factor or consequence of altered palmitoylation of PLP, the fact that PLP is normally thioacylated has important implications for EAE and MS. Although palmitic acid attached to peptide via a stable amide bond can enhance CTL and Ab responses to the peptide (16, 17, 18, 19), a recent study using a canine parvovirus model showed that peptides palmitoylated via the much more labile thioester bond enhanced Ab production even more than did N-palm peptides (20). Thus, thioacylation of PLP may have immunological consequences due to the release of S-palm PLP peptides during demyelination and their subsequent enhanced uptake and presentation to cells of the immune system.

The results of the present study confirm that S-palm peptides can induce greater Ab responses than nonthioacylated peptides. They also show that immunization with S-palm PLP lipopeptides can enhance autoimmune CD4+ Th cell reactivity. For S-palm-PLP139–151, this enhancement occurs in both the induction and effector phases. Mice immunized with the S-palm-PLP139–151 show increased responses to all three peptides compared with mice immunized with nonacylated peptide, indicating effects on the induction phase. Furthermore, the proliferative response against lower concentrations of S-palm-PLP139–151 of LNC from mice immunized with either nonacylated or S-palm PLP139–151 is consistently greater than the response to the nonacylated peptide, indicating that acylation also influences the effector phase.

By contrast, the enhancement in the effector phase is not as clear for S-palm-PLP104–117. If the S-palm peptides enter the cell by endocytic mechanisms involving passage through lysosomes, then it would be expected that the acyl groups would be removed from the peptides by palmitoyl protein thioesterases, which are a major component of the lysosome (29). However, it has recently been shown that small hydrophobic or lipid-containing molecules can enter macrophages via several other pathways (reviewed in Ref. 30), although it is not yet known how organelles involved in these pathways interact with MHC class II-containing compartments. Therefore, the possibility exists that the peptide might bind to MHC class II molecules with the lipid side chain still attached. This could potentially affect recognition of the peptide by T cells. It is known that the threonine residue at position 117 of peptide PLP104–117 is critical for encephalitogenicity in SJL/J mice (10), and thus it would not be expected that palmitoylation of residue Cys108 would directly influence the formation of the trimolecular complex and the recognition of this peptide. However, the bulky fatty acid might induce an altered conformation of the peptide. This could result in S-palm-PLP104–117 acting as a partial agonist. Several previous studies (31, 32) have found that expansion of T cells on a partial agonist can lead to stronger responses against the agonist peptide, similar to the situation we have described in this study, in which LNC from mice immunized with S-palm-PLP104–117 showed an increased response to the nonacylated peptide, but not to S-palm-PLP104–117. Furthermore, there may also be differences in the ability of various types of APC to take up, process, and present this S-palm peptide. In particular, APC in the LNC preparations used in the proliferation assays may not process and present S-palm peptides via the same pathways as APC that process and present the encephalitogenic palmitoylated peptides in vivo.

The importance of the type of linkage between the peptide and the palmitic acid for enhancement of Th cell responses was investigated by comparing N-palm peptides with a stable amide linkage to S-palm peptides, in which there is a labile linkage between peptide and fatty acid. Rather than enhancing the immune responses, the N-palm peptides appeared to have some suppressive effects. A similar suppressive effect has been reported in EAE studies using N-palm peptides of MBP (33, 34, 35). N-palm peptides with acyl chain formulations containing from one to three palmitic acid residues have been used to induce CD8+ CTL responses in several other systems (16, 17, 18, 19, 36, 37, 38, 39, 40, 41, 42). Although these N-palm peptides show similar CTL-enhancing properties, irrespective of the number of palmitic acid residues attached, they appear to exert their effects by different mechanisms (18, 39, 40, 41, 42). For peptides palmitoylated by attachment of a single palmitic acid moiety via an amide bond at the N terminus, the lipid tail may facilitate passive translocation of the peptide through the plasma membrane into the cytosol, where it could enter the MHC class I pathway (39, 40). Therefore, it seems likely that the poor immunogenicity of the N-palm PLP and MBP peptides, and their immunosuppressive effects, may be due to the peptide entering a MHC class I presentation pathway. Because the epitopes used in the studies are MHC class II epitopes that may not bind to MHC class I with high affinity, this might produce no effective response. Alternatively, they may stimulate a weak MHC class I-restricted response in vivo, as suggested by the data in Table IIGo. Such a response might down-regulate the CD4+ response.

The observation that S-palm peptides, in contrast to N-palm peptides, preferentially induce Th cell responses may be useful in the design of peptide-based vaccines. For such usage, the mechanisms by which S-palm peptides are taken up by APC must be determined. As noted above, it is possible that different types of APC may take up S-palm peptides by different pathways. Furthermore, it is not yet known whether the acyl side chain needs to be located within the immunogenic epitope or merely in the vicinity of the epitope of interest to induce the immune-enhancing effects. In addition, it has recently been demonstrated that some N-palm lipopeptides, but not the corresponding nonacylated peptides, can activate macrophages via CD14 (41) or Toll-like receptor 2 (42) pathways, suggesting that the lipid moiety itself can promote interaction with these receptors. It remains to be determined whether S-palm peptides can also activate APC via pathways of the innate immune system.

Thioacylation might help to explain the dominance of PLP139–151 in demyelinating diseases in SJL/J mice. When SJL/J mice are immunized with an equimolar mixture of nonacylated encephalitogenic peptides PLP139–151 and PLP178–191, responses to both peptides are equally strong (10, 23). By contrast, if EAE is induced by immunization of SJL/J mice with whole spinal cord homogenate, which contains many potential autoantigens (4, 10, 43, 44, 45), including PLP with covalently attached fatty acids, the dominant immune response is to PLP139–151 (46). Additionally, if EAE is induced in SJL/J mice with another autoantigen such as MBP, or if these mice are infected with Theiler’s murine encephalomyelitis virus and allowed to recover, PLP139–151 is the first new epitope against which autoreactivity subsequently develops (47, 48). The myelin breakdown products generated as a consequence of tissue injury in EAE and in Theiler’s virus encephalitis would most likely contain PLP with the acyl chains still covalently attached. We postulate that the presence of the thioacyl side chain in these conditions may skew the response toward naturally thioacylated peptides such as PLP139–151. It has also been shown that SJL/J mice naturally have a high precursor frequency of cells potentially responsive to PLP139–151, which appears to be due, in part at least, to the presence of DM-20 (which does not contain the PLP139–151 epitope), but not PLP, in the thymus (49, 50). However, whether a relationship also exists between thioacylation and the development of the repertoire is presently unknown.

PLP is not the only well-characterized autoantigen that is known to be thioacylated. For example, GAD-65, P0, erythrocyte band 3, and rhodopsin, putative autoantigens for insulin-dependent diabetes mellitus, autoimmune neuritis, autoimmune hemolytic anemia, and autoimmune uveoretinitis, respectively, are all thioacylated (51, 52, 53, 54). An increased uptake of thioacylated peptide Ags and/or increased activation of APC due to the presence of the thioacyl side chain might result in a greater tendency for autoreactivity to spread to thioacylated Ags. If thioacylated lipopeptides act as natural adjuvants by stimulating APC through receptors of the innate immune system, then it may be that polymorphisms in some of these receptors, e.g., the Toll-like receptors, which are thought to be highly polymorphic in humans (55), could correlate with development of autoreactivity to particular Ags, and may increase the susceptibility of individuals to the development of chronic autoimmune disease. The relevance of the present findings to human disease, particularly MS, remains to be determined.


   Acknowledgments
 
We thank Diane Muller for technical assistance, and Dr. Marjorie Lees for critical reading of the manuscript.


   Footnotes
 
1 This work was supported in part by the National Health and Medical Research Council of Australia (to J.M.G.), Association pour la Recherche sur la Sclérose en Plaques (to B.D. and E.T.), Association Francaise des Femmes Diplômées des Universités (to B.D.), and National Multiple Sclerosis Society Grant RG 3055 (to R.A.S.). Back

2 Address correspondence and reprint requests to Dr. Judith Greer, Neuroimmunology Research Unit, Department of Medicine, University of Queensland, Clinical Sciences Building, Royal Brisbane Hospital, Herston QLD 4029, Australia. E-mail address: j.greer{at}medicine.uq.edu.au Back

3 Current address: Dictagene S.A., Chemin des Croisettes 22, CH-1066 Epalinges, Switzerland. Back

4 Abbreviations used in this paper: PLP, proteolipid protein; EAE, experimental autoimmune encephalomyelitis; LNC, lymph node cell(s); MBP, myelin basic protein; MS, multiple sclerosis; N-palm, N-palmitoylated; S-palm, thiopalmitoylated; SI, stimulation index. Back

Received for publication November 27, 2000. Accepted for publication March 22, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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