Allelic variations in rat MHC class II binding of myelin basic protein peptides correlate with encephalitogenicity

Katrien L. de Graaf1,2, Robert Weissert1,2, Peter Kjellén3, Rikard Holmdahl3 and Tomas Olsson1

1 Neuroimmunology Unit, Center of Molecular Medicine L8:04, Karolinska Hospital, 17176 Stockholm, Sweden
2 Department of Neurology, University of Tübingen, 72076 Tübingen, Germany
3 Department of Cellular and Molecular Biology, Section for Medical Inflammation Research, Lund University, 22100 Lund, Sweden

Correspondence to: K. L. de Graaf, Department of Neurology, Auf der Morgenstelle 15, University of Tübingen, 72076 Tübingen, Germany


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The impact of the strength and promiscuity of the self peptide–MHC class II interaction on susceptibility to autoimmune disease is uncertain. Here we studied allelic differences in the affinity of rat MHC class II molecules for myelin basic protein (MBP) peptides spanning from position 63 to 106. Predominantly peptides from this region are immunogenic in the rat and the MHC class II region determines if the response is disease promoting or disease protective. Strikingly, RT1.B (DQ-like) molecules showed much more allelic variation of MBP peptide binding than RT1.D (DR-like) molecules. Moderate to strong binding of particular MBP peptides correlated with their previously documented encephalitogenicity. Moreover, the differences in disease susceptibility to certain MBP peptides observed in the different rat strains were clearly reflected in the allelic diversity of the peptide binding profiles. In conclusion our findings demonstrate that disease-inducing stretches of MBP generally comprise good binding peptides.

Keywords: antigen, antigen binding, epitope, experimental autoimmune encephalomyelitis, MHC, multiple sclerosis, peptide, rodent


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Susceptibility to autoimmune diseases such as insulin-dependent diabetes mellitus, rheumatoid arthritis and multiple sclerosis (MS) is associated with specific MHC haplotypes (1). Although not formally proven, these associations might well arise from the allelic differences in presentation of self or self-mimicking foreign peptides by MHC class I or class II molecules to autoagressive T cells, in turn leading to organ-specific tissue inflammation. In addition to such peripheral events, preferences in MHC peptide binding might also affect the shaping of the T cell repertoire in the thymus leading to susceptibility to or protection from autoimmune disease (2). Despite these hypotheses, there are little experimental data on how allelic variations in peptide binding relate to immunogenicity and disease-inducing potential of peptides derived from autoantigens in well-defined animal models. This matter is not trivial since better definition of allelic variations in MHC class II peptide binding in relation to disease-inducing potential would enable a better understanding of MHC associations in general, and give hints on the utility of such studies in designing selective immunotherapies targeting the trimolecular complex consisting of MHC molecule, TCR and peptide.

Experimental autoimmune encephalomyelitis (EAE) represents a prototype autoimmune disease and is an animal model for MS. The autoagressive attack is mainly mediated by CD4+ T cells (3) which can be activated in susceptible mouse or rat strains upon immunization with central nervous system proteins such as MBP, proteolipid protein or myelin oligodendrocyte glycoprotein. A long-standing goal has been to exactly define the autoantigen which is relevant for inducing and/or perpetuating autoimmune disease, and in addition to define which epitopes within this particular autoantigen are encephalitogenic. In EAE this is dependent on the MHC haplotype (46), and the major genetic influence most likely is exerted by MHC class I and class II molecules since they are the peptide-presenting elements and display allelic diversity. Among autoantigens in autoimmune neuroinflammatory disease most interest so far has been devoted to MBP. In particular, peptides from the region between MBP 84 to 102 have been of interest since they can bind to the MS-associated DR2 molecule and since many T cells in the CNS of DR2+ MS patients are specific for these peptides (7,8).

In the LEW rat, immunization with the RT1.Bl (equivalent for I-A in the mouse or HLA-DQ in humans)-restricted immunodominant MBP 68–88 leads to a monophasic self-limiting disease (9,10). A second minor RT1.Dl (equivalent to I-E in mice or HLA-DR in humans)-restricted epitope, MBP 89–101 (11), also has encephalitogenic properties in LEW rats. Studies with MHC congenic rat strains on the LEW background revealed that encephalitogenicity of both these peptides is strongly dependent on the allelic variations in the MHC region and use of intra-MHC recombinant congenic strains mapped these influences to the MHC class II region (1214).

If indeed the MHC haplotype influence on EAE is due to allelic variations in MHC class II molecules, it is still not clear how MBP peptide–MHC class II interactions would relate to encephalitogenicity. To study this issue in more detail, we purified RT1.B and RT1.D molecules from four different rat MHC haplotypes (RT1a, RT1l, RT1n and RT1u) and used them in an MHC class II peptide binding assay. We measured the relative affinity of 13mer stepped MBP peptides covering the MBP 63–106 region for the purified MHC class II molecules and then compared findings in the binding assay with disease-inducing capacity in the respective rat strains.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Synthetic peptides
The N-terminally biotinylated tracer peptides MBP 87–99, MBP 83–97, CLIP 97–120 and P2 58–81 and their non-biotinylated equivalents (Table 1Go) were synthesized by the F-moc/HBTU strategy (Dr Å. Engström, Department of Medical and Physiological Chemistry, University of Uppsala, Uppsala, Sweden). Peptides were purified by reversed-phase chromatography and subsequently analyzed by plasma desorption mass spectroscopy. The stepped 13mer MBP peptides were similarly synthesized and generously provided by Ferring (Malmö, Sweden).


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Table 1. Peptide sequences
 
Purification of RT1.B and RT1.D molecules
The different RT1.B and RT1.D molecules were purified from MHC congenic LEW rat (LEW, LEW.1A, LEW.1N and LEW.1W) thymic and splenic tissues by affinity chromatography using OX-6 (specific for RT1.B molecules) and OX-17 (specific for RT1.D molecules) antibodies as described previously (16). Briefly, tissues were lysed in PBS containing 1% NP-40 (Boehringer, Mannheim, Germany) in the presence of a cocktail of protease inhibitors. The lysates were cleared of nuclei and debris by centrifugation at 50,000 g for 60 min and subsequent passage over a 45 µm filter. The cleared lysates were cycled over OX-6- and OX-17-coupled CNBr-activated Sepharose-4B (Pharmacia, Uppsala, Sweden) columns. The columns were washed with 20 column volumes of PBS/0.1% SDS/0.5% NP-40, three column volumes of PBS/0.05% NP-40 and 3 column volumes of PBS/1% octyl-ß-D-glucopyranoside (Sigma, St Louis, MO), and finally bound MHC molecules were eluted with 0.05 M diethylamine, pH 11/0.15 M NaCl/0.1% octyl-ß-D-glucopyranoside. After neutralization with 2 M Tris–HCl, pH 6.3, the purity of the eluted proteins was assessed by SDS–PAGE and subsequent silver staining. The presence of stable MHC class II complexes for each of the haplotypes was confirmed by running the proteins in SDS–PAGE without denaturation through boiling. The protein content was measured with the BCA protein assay (Pierce, Rockford, IL) using BSA as a standard.

Peptide binding assay
Relative affinities of MBP peptides for purified RT1.B and RT1.D molecules were assessed by an inhibition ELISA based on a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA; Wallac, Turku, Finland) as described previously (17). Initially, biotinylated tracer peptides were used in a direct binding assay to establish optimal binding conditions for each of the purified RT1.B and RT1.D molecules.

In the inhibition ELISA assay, RT1.B and RT1.D (50–100 nM) molecules were incubated with fixed amounts of their respective tracer peptides (10–50 nM) (Table 2Go) in the presence of various concentrations (10-fold dilutions between 1 nM and 100 µM) of the unlabeled MBP peptides. Inhibition studies were routinely performed at pH 5, since pH titration experiments had shown that this was the optimal pH for binding of the tracer to most of the purified MHC molecules. The binding buffer consisted of a carbonate buffer titrated to pH 5 containing 2 mM EDTA, 0.01% azide, 0.1 mM PMSF and 0.1% Nonidet P-40 (Boehringer). After an incubation of 48 h at room temperature, the peptide–MHC complexes were transferred to antibody coated (OX-6 or OX-17) ELISA plates (FluoroNunc; Nunc, Roskilde, Denmark) to remove the excess of non-bound peptides. Europium-labeled streptavidin (Wallac) was added to the plates and incubated for 1 h at room temperature. Finally, the plates were treated with an enhancement solution (Wallac), which releases chelated europium from streptavidin and forms a highly fluorescent miscellar solution that can be measured by using a DELFIA fluorometer (Wallac).


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Table 2. Tracer peptides in the inhibiiton ELISA
 
The concentration yielding 50% inhibition of binding of the tracer peptide (IC50) was measured by plotting the percentage of inhibition versus the concentration of MBP peptide. Peptides were tested in two to three independent experiments.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Definition of tracer peptides for the purified MHC class II molecules
In order to perform competitive peptide binding assays with the affinity-purified RT1.B and RT1.D molecules we first defined tracer peptides for the purified MHC class II molecules. For the RT1.D molecules we selected candidate tracer peptides, such as MBP 87–99 or MBP 83–97, which have been described as predominantly recognized by T cells in either rats or humans and are known to be RT1.D or HLA-DR restricted (8,11). Both peptides bound with the RT1.Dn, RT1.Da and RT1.Dl alleles (Table 2Go), and were consequently used to perform inhibition assays. However, since neither of them bound with sufficient affinity to the RT1.Du allele, we used a biotinylated CLIP 97–120 peptide comprising a supermotif which enables this peptide to interact with all MHC class II molecules (18). The MBP 83–97 peptide was useful as a tracer for the purified RT1.Ba and RT1.Bn molecules as well. As tracer for the RT1.Bl molecule we were able to test several candidate peptides, since an RT1.Bl peptide binding motif has been proposed by two independent groups (19,20). We decided to use the P2 58–81 peptide, an autoantigenic peptide derived from the peripheral nerve protein P2 fitting the published RT1.Bl motif, since it also displayed binding to the RT1.Bu molecule.

Measurement of the relative affinities of 13mer stepped MBP peptides for purified rat MHC class II molecules
The relative affinity of the stepped MBP peptides for each of the purified RT1.D alleles was measured and expressed as the IC50 value (Fig. 1Go). Peptides which bound well to RT1.D molecules, displaying IC50 values <10 nM could be detected for the RT1.Da and the RT1.Dl alleles. Peptides with intermediate binding strength with IC50 values <1 µM were found for the RT1.Dn and RT1.Du alleles, while other peptides did not display any binding even at concentrations as high as 100 µM. Most binding peptides were found in the region surrounding the MBP 87–99 epitope and the MBP binding stretches were largely overlapping in the four different RT1.D molecules studied.



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Fig. 1. Binding of MBP peptides to purified RT1.D molecules. IC50 values for each of the peptides were derived from the inhibition curves obtained by ELISA as described in Methods. The tracer peptides for the different RT1.D alleles are shown in Table 2Go.

 
In contrast to the RT1.D molecules, each of the studied RT1.B molecules was characterized by a distinct binding profile, thus showing more allelic diversity (Fig. 2Go). The RT1.Ba molecule displayed the most promiscuous binding of MBP peptides with several peptides binding with IC50 values as low as 50 nM. For the RT1.Bl molecule, binding was limited to the immunodominant RT1.Bl-restricted MBP 62–89 region. Intermediate binding affinities were revealed for this allele with IC50 values in the micromolar range. The profile of peptides binding to the RT1.Bn molecule showed more similarity to that observed in the RT1.Dn molecules. Peptide binding was concentrated to the MBP 87–99 region and some of the peptides bound in this region with IC50 values < 1 µM. For the RT1.Bu molecule just a few weakly binding peptides (IC50 > 10 µM) could be detected in the MBP 62–89 region.



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Fig. 2. Binding of MBP peptides to purified RT1.B molecules. IC50 values for each of the peptides were derived from the inhibition curves obtained by ELISA as described in Methods. The tracer peptides for the different RT1.B alleles are shown in Table 2Go.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The issue we addressed with this study is how MHC–peptide binding relates to autoimmune T cell responses and disease outcome in an MBP-induced EAE model. By common sense, an MBP peptide not binding to MHC molecules will not induce disease since it cannot be presented to T cells. If binding of MBP peptides to the proper MHC molecules does occur, lack of disease induction can be caused by holes in the T cell repertoire due to inactivation of high-avidity T cells by endogenously expressed MBP (21). Alternatively, activation of peptide-specific regulatory T cells secreting disease down-regulatory cytokines or peripheral tolerization of T cells might lead to disease protection. Only in the case of recruitment and activation of autoreactive T cells producing inflammatory cytokines will EAE ensue. To study the role of the MHC molecules in these processes in more detail, we analyzed the binding data obtained for the four different rat MHC class II haplotypes in regard to previously published results obtained in EAE actively induced with MBP encompassing the MBP 63–106 region.

Both DA and LEW rats are susceptible to EAE upon immunization with the RT1.B-restricted MBP 68–86 peptide (22). The minimal encephalitogenic peptide for LEW rats, the RT.Bl-restricted MBP 73–86 peptide (23,24), does not induce disease in DA rats. The encephalitogenic epitopes for DA rats in the MBP 68–86 region have been mapped to MBP 63–76, MBP 66–81 and to the C-terminal residues MBP 79–86 (25,26). Clearly, the non-overlapping character of the encephalitogenic epitopes for LEW and DA rats parallels the RT1.Ba and RT1.Bl binding patterns, which do not overlap either (Table 3Go).


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Table 3. Correlation of encephalitogenicity of MBP peptides to affinity for RT1.B and RT1.D molecules in rats with an EAE susceptible non-MHC background genome
 
The existence of both regulatory and disease-inducing MBP peptides has been demonstrated previously (13,14). The MBP 62–89 peptide induces a disease-promoting Th1 response in LEW rats with the RT1a and RT1l haplotypes, while a protective Th2 response is elicited in connection with the RT1u haplotype. Both the RT1.Ba and the RT1.Bl alleles are capable of binding peptides within the MBP 62–89 region with high to intermediate affinity (Table 3Go). In contrast, RT1.Bu as well as RT1.Du molecules can only bind peptides in this region with very low affinity (IC50 > 10 µM). Interestingly, it has been shown that the interaction between TCR, peptide and MHC molecule can determine Th1/Th2 dominance in certain defined experimental systems (28,29).

Upon immunization with the subdominant MBP 87–99 peptide, LEW.1W-derived splenocytes do not respond in a measurable way (14). This non-responsiveness is in complete correlation with the lack of binding affinity for RT1.Bu and RT1.Du molecules (IC50 > 100 µM) (Table 3Go). In contrast, LEW rats with the MHC class II alleles RT1l, RT1a and RT1n develop disease after immunization with MBP 87–99. The restriction elements for MBP 87–99 in the RT1l and RT1a haplotypes are the RT1.Dl and RT1.Ba molecules respectively, and relatively good binding of this peptide is observed in both cases (IC50 10–100 nM).

The pathogenic importance of the affinity of autoantigenic peptides to the class II molecule has been a continuous point of debate. Low-affinity binding is compatible with the idea that autoreactive T cells escape tolerance (30), while high-affinity binding of autoantigens would require breaking of tolerance or regulatory recognition. Our study shows that the elicitation of an immune response, whether of the Th1 or the Th2 type, corresponds to the presence of T cell determinants in peptides which are capable of binding to either RT1.B or RT1.D molecules with IC50 values <100 µM. These results are in line with previously published MBP peptide binding studies in mice as well as in human, where it has been shown that high to intermediate binding affinity is a necessary but not sufficient characteristic of encephalitogenic determinants (8,31).

By analyzing the experimental data in more depth we observed isotype-dependent differences in the RT1.B and RT1.D molecule MBP peptide binding patterns. Whereas the RT1.B molecules displayed striking allelic differences in the spectrum of binding peptides, the RT1.D molecules bound similar MBP stretches, mainly in the MBP 87–99 region. The importance of this sequence has been amply documented in humans, where it was shown that MBP epitopes in this region are recognized in the context of several MS associated HLA-DR types (32). The overlapping of the RT1.D binding patterns is in line with recent data obtained by Southwood et al. (33) showing that several common HLA-DR types share largely overlapping peptide binding repertoires characterized by the presence of a common P1–P6 supermotif. This might have a structural basis since the RT1.D locus is less polymorphic than the RT1.B locus (34,35). Thus, as in mice and humans the Da gene is largely invariant, whereas in the rat the Db gene also has limited polymorphism. In fact, the basis for selecting the RT1a, RT1l, RT1n and RT1u alleles in this study was that these are polymorphic at the RT1.D locus, whereas most other alleles are identical to one of these. Although no functional differences have been reported for RT1.B and RT1.D molecules so far (36), the differences in peptide binding abilities and the different degree of polymorphism suggest differences in their biological role. One interesting possibility is that disease association with an RT1.B molecule might lead to a more chronic disease as a consequence of the larger number of encephalitogenic MBP peptides which can bind and be presented to T cells (37,38). While human MS is associated to a particular HLA class II haplotype, i.e. DRB1*1501, DQA1*0102 and DQB1*0602, association studies have suggested several class II molecules from both DR and DQ loci (3941). Possibly, several of them play a role as binders of various myelin protein-derived peptides as has been shown to occur in the rat.

In conclusion, the present study demonstrates that differences in disease susceptibility to certain MBP peptides in different rat strains can partially be explained by allelic diversity of the MHC class II peptide binding patterns. Moreover, although the role of the MHC class II molecule in the MHC class II associations to EAE is still not formally proven, our data strongly argue for a crucial function of the MHC class II molecule itself in the observed effects.


    Acknowledgments
 
This study was supported by grants from the Swedish Society for Neurologically Disabled (NHR), the Swedish Medical Research Council, the Petrus and Augusta Hedlund Foundation and the Deutsche Forschungsgemeinschaft (We 1947/1-1, 2-1).


    Abbreviations
 
DELFIA dissociation-enhanced lanthanide fluoroimmunoassay
EAE experimental autoimmune encephalomyelitis
IC50 concentration of the test peptide yielding 50% inhibition of binding of the tracer peptide
MBP myelin basic protein
MS multiple sclerosis
RT1 MHC of rat

    Notes
 
Transmitting editor: L. Steinman

Received 11 May 1999, accepted 25 August 1999.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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