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The Journal of Immunology, 2004, 173: 2792-2802.
Copyright © 2004 by The American Association of Immunologists

MHC Class II Isotype- and Allele-Specific Attenuation of Experimental Autoimmune Encephalomyelitis1

Katrien L. de Graaf*, Silvia Barth*, Martin M. Herrmann*, Maria K. Storch{ddagger}, Christoph Otto§, Tomas Olsson, Arthur Melms*, Günther Jung{dagger},||, Karl-Heinz Wiesmüller{dagger},|| and Robert Weissert2,*

* Experimental Neuroimmunology Laboratory, Department of General Neurology, Hertie Institute for Clinical Brain Research, and {dagger} Department of Organic Chemistry, University of Tübingen, Tübingen, Germany; {ddagger} Department of Neurology, University of Graz, Graz, Austria; § Experimental Transplantation Immunology, Department of Surgery, University of Würzburg, Würzburg, Germany; Neuroimmunology Unit, Center for Molecular Medicine, Karolinska Hospital, Stockholm, Sweden; and || EMC Microcollections, Tübingen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Most autoimmune diseases are associated with certain MHC class II haplotypes. Autoantigen-based specific immune therapy can lead either to beneficial or, in the context of inflammatory conditions, detrimental outcomes. Therefore, we designed a platform of peptides by combinatorial chemistry selected in a nonbiased Ag-independent approach for strong binding to the rat MHC class II isotype RT1.Dn allelic product of the RT1n haplotype that is presenting autoantigen in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in LEW.1N rats. Peptide p17 (Ac-FWFLDNAPL-NH2) was capable of suppressing the induction of and also ameliorated established experimental autoimmune encephalomyelitis. MHC class II isotype and allele specificity of the therapeutic principle were demonstrated in myelin basic protein-induced experimental autoimmune encephalomyelitis in LEW rats bearing the RT1l haplotype. A general immunosuppressive effect of the treatment was excluded by allogeneic heart transplantation studies. In vitro studies demonstrated the blocking effect of p17 on autoantigenic T cell responses. We thus demonstrate a rational design of strong MHC class II-binding peptides with absolute isotype and allele specificity able to compete for autoantigenic sequences presented on disease-associated MHC class II molecules.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Most autoimmune diseases are associated with certain MHC class II isotype allelic variants (1). Without much doubt, this is due to presentation of autoantigenic peptides on certain MHC class II molecules (2). A self-reactive T cell repertoire reactive with the presented peptide fragments must be available for development of autoimmunity (3). Theoretically, treatment of autoimmune diseases in an immunoselective manner would be possible by targeting particular allelic variants of MHC class II isotypes. But, dependent on the circumstances, autoantigen-based specific immunotherapy can lead to a beneficial or a detrimental outcome (4, 5). The outcome is dependent on the individual T cell repertoire and inflammatory stimuli. Altered peptide ligands use an autoantigenic peptide with minor alterations compared with the native self peptide. Such therapy has worsened disease in a number of multiple sclerosis (MS)3 patients (6). Most likely, this is due to the capacity of an altered peptide ligand to function as antagonist, agonist, and superagonist, depending on the particular TCR (7). Targeting of dendritic cells with autoantigen can lead to tolerance (4). This can be reverted in the context of inflammation in which dendritic cells can present autoantigen in such a way that autoimmunity is induced (5, 8). Therefore, a selective targeting of certain allelic variants of MHC class II isotypes with sequences without any structural similarity to self Ags would possibly be a way to prevent unwanted autoimmunity.

MS is an inflammatory disease of the CNS with demyelination and axonal and neuronal loss (9). Currently, available treatments are only modestly effective and there is need for improved therapies that could stop further disease development. MS is a complex genetic disease that is strongly associated with the isotypes and alleles HLA-DR2a, DR2b, and DQ6 in U.S. Americans and Northern Europeans (10). Understanding of immunogenetic mechanism governed by MHC genes may be studied in rodent models of MS. Hereby, inbred rat strains induced to develop experimental autoimmune encephalomyelitis (EAE) are important tools. Susceptibility or resistance in many of these models is associated with the MHC (RT1 in the rat) class II gene products (11). This is not surprising in view of the key role of MHC class II molecules in triggering CD4+ T cells by presenting restricted sets of peptides to the TCR. Little is known about the structural characteristics of rat MHC class II molecules. Currently, only the RT1.Bl ligand-binding motif, an HLA-DQ-like molecule of the LEW (RT1l) rat, has been described (12, 13). Combinatorial peptide libraries have been successfully applied in the past to examine different aspects of MHC class I and II molecule interactions with peptide (14, 15). Such libraries offer the distinct advantage of being able to quantitatively assess the contribution of each amino acid residue in each position for interaction with the peptide-binding groove of the analyzed MHC class II isotype (RT1.B and RT1.D) allelic variants.

The extracellular domain of myelin oligodendrocyte glycoprotein (MOG 1–125) and its encephalitogenic core sequence, MOG 91–108, induce a very MS-like lesional spectrum in the CNS with inflammation, demyelination, and axonal loss in LEW.1N (RT1n) rats (16, 17, 18). Therefore, we used this model for our investigations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptide libraries and peptides

Synthetic acetylated nonapeptide amide libraries, as well as defined acetylated nonapeptide amides and biotinylated peptide amides were prepared by fully automated solid-phase peptide synthesis using 9-fluorenylmethoxycarbonyl/tert.-butyl (Fmoc/tBu) chemistry and analyzed by HPLC and electrospray ionization mass spectrometry (19). Biotinylated CLIP peptide 97–120 (LPKSAKPVSPMRMATPLLMRPSMD) was obtained by elongating the peptide with two spacer amino acids, followed by biotin using a coupling method. p17-FITC was obtained from EMC Microcollections. Copolymer-1 (COP-1) was obtained from TEVA Pharma (Kirchzarten, Germany).

Purification of MHC molecules and peptide-binding studies

RT1.Dn, RT1.Bn, RT1.Dl, and RT1.Bl molecules were purified from thymic and splenic tissue from LEW.1N (RT1n) rats and LEW (RT1l) rats by affinity chromatography using the mAbs OX-17 (anti RT1.D) and OX-6 (anti RT1.B), as previously described (18). Binding assays were performed with a competitive ELISA based on a dissociation-enhanced lanthanide fluoroimmunoassay (Wallac, Turku, Finland) (18). For the competitive ELISA, a 50 nM solution of RT1.D molecules was incubated for 48 h at 37°C with 100 nM biotinylated CLIP 97–120 and 2 µM acetylated nonapeptide amide sublibrary or with various concentrations of competitor peptides. The concentration of totally randomized acetylated nonapeptide library Ac-X9-NH2 yielding 30% competition was used for measuring the competition with the acetylated nonapeptide amide libraries (Fig. 1a). Competitor peptides were measured at various concentrations ranging from 1 nM to 100 µM. The IC50 of a peptide was defined as the concentration of peptide necessary for the inhibition of binding of the tracer peptide by 50%.



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  		FIGURE 1. Binding of Ac-X9-NH2 acetylated amide sublibraries and predicted peptides to RT1.Dn. a, The competition of Ac-X9-NH2 with biotinylated CLIP 97–120 for binding to purified RT1.Dn molecules was measured. b, The competition of acetylated nonapeptide amide sublibraries with biotinylated CLIP 97–120 for binding to purified RT1.Dn molecules was assessed. The relative competition (= percentage of competition of Ox/mean percentage of competition of O1–9) of 4 defined aa (N, P, Y, and W) in their respective sequence positions is shown. c, The competition of predicted peptides (Table II) with biotinylated CLIP 97–120 for binding to purified RT1.Dn molecules was analyzed. d, Binding of truncated p17 variants. e, Binding of alanine (A)-substituted p17 variants. f, Binding of lysine (K)-substituted p17 variants. g, Binding of p17-FITC to intact B cells. p17-FITC was competed out with unlabeled p17 or p45.

 
Rat rMOG 1–125

Rat rMOG 1–125 (rrMOG 1–125) was expressed in Escherichia coli and purified by chromatography (16).

Rats, immunization protocol, and scoring of EAE

Rats were obtained from H. Hedrich (Central Animal Laboratory, Hannover Medical School, Hannover, Germany). They were bred and kept under specific pathogen-free conditions. Two to four rats were housed per cage and obtained food and water ad libitum. Female rats between age 8 and 10 wk were used for all experiments. All experiments were approved by the regional boards in Tübingen and Würzburg, Germany.

Rats were injected intradermally at the base of the tail either with 100 µg of MOG 91–108, myelin basic protein (MBP) 63–88, or MBP 85–99, or with 20 µg of rrMOG 1–125. For blocking experiments, 100 µg of p45, p17, p79, or COP-1 was added. The Ags in a total volume of 100 µl were mixed with 100 µl of CFA (1:1). A total of 100 µl of CFA consisted of IFA (Sigma-Aldrich, St. Louis, MO) and 200 µg (for rrMOG 1–125-induced EAE) or 500 µg (for MOG 91–108-induced EAE) of heat-inactivated Mycobacterium tuberculosis (strain H37 RA; Difco Laboratories, Detroit, MI). For treatment of ongoing disease, LEW.1N (RT1n) rats were immunized with 50 µg of rrMOG 1–125 in IFA. On day 11 postimmunization (p.i.), rats received a single injection of 500 µg of p45 or p17 in 500 µl of IFA i.p. Rats were examined daily for signs of EAE and weighed from day 7 p.i. until sacrifice. The clinical scoring was as follows: 0 = no illness; 1 = tail weakness or paralysis; 2 = hind leg paraparesis or hemiparesis; 3 = hind leg paralysis or hemiparalysis; 4 = tetraparesis or moribund; 5 = death.

Cellular assays and elution of cells from the CNS

Mononuclear cells (MNC) from draining lymph nodes, spleen, and CNS were obtained, as described (18). B cells from the spleens of naive rats were purified by magnetic cell separation against CD45RA (Miltenyi Biotec, Bergisch Gladbach, Germany). Subsequently, cytofluorometric analysis by FACS with anti-RT1.D mAb (Ox-17) and p17-FITC (1 µM) was performed. Competition experiments were performed with unlabeled p17 (200 µM) and p45 (200 µM).

Enumeration of IFN-{gamma}-secreting cells by ELISPOT was performed, as described (18). To assess the MHC class II restriction of the generated IFN-{gamma} responses, 10 µg/ml OX-6 (anti-RT1.B), OX-17 (anti-RT1.D), or Tib191 (isotype-matched control) was added to the cells together with the Ag.

CNS-infiltrating cells were eluted from the CNS of diseased rats by density gradient centrifugation, as described (18). A total of 1 x 104 cells was incubated with 1 x 105 irradiated (30 Gy) thymocytes with or without Ags. Specificity was assessed by ELISPOT for IFN-{gamma}-secreting cells.

Determination of MOG-specific Abs

The determination of rrMOG 1–125-specific IgG was performed, as described (18). The OD was read at 405 nm.

cDNA synthesis and quantification of cytokine mRNA levels using real-time PCR

Quantitative real-time PCR and primer design were performed, as described (18). Relative quantity of mRNA levels was determined using the {Delta}{Delta}CT method. The amount of mRNA in each sample was calculated as the ratio between the amount of cytokine mRNA and the amount of GAPDH mRNA in this sample. For cells without Ag or Ag-restimulated cells, the cytokine/GAPDH ratio of the samples from the rrMOG 1–125-immunized rats was set to 1, and the ratio of the samples from the p17 or p17 and rrMOG 1–125-coimmunized rats was calculated relative to this sample.

Transfer of spleen cells

LEW.1N (RT1n) rats were immunized with either 100 µg of p45 or p17 in 500 µg of CFA at the base of the tail. On day 9 p.i., spleen MNC were isolated and 107 cells were transferred i.v. into naive LEW.1N (RT1n) rats that were subsequently immunized with 50 µg of rrMOG 1–125 in 200 µg of CFA.

Cardiac transplantation

LEW (RT1l) rats served as recipients, and WF (RT1u) rats as donors. Heterotopic cardiac transplantation was performed according to the method of Ono and Lindsey (20). The allograft function was monitored by daily transabdominal palpation of cardiac contractions. Graft rejection was considered as the complete cessation of palpable cardiac contractions, which was then confirmed histologically.

Histopathology

Histological evaluation was performed on paraformaldehyde-fixed, paraffin-embedded sections of brains and spinal cords on days 14 and 19 p.i. (16, 17, 21). Paraffin sections were stained with H&E and Luxol fast blue to assess inflammation and demyelination. An inflammatory index was calculated from the number of perivascular inflammatory infiltrates of each rat on an average of 15 complete cross sections of spinal cord. The degree of demyelination was evaluated for brain and spinal cord sections separately and semiquantitatively described and scored (16, 17, 21).

Statistical analysis

Student’s t test was used for normally distributed variables. When the data did not fulfill the criteria of being normally distributed, nonparametric statistics (Mann-Whitney U test) were used. Values of p were adjusted for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MHC class II restriction of the MOG-specific T cells

To find selective isotype- and allele-specific MHC class II-binding peptides for the LEW.1N (RT1n) rat, we had to assess the restriction of the encephalitogenic T cell response toward the main encephalitogenic determinant within MOG 1–125 in LEW.1N (RT1n) rats, MOG 91–108. There is no detectable ex vivo T cell response toward MOG 91–108 from peripheral lymphoid tissue after EAE induction with rrMOG 1–125 or MOG 91–108 (18). Therefore, we eluted infiltrating cells from the CNS on day 12 p.i. (n = 4). A restriction analysis by ELISPOT of the T cell response to MOG 91–108 was performed by adding mAbs OX-6 (anti-DQ) or OX-17 (anti-DR) together with Ag. The MOG 91–108-specific T cell response was reduced to ~30% by OX-17 (anti-RT1.D) as compared with OX-6 (anti-RT1.B) (Fig. 2a). Next, we assessed the restriction of the rrMOG 1–125 T cell response in the periphery by blocking with the mAbs OX-6 or OX-17 (n = 4). The T cell response was reduced by addition of OX-17 (anti-RT1.D) to ~50%, but not by addition of the OX-6 (anti-RT1.B) (Fig. 2b). These experiments indicated that the RT1.Dn molecule is the restriction element of the encephalitogenic T cell response in rrMOG 1–125-induced EAE.



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  		FIGURE 2. MHC restriction of the encephalitogenic T cell response. a, The intracerebral encephalitogenic MOG 91–108-specific T cell response in LEW.1N (RT1n) rats is RT1.Dn restricted, as was demonstrated by inhibition of the T cell response by addition of the mAb OX-17 (anti-RT1.D) as compared with mAb OX-6 (anti-RT1.B) (n = 4). b, Similar results were obtained for the peripheral T cell response against MOG 1–125 in LEW.1N (RT1n) rats that was RT1.Dn restricted as well (n = 4).

 
Competition of acetylated nonapeptide amide libraries for binding to RT1.D molecules

The peptide-binding pattern of the RT1.Dn molecule was assessed by using combinatorial peptide libraries. In a first step, a completely randomized acetylated nonapeptide amide library (Ac-X9-NH2; X: 19 L amino acids excluding cysteine (C)) was tested for its capacity to bind to RT1.Dn in a competition ELISA. The randomized library readily competed for binding to RT1.Dn, and addition of 100 µM Ac-X9-NH2 inhibited binding of the biotinylated tracer peptide (rat CLIP 97–120, LPKSAKPVSPMRMATPLLMRPSMD) to RT1.Dn up to 90% (Fig. 1a). Because Ac-X9-NH2 bound satisfactorily and because the use of longer combinatorial libraries would probably complicate the obtained data due to translational invariance, acetylated nonapeptide amide sublibraries were selected for carrying out the subsequent competition experiments (14).

A total of 171 nonapeptide sublibraries (9 sequence positions x 19 aa, excluding C) was screened in a competition ELISA to elucidate the binding patterns of the RT1.Dn molecule. A concentration of 50 nM RT1.Dn in combination with 100 nM biotinylated CLIP 97–120 and 2 µM of each sublibrary was applied for the assays. We compared the competition of the 171 sublibraries, each characterized by 1 defined aa shifted over the 9 different positions, by measuring the relative competition of this amino acid at all 9 positions (relative competition = percentage of competition of Ox/mean percentage of competition of O1–9) (Fig. 1b). Because in some cases (for example, for F) the differences in relative competition values were not incisive, measurements were confirmed in at least two independent experiments (data not shown). Moreover, frequently, amino acid residues with similar structural properties (for example, the sublibraries with F, W, and Y in defined positions) clearly showed comparable tendencies in the relative competition values (Fig. 1b). Thus, the effect of each amino acid in its respective position was termed as favorable, indifferent, or unfavorable.

Aromatic amino acids on P1, P2, and P3 of the sublibraries favored binding to RT1.Dn (Table I). Contrarily, aromatic residues rather inhibited binding if present at P5–P9. Both D and N enhanced binding at P4, P5, and P6, whereas P showed significant competition at P6, P7, and P8. Residues with aliphatic side chains such as I, L, M, V, as well as G were favorable at P9, and I, L, and M were also well tolerated at P4.


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  		Table I. Influence of defined amino acid residues on competitive binding to RT1.Dn

 
Competition of acetylated and amidated peptides to RT1.Dn molecules

To test the generated database (Table I) for its predictive value for high and low affinity binders, two sets of peptides were synthesized: the first set of peptides consisted of randomly selected combinations of favorable amino acids for each of the nine positions. The second set of peptides was made up of randomly selected combinations of unfavorable amino acids for each of the nine positions. IC50 values were measured for all defined acetylated nonapeptide amides (Table II). All peptides belonging to the set of favorable peptides showed significant binding and ranged from very high affinity ligands (IC50 = 2 nM for p17) to low affinity ligands (IC50 = 89 µM for p19). In contrast, 70% of the unfavorable peptides did not show any competition at all, even at peptide concentrations of 100 µM (Fig. 1c). The binding of p17 was RT1.Dn isotype and allele specific: p17 neither bound RT1.Bn molecules (IC50 > 100 µM), nor RT1.Dl (IC50 = 12 µM) and RT1.Bl (IC50 > 100 µM) molecules (Table III).


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  		Table II. IC50 values of acetylated nonapeptide amides for binding to RT1.Dn and prediction of binding propertiesa

 

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  		Table III. IC50 values of acetylated nonapeptide amides (p17 and p79) for binding to RT1.Dn, RT1.Bn, RT1.Dl, and RT1.Bl

 
All shortened variants of p17 indicated lower binding capabilities compared with p17 (Fig. 1d). Truncated peptides from the C-terminal, from the N-terminal parts, as well as from the C- and N-terminal parts of p17 revealed a minimum sequence of 6 aa for binding. C-terminal truncations resulted in stronger loss of binding compared with N-terminal truncations (Fig. 1d). Binding was strongly improved by acetylation at the N-terminal part of the shortened peptides (Fig. 1d). Subsequently, we substituted all positions in the p17 peptide sequence by an alanine and lysine scan and found that binding to RT1.Dn was dramatically inhibited in case of substituting positions 3 and 6 (Fig. 1, e and f).

Binding of p17 to MHC class II RT1n molecules on live B cells

To analyze whether live APCs could present p17 on MHC class II molecules, we purified B cells from spleen. These were purified and stained for anti-RT1.D-PE (mAb Ox-6) and p17-FITC on the cell surface (Fig. 1g). Binding of p17 to RT1.Dn-positive cells was 20.5% (arbitrary threshold level set). Coincubated unlabeled p17 in 200-fold excess reduced binding of p17-FITC to 3.8%. Coincubated unlabeled p45 in 200-fold excess did not reduce binding of p17-FITC with 23.1% positive cells.

Immunogenicity of high affinity ligands in LEW.1N (RT1n) rats

To determine whether treatment of LEW.1N (RT1n) rats with synthetic high affinity ligands could prime for T cell reactivity to the peptides, rats were immunized with the four peptides showing the lowest IC50 values (p2, p3, p11, and p17) (n = 3 for each peptide). On day 12 p.i., the recall responses to these peptides in lymph node suspensions were evaluated by measuring the IFN-{gamma} secretion using an ELISPOT assay (Fig. 3a). To test the restriction of the response, mAbs OX-6 (anti-RT1.B), OX-17 (anti-RT1.D), or Tib 191 (an isotype-matched control) were added to the culture medium (Fig. 3b) (n = 4 each peptide). The four different peptides raised striking IFN-{gamma} responses, indicating that all investigated peptides were immunogenic and represented T cell agonists. As shown by the blocking experiments with mAb against RT.1B and RT1.D molecules, the responses elicited to the peptides were RT1.D restricted (Fig. 3b). The number of IFN-{gamma}-secreting spots for the tested peptides did not clearly correlate to the IC50 values.



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  		FIGURE 3. Immunogenicity and restriction of T cell responses to high affinity ligands. a, LEW.1N (RT1n) rats were immunized with the high affinity-binding peptides p2, p3, p11, and p17, and T cell responses were assessed by IFN-{gamma} ELISPOT (each n = 3). b, To test the restriction of the peptide responses, mAbs OX-6 (anti-RT1.B), OX-17 (anti-RT1.D), or Tib 191 (isotype matched, specific for irrelevant control Ag) were added to the cell cultures (each n = 4).

 
Amelioration of MOG-EAE in LEW.1N (RT1n) rats

Next, the newly defined synthetic nonapeptides were investigated for their capacity to inhibit the induction of EAE in vivo. Because p17 (Ac-FWFLDNAPL-NH2) had the lowest IC50 value for binding to RT1.Dn and therefore the highest affinity for the RT1.Dn molecule (Table II), this peptide was selected for testing its ability to inhibit MOG 91–108- and rrMOG 1–125-induced EAE in LEW.1N (RT1n) rats in which the encephalitogenic response to MOG 91–108 is RT1.Dn restricted (Fig. 2). Nonapeptide p45 (Ac-NHPSPKYLW-NH2) was used as a control, because this was one of the peptides lacking any measurable affinity for RT1.Dn (IC50 > 100 µM; Table II).

First, LEW.1N (RT1n) rats were immunized with the encephalitogenic MOG 91–108 peptide in CFA mixed with PBS alone (n = 8), p45 (n = 11), or p17 (n = 11) in PBS. Coadministration of p17 together with MOG 91–108 significantly prevented MOG 91–108-induced EAE compared with PBS alone (p < 0.001, sum score, t test) or p45 (p < 0.001, sum score, t test) (Table IV, group 1). None of the p17-treated rats developed any clinical signs of EAE.


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  		Table IV. Disease course of rats treated with high affinity ligands

 
Second, LEW.1N (RT1n) rats were coimmunized with rrMOG 1–125 in CFA mixed with either p45 (n = 4), p17 (n = 12), or PBS alone (n = 8). Peptide p17 strongly ameliorated EAE in rrMOG 1–125-immunized rats compared with p45 (p < 0.001, sum score, t test) or PBS alone (p < 0.001, sum score, t test) (Table IV, group 2). To test whether competition at the level of MHC class II binding was the only mechanism responsible for EAE attenuation, LEW.1N (RT1n) rats were injected with p17 and rrMOG 1–125 in CFA at different sites of the body (immunization with rrMOG 1–125 at tail base, p17 application in CFA at the neck). This treatment also resulted in amelioration of EAE (p17 treatment at different site compared with PBS control, p < 0.002, sum score, t test) (Table IV, group 2). Due to extreme disease severity present in LEW.1N (RT1n) rats immunized with rrMOG 1–125 in CFA, the experiments had to be terminated at day 15 p.i. We followed additional rats coimmunized with rrMOG 1–125 and p17 dimer in CFA at the same site up to 40 days. These rats were completely protected from EAE (Table IV, group 2). Histopathology of PBS, p45, and p17, and rrMOG 1–125 in CFA-coimmunized rats indicated absence of lesions in the CNS of p17-treated rats. In contrast, inflammation and widespread inflammation were present in rats treated with p45 or PBS (Fig. 4, a–c). On day 14 p.i., LEW.1N (RT1n) immunized with rrMOG 1–125 in CFA and coimmunized with p45 or PBS alone showed inflammation (inflammatory index in p45 group (n = 4) 0.9 and in PBS group (n = 4) 0.79) and large demyelinating lesions. In contrast, LEW.1N (RT1n) rats coimmunized with rrMOG 1–125 in CFA together with p17 did not have inflammatory or demyelinated lesions in the CNS (n = 4). LEW.1N (RT1n) rats coimmunized with rrMOG 1–125 in CFA together with p17 and perfused on day 19 p.i. did not show lesions either (n = 4; data not shown).



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  		FIGURE 4. Histopathology of LEW.1N (RT1n) rats immunized with rrMOG 1–125 and coimmunized with either PBS (a), p45 (b), or p17 (c) on day 14 p.i. is depicted. One representative example of four histopathologically analyzed rats per group is shown. Only coimmunization with p17, but not p45 or PBS of MOG 1–125-immunized LEW.1N (RT1n) rats resulted in the prevention of lesion formation, as indicated by Luxol fast blue myelin staining.

 
Third, we assessed the effect of treatment with p45 or p17 after onset of EAE. Therefore, we treated LEW.1N (RT1n) rats that had been actively induced with rrMOG 1–125 in IFA on day 11 p.i. with a single dose of either p45 in IFA (n = 10) or p17 (n = 17) in IFA. Treatment with p17 resulted in strong amelioration of EAE compared with the p45-treated group (p = 0.002, sum score, t test) (Table IV, group 3).

Immune responses in EAE

We assessed the numbers of rrMOG 1–125-, MOG 91–108-, p45-, and p17-specific IFN-{gamma}-secreting cells in the different groups immunized with rrMOG 1–125 and peptides in CFA (Fig. 5a). COP-1 is a synthetic copolymer composed of tyrosine (Y), glutamic acid (E), alanine (A), and lysine (K) with an average length of 40–100 aa. COP-1 mimics the physicochemical properties of MBP and is used for the immunomodulatory treatment of MS (22). One suggested mechanism of action of COP-1 is blockade of presentation of autoantigen on MHC class II molecules; the capability of COP-1 to inhibit rrMOG 1–125-induced immune responses was assessed as well (Fig. 5a).



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  		FIGURE 5. Influence of p17 and p45 on autoimmune T and B cell responses. a, Numbers of IFN-{gamma}-secreting cells in LEW.1N (RT1n) rats coimmunized with either rrMOG 1–125 and p45, rrMOG 1–125 and p17, or rrMOG 1–125 and COP-1 in CFA. b, Numbers of IFN-{gamma}-secreting cells in LEW.1N (RT1n) rats with rrMOG 1–125-induced EAE treated with either p17 or p45 in IFA at day 11 p.i. c, MNC from LEW.1N (RT1n) rats immunized with either rrMOG 1–125 alone, p17 alone, or rrMOG 1–125 and p17 together were cultured for 48 h with either no Ag (upper panel), rrMOG 1–125 (middle panel), or p17 (lower panel). Subsequently, mRNA expression for IL-4 and IL-10 was detected by real-time quantitative PCR. There were increased mRNA values after restimulation with p17 for IL-4 in p17-immunized LEW.1N (RT1n) rats (p = 0.004), and a trend for rats coimmunized with p17 and rrMOG 1–125 (p = 0.067). A similar increase was observed after restimulation with p17 for IL-10 in p17-immunized (p = 0.01) and rrMOG 1–125- and p17-coimmunized rats (p = 0.007). *, Indicates statistical significance.

 
Coimmunization of rrMOG 1–125 together with p17 led to strong decrease of the T cell response in the form of IFN-{gamma}-secreting cells toward rrMOG 1–125 as compared with coimmunization with p45. Coimmunization of rrMOG 1–125 together with COP-1 led to an intermediate reduction of the number of rrMOG 1–125-specific IFN-{gamma}-secreting cells. There were high numbers of p17-reactive IFN-{gamma}-secreting cells in rats that had been coimmunized with rrMOG 1–125 together with p17. Peptide p45-induced IFN-{gamma} secretion was not detectable after coimmunization of rrMOG 1–125 together with p45. There were COP-1-reactive IFN-{gamma}-secreting cells after coimmunization of rrMOG 1–125 together with COP-1. IFN-{gamma}-secreting cells against MOG 91–108 were not detected in peripheral lymphoid tissue in any of the three different analyzed groups (each group, n = 5).

Next, we assessed the Ab response level against rrMOG 1–125 in rats coimmunized with rrMOG 1–125 together with p45, p17, or COP-1 in CFA (each group, n = 5). Only coimmunization with p17 led to a significant decrease of the Ab titers (p < 0.05) (data not shown).

Immune responses of rats immunized with rrMOG 1–125 in IFA and treated on day 11 p.i. with p17 or p45 in IFA were analyzed from inguinal lymph nodes (n = 5 each). The application of p17 or p45 in IFA led to the nearly complete absence of T cell responses to p17. rrMOG 1–125-specific T cell responses were drastically reduced in the p17-treated LEW.1N (RT1n) rats (Fig. 5b).

Subsequently, by real-time quantitative PCR, we assessed the expression of IL-4 and IL-10 in MNC from lymph nodes that had been restimulated in vitro for 48 h with either no Ag, rrMOG 1–125, or p17 in rrMOG 1–125-immunized, p17-immunized or p17- and rrMOG 1-125-coimmunized MOG-coimmunized LEW.1N (RT1n) rats (n = 4 each) (Fig. 5c). In contrast to rrMOG 1–125-immunized rats, p17-immunized and rrMOG 1–125- and p17-coimmunized rats showed a significant up-regulation of IL-4 (restimulation with p17: rats immunized with rrMOG 1–125 compared with rats immunized with p17, p = 0.004; rats immunized with rrMOG 1–125 compared with rats immunized with rrMOG 1–125, and p = 0.067) and IL-10 (restimulation with p17: rats immunized with rrMOG 1–125 compared with rats immunized with p17, p < 0.01; rats immunized with rrMOG 1–125 compared with rats immunized with rrMOG 1–125 and p17, p = 0.007) after restimulation with p17, but not with rrMOG 1–125 (NS, t test).

Transfer of p45- and p17-specific T cells

Next, we assessed the potential transferability of the protective effect of p17-exposed cells. Therefore, we immunized LEW.1N (RT1n) rats with p17 (n = 9) or p45 (n = 9) in CFA, and subsequently transferred 5 x 107 spleen cells to naive LEW.1N (RT1n) rats that we subsequently induced for EAE with rrMOG 1–125 in IFA. Transfer of neither p45- nor p17-reactive spleen cells resulted in significant amelioration of EAE (NS, sum score, t test) (data not shown).

Attenuation of MBP-EAE in LEW (RT1l) rats by p79

In synergy with studies for RT1.Dn, we also created a binding pattern for RT1.Dl of LEW (RT1l) rats and subsequently defined single peptides, and performed binding studies for strong or weak binding of acetylated nonapeptide amides (Tables Vand VI). p79 (Ac-FWYIAIQDE-NH2) bound with an IC50 of 57 nM to RT1.Dl, but not RT1.Bl molecules (IC50 > 100 µM) nor to RT1.Dn (IC50 > 100 µM) and RT1.Bn (IC50 > 100 µM) molecules (Table III). The T cell response in LEW (RT1l) rat EAE induced with MBP 85–99 is RT1.Dl restricted (23). In contrast, EAE induced in LEW (RT1l) rats with MBP 63–88 is RT1.Bl restricted (23). To evaluate the isotype- and allele-specific amelioration of EAE, we coimmunized groups of LEW (RT1l) rats with either MBP 85–99 alone or together with p79 (Table IV, group 4) and MBP 63–88 or MBP 63–88 together with p79 (Table IV, group 5) (each group, n = 5) (Table IV, group 5). Only in LEW (RT1l) rats immunized with MBP 85–99 significant amelioration of EAE by coadministration of p79 could be observed (p = 0.007, sum score, t test) (Table IV, group 4). Additionally, we tested the isotype and allele specificity of p17 (RT1.Dn binder) in LEW (RT1l) rats. Neither treatment of p17 of LEW (RT1l) rats immunized with MBP 85–99 (RT1.Dl restricted) (Table IV, group 6) nor MBP 63–88 (RT1.Bl restricted) (Table IV, group 7) ameliorated EAE (each group, n = 5). Therefore, we conclude that disease suppression with p17 or p79 in MOG-induced EAE in LEW.1N rats and MBP-induced EAE in LEW rats was MHC class II isotype and allele specific.


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  		Table V. Influence of defined amino acid residues on competitive binding to RT1.Dl

 

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  		Table VI. IC50 values of acetylated nonapeptide amides for binding to RT1.Dl and prediction of binding propertiesa

 
Allogeneic heart transplantation studies

To exclude a general immunosuppressive effect of the functional nonapeptide high affinity ligands, LEW (RT1l) rats were transplanted with hearts from WF (RT1u) rats. LEW (RT1l) rats carrying heterotopic WF hearts were treated with p79 (n = 4), a syngeneic control peptide (n = 7), or PBS (n = 6). There was no significant delay of heart function in LEW (RT1l) rats after treatment with p79 (6.5 ± 0.58 days) as compared with control peptide (7 ± 0.5 days) or PBS (6.5 ± 0.5 days). These results argue against general immunosuppressive properties of p79 in LEW (RT1l) rats.

In vitro blocking of IFN-{gamma} reduction of MOG-specific T cells by p17

Finally, we tested whether MOG-specific T cells eluted from the CNS could be blocked by p17. T cells were eluted from the CNS of diseased LEW.1N (RT1n) rats and incubated with irradiated thymocytes in the presence or absence of MOG 91–108 alone or MOG 91–108 and p17 in parallel. Addition of 10 or 50 µg of p17 led to a significant reduction of the numbers of MOG 91–108-specific IFN-{gamma}-secreting cells, as shown in Fig. 6 (n = 6 each; MOG 91–108 vs MOG 91–108 plus p17, 10 µg/ml, p < 0.05; MOG 91–108 vs MOG 91–108 plus p17, 50 µg/ml, p < 0.01).



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  		FIGURE 6. Effects of p17 on IFN-{gamma} secretion of T cells eluted from the CNS of MOG-immunized LEW.1N (RT1n) rats in the presence of Ag. T cells were eluted from the CNS of LEW.1N (RT1n) rats on day 9 p.i., and subsequently restimulated with irradiated thymocytes in the presence of Ag. Subsequently, ELISPOT analysis for enumeration of IFN-{gamma}-secreting cells was performed. Addition of p17 to MOG 91–108-restimulated T cells led to reduction of numbers of MOG 91–108-specific IFN-{gamma}-secreting cells (n = 6 each; MOG 91–108 vs MOG 91–108 plus p17, 10 µg/ml, p < 0.05; MOG 91–108 vs MOG 91–108 plus p17, 50 µg/ml, p < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that MOG 91–108- and MOG 1–125-induced EAE in LEW.1N (RT1n) rats can be prevented or ameliorated by a synthetic high affinity RT1.Dn ligand, p17 (Ac-FWFLDNAPL-NH2), which is unrelated to MOG. In synergy, MBP 85–99 (RT1.Dl restricted)-, but not MBP 63–88 (RT1.Bl restricted)-induced EAE in LEW (RT1l) rats could be ameliorated by the MBP-nonrelated RT1.Dl high affinity ligand p79 (Ac-FWYIAIQDE-NH2), but not p17, demonstrating the isotype and allele specificity of the intervention. A database search did not reveal any sequence similarity to self molecules for these ligands (data not shown). The newly designed high affinity ligands p17 as well as p2, p3, and p11 were highly immunogenic if injected in CFA in LEW.1N (RT1n) rats. The presentation of these acetylated peptide amides was limited to RT1.Dn because only coculture with mAb OX-17 (anti-RT1.D), but not mAb OX-6 (anti-RT1.B) resulted in blocking of T cell responses.

Although these synthetic peptides are expected to be nonself to the T cell repertoire of the LEW.1N (RT1n) rat, it was not excluded that the peptides could mimic unknown self peptides with thymic deletion of the reactive T cell repertoire as a consequence (24). The presence of a T cell repertoire to each of these peptides indicates that presented self-unrelated high affinity ligands could be important for the control of autoimmunity in general. First, by competition for presentation at the immunological synapse they can prevent the display of autoantigen-derived peptides to T cells (25, 26, 27); second, they can bias the T cell repertoire toward high avidity T cells, filling up empty space in the repertoire, and suppress low avidity T cell response that could be associated with autoimmunity (28). Third, they can induce T cells with a regulatory phenotype expressing IL-4 and IL-10.

Potentially, the induction of immune responses against high affinity ligands could result in autoimmunity by molecular mimicry at least in conjunction with a certain T cell repertoire (29). Our study underscores such a scenario, because we demonstrate that in the context of an inflammatory stimulus (CFA) all investigated high affinity MHC class II ligands raised immune responses. Nevertheless, this possibility is very low due to the extremely high number of nonself sequences the body is encountering in its life in the context of host defense mechanisms that do not result in autoimmunity.

This is the first report on the peptide-binding specificities of RT1.D molecules. We gathered information on the RT1.Dn- and RT1.Dl-binding specificities using combinatorial peptide libraries. Various human HLA-DR and mouse H2-E molecules have been crystallized with and without peptides in the peptide-binding groove, and structural analysis revealed binding of a nonapeptide core sequence with anchor residues at positions 1, 4, 6, and 9 (30, 31). Moreover, pocket 1 (P1) has been shown to be preferentially occupied by aromatic and aliphatic residues. We also found a preference for aromatic amino acids in position 1 of the HLA-DR-like RT1.Dn molecule. Although the approach described in this work did not allow assigning anchor positions in homology with HLA-DR and H2-E molecules, we found that positions 3 and 6 of p17 (Ac-FWFLDNAPL-NH2) were most important for binding. A or K substitutions at these positions resulted in dramatic reduction of binding. Truncated hexapeptide variants of p17 still bound to RT1.Dn. Further truncations resulted in loss of binding. Acetylation at the N-terminal part of the peptides strongly enhanced binding.

In the past, several studies have addressed the possibility of inhibiting experimentally induced autoimmunity in rodents with Abs or peptides inhibiting the presentation of the autoantigen at the MHC level. Steinman et al. (32) have shown in SJL/J (H-2s) mice that blockade of MHC class II presentation with a mAb can lead to prevention of EAE. Subsequently, Wraith et al. (33) have demonstrated that a peptide based on rat MBP Ac1–11 (Ac-ASQKRPSQRHG) substituted on position 4 with alanine can be used to attenuate EAE induced with MBP Ac1–11 in (PL/J x SJL)F1 (H-2u+s) mice. This approach has been followed by others (34, 35). However, if the competitor peptide is structurally closely related to the pathogenic peptide, other mechanisms than inhibiting presentation of autoantigen-derived peptide at the level of the MHC class II molecules play a critical role.

T cell determinants from known self or nonself proteins were used that were unrelated to the autoantigenic peptide sequence to inhibit induced autoimmune disease (27, 36). Hurtenbach et al. (27) defined a ligand (yTYTVHAAHAYTYt, small letters indicate D amino acids) selected by in vitro inhibition studies against an hen egg lysozyme peptide 8–29-specific T cell hybridoma restricted to the MHC class II allele H2-Ag7 of the diabetes-prone NOD mouse that inhibited onset of diabetes in vivo. This ligand bound with an IC50 of ~1 µM to H2-Ag7. In such approaches, the selection of the competing determinant strictly depends on the availability of information on relevant T cell epitopes. Single amino acid modifications of the selected peptides to improve binding efficiency do not necessarily generate ligands with very high affinity. Our approach has the definitive advantage that it guarantees the generation of ligands with the highest possible affinity and reduces the risk of generating undesired altered peptide ligand effects. Additionally, acetylation and amidation further improved binding and stabilization against exopeptidases. Moreover, the small size of the peptides of 9 aa offers the important advantage that binding is restricted to a minimum of core residues and is therefore highly isotype and allele specific.

MOG-induced EAE in rats mimics many hallmarks of MS, such as CNS lesions with demyelination and axonal and neuronal loss (16, 17, 18, 21, 37, 38). This model is therefore highly relevant for investigations of disease pathogenesis and treatment strategies for MS. Interestingly, in LEW.1N (RT1n) rats, MOG 91–108 does not induce an ex vivo detectable T cell response in peripheral lymphoid tissue, but a strong cellular response is detectable in the CNS (18). Upon coimmunization of MOG 1–125 or MOG 91–108 and p17 in CFA in LEW.1N (RT1n) rats, disease was completely abrogated, indicating that there was prevention of intracerebral autoimmunity. We have demonstrated that p17 binds to intact MHC class II molecules on the cell surface. Our results indicate that p17 with an IC50 of 2 nM efficiently competes with MOG 91–108 for presentation on RT1.Dn, because MOG 91–108 has a higher IC50 of 30 nM.

We performed in vitro studies of T cells that had been eluted from the CNS and subsequently exposed to Ag in the presence of irradiated thymocytes. The addition of p17 led to a reduction of the MOG 91–108-specific T cell responses. These data underscore that p17 is competing at the immunological synapse for presentation of autoantigen. In light of these results in vitro, our in vivo studies would also strongly indicate that p17 acts by competition for presentation of autoantigen on APC.

Not only is p17 capable of blocking disease upon coimmunization with either MOG 91–108 or rrMOG 1–125, rrMOG-induced EAE could also be treated by the i.p. application of p17. Moreover, application of the encephalitogen and p17 at the same time point, but at different sites of the body, led to a significant attenuation of EAE. This cannot solely be explained by competition at the level of the restrictive MHC class II molecule. Although coimmunization and treatment of EAE with p17 led to a clear reduction of IFN-{gamma}-secreting cells in response to rrMOG 1–125, IL-4 and IL-10 measurements did not indicate a dramatic Th1/Th2 shift. Moreover, the protective effect of p17 could not be transferred. p17 as well as the other high affinity ligands tested raised a considerable IFN-{gamma} response upon immunization. Therefore, it can be assumed that disease protection in the context of inflammation not only occurred because of competition at the MHC level, but also because of the immune response generated against p17 with presence of high avidity T cells suppressing the low avidity MOG-specific self-reactive T cell repertoire (28).

There is a multitude of mechanisms discussed in the context of the action of COP-1 (22). Recently, it has been shown that modified COP-1 variants improved clinical efficiency (39). The variants with the best clinical effect showed improved fitting to MHC class II molecules. Our study regarding the effects of p17 would underscore that the mechanism of COP-1 in MS is competition for binding at the level of MHC class II presentation. In contrast to COP-1 and its variants, p17 and related nonamer peptides have a nearly absolute MHC class II isotype and allele specificity and show improved binding. As we have demonstrated for p17 and COP-1 at equimolar concentrations, p17 and related peptides are of superior efficiency compared with COP-1 to reduce MOG-specific immune responses.

We have demonstrated that MOG-induced EAE in rats can be attenuated by highly selective isotype- and allele-specific intervention, targeting disease-associated MHC class II molecules at the immunological synapse. We did not observe undesired side effects such as anaphylactic reactions, enhanced autoimmunity, or general immunosuppression. Therefore, peptides or peptidomimetics selectively intervene with presentation of autoantigenic peptides on disease-associated MHC class II isotype allelic variants. The therapeutic potential of this strategy merits further investigations in clinical trials for treatment of autoimmune diseases of humans.


   Acknowledgments
 
We thank Hans-Georg Rammensee for very valuable comments on the manuscript.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported in part by grants from the Deutsche Forschungsgemeinschaft (DFG We 1947/2-3 and Sonderforschungsbereich 510) and by EMC Microcollections. R.W. holds a Heisenberg fellowship of the Deutsche Forschungsgemeinschaft (We 1947/4-1). Back

2 Address correspondence and reprint requests to Dr. Robert Weissert, Experimental Neuroimmunology Laboratory, Department of General Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, Hoppe-Seyler-Strasse 3, 72076 Tübingen, Germany. E-mail address: robert.weissert{at}uni-tuebingen.de Back

3 Abbreviations used in this paper: MS, multiple sclerosis; COP-1, copolymer-1; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; MNC, mononuclear cell; MOG, myelin oligodendrocyte glycoprotein; p.i., postimmunization; rrMOG, rat rMOG. Back

Received for publication September 5, 2003. Accepted for publication June 1, 2004.


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