Four different synthetic peptides of proteolipid protein induce a distinct antibody response in MP4-induced experimental autoimmune encephalomyelitis

YCLIM-07470; No. of pages: 14; 4C: Clinical Immunology (2015) xx, xxx–xxx

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Clinical Immunology www.elsevier.com/locate/yclim

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Mascha S. Recks a,1 , Nicolai B. Grether b,1 , Franziska van der Broeck c , Alla Ganscher b , Nicole Wagner b , Erik Henke b , Süleyman Ergün b , Michael Schroeter d , Stefanie Kuerten b,⁎

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Department of Anatomy II (Neuroanatomy), University of Cologne, Kerpener Straβe 62, 50924 Cologne, Germany Department of Anatomy and Cell Biology, University of Wuerzburg, Koellikerstr. 6, 97070 Wuerzburg, Germany c Department of Anatomy I, University of Cologne, Joseph-Stelzmann-Str. 9, 50931 Cologne, Germany d Department of Neurology, University of Cologne, Kerpener Str. 62, 50924 Cologne, Germany

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Four different synthetic peptides of proteolipid protein induce a distinct antibody response in MP4-induced experimental autoimmune encephalomyelitis

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Received 16 April 2015; accepted with revision 30 April 2015

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Abstract Here we studied the autoantibody specificity elicited by proteolipid protein (PLP) in MP4-induced experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis (MS). In C57BL/6 (B6) mice, antibodies were induced by immunization with one of the two extracellular and by the intracellular PLP domain. Antibodies against extracellular PLP were myelin-reactive in oligodendrocyte cultures and induced mild spinal cord demyelination upon transfer into B cell-deficient JHT mice. Remarkably, also antibodies against intracellular PLP showed binding to intact oligodendrocytes and were capable of inducing myelin pathology upon transfer into JHT mice. In MP4-immunized mice peptide-specific TH1/TH17 responses were mainly directed against the extracellular PLP domains, but also involved the intracellular epitopes. These data suggest that both extracellular and intracellular epitopes of PLP contribute to the pathogenesis of MP4-induced EAE already in the setting of intact myelin. It remains to be elucidated if this concept also applies to MS itself. © 2015 Published by Elsevier Inc.

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Animal models; Antibodies; Autoimmunity; B cells; Multiple sclerosis

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KEYWORDS

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Abbreviations: B6, C57BL/6; CNS, central nervous system; MBP, myelin basic protein; MP4, MBP–PLP fusion protein; MS, multiple sclerosis; PLP, proteolipid protein. ⁎ Corresponding author at: Department of Anatomy and Cell Biology, University of Wuerzburg, Koellikerstr. 6, 97070 Wuerzburg, Germany. Fax: + 49 93131820871. E-mail address: [email protected] (S. Kuerten). 1 M.S.R. and N.B.G. contributed equally to this work.

http://dx.doi.org/10.1016/j.clim.2015.04.020 1521-6616 © 2015 Published by Elsevier Inc. Please cite this article as: M.S. Recks, et al., Four different synthetic peptides of proteolipid protein induce a distinct antibody response in MP4-induced experimental autoimmune encephalomyelitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.020

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Multiple sclerosis (MS) is thought to be a chronic autoimmune disorder of the central nervous system (CNS) affecting approximately two million people worldwide [1]. The etiology of the disease is still unclear. Due to the limited availability of tissue from MS patients that can be used for mechanism-oriented analyses, animal models of MS are often studied. The most commonly used animal model for MS is experimental autoimmune encephalomyelitis (EAE) that is inducible in susceptible animal strains either by active immunization with myelin components or the passive transfer of autoreactive T cells [2,3]. In addition, spontaneous models exist that make use of genetically-modified mice [4]. EAE is traditionally considered to be a T cell-mediated model [5]. Initially, EAE was provoked by immunization with whole spinal cord homogenate. Subsequently, defined encephalitogenic proteins of the CNS were identified and today immunization involves single peptides derived from CNS proteins. While immunizations with single short peptides typically trigger T cell responses, the induction of pathogenic B cell responses in EAE requires proteins [6]. To this end, the contribution of B cells to the immune pathogenesis of MS has not been studied well. Only recently, evidence for a crucial role of B cells in MS has accumulated. Antibody and complement depositions are frequently found in CNS lesions of patients with MS [7]. Plasmapheresis has been shown to be successful in a fraction of patients [8] and the therapeutic efficacy of the anti-CD20 antibody rituximab has been demonstrated [9]. In addition, B cells have been shown to form aggregates in the meninges of patients with secondary progressive MS, and these aggregates have been linked to an earlier disease onset and more severe disease progression both clinically and histologically [10, 11]. To our knowledge, three mouse models reflecting the B cell component of MS are available. One model is the human myelin oligodendrocyte glycoprotein (hMOG)-induced EAE in C57BL/6 mice that has been used to study mechanisms of antibody encephalogenicity [12, 13]. In addition, there is one spontaneous EAE model that relies on the presence of a T cell receptor specific for MOG 92-106 [4]. In this model it was demonstrated that MOG-transgenic T cells can expand MOG-specific B cells that subsequently secrete antibodies which differ from the T cell target antigen in their specificity. In 2006, we introduced MP4-induced EAE in C57BL/6 (B6) mice [14]. MP4 is a fusion protein that is comprised of the human myelin basic protein (MBP) and the three hydrophilic domains of proteolipid protein (PLP). It has been shown to be highly effective in suppressing EAE caused by multiple neuroantigenic epitopes when administered systemically [15]. In the study by Elliott et al. SJL/J mice were actively immunized with PLP: 139–151 or MP4. Alternatively, passive EAE was induced by the adoptive transfer of MBP- and/or PLP: 139–191-specific T cells. Intravenous treatment with MP4 was successful in preventing EAE and in the treatment of established EAE. Mice treated with MP4 were also resistant to the reinduction of EAE after immunization with PLP: 139–151 and rechallenge. The B cell dependence of MP4-induced EAE was demonstrated in B cell-deficient mice that were resistant to the disease induced by MP4 as well as by the infiltration of B cells into the CNS that developed B cell aggregates and consecutively tertiary lymphoid organs [14, 16, 17]. We have also previously reported on

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the antibody dependence of MP4-induced EAE. On the one hand, transfer of MP4-reactive serum into MP4-immunized B cell-deficient mice restored the disease to the level of the wild-type mice [18]. On the other hand, the deposition of antibodies and complement components was evident in the CNS of MP4-immunized mice in association with demyelinated lesions [19]. While C57BL/6 mice are resistant to disease induced by MBP, PLP is pathogenic [20, 21]. MBP is an entirely intracellular protein. On the contrary, PLP possesses two extracellular domains that are easily accessible to autoreactive immune cells and autoantibodies. One major shortcoming has been, however, that PLP is a hydrophobic protein, which has limited experimentation with the whole molecule. In C57BL/6 mice the encephalitogenic determinant for T cells has been shown to be the PLP peptide 178–191 that is located on the second extracellular loop of the MP4 molecule [20]. It is unclear, which domain of the MP4 molecule is responsible for the induction of the B cell component in the model. Overall, studies have focused on defining encephalitogenic T cell determinants within the antigens used for EAE, while studies of B cell targets are scarce. The aim of this study was to investigate the specificity of the MP4-induced antibody response and to characterize its pathogenic contribution to the disease. To this end, peptides covering the two extracellular and the intracellular domain of the PLP part of MP4 were synthesized and their capacity to induce an encephalitogenic autoantibody response was investigated. In the following, we demonstrate a differential involvement of the PLP-derived domains of MP4 in the development and histopathology of EAE.

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1. Introduction

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Figure 1 Localization of the different PLP epitopes within the two extracellular and the intracellular loop of the MBP–PLP fusion protein MP4. For active immunization of B6 mice and for passive transfer experiments of B cell-deficient JHT mice, five peptides were synthesized: the three extracellular epitopes PLP peptide 36–70 (Extra I), PLP peptide 178–208 (Extra II) and PLP peptide 209–238 (Extra III) as well as the two intracellular epitopes PLP peptide 88–125 (Intra I) and PLP peptide 126–155 (Intra II).

Please cite this article as: M.S. Recks, et al., Four different synthetic peptides of proteolipid protein induce a distinct antibody response in MP4-induced experimental autoimmune encephalomyelitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.020

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Antibody epitopes in EAE

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Female C57BL/6 (B6) mice were purchased from Janvier (Saint Berthevin Cedex, France). B cell deficient J T mice H were originally created by Klaus Rajewsky and a kind gift from Ari Waisman (University of Mainz, Germany). All mice

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2.1. Mice

were 6–8 weeks old at the time of treatment. The mice were maintained at the animal facilities of the Department of Anatomy, University of Cologne and the Department of Anatomy and Cell Biology, University of Wuerzburg, under specific pathogen-free conditions. All treatments were performed according to an approved protocol and complied with the institutional guidelines (permission no. 2011.A276 and 114/13, principal investigator: Stefanie Kuerten).

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2. Materials and methods

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Figure 2 IgG1 and IgG2a antibody titers after immunization of B6 mice with PLP peptides. Wild-type B6 mice were immunized with 100 μg of one of the five PLP peptides Extra I (A; n = 17), Extra II (B; n = 11), Extra III (C; n = 28), Intra I (D; n = 10) and Intra II (E; n = 13) in CFA and injected with pertussis toxin on days 0 and 2. ELISA assays were performed at the time points indicated by the circles in the panels. Means ± SD are shown. Please cite this article as: M.S. Recks, et al., Four different synthetic peptides of proteolipid protein induce a distinct antibody response in MP4-induced experimental autoimmune encephalomyelitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.020

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2.3. Enzyme-linked immunosorbent assay (ELISA)

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ELISA plates (Thermo Scientific, Schwerte, Germany) were coated overnight with MP4 or PLP peptides at 10 μg/ml diluted in PBS. Plates were blocked with 1% bovine serum albumin (BSA) (PAA Laboratories GmbH, Cölbe, Germany) or milk powder (Heirler Cenovis GmbH, Radolfzell, Germany) in PBS for 2 h at room temperature. The plates were incubated with serum at 4 °C overnight. All serum samples were diluted 1:400 in 1% BSA or 0.1% milk powder solution containing 0.05% Tween 20 detergent. Biotinylated anti-mouse IgG1 and

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OLN-93 cells [22] were cultured in Dulbecco's Modified Eagle Medium (DMEM; Life Technologies, Darmstadt, Germany) containing 10% fetal calf serum (FCS) (Life Technologies) and 0.5% penicillin–streptomycin (Sigma) at 37 °C and 5% CO2. In order to favor cell differentiation before immunocytochemistry (ICC), cells were split onto poly-L-ornithine (Sigma)-coated cover slips and grown for one day in DMEM containing 10% FCS. Afterwards, the amount of FCS was reduced to 0.5% and cells were grown for another two days.

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Cells were incubated with purified IgG antibodies against MP4 or the peptides Extra II, Intra I or Intra II at room temperature for 2 h. IgG purified from the sera of non-immunized mice was used as a negative control. Incubation in the absence of primary antibody served as an additional negative control (blank). All antibodies were used at a concentration of 100 μg/ml diluted in PBS and were obtained using a mouse antibody purification kit (Abcam, Cambridge, UK). Rabbit anti-mouse β-tubulin antibody (Covance, Princeton, NJ) was used at a dilution of 1:1000. Goat anti-mouse Cy2 and goat anti-rabbit Cy2 antibodies (Dianova, Hamburg, Germany) were used as secondary antibodies at dilution of 1:500 in PBS. Cells were then fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min. DAPI (Roche, Mannheim, Germany) was used as a nuclear counterstain at 1:1000 dilution and Alexa Fluor® 594-conjugated wheat germ agglutinin (WGA) (Life Technologies) at 1:200 dilution for membrane staining. Cover slips were mounted in n-propyl gallate (Sigma) and stored protected from light at 4 °C.

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The MBP–PLP fusion protein MP4/Apogen (containing the 21.5 kD isoform of human MBP and the three hydrophilic domains of PLP) was obtained from Alexion Pharmaceuticals (Cheshire, CT). The MP4 peptides Extra 1 (GHEALTGTEKLIETYF SKNYQDYEYLINVIHAFQY), Extra II (FNTWTTCQSIAFPSKTSASIGSL CADARMYG), Extra III (VLPWNAFPGKVCGSNLLSICKTAEFQMTFH), Intra I (AEGFYTTGAVEQIFGDYKTTICGKGLSATVTGGQKGRG) and Intra II (SRGQHQAHSLERVCHCLGKWLGHPDKFVGI) (Fig. 1) were synthesized by Genscript (Piscataway, NJ, USA). Incomplete Freund's adjuvant (IFA) was prepared as a mixture of mannide monooleate (Sigma-Aldrich, St. Louis, MO) and paraffin oil (EM Science, Gibbstown, NJ). Complete Freund's adjuvant (CFA) was obtained by mixing Mycobacterium tuberculosis H37 RA (Difco Laboratories, Franklin Lakes, NJ) at 5 mg/ml into IFA. For active immunization, B6 mice were immunized subcutaneously in both sides of the flank with a total dose of 200 μg MP4 or 100 μg of the respective PLP peptide in CFA. Pertussis toxin (List Biological Laboratories, Hornby, ON, Canada) was given at 200 ng per mouse on the day of immunization and 48 h later. For passive transfer experiments, the protocol of Lyons et al. [12] was used with JHT mice receiving four 150 μl-injections of pooled antisera at three-day intervals for a total of 600 μl starting on day 0 of the immunization. The donor sera had been obtained from wild-type C57BL/6 mice after immunization with the PLP peptides at 100 μg/mouse or MP4 at 200 μg/mouse in CFA. The presence or absence of antigen-specific antibodies in the serum samples was confirmed by ELISA. Clinical assessment of EAE was performed daily according to the following criteria: (0), no disease; (1), floppy tail; (2), hind limb weakness; (3), full hind limb paralysis; (4), quadriplegia; (5), death. Mice that were in between the clear-cut gradations of clinical signs were scored intermediate in increments of 0.5.

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IgG2a antibodies (BD Biosciences, Heidelberg, Germany) diluted 1:400 were used as detection antibodies. All plates were developed with tetramethylbenzidine substrate (eBioscience, Frankfurt am Main, Germany) after incubation with streptavidin–horseradish peroxidase (eBioscience) at 1:1000 dilution. The reaction was stopped with 0.16 M sulphuric acid and the optical density (OD) in the wells was read at 450 nm using a FLUOstar Omega microplate reader (BMG Labtech GmbH, Ortenberg, Germany) or a Perkin Elmer (Waltham, MA) Victor 3 1420 Multilabel Counter with Wallac 1420 software version 3.00 revision 5.

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Table 1 Mouse strain

Treatment

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Mean day of onset

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Mean maximal disease severity

CNS pathology

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0/17 10/11

N/A 19.7 ± 9.5

N/A 2.6 ± 0.2

N/A 2.8 ± 0.3

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0/28 0/13 0/10

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N/A Inflammation, demyelination and axonal loss N/A N/A N/A

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iz immunized, Extra 1 PLP peptide 36–70, Extra II PLP peptide 178–208, Extra III PLP peptide 209–238, Intra I PLP peptide 88–125, Intra II PLP peptide 126–155.

Development of EAE after immunization with different EAE peptides.

Please cite this article as: M.S. Recks, et al., Four different synthetic peptides of proteolipid protein induce a distinct antibody response in MP4-induced experimental autoimmune encephalomyelitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.020

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Sections were first observed with a Keyence BZ-9000 fluorescence microscope (Neu-Isenburg, Germany). Cells were then

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Cells were fixed in 2% PFA and permeabilized with 0.1% Triton-X-100 for 10 min. Subsequently, cells were incubated with primary and secondary antibodies, stained with DAPI and WGA, mounted and stored as described above.

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2.6. ICC on fixed and permeabilized cells

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For the exclusion of unspecific binding, cells were incubated with FcRγIII/II-blocking solution (purified NA/LE rat anti-mouse CD16/CD32; BD Biosciences) at room temperature for 30 min. For blocking of clathrin-mediated endocytosis, cells were incubated with Pitstop 2® reagent (Abcam), Pitstop 2® control solution or with serum-free DMEM according to the vendor's instructions. Further steps were performed as described above, but with the following modifications: antibodies were diluted in serum-free DMEM and incubation of antibodies was performed at 37 °C and 5% CO2 for 30 min. Cells were counterstained with DAPI at a dilution of 1:10,000.

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Figure 3 CNS histopathology in B6 mice after immunization with PLP peptides. B6 mice were immunized with 100 μg of one of the five PLP peptides Extra I (A; n = 17), Extra II (B; n = 11), Extra III (C; n = 28), Intra I (D; n = 13) and Intra II (E; n = 10) in CFA and injected with pertussis toxin on days 0 and 2. CNS histopathology was assessed on methylene blue-stained semi-thin sections 133 days after immunization. Panel F refers to the spinal cord of a non-immunized mouse. Representative images are shown. The scale bars represent 80 μm. Please cite this article as: M.S. Recks, et al., Four different synthetic peptides of proteolipid protein induce a distinct antibody response in MP4-induced experimental autoimmune encephalomyelitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.020

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Please cite this article as: M.S. Recks, et al., Four different synthetic peptides of proteolipid protein induce a distinct antibody response in MP4-induced experimental autoimmune encephalomyelitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.020

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On day 14 after immunization with MP4, the spleens of B6 mice were removed, disintegrated mechanically and filtered through a 70 μm nylon cell strainer (BD Falcon, Heidelberg, Germany). After washing the cells with RPMI-1640 (Biochrom AG, Berlin, Germany) and counting them with acridine orange (0.1%, Sigma)/ethidium bromide (0.1%, Serva, Heidelberg, Germany), cells were resuspended in HL-1 (Lonza, Cologne, Germany) supplemented with 1% L-glutamine (Sigma) and 1% penicillin/streptomycin (Sigma). ELISPOT plates (Merck KGaA, Darmstadt, Germany) were coated overnight with the capture antibodies rat anti-mouse interferon (IFN)-γ (final concentration 4 μg/ml, clone AN-18, eBioscience) and rat anti-mouse interleukin (IL)-17 (final concentration 3 μg/ml, clone TC-11-18H10, BD Biosciences, San Diego, CA) in PBS. Plates were washed with PBS, and blocked with 1% BSA in PBS for 2 h at room temperature. Cells were plated at 1 × 106 cells/well. Cells were incubated either with medium or PLP peptides (final concentration: 15 μg/ml) at 7% CO2 and 37 °C for 24 h. Plates were washed and incubated with biotin-conjugated anti-IFN-γ (1 μg/ml; eBioscience, clone R4-6A2) or anti-IL-17 (1 μg/ml; eBioscience, clone TC-11-8H4.1) at 4 °C overnight. After washing, plates were incubated for 2 h with streptavidinconjugated alkaline phosphatase (1:1000; Dako, Glostrup, Denmark). Plates were developed with Vector Blue (Vector Laboratories, Burlingame, CA) solution according to the vendor's instructions. Plates were air-dried overnight and spots were counted with an ImmunoSpot Series 6 Analyzer (Cellular Technology Limited, Shaker Heights, OH). All results were medium-subtracted and normalized to 106 cells per well.

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2.10. Statistical analysis

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Differences in disease incidence were calculated using Fisher's Exact Test. For the evaluation of differences in disease onset and clinical EAE scores one-way ANOVA was used. Differences in the extent of spinal cord histopathology and cytokine production were assessed by Student's t-test. In case the Normality Test or the Equal Variance Test failed, the Mann–Whitney U rank-sum test was used. Statistical significance was determined with SigmaPlot, version 12.0 software (SPSS Inc., Chicago, IL) and set at p ≤ 0.05.

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2.9. Enzyme-linked immunospot assays (ELISPOT)

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At the time point of sacrifice, mice were deeply anesthetized with CO2 and perfused intracardially with 4% PFA/4% glutaraldehyde in 0.1 M PBS (pH = 7.4) (Serva Electrophoresis GmbH, Heidelberg, Germany). The tissue was post-fixed at 4 °C in PFA-GA for at least 24 h before spinal cords were carefully removed from the vertebral canal. The lumbar part was cut off with sharp blades, subdivided into three equidistant parts and post-fixed in PFA-GA for 24 h at 4 °C. Specimens were rinsed in cacodylate buffer (pH = 7.35) three times and treated with 1% osmium tetroxide on ice for 4 h. The tissue was rinsed again in cacodylate buffer and dehydrated in a graded series of ethanol before overnight treatment with 1% uranyl acetate in 70% ethanol for contrast enhancement. On the next day, the specimens were embedded in epon (Serva Electrophoresis GmbH) and polymerized at 60 °C for 72 h. Of each epon-embedded transverse spinal cord sample, 1 μm thick semi-thin sections and 80 nm thick ultra-thin sections were cut on a Leica Ultracut UCT ultramicrotome (Leica Microsystems, Wetzlar, Germany). Semi-thin sections were stained with methylene blue and observed with a Leica DM LB2 microscope (Leica Microsystems), Zeiss camera and software (Carl Zeiss AG, Oberkochen, Germany). Ultra-thin sections were mounted on 150 mesh Formvar-coated copper grids (Electron Microscopy Sciences, Hatfield, PA) and contrasted with 1% uranyl acetate (Plano GmbH, Wetzlar, Germany) and Reynold's lead citrate solution (Merck KGaA). Sections were examined on a Zeiss EM 902 A transmission electron microscope (Carl Zeiss AG) at 80 kV acceleration voltage. Images were taken with a digital EM camera (MegaView III, Olympus Soft Imaging Systems GmbH, Münster, Germany). Ten images were obtained from the ventrolateral tract in each of the three equidistant lumbar spinal cord segments at 3000 × and 10 images at 7000 ×, resulting in a total of 60 images per mouse. Image analysis was conducted with the Image-Pro Plus software (Version 6.0, Media Cybernetics, Bethesda, MD). Histological examination of spinal cord pathology was done blinded as to the clinical score and immunization status of the animals. For analysis of myelin pathology, the g-ratio (axon diameter divided by nerve fiber diameter) [23, 24] was measured for each nerve fiber. In order to determine the optimal value of the g-ratio, non-immunized JHT (n = 5) control mice were evaluated. The mean value was calculated for each control mouse and the three-sigma limit was assessed from the mean values of all five control mice. Hence, the optimal g-ratio for the ventrolateral tract was

0.37–0.91. Nerve fibers displaying a g-ratio below the optimal range were classified as demyelinating (i.e. nerve fibers with a broadened, swollen myelin sheath) and nerve fibers with a g-ratio of 1 as demyelinated (i.e. complete loss of the myelin sheath). Thereby, the number of normal, demyelinating and demyelinated nerve fibers/mm2 was calculated for each mouse. Besides myelin pathology, the amount of axonal pathology was assessed by counting the number of axolytic nerve fibers/mm2.

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2.8. Analysis of semi and ultrathin sections of the spinal cord

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observed with a confocal microscope (Nikon A1R MP), and z-stacks were generated. Different channels were merged with the Image J 1.46r software (National Institutes of Health, Bethesda, USA).

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Figure 4 Immunocytochemistry of oligodendrocytes stained with antibodies against MP4 and the different PLP peptides. The oligodendrocyte cell line OLN-93 was incubated with purified IgG from non-immunized control mice (A), or from MP4- (B), Extra II- (C), Intra I(D) and Intra II-immunized mice (E). Antibody against β-tubulin was used as a control (F). DAPI was used as nuclear counterstain and wheat germ agglutinin (WGA) for visualizing the cell membrane. Representative confocal images for a total of n = 8 experiments are shown. Scale bars represent 20 μm. Please cite this article as: M.S. Recks, et al., Four different synthetic peptides of proteolipid protein induce a distinct antibody response in MP4-induced experimental autoimmune encephalomyelitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.020

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3.3. The capacity of PLP-induced antibodies to bind to the oligodendrocyte surface In order to evaluate the cross-reactivity of the PLP-peptide induced antibodies with PLP in its native conformation in the oligodendrocyte/myelin continuum we stained living oligodendrocytes with purified IgG antibodies against MP4 or individual PLP peptides. The antibodies were obtained from n = 6 MP4-, n = 7 Extra II-, n = 6 Intra I- and n = 8 Intra II-immunized mice. All antibodies were reactive to the respective peptides and to native MP4 as confirmed by ELISA. In addition, we purified control IgG from the sera of n = 10 non-immunized wild-type B6 mice. The oligodendrocyte cell line OLN-93 was used, which was originally derived from spontaneously transformed cells in primary rat brain glial culture [22] and resembles postnatal cultured rat brain oligodendrocytes in its morphological features and antigenic properties. Purified IgG from serum of non-immunized mice did not show any myelin-specific binding (Fig. 4A). Strong

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In order to investigate the potential encephalitogenicity of the antibodies induced by the different PLP peptides, we performed serum transfer experiments and subsequent histological analysis in B cell-deficient JHT mice. To this end, B cell-deficient JHT mice were immunized with 200 μg MP4 in CFA and pertussis toxin was given as above. The mice then received four injections of 150 μl pooled serum containing antibodies against MP4 or the different PLP peptides on days 0, 4, 8 and 12 after immunization. Of the JHT mice that received MP4-specific serum 5 of 6 developed severe EAE, while one mouse showed transient disease with a clinical score of 1.0. Conversely, anti-Extra II antibodyinjected JHT mice (n = 5) did not show any clinical signs of EAE. In JHT mice that received serum containing antibodies specific for the two intracellular PLP epitopes, severe clinical EAE developed in 2 of 5 mice that received Intra I peptide-specific serum and in 1 of 5 mice in the Intra II group. However, disease onset was delayed in comparison to MP4 serum-transferred mice. Two mice that received Intra I-specific antibodies and one Intra II serum-transferred mouse developed mild disease with a clinical score of 0.5. Table 2 summarizes the data on disease incidence, onset and severity following serum transfer into JHT mice and Table 3 refers to the corresponding statistical analysis. On days 18, 25 (MP4) or 50 (Extra II, Intra I, Intra II) after immunization, JHT mice were sacrificed and ultrastructural analysis of the spinal cord was performed. For analysis of myelin pathology, the g-ratio (axon diameter divided by nerve fiber diameter) was measured for each nerve fiber after establishment of the optimal g-ratio range in non-immunized control mice (see the Materials and methods section for details). As shown in Fig. 6A, mice from all passive transfer groups displayed increased numbers of nerve fibers with axonal and/or myelin pathology/mm2 when compared to control mice. Interestingly, slight myelin pathology was evident in Extra II-transferred JHT mice although clinically evident EAE was not observed. The same applied to mice that received either Intra I or Intra II peptide-specific serum, but did not

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3.2. Only mice immunized with the peptide Extra II develop EAE both clinically and histopathologically To investigate whether the induction of peptide-specific antibodies was associated with the development of EAE, we immunized B6 mice with 100 μg of Extra I, Extra II, Extra III, Intra I or Intra II peptide. The development of EAE was monitored over a period of 133 days and is shown in Table 1. While 10 of 11 mice immunized with Extra II developed EAE on day 19.7 ± 9.5 after immunization with a mean maximal score of 2.8 ± 0.3, none of the mice immunized with any of the other peptides showed any signs of the disease. This finding was confirmed by histological analysis of methylene-blue stained semi-thin sections obtained from the lumbar part of the spinal cord on day 133 after immunization. While inflammation of the anterolateral tract of the spinal cord was evident in Extra II-immunized mice (Fig. 3B), the spinal cord of Extra I-, Extra III-, Intra I- and Intra II-immunized mice resembled the one of non-immunized control mice (Figs. 3A and C–F).

3.4. Encephalitogenicity of antibodies induced by the extracellular and intracellular domains of PLP

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binding to the oligodendrocyte surface was observed after incubation with MP4-specific antibodies (Fig. 4B). A similar staining pattern was achieved with antibodies against the Extra II peptide (Fig. 4C). Surprisingly, we also observed membrane-associated binding to intact oligodendrocytes after incubation with Intra I- or Intra II-specific antibodies (Figs. 4D, E). This staining could not be blocked when inhibiting endocytosis by Pitstop 2® reagent (data not shown). When oligodendrocytes were fixed and permeabilized prior to immunocytochemistry, antibodies against MP4, Extra II, Intra I and Intra II peptides all stained internal antigens in the cell bodies of the oligodendrocytes (Figs. 5A–E). In order to survey the integrity of the cells during immunocytochemistry, we also stained both living and permeabilized cells with an anti-β-tubulin antibody. Since only permeabilized cells showed an intracellular staining of β-tubulin, we conclude that the staining observed with antibodies against the peptides Intra I and Intra II was not related to damage of the cell membrane (Figs. 4F and 5F).

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Wild-type B6 mice were immunized with 100 μg of one of the five PLP peptides Extra 1 (n = 17), Extra II (n = 11), Extra III (n = 28), Intra I (n = 10) or Intra II (n = 13) in CFA (Fig. 1). Pertussis toxin was injected on days 0 and 2. Serum was collected every 14 days and screened for the respective PLP peptide-specific IgG1 and IgG2a antibodies by serum ELISA. The antibody response to PLP immunization in C57BL/6 mice is shown in Fig. 2. While the peptides Extra II, Intra I and Intra II induced strong responses that covered both the IgG1 and IgG2a isotype (with 9 of 11, 10 of 10 and 13 of 13 mice responding, respectively), the Extra I peptide failed to induce an antibody response and only transiently induced antibodies in 5 of 28 mice. In another group of n = 18 mice immunized with the Extra III peptide no Extra III-specific antibodies were detectable.

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3.1. Antibodies are induced by both the extracellular and intracellular domains of PLP

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Please cite this article as: M.S. Recks, et al., Four different synthetic peptides of proteolipid protein induce a distinct antibody response in MP4-induced experimental autoimmune encephalomyelitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.020

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3.5. The MP4-specific T cell response targets both extra- and intracellular PLP

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Here we show that immunization with peptides derived from both the second extra- and the intracellular domain of PLP induced peptide-specific antibodies in C57BL/6 mice. After transfer of serum containing antibodies against the different PLP peptides into MP4-immunized B cell-deficient JHT mice differences in the clinical disease development and CNS histopathology were observed. While JHT mice that received Extra II-specific serum did not display any clinical disease, these mice showed demyelinated nerve fibers in the spinal cord. Transfer of antibodies directed against the intracellular domain of PLP was capable of inducing both EAE and

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detectable with a bias towards the peptide Intra II, which was significant for IFN-γ.

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display severe clinical EAE. Representative images are shown in Figs. 6B–F.

To analyze the peripheral antigen-specific T cell response to the different extra- and intracellular PLP epitopes in MP4-induced EAE, B6 mice (n = 6) were immunized with 100 μg MP4 in CFA with pertussis toxin given on the day of immunization and 48 h later. On day 14 after immunization, mice were sacrificed and splenocytes were assessed for their MP4-specific TH1 and TH17 response in IFN-γ and IL-17 ELISPOT assays (Fig. 7). Our data show that the extracellular epitopes triggered significantly stronger antigen-specific TH1/TH17 responses than the intracellular PLP domains. Responses against the two intracellular peptides were

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Figure 5 Immunocytochemistry of oligodendrocytes stained with antibodies against MP4 and the different PLP peptides after fixation and permeabilization. The oligodendrocyte cell line OLN-93 was fixed with 4% PFA and permeabilized with 0.1% Triton-X prior to incubation with purified IgG from non-immunized control mice (A), or from MP4- (B), Extra II- (C), Intra I- (D) and Intra II-immunized mice (E). Antibody against β-tubulin was used as a control (F). DAPI was used as nuclear counterstain and wheat germ agglutinin (WGA) for visualizing the cell membrane. Representative confocal images for a total of n = 2–4 experiments are shown. Scale bars represent 20 μm.

t2:1 t2:2 t2:3

Table 2

Mouse strain

Treatment

Disease incidence

Mean day of onset

Mean maximal disease severity

t2:4 t2:5 t2:6 t2:7

JH T JH T JH T JH T

MP4 MP4 MP4 MP4

6/6 0/5 4/5 2/5

13.0 ± 0.0 N/A 32.5 ± 7.7 33.0 ± 7.1

2.5 ± 0.8 N/A 1.6 ± 1.2 1.5 ± 1.4

t2:8 t2:9

iz immunized, Extra 1 PLP peptide 36–70, Extra II PLP peptide 178–208, Extra III PLP peptide 209–238, Intra I PLP peptide 88–125, Intra II PLP peptide 126–155, MP4 MBP–PLP fusion protein.

Development of clinical EAE after transfer of MP4- or PLP peptide-reactive serum into MP4-immunized JHT mice.

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+ + + +

MP4 serum Extra II serum Intra I serum Intra II serum

Please cite this article as: M.S. Recks, et al., Four different synthetic peptides of proteolipid protein induce a distinct antibody response in MP4-induced experimental autoimmune encephalomyelitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.020

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Table 3

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MP4 vs. Extra II MP4 vs. Intra I MP4 vs. Intra II Extra II vs. Intra I Extra II vs. Intra II Intra I vs. Intra II

N/A b 0.01 b 0.01 N/A N/A n.s.

b 0.01 b 0.01 b 0.01 b 0.01 n.s. n.s.

F

Determined by Fishers' exact test. Determined by one-way ANOVA.

the manifestation of new lesions partly depends on the composition of the myelin sheath and the prevalence of epitopes that are recognized by autoreactive cells in the CNS. Along these lines, Berger et al. have previously demonstrated

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histopathological alterations in the spinal cord, although only one mouse in each group developed severe disease. In MS patients lesions are typically disseminated within the CNS and their location is unpredictable. It is conceivable that

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Statistical analysis for comparison of disease parameters in JHT mice after respective serum transfer.

Figure 6 Antibodies specific for extra- and intracellular PLP domains are capable of inducing CNS histopathology in JHT mice. Passive transfer of serum containing antibodies specific for different PLP peptides into B cell-deficient JHT mice was performed as described in the Materials and methods section. The lumbar spinal cords were embedded in epon and ultra-thin sections were cut for electron microscopic analysis. Images were taken at 3000 × magnification and the number of nerve fibers/mm2 displaying axonal and/ or myelin pathology was calculated for each epitope (A). Representative images for MP4 (B; n = 6), PLP Extra II (C; n = 3), Intra I/II (D and E; n = 6) as well as for non-immunized control mice (F; n = 5) are shown at 3000 × magnification. *p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001. Please cite this article as: M.S. Recks, et al., Four different synthetic peptides of proteolipid protein induce a distinct antibody response in MP4-induced experimental autoimmune encephalomyelitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.020

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II-specific serum although clinical disease was not observed. Surprisingly, staining of the oligodendrocytes was also observed with antibodies against Intra I and Intra II. Comparing our staining pattern to previous descriptions, antibodies against Intra I and Intra II were neither localized within the rough endoplasmatic reticulum nor the endosomal compartment [43, 44]. Accordingly, the staining could not be blocked by using the Pitstop 2® reagent, which inhibits clathrin-mediated endocytosis (data not shown). The antibody binding rather seemed to be localized within the myelin-like membrane of the cultured oligodendrocytes itself. These results could explain the development of EAE and CNS histopathology in JHT mice that were transferred with Intra I/II-specific serum. At this point, it is unclear why the antibodies directed against Intra I and Intra II were capable of binding to intact myelin. Previous studies have shown that incorporation of anti-PLP IgM into the myelin sheath during development can cause “wide-spaced” or “expanded myelin”, focal demyelination and remyelination [45]. In patients with demyelinating polyneuropathy penetration of anti-myelin associated glycoprotein (MAG) antibodies into myelinated peripheral nerve fibers and widening of the myelin sheath was observed. However, the pathogenic mechanisms of antibody penetration and subsequent nerve fiber damage have remained unclear [46]. To our knowledge, there is currently no report in the literature on the penetration of anti-PLP antibodies into the myelin sheath and future studies are clearly needed to elucidate the mechanisms behind the results reported here. Our data may suggest that there is no single MP4/ PLP-specific B cell domain that determines the B cell/ antibody dependence in the model, but that the B cell/ antibody component of the disease is likely to be due to a combination of responses against both extracellular and intracellular epitopes. Our data demonstrate that the tissue pathology induced by PLP peptide-specific antibodies was frequently only subtle. It is conceivable that the individual domains by themselves induce pathologic changes within the CNS that are below a certain threshold to induce clinical symptoms. However, when combined in the setting of immunization with the whole MP4 molecule, tissue damage is apparently severe enough to cause clinically evident EAE.

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that the antigen specificity of T cells determines the topography of CNS lesion development in adoptive transfer EAE experiments [25]. The influence of autoantibody specificity on lesion topography is less clear. Antibody depositions are frequently found in brain lesions of MS patients [7], but the specificity of these antibodies is still unclear [26–29]. While previous reports failed to provide evidence for binding of antibodies derived from MS patients to any of the prevalent myelin antigens including MOG, PLP, or MBP [28], a recent study used antigen arrays and identified antibodies of the IgG subclass against myelin antigens including PLP in 59% of cases [29]. The general thought is that extracellular domains of a CNS antigen are more likely to initiate an antibody response against the respective antigen than intracellular domains, while the latter are rather involved in the course of the disease as a consequence of tissue damage and epitope spreading [30–33]. The pathogenicity of antibodies recognizing surface exposed PLP B cell epitopes has been studied before. Screening polyclonal rabbit anti-rat and mouse anti-human PLP antisera, amino acid residues 200–217 have been shown to be an important antibody recognition site [34]. Another study identified the carboxyl-terminal amino acid, phenylalanine276, as a critical requirement for antibody recognition of this epitope in the Lewis rat [35]. Contrasting the traditional notion of primary antibody binding to extracellular epitopes in autoimmune diseases, antibodies directed against the intracellular antigen hnRNP A1 have only recently been found to penetrate into neuronal cells through clathrin-mediated endocytosis, causing depletion of cellular ATP and apoptosis [36]. Other antibodies including anti-AQP4, anti-U1RNP, anti-dsDNA, anti-Ro/SSA, anti-La/SSB and anti-Hu have also been reported to penetrate cells in vitro or in vivo [37–42]. Overall, the mechanisms of recognition of intracellular epitopes have remained unclear. To further explain our results, we stained oligodendrocyte cultures with antibodies directed against the respective PLP peptides. Extra II-specific antibodies bound to the surface of the oligodendrocytes, which could explain the presence of demyelination in JHT mice that received Extra

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Figure 7 Antigen-specific T cells target both the extra- and intracellular domains of PLP in MP4-immunized mice. B6 mice (n = 6) were immunized with MP4 and ELISPOT assays for the detection of PLP peptide-specific production of IFN-γ and IL-17 by splenocytes were performed on day 14. All results are medium-subtracted. *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Please cite this article as: M.S. Recks, et al., Four different synthetic peptides of proteolipid protein induce a distinct antibody response in MP4-induced experimental autoimmune encephalomyelitis, Clin. Immunol. (2015), http://dx.doi.org/10.1016/j.clim.2015.04.020

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We would like to thank Jolanta Kozlowski for technical assistance and Michael Christof for help with the figure design. This work was supported by a research grant from

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the German Research Foundation (DFG) (project KU 2760/21) and the Deutsche Multiple Sklerose Gesellschaft (DMSG) [grants to S.K.]. N.B.G. was supported by the Koeln Fortune Program/Faculty of Medicine, University of Cologne.

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In MS it has been demonstrated that the transition from relapsing-remitting to secondary progressive MS and the development of irreversible deficits in patients is associated with accumulating nerve fiber damage [47, 48]. Therefore, it is tempting to speculate that also in MS a multitude of antibody specificities contributes to the disease and that the sum of pathological events induced by these specificities accounts for the gross picture of CNS histopathology that is responsible for the clinical deficits in patients [49–51]. Along these lines, Robinson et al. used “myelin proteome” microassays for profiling the evolution of autoantibody responses in EAE and they showed that an increased diversity of autoantibody responses in acute EAE was predictive of a more severe clinical course of EAE. In addition, in chronic EAE, extensive intra- and intermolecular epitope spreading of autoreactive B cell responses was evident. As a conclusion, Robinson et al. suggested the use of proteomic monitoring of autoantibody responses as a useful tool for the development of disease-specific tolerizing DNA vaccines that could be tailored for individual patients [52]. Future studies will also have to address the relationship of protective and pathogenic B cell to T cell epitopes. A study by Tan et al. tested the capacity of IFN-γ-treated astrocytes from SJL/J mice to process and present encephalitogenic PLP epitopes from purified PLP and from MP4 to PLP epitope-specific T cell lines and hybridomas [53]. The treated astrocytes processed both MP4 and PLP for activation of a PLP: 139–151-specific T cell line/hybridoma. In contrast to SJL/J splenocytes, no activation of T cell lines/ hybridomas specific for the subdominant PLP: 56–70 and/or PLP: 178–191 residues was observed. In our present study we tested the recognition of the different PLP epitopes by T cells in MP4-immunized mice. While strong TH1/TH17 responses were detected against the extracellular PLP peptides, we also observed antigen-specific production of IFN-γ and IL-17 upon stimulation of splenocytes with intracellular epitopes. These data underline the notion that both extra- and intracellular epitopes of PLP are involved in MP4-induced EAE. While the exact role of autoantibodies is far from being fully understood in patients with MS, the data presented here highlight the role of B cells and autoantibodies in the disease pathogenesis. The early diversification of the autoimmune response underlines the need for therapeutic intervention at the earliest possible time point in order to increase the chances for delaying disease progression. The further improvement of tools to monitor the autoimmune response in patients along with the development of individual patient-adjusted treatment options will be a primary goal that needs to be achieved by future research.

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