TY - JOUR
AU - 't Hart, B A
AB - Summary Current therapies for multiple sclerosis (MS), a chronic autoimmune neuroinflammatory disease, mostly target general cell populations or immune molecules, which may lead to a compromised immune system. A more directed strategy would be to re-enforce tolerance of the autoaggressive T cells that drive tissue inflammation and injury. In this study, we have investigated whether the course of experimental autoimmune encephalomyelitis (EAE) in mice and marmosets can be altered by a potent tolerizing fusion protein. In addition, a multi-parameter immunological analysis was performed in marmosets to assess whether the treatment induces modulation of EAE-associated cellular and humoral immune reactions. The fusion protein, CTA1R9K-hMOG10–60-DD, contains a mutated cholera toxin A1 subunit (CTA1R9K), a dimer of the Ig binding D region of Staphylococcus aureus protein A (DD), and the human myelin oligodendrocyte glycoprotein (hMOG) sequence 10–60. We observed that intranasal application of CTA1R9K-hMOG10–60-DD seems to skew the immune response against myelin oligodendrocyte glycoprotein (MOG) towards a regulatory function. We show a reduced number of circulating macrophages, reduced MOG-induced expansion of mononuclear cells in peripheral blood, reduced MOG-induced production of interleukin (IL)-17A in spleen, increased MOG-induced production of IL-4 and IL-10 and an increased percentage of cells expressing programmed cell death-1 (PD-1) and CC chemokine receptor 4 (CCR4). Nevertheless, the treatment did not detectably change the EAE course and pathology. Thus, despite a detectable effect on relevant immune parameters, the fusion protein failed to influence the clinical and pathological outcome of disease. This result warrants further development and improvement of this specifically targeted tolerance inducing therapy. EAE, marmoset, multiple sclerosis, tolerance Introduction Multiple sclerosis (MS) is a chronic autoimmune neuroinflammatory disease characterized by inflammation, demyelination and neurodegeneration in the white and grey matter of the central nervous system (CNS). T cells specific for myelin proteins, myelin oligodendrocyte glycoprotein (MOG) in particular, play an important pathogenic role in the perpetuation of MS. The T cell response in MS is directed mainly against three domains in the extracellular part of MOG, i.e. 1–22, 34–56 and 64–96 [1–3]. Several current therapies are based on biological molecules (monoclonal antibodies, cytokines and cytokine antagonists) that broadly target putative pathogenic cytokines or cell populations, rather than the autoantigen-specific T cell population. A new method to induce specific tolerance has been developed based on intranasal application of a recombinant fusion protein combining a mutated cholera toxin A1-subunit (CTA-1), a MOG-derived peptide, and a dimer of the Ig binding D region of Staphylococcus aureus protein A. The fusion protein, without the MOG peptide, was first developed to circumvent the toxic side effects of the very powerful cholera toxin adjuvant [4,5]. To this end, CTA1 was genetically fused via its C-terminus to the immunoglobulin (Ig) binding D dimer (CTA1-DD); the construct was expressed in Escherichia coli. The CTA1-DD was found to be non-toxic and to display strong adjuvant/immune stimulatory activity [6]. However, when the CTA1-subunit was mutated by site-directed mutagenesis (R9K substitution) the enzymatic effect and the adjuvant function were lost, while the fusion protein exerted a tolerance promoting effect in vivo against peptides that are inserted between CTA1 and the DD region [5]. In mice, intranasal application of the mutated fusion protein containing peptides of ovalbumin or type II collagen induced reduction of the proinflammatory response and marked up-regulation of interleukin (IL)-10 expression [7,8]. In addition, disease incidence and pathology were reduced in a mouse collagen-induced arthritis model [7]. The finding that IL-10-deficient mice failed to develop tolerance after intranasal treatment with ovalbumin-containing fusion protein [8] demonstrates that tolerance induction was mediated by IL-10. Experimental autoimmune encephalomyelitis (EAE) in the common marmoset (Callithrix jacchus) is a well-validated animal model for MS that can be induced by immunization with recombinant human (rh)MOG emulsified in incomplete Freund's adjuvant (IFA). The ensuing disease course is characterized by lesions with inflammation and demyelination within the CNS white and grey matter [9]. The 100% disease incidence after immunization of marmosets with rhMOG maps to the Caja-DRB*W1201 restricted activation of T helper type 1 (Th1) cells specific for MOG24–36 [10]. In addition, T cells specific for MOG34–56 have been implicated in the disease progression to clinically evident EAE [11,12]. These T cells have an effector memory phenotype and cytotoxic function triggered by the MOG40–48 epitope presented by Caja-E molecules [13]. As both MOG24–36 and MOG34–56 play an important role in the marmoset EAE model, a fusion protein was made that expressed residues 10–60 of human MOG. The aim of this study was to investigate whether the EAE course in mice and marmosets can be altered by intranasal administration of the T cell tolerizing fusion protein CTA1R9K-hMOG10–60-DD. In addition, we performed a multi-parameter immunological analysis in marmosets to assess whether the treatment induces modulation of EAE-associated cellular and humoral immune reactions. Material and methods Animals Specific pathogen-free C57BL/6N female mice (Harlan, Venray, The Netherlands) were between 8 and 12 weeks of age when used in the experiment. Food and water were provided ad libitum. The common marmoset monkeys (Callithrix jacchus) used in this study were purchased from the outbred colony kept at the Biomedical Primate Research Centre (BPRC, Rijswijk, The Netherlands). Only marmosets that were declared healthy after the veterinarian's physical, haematological and biochemical check-up were included. Monkeys were pair-housed in spacious cages and remained under intensive veterinary supervision throughout the study. The daily diet consisted of commercial food pellets for New World monkeys (Special Diet Services, Witham, Essex, UK), supplemented with raisins, peanuts, marshmallows, biscuits and fresh fruit. Drinking water was provided ad libitum. According to Dutch law on animal experimentation, all study protocols and experimental procedures were reviewed and approved by the Institute's Ethics Committee. Fourteen male marmoset monkeys were divided equally and randomly over a placebo-treated and CTA1R9K-hMOG10–60DD-treated group. The placebo-treated group had an average (± standard deviation) body weight of 356 ± 36 g and an average age of 37 ± 7 months. The CTA1R9K-hMOG10–60DD-treated group had an average body weight of 360 ± 26 g and an average age of 47 ± 13 months. EAE induction in mice Mice received 100 μg MOG35–55 (MD Bioproducts, Zurich, Switzerland) and 100 μg PLP178–191 (Pepscan, Lelystad, The Netherlands) dissolved in phosphate-buffered saline (PBS) and 1:1 emulsified in complete Freund's adjuvant (CFA; MD Bioproducts, Zurich, Switzerland) containing 7 mg/ml Mycobacterium tuberculosis. A total volume of 100 μl was injected subcutaneously (s.c.) on each side of the tail base. The mice were also given 200 ng pertussis toxin (List Biological Laboratories, Campbell, CA, USA) intraperitonally (i.p.). Another 200 ng of pertussis toxin was given 2 days later. Mice were weighed three times a week and were scored daily as follows: 0 = no overt signs of disease; 1 = limp tail or hind limb weakness (only one); 2 = limp tail and hind limb weakness; 3 = lartial hind limb paralysis; 4 = lomplete hind limb paralysis; and 5 = moribund state; death by EAE: euthanasia for humane reasons. RhMOG/IFA-induced EAE in marmosets EAE in marmosets was induced with a recombinant protein encompassing the extracellular domain of human MOG, i.e. residues 1–125 (rhMOG), which was produced in E. coli and purified as described previously [1], kindly provided by Dr A. Jagessar (BPRC). The inoculum contained 100 μg rhMOG in 200 μl PBS and was emulsified in 200 μl IFA (Difco Laboratories, Detroit, MI, USA) by gentle stirring for at least 1 h at 4°C. The emulsion was injected at four locations (100 μl per spot) into the dorsal skin under alfaxalone anaesthesia (10 mg/kg; alfaxan; Vetoquinol, Den Bosch, The Netherlands). Clinical signs were scored daily by two independent observers using a previously described semi-quantitative scale [14]. Briefly, 0 = no clinical signs; 0·5 = apathy, altered walking pattern without ataxia; 1 = lethargy, tail paralysis, tremor; 2 = ataxia, optic disease; 2·25 = monoparesis; 2·5 = paraparesis, sensory loss; and 3 = para- or hemiplegia. Overt neurological deficit starts at score 2. For ethical reasons monkeys were killed once paresis of one or more limbs (score ≥2·5) was observed. Body weight measurements of conscious monkeys, which is used a surrogate disease marker, were performed twice per week. Treatment The test substance CTA1R9K-hMOG10–60-DD is a fusion protein of cholera toxin A1 with an arginine to lysine substitution at position 9 (CTA1R9K), human MOG residues 10–60 and a dimer of the Ig binding D region of S. aureus protein A (DD) expressed in E. coli BL-21 cells and purified as described previously [7]. Mice were treated intranasally with PBS (n = 10) or CTA1R9K-hMOG10–60-DD (5 μg per dose; n = 10) on days 0, 2, 10, 12 and 14. Intranasal treatment of marmosets was performed according to Smith et al. [15]. Briefly, the animal was held in a horizontal position and PBS or test compound was first administered into one nostril. After 30 s, the same volume was administered into the other nostril, after which the animal was held horizontally for another 30 s. Treatment was given at post-sensitization days −2, 0, 2 and 7 followed by weekly treatments. Control marmosets received 50 μl PBS per nostril. CTA1R9K-hMOG10–60-DD was given as 50 μl per nostril in a concentration of 100 μg/100 μl, aiming at a dose of approximately 250 μg/kg. Necropsy Monkeys selected for necropsy were first deeply sedated by intramuscular injection of alfaxalone (10 mg/kg). The maximum volume of blood was collected into ethylenediamine tetraacetic acid (EDTA) vacutainers and the marmoset was euthanized subsequently by infusion of pentobarbital sodium (Euthesate®; Apharmo, Duiven, The Netherlands). Half the brain and representative pieces of spinal cord from cervical, thoracic and lumbar regions were stored in formalin and the other half was snap-frozen in liquid nitrogen. Spleen and lymph nodes were collected aseptically and cut into four pieces, which were processed for cell culture, fixed in buffered formalin, snap-frozen in liquid nitrogen or processed for RNA extraction. Lymph nodes of several body compartments were harvested: axillary lymph nodes (ALN) and inguinal lymph nodes (ILN) drain the immunization sites of the back; cervical lymph nodes (CLN) and lumbar lymph nodes (LLN) drain the CNS, i.e. brain and spinal cord, respectively; submandibular lymph nodes (SLN) drain the nose, where the treatment had been applied; mesenteric lymph nodes (MLN) drain the intestine, which is a potential site for tolerance induction. During active EAE, myelin-loaded antigen-presenting cells can be found in CLN [16]. The numbers of mononuclear cells (MNC) obtained from CLN and SLN are low, and these LNs were therefore included in selected assays. MRI The brain hemispheres that were fixed in 4% buffered formalin for 14 days were transferred into buffered saline containing sodium azide to stabilize magnetic resonance (MR) relaxation time characteristics [17]. Post-mortem magnetic resonance imaging (MRI) was performed on a 9·4 T horizontal bore nuclear magnetic resonance (NMR) spectrometer (Agilent, Santa Clara, CA, USA), equipped with a quadrature coil (RAPID, Biomedical, Rimpar, Germany). Formalin-fixed brains were submerged in a non-magnetic oil (Galden; perfluorinated polyether; Solvay Solexis, Weesp, The Netherlands) to prevent unwanted susceptibility artefacts. On a sagittal scout image, 41 contiguous coronal slices of 0·75 mm were defined covering the complete brain (field of view = 25 × 25 mm; matrix = 256 × 256; voxel volume = 7·15 × 10−3 mm3, two transitions). The following MRI data sets were collected: T2 maps. These maps were calculated by a mono-exponential fit of six spin echo images with increasing echo time (TE). Repetition time (TR) = 4000 ms; TE = 10 + 5 × 10 ms. Magnetization transfer ratio (MTR) maps. Maps were calculated from two T1-weighted (T1W) spin echo images with and without a magnetization transfer-saturation pulse. TR = 1675 ms; TE = 23 ms; MT-pulse: 8·19 ms Gaussian-shaped pulse, nominal flip angle 1000, offset −9·4 kHz. MTR values display the decrease of signal intensity as a result of the MT pulse. Fluid attenuated inversion recovery (FLAIR) images. Fast spin echo sequence; TR = 4000 ms; echo train length = 4; echo spacing = 10 ms with an effective TE of 20 ms; inversion time (TI) = 725 ms to suppress signal from cerebral spinal fluid. White matter attenuated inversion recovery (WAIR) images. Similar to the FLAIR images, only TI was now chosen in such a way that the signal from white matter was suppressed. This was explored in a low-resolution experiment. Found TI values varied between 475 and 525 msec. Calculations of T2 and MTR images were performed with a homemade software package developed in matlab version 11b (The Mathworks Inc., Natick, MA, USA). All lesion areas were outlined in the measured hemisphere using the freely available Medical Image Processing, Analysis and Visualization (mipav version 7·0·0; National Institutes of Health, Bethesda, MD, USA) software package. Haematology Fully automated haematology analysis on EDTA venous blood samples was performed on a Sysmex XT-2000i analyser. IgM and IgG levels against MOG Plasma samples were prepared from whole blood (about 40% volume) and stored in aliquots at −20°C. Antibody responses to rhMOG and MOG peptides (14–36, 24–46, 34–56, 44–66, 54–76) were analysed by ELISA, as described previously [11]. ELISA data were normalized against a standard curve of a plasma pool (made with necropsy plasma of three EAE marmosets from a previous study). The antibody content in the pooled plasma was defined at 2500 arbitrary units (AU) and newly collected ELISA data were fitted to a four-parameter hyperbolic function, using the home-made adamsel program developed by Dr E. Remarque (BPRC). T cell proliferation Peripheral blood mononuclear cells (PBMC) were prepared according to standard procedures by density gradient centrifugation on lymphocyte separation medium (Axis-Shield PoC AS, Oslo, Norway). A second source of MNC used for cellular immunology tests were the spleen and LNs (prepared as described above). In-vitro responses against rhMOG and MOG peptides (14–36, 24–46, 34–56, 44–66, 54–76) were assessed in short-term proliferation assays. PBMC or lymphoid cells (2 × 105/well) were seeded into 96-well round-bottomed plates and cultured with rhMOG (5 μg/ml) or MOG peptides (5 μg/ml) or without stimulant as negative control. Ovalbumin (5 μg/ml) and concanavalin A (ConA) (5 μg/ml) were included as extra negative and positive controls. After 48 h, 50 μl of supernatant was removed from each well and stored (−20°C) for cytokine analysis. [3H]-Thymidine, 0·5 μCi/well, was added to the culture and incorporation of radiolabel was determined 18 h later using a matrix 9600 β-counter (Packard 9600; Packard Instrument Company, Meriden, CT, USA). Results are expressed as stimulation index, which is calculated by dividing the counts per minute (cpm) of stimulated cells by the cpm of unstimulated cells. A stimulation index above 2 is considered as a positive proliferative response. Flow cytometry The procedures and reagents used for the phenotypical characterization of marmoset MNC by flow cytometry have been described previously [18]. Briefly, cells were stained with the violet viability stain (Invitrogen, Molecular Probes, Carlsbad, CA, USA) to exclude dead cells. Aspecific staining was blocked by FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany). Subsequently, cells were incubated with CD3 (SP43-2), CD56 (NCAM 16·2) (both from BD Biosciences, San Diego, CA, USA); PD-1 (CD279; e-Bioscience, San Diego, CA, USA); CD20 (H299; Beckman Coulter, Fullerton, CA, USA); CCR4 (205410; R&D Systems, Minneapolis, MN, USA); CD40 (B-B20; Abcam, Cambridge, UK), CD4 (MT310; Dako, Glostrup, Denmark) and CD8 (LT-8; Serotec, Düsseldorf, Germany). Cells were fixed in 1% cytofix (BD Biosciences). Flow cytometric analysis was performed on a fluorescence activated cell sorter (FACS) LSRII (BD Biosciences) using FlowJo software (Treestar, Ashland, OR, USA). Cytokine detection with enxyme-linked immunosorbent assay (ELISA) Cytokines were measured in supernatant of cell cultures that were stimulated for 48 h with rhMOG or MOG peptides. ELISAs to detect interleukin (IL)-17A and interferon (IFN)-γ were performed according to the manufacturer's instructions (UCytech, Utrecht, The Netherlands). Quantitative PCR RNA was isolated from whole blood using the ZR whole-blood RNA mini prep kit (Zymogen, Irvine, CA, USA). Small pieces of spleen and lymph nodes collected at necropsy were stored snap-frozen in −80°C. The isolation of RNA and the quantitative polymerase chain reaction (qPCR) assay were performed as described previously [18]. Briefly, RNA was isolated using RNeasy minikit (Qiagen, Hilden, Germany) and cDNA was synthesized using RevertAid First Strand cDNA synthesis Kit (Fermentas, St Leon-Rot, Germany). Expression levels of mRNA were determined by qPCR using iTaq supermix and the CFX96 real-time system (Bio-Rad, Hercules, CA, USA). Transcript levels were normalized against the reference gene Abelson (ABL) [19]. Statistics Data are presented of individual animals and as group mean. Statistical analysis was performed using Prism version 6·0b for Mac OS X. Survival was analysed using the log-rank test. Other data were analysed using the Mann–Whitney U-test. P < 0·05 was considered statistically significant. Results Intranasal application of CTA1R9K-hMOG10–60-DD ameliorated clinical symptoms in mice The clinical efficacy of CTA1R9K-MOG10–60-DD fusion protein was first tested in a murine EAE model. Figure 1 shows that treatment with CTA1R9K-hMOG10–60-DD significantly reduced the mean maximum score (3·9 for the control group and 3·0 for the treated group) and the frequency of mice with EAE symptoms. In addition, mice treated with CTA1R9K-hMOG10–60-DD recovered better from body weight loss (Fig. 1c). The cumulative EAE score was reduced significantly in the treated group (Fig. 1d). Fig. 1 Open in new tabDownload slide Treatment with CTA1R9K-hMOG10–60-DD ameliorates the disease in mice. Mice were treated intranasally with CTA1R9K-hMOG10–60-DD (5 μg per dose) on days 0, 2, 10, 12 and 14. Shown (mean ± standard error of the mean) are the experimental autoimmune encephalomyelitis (EAE) score (a), the frequency of mice with a score of 1 or higher (b) and the body weight loss compared to day −1 (c). The x-axis represents the day after immunization. (d) The cumulative EAE score. Each dot represents one mouse. Statistical significance (P < 0·05; t-test) is indicated by an asterisk. Fig. 1 Open in new tabDownload slide Treatment with CTA1R9K-hMOG10–60-DD ameliorates the disease in mice. Mice were treated intranasally with CTA1R9K-hMOG10–60-DD (5 μg per dose) on days 0, 2, 10, 12 and 14. Shown (mean ± standard error of the mean) are the experimental autoimmune encephalomyelitis (EAE) score (a), the frequency of mice with a score of 1 or higher (b) and the body weight loss compared to day −1 (c). The x-axis represents the day after immunization. (d) The cumulative EAE score. Each dot represents one mouse. Statistical significance (P < 0·05; t-test) is indicated by an asterisk. No effect of intranasal CTA1R9K-hMOG10–60-DD application on clinical and pathological presentation in the marmoset Next, we tested whether we could translate this positive effect of CTA1R9K-hMOG10–60-DD to the immunologically more complex EAE model in common marmosets. Two important clinical parameters in the marmoset model are clinical EAE scores and body weight loss. It should be noted that only clinical EAE scores equal to or above 2 represent overt neurological deficit. In the control group, all seven marmosets developed EAE score 2 between post sensitization day (psd), i.e. days after immunization, 23 and 55. In the treatment group, all seven marmosets developed clinical score 2 between psd 28 and 65. Despite the slight delay, the differences between the control and CTA1R9K-hMOG10–60-DD-treated group observed in the disease-free survival (until score 2) and total survival (Fig. 2) were not significant. Fig. 2 Open in new tabDownload slide Treatment with CTA1R9K-hMOG10–60-DD does not modulate the disease course in marmosets. Marmosets were treated at days −2, 0, 2 and 7 followed by weekly treatments. All marmosets developed clinical symptoms of experimental autoimmune encephalomyelitis (EAE). Shown are the disease-free survival, i.e. until score 2 (a) and total survival, i.e. until the day of killing with a score equal to or higher than 2 (b). The x-axis represents the day after immunization. No significant differences (log-rank test) were observed between control and treated marmosets. Fig. 2 Open in new tabDownload slide Treatment with CTA1R9K-hMOG10–60-DD does not modulate the disease course in marmosets. Marmosets were treated at days −2, 0, 2 and 7 followed by weekly treatments. All marmosets developed clinical symptoms of experimental autoimmune encephalomyelitis (EAE). Shown are the disease-free survival, i.e. until score 2 (a) and total survival, i.e. until the day of killing with a score equal to or higher than 2 (b). The x-axis represents the day after immunization. No significant differences (log-rank test) were observed between control and treated marmosets. The pathological presentation of the model is complex and variable between individual animals. The global pathological picture obtained with MRI showed white matter lesions in four of the seven control marmosets and grey matter lesions in three of the seven control marmosets. In the CTA1R9K-hMOG10–60-DD-treated group, white matter lesions were detected in four marmosets and grey matter lesions were detected in two marmosets. With respect to total lesion volume and lesion T2 and MTR characteristics, no significant differences were observed (data not shown). More detailed pathology examination with histology revealed that demyelination was present in the brain of four control marmosets and three treated marmosets. Demyelination was observed in the spinal cord of all marmosets, except in one monkey treated with the fusion protein. Demyelination in the optic nerve was detected in five marmosets of both groups. Inflammation was observed in the brain of five control marmosets and in three treated marmosets as well as in the meninges of three treated marmosets. In the spinal cord, inflammation was detected in all control marmosets and in five treated marmosets. Quantification of the histological parameters showed no significant differences in the percentage of demyelination or the inflammatory index between control and treated monkeys (data not shown). Intranasal application of CTA1R9K-hMOG10–60-DD did not impair anti-MOG antibody induction The levels of IgM and IgG against rhMOG and MOG peptides were analysed in bi-weekly collected plasma and at necropsy. In both control and treated marmosets, a peak IgM level against rhMOG was observed at psd 14 or 28 (Supporting information, Fig. S1A). No significant difference between the two groups was observed during EAE development (Supporting information, Fig. S1B). At necropsy, IgM levels against MOG44–66, but not against rhMOG or MOG54–76, were significantly lower in the CTA1R9K-hMOG10–60-DD-treated group (Supporting information, Fig. S1C). In both control and treated marmosets, we detected IgG reactivity against rhMOG, MOG24–46, and MOG54–76. No significant differences were observed between the control and treatment groups during the study course or at necropsy (Supporting information, Fig. S1). Intranasal application of CTA1R9K-hMOG10–60-DD dampens the EAE-associated increase of circulating monocytes Before immunization and at necropsy, the numbers of white blood cells and leucocyte subpopulations were analysed in blood. No differences were observed between the numbers of white blood cells, neutrophils or lymphocytes in control and treated marmosets (data not shown). In the control marmosets, the number of circulating monocytes increased twofold after immunization. However, in the treated marmosets the number of circulating monocytes remained stable or even decreased (Fig. 3a). The ratio of necropsy versus pre-immunization was significantly different between the control and treatment groups (Fig. 3b). Fig. 3 Open in new tabDownload slide The number of monocytes does not increase during experimental autoimmune encephalomyelitis (EAE) in treated marmosets. (a) The monocyte number measured before the study (baseline) is compared to the monocyte number measured at necropsy. (b) The ratio of the number of monocytes measured at necropsy and before immunization is shown. Statistical significance (P < 0·05; t-test) is indicated by an asterisk. Fig. 3 Open in new tabDownload slide The number of monocytes does not increase during experimental autoimmune encephalomyelitis (EAE) in treated marmosets. (a) The monocyte number measured before the study (baseline) is compared to the monocyte number measured at necropsy. (b) The ratio of the number of monocytes measured at necropsy and before immunization is shown. Statistical significance (P < 0·05; t-test) is indicated by an asterisk. Reduction of MOG10–60-induced proliferation in PBMC after treatment with CTA1R9K-hMOG10–60-DD The effect of CTA1R9K-hMOG10–60-DD on MNC proliferation was assessed by culturing MNC with or without stimulation by rhMOG or MOG peptides, the mitogen ConA or the irrelevant protein antigen ovalbumin (OVA). Proliferation against the immunizing protein rhMOG was observed in all marmosets, mainly in the spleen and ALN. Although MOG-induced proliferation in PBMC was low in both groups, which is always observed in the marmoset EAE model, it was significantly lower in CTA1R9K-hMOG10–60-DD-treated marmosets compared to control marmosets. This was observed only for MOG peptides within the 10–60 domain (Fig. 4), but not for rhMOG (Fig. 4) or MOG74–96 (data not shown), neither for OVA or ConA (data not shown). Moreover, the effect of treatment was observed only at the peak of the disease (necropsy) and not in the bi-weekly blood samples (data not shown). This effect was not attributable to the day of euthanasia, as this did not differ significantly between control and treatment groups. As the background proliferation was comparable between both groups (data not shown), the conclusion is warranted that the reduced proliferation was MOG peptide-specific. No differences were observed in the spleen or lymph nodes (Fig. 4). Fig. 4 Open in new tabDownload slide Myelin oligodendrocyte glycoprotein (MOG)-specific proliferation is reduced in the peripheral blood mononuclear cells (PBMC) of treated marmosets. PBMC or mononuclear cells obtained from lymphoid organs were cultured with MOG peptides. The stimulation index is calculated by dividing the counts per minute (cpm) of stimulated cells by the cpm of non-stimulated cells. A stimulation index above 2 is considered proliferation. The proliferation is shown for individual marmosets, each indicated as one dot, with a horizontal bar as the average per group. Statistical significance (P < 0·05; t-test) is indicated by an asterisk. Fig. 4 Open in new tabDownload slide Myelin oligodendrocyte glycoprotein (MOG)-specific proliferation is reduced in the peripheral blood mononuclear cells (PBMC) of treated marmosets. PBMC or mononuclear cells obtained from lymphoid organs were cultured with MOG peptides. The stimulation index is calculated by dividing the counts per minute (cpm) of stimulated cells by the cpm of non-stimulated cells. A stimulation index above 2 is considered proliferation. The proliferation is shown for individual marmosets, each indicated as one dot, with a horizontal bar as the average per group. Statistical significance (P < 0·05; t-test) is indicated by an asterisk. CTA1R9K-hMOG10–60-DD treatment reduced IL-17A and increased IL-4 and IL-10 in response to MOG Levels of IL-17A and IFN-γ were analysed in the 48-h culture supernatants of PBMC and lymphoid organs collected at necropsy. Although antigen-induced proliferation of PBMC obtained at necropsy was decreased significantly in CTA1R9K-hMOG10–60-DD treated marmosets (Fig. 4), we observed no effect of the treatment on IL-17A (Fig. 5a) and IFN-γ (Supporting information, Fig. S2A) production in PBMC. In the spleen of CTA1R9K-hMOG10–60-DD treated marmosets, the production of IL-17A was lower in response to rhMOG (P = 0·0670) and MOG34–56 (P = 0·0693) compared to control animals (Fig. 5a), whereas IFN-γ was not changed (Supporting information, Fig. S2A). No effect of the treatment was observed in the ALN, ILN (Fig. 5a and Supporting information, Fig. S2A), LLN or MLN (data not shown). Fig. 5 Open in new tabDownload slide Myelin oligodendrocyte glycoprotein (MOG)-specific production of interleukin (IL)-17A is reduced, whereas IL-4 and IL-10 are increased. (a) Supernatant of marmoset cell cultures from Fig. 4 were analysed for the secretion of IL-17A. The cytokine levels are shown for individual marmosets, each indicated as one dot, with a horizontal bar as the average per group. (b) Cells from the axillary lymph node were stimulated with recombinant human (rh)MOG, MOG24–46 or MOG34–56 for 24 or 48 h. RNA was isolated and mRNA levels were determined by quantitative polymerase chain reaction (qPCR). The fold increase of stimulated cells compared to non-stimulated cells is shown for IL-4 and IL-10. Each dot represents one marmoset. Statistical significance (P < 0·05; t-test) is indicated by an asterisk. Fig. 5 Open in new tabDownload slide Myelin oligodendrocyte glycoprotein (MOG)-specific production of interleukin (IL)-17A is reduced, whereas IL-4 and IL-10 are increased. (a) Supernatant of marmoset cell cultures from Fig. 4 were analysed for the secretion of IL-17A. The cytokine levels are shown for individual marmosets, each indicated as one dot, with a horizontal bar as the average per group. (b) Cells from the axillary lymph node were stimulated with recombinant human (rh)MOG, MOG24–46 or MOG34–56 for 24 or 48 h. RNA was isolated and mRNA levels were determined by quantitative polymerase chain reaction (qPCR). The fold increase of stimulated cells compared to non-stimulated cells is shown for IL-4 and IL-10. Each dot represents one marmoset. Statistical significance (P < 0·05; t-test) is indicated by an asterisk. To test MOG-induced production of anti-inflammatory cytokines, MNC isolated from the ALN were stimulated with MOG24–46, MOG34–56 or rhMOG. After 24- or 48-h culture, MNC were harvested and mRNA was extracted for analysis by qPCR, as no ELISA reagents are available that cross-react with marmoset IL-4 or IL-10. Figure 5b shows that the mRNA expression levels of IL-4 and IL-10 were increased in response to stimulation with MOG24–46 and MOG34–56, and that this increase was significantly higher in CTA1R9K-hMOG10–60-DD-treated marmosets compared to control marmosets. In addition, we determined the mRNA levels for a selection of EAE-related cytokines in the different lymphoid compartments. We observed that mRNA expression of several cytokines were increased in the spleen of CTA1R9K-hMOG10–60-DD-treated marmosets, i.e. IFN-γ (P = 0·0530), IL-17A (P = 0·0513) and IL-17F (P = 0·0175), IL-2 (P = 0·0530) and transforming growth factor (TGF)-β3 (Supporting information, Fig. S2B). In addition, in the ILN of CTA1R9K-hMOG10–60-DD-treated marmosets, IFN-γ, IL-2, IL-4, IL-15, TGF-β2 and TGF-β3 were significantly increased (Supporting information, Fig. S2B), no differences were observed for these cytokines in the ALN, LLN, CLN, MLN and SLN (data not shown). CTA1R9K-hMOG10–60-DD treatment induced increased expression of CCR4 and programmed cell death-1 During the course of the disease and at necropsy, the percentages of different subsets of B cells and T cells were determined by flow cytometry analysis. During the disease course, no significant changes were observed for T cells (CD3+) or B cells (CD20+) (data not shown). At necropsy, the percentage of CD20+CD40+ B cells was reduced significantly in the spleen of CTA1R9K-hMOG10–60-treated marmosets. No effect of the treatment was found in the percentage of CD20+ cells, either expressing or not expressing CD40, in PBMC or the other secondary lymphoid organs (Fig. 6). Fig. 6 Open in new tabDownload slide The percentage of programmed death-1 (PD-1) and CC chemokine receptor 4 (CCR4)-expressing cells is increased in treated marmosets. The expression of several markers on peripheral blood mononuclear cells (PBMC) or mononuclear cells obtained from lymphoid organs were analysed by flow cytometry. Cells were first gated on living lymphocytes. Shown are the percentages of the population after the/within their parent gate, which is indicated before the /. Each dot represents one marmoset. Statistical significance (P < 0·05; t-test) is indicated by an asterisk. Fig. 6 Open in new tabDownload slide The percentage of programmed death-1 (PD-1) and CC chemokine receptor 4 (CCR4)-expressing cells is increased in treated marmosets. The expression of several markers on peripheral blood mononuclear cells (PBMC) or mononuclear cells obtained from lymphoid organs were analysed by flow cytometry. Cells were first gated on living lymphocytes. Shown are the percentages of the population after the/within their parent gate, which is indicated before the /. Each dot represents one marmoset. Statistical significance (P < 0·05; t-test) is indicated by an asterisk. The percentages of total CD3–, CD3+, CD3+CD4+ or CD3+CD8+ cells were not changed significantly (data not shown). Cells expressing programmed cell death-1 (PD-1; CD279) were increased significantly in the CD3– as well as CD3+CD4+ and CD3+CD8+ populations of CTA1R9K-hMOG10–60-treated marmosets (Fig. 6). Within the CD3+CD4–, but not CD3+CD4+ cells, we observed an increase in the percentage of cells expressing CCR4, which was significant for the spleen (Fig. 6). Discussion The aim of this study was to test whether the tolerogenic effect of intranasal CTA1R9K-hMOG10–60-DD observed in a mouse EAE model could be replicated in the common marmoset EAE model. In the marmoset EAE model, the treatment did not reduce the clinical course or CNS pathology significantly despite several immunological changes. Intranasal application with CTA1R9K-hMOG10–60-DD induced a reduction of the number of circulating monocytes, a reduction of MOG-specific proliferation in peripheral blood, reduced production of IL-17A in spleen and an increase in IL-4 and IL-10 in response to MOG. In addition, the percentages of cells expressing PD-1 and CCR4 were increased after treatment. However, the treatment did not alter MOG-specific IgM and IgG levels. Moreover, levels of rhMOG-induced production of IFN-γ were equal between both groups, and mRNA expression levels of several proinflammatory cytokines were increased in secondary lymphoid organs after treatment. At the mRNA level, we observed an increase in the proliferation/homeostasis cytokines IL-2, IL-7 and IL-15, the Th1 marker IFN-γ, the Th17 marker IL-17F and the Th2 marker IL-4 in the lymphoid organs. These findings are indicative for a higher T cell activation state by the treatment. However, this effect may also be explained by retention of T cells in the spleen and ILN. We hypothesize that in the control marmosets the cytokine-producing cells egressed from spleen and LN to the CNS, whereas in treated marmosets a certain proportion may stay in spleen and LN. This may also explain the reduced MOG-induced proliferation of blood T cells of treated animals. In addition, the observation that the percentages of MNC expressing CCR4 and PD-1 (CD279) are increased may suggest that the intranasal application of the fusion protein CTA1R9K-hMOG10–60-DD elicits an immunosuppressive response. It has been described that human CD8+ T cells that express CCR4 do not produce perforin or granzyme [20], suggesting that these cells may be immature memory T cells rather than cytotoxic T cells. In addition, these T cells produce Th1 as well as Th2 cytokines [20]. The increase of CCR4+ cells within the CD4– compartment may hint at a change from cytotoxic T cells towards more regulatory T cells. This possibility complies with the increase of both Th1 and Th2 cytokines we observed at the mRNA level. In addition, it has been demonstrated that PD-1 plays a crucial role in the regulation of EAE, as blocking PD-1 made EAE in mice more severe [21]. The observation that the increased percentage of PD-1+ cells could not halt the disease may be explained by inhibition of PD-1 by the signal transducer and activator of transcription-5 (STAT-5) activating cytokines IL-2, IL-7 and IL-15, which were increased at the mRNA level by the treatment, as has been shown previously for human T cells [22]. In conclusion, we report that immune tolerization via intranasal application of a new fusion protein, CTA1R9K-hMOG10–60-DD, is well tolerated and exerts biological effects in mice and marmosets. The treatment dampened the inflammatory response in EAE and stimulated the expression of IL-4, IL-10 and PD-1 that potentially modulate the pathogenic immune response. This effect was, nevertheless, insufficient for mitigating the development of clinically evident EAE in marmosets. We believe that the reported data are encouraging, and are optimistic about the clinical potential of this novel tolerance-targeting strategy using high-affinity T cell epitopes incorporated in the CTA1R9K-peptide-DD platform. Acknowledgement The authors would like to thank Dr Ed Remarque for statistical advice. Disclosure N. L. is the CEO of Toleranzia AB and the founder of the company. He is also the inventor of the CTA1R9K-peptide-DD tolerance-inducing platform. M. V. is employed by Toleranzia AB and is a shareholder of the company. R. A. and G. R. are full-time employees of Synthon Biopharmaceuticals BV, Nijmegen, The Netherlands. Author contributions Y. S. K. and N. v. D. performed the in-vivo study in marmosets and the ex-vivo analysis. B. 't H. supervised the marmoset study. Y. S. K. and B. 't H. wrote the manuscript. E. B. performed the MRI studies. N. L. is the inventor of the tolerizing fusion protein. N. L., M. V., R. A. and G. 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TI - Immune modulation by a tolerogenic myelin oligodendrocyte glycoprotein (MOG)10–60 containing fusion protein in the marmoset experimental autoimmune encephalomyelitis model
JF - Clinical & Experimental Immunology
DO - 10.1111/cei.12487
DA - 2015-03-10
UR - https://www.deepdyve.com/lp/oxford-university-press/immune-modulation-by-a-tolerogenic-myelin-oligodendrocyte-glycoprotein-BqzgaeROwt
SP - 28
EP - 39
VL - 180
IS - 1
DP - DeepDyve
ER -