Supplemental Data

Appendix e-1

Cell-based assay for detection of antibodies to cell surface MOG in serum and CSF

We used FACS analysis to detect antibody binding of patient serum IgG to surface MOG transduced in HEK293 cells as we have previously described. 1-5 Briefly, cells were harvested using versene (Invitrogen) and washed in PBS supplemented with 2% FBS (PBS/FBS). 50,000 cells were then incubated with serum at a 1:50 dilution, or neat CSF in V-bottom plate (Corning) for 30 min at room temperature. Cells were then washed three times with 200l PBS/FBS, and incubated with Alexa Fluor 647-conjugated goat anti-human IgG secondary antibody (Invitrogen) for 30 min at room temperature. Cells were washed three times with PBS/FBS, and then resuspended in 50µl PBS/FBS before analysis. Before acquisition, viability dye 7-AAD (BD Biosciences) was added to the cells to exclude dead cells. A total of 10,000 events/well were recorded on a BD LSRII instrument equipped with a high-throughput sampler (BD Biosciences). Data analysis was performed using Flow Jo software (TreeStar, Ashland, OR, USA) and Excel. Binding was expressed as mean fluorescence intensity (MFI) as previously described. 1, 4, 5 Levels of antibody binding in ZsGreen-positive transfected cells were expressed as DMFI. DMFI was determined by the subtraction of MFI obtained with HEK293Ctl cells from the MFI obtained with HEK293MOG+ cells. An DMFI greater than mean + 3 standards deviations of values of the healthy control samples was considered positive. Each experiment was performed at least three times. Cell-based assays were performed by blinded investigator and data was unblinded in order to calculate the threshold of positivity. Representative dot plot out of three experiments is shown. Samples were considered positive if they were above threshold at least two times out of three repeated experiments. All cell-based assays were optimized on prior assessment of antigen surface expression. Surface expression on trasnduced cells was analyzed by flow cytometry after staining with a mouse monoclonal anti-human extracellular MOG (Clone 818C5, kind gift of Prof C. Linington) in combination with an Alexa Fluor 647-conjugated appropriate secondary antibody (Invitrogen). Dot plots shown in figures 1 and 4 were generated using Prism software version 4.0b (GraphPad Software, Inc, La Jolla, CA, USA).

The intra-assay variation is summarized as follows:

Group / 3 out of 3 above mean + 3SD threshold / 2 out of 3 above mean + 3SD threshold / 1 out of 3 above mean + 3SD threshold / 0 out of 3 above mean + 3SD threshold
MOG antibody-positive DEM (n=31) / 28 / 3 / 0 / 0
MOG antibody- negative DEM (n=42) / 0 / 0 / 6 / 36
CTL (n=24) / 0 / 0 / 0 / 24
CTL (n=57, not shown / 0 / 0 / 0 / 57

Pathogenic effects and Immunocytochemistry on human oligodendroglial cells

Human oligodendroglial MO3.13 cells were cultured as previously described. 6 Transduced MO3.13MOG+ and MO3.13Ctl cells were immunolabeled live, fixed with 4% paraformaldehyde, and fixed/permeabilized (0.3% Triton X). Cells were then incubated with primary antibodies, protein G-purified human IgG, and/or human anti-MOG IgG antibody. Cells were then washed and incubated with a secondary Alexa Fluor 647-conjugated anti human IgG antibody, or appropriate secondary antibodies (Invitrogen). Antibodies used in this study were anti-MBP (Millipore), -Oligodendrocyte marker O4 (O4, Millipore), -b-tubulin (marker of cytoskeleton microtubule, Clone DSHB, The University of Iowa Hybridoma Bank), -MOG (818C5 clone), -c-series ganglioside-specific antigen A2B5 (A2B5, Millipore), -Galactocerebroside (GalC, Millipore), -Vimentin (Dako, Glostrup, Denmark), and -2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase, Millipore). We also used fluorochrome conjugated-phalloidin to vizualize filamentous actin (Life Technologies).

In order to visualize effects of protein G-purified human IgGs on single cells, MO3.13 cells were seeded at low density and incubated with 6mg of protein G-purified human IgGs from patient or control sera for 45 min at room temperature followed by goat anti-human IgG secondary antibody for 15 min at room temperature. 7, 8 After washing, cells were fixed and permeabilized, and cytoskeleton filamentous actin (F-actin, marker of cytoskeleton thin filaments) and b-tubulin (marker of cytoskeleton microtubule) were quantified using 3-D deconvolution microscopy. MO3.13 cells were visualized through 100X 1.4 NA and 60X 1.4 NA oil immersion lenses with an inverted Olympus IX-70 microscope (DeltaVision Core, Applied Precision, Issaquah, USA) and a photometrics CoolSnap QE camera. Images were acquired as 0.15µm-thick 40 serial optical sections, then deconvolved using DeltaVision SoftWoRx software, version 5.0.0, and volume projections of the entire Z-series were generated and overlaid using ImageJ software version 1.48b (National Institute of Health, Bethesda, USA). Loss of cytoskeleton organization was quantified using ImageJ software version 1.48b (National Institute of Health, Bethesda, USA). As F-actin and b-tubulin immunolabelings seemed to be decreased in the cytoplasm after incubation with IgG from MOG antibody-positive patients, we sought to quantify this effect. The disorganization of the thin filaments and microtubule were calculated by measuring the average fluorescence intensity of F-actin and b-tubulin per unit volume of the entire cell (delimited by differential interference contrast or DIC image), of the cytoplasm (delimited by DIC and DAPI images, nucleus excluded) and of the perinuclear region (delimited by DAPI image) after all Z-series were summed. 9 We calculated the relative enrichment (RE) within the cytoplasm using the formula: Average fluorescence Intensity cytoplasm/Average fluorescence Intensity entire cell, and the relative enrichment (RE) within the perinuclear region using the formula: Average fluorescence Intensity perinuclear region/Average fluorescence Intensity entire cell. Then we calculated the ratio between cytoplasm and perinuclear region. 9 We expressed results as percentage of healthy control in fixed conditions. Scatter plots in figure 4 show results obtained from 40 cells using two healthy controls and two MOG antibody-positive purified IgG from 3 independent experiments. Dotted lines were drawn using differential interference contrast (DIC) images. Panel A and B in figure 4 are Z-series maximum intensity projections, whereas all quantification experiments have been performed on Z-series summed images. Additionally, images from Alexa Fluor 647-conjugated secondary antibody staining were pseudo-colored in green for clarity. Importantly, all images were taken and analysed using the same parameters (time exposure, deconvolution, and scaling).

HLA-DRB1*1501 Genotyping

DNA was extracted from patient saliva using a saliva-specific DNA extraction kit according to the manufacturer’s instructions (Orangene, DNA genotek). Saliva samples were collected from 40 DEM patients. The concentration of the extracted DNA was quantified by Nanodrop 2000, it was aliquoted and stored long term at -20°C and at -4°C. In order to genotype the patients for HLA-DRB1*1501, Polymerase Chain Reaction (PCR) was used. A set of primers were designed to flank a single nucleotide polymorphism (SNP) called rs9271366 which lies on chromosome 6 at position 32,694,832. rs9271366 lies upstream of the allelic variation (A → G), and can be used to tag it. 10 Patients with the allelic A are negative for HLA-DRB1*1501, whereas patients with the allelic G are HLA-DRB1*1501-positive. DNA was amplified using Polymerase Chain Reaction (PCR). 10-20 ng of each DNA sample was added to a reaction mixture containing Buffer D (Roche), HLA DRB1 Forward 1 primer (5’-TGTGGCCAAGACTAGCAT-3’), HLA DRB1 Reverse 1 primer (5’-AAATGTTGAGATTCAGAGAGACCA-3’), Taq DNA polymerase (Invitrogen, USA), and diethylpyrocarbonate (DEPC) H2O (Bioline Reagents Ltd, UK). PCR reactions were run at: 95°C for 2 minutes; 5 cycles of 30 seconds at 95°C, 64°C and 72°C; 35 cycles 72°C of 30 seconds at 95°C, 60°C and 72°C; and 10 minutes at 72°C, samples were then held at 4°C. PCR samples were then visualised by 1.5% agarose gel electrophoresis. DNA products should be approximately 237 base pairs. Samples were then purified with an exosap reaction to purify the amplified DNA. Shrimp Alkaline Phosphatase (SAP, 1 u/µL) and Exonuclease 1 (New England Biolabs, USA) were added to each PCR tube containing each sample. The samples underwent PCR at 37°C for 30 minutes and 80°C for 20 minutes. Samples were then ready to be sequenced by the Australian Genomic Research Facility (AGRF, Westmead) using a Forward Sequencing HLA-DRB1 1501 primer (forward: 5’- CAAGACTAGCATTGTCTTCAG-3’) and HLA DRB1 Reverse 1 primer (5’-AAATGTTGAGATTCAGAGAGACCA-3’), DEPC H2O, and DMSO (Sigma Aldrich, USA). Sequences were analyzed for presence or absence of the SNP using the software Sequencher.

Appendix e-2

Patient A, a 14 year old female presented with clinical and radiological longitudinal extensive transverse myelitis with asymptomatic brain lesions (figure 2A). Despite steroids and intravenous immunoglobulin, the patient had a cerebellar relapse at 3 months, and then a further ON episode at six months. The ESR was over 90mm/hr during each episode, and CSF oligoclonal bands, and serum AQP4 and lupus antibodies were negative on two occasions. The patient was started on MMF and has remained in remission for two years, the ESR is now 30 mm/hr, and MRI shows near complete resolution of the lesions and no new lesions on annual MRI follow-up (figure 2C). Patient B, a 5 year old male presented with clinical and radiological ADEM, negative CSF oligoclonal bands and a raised ESR of 36 mm/hr (figure 2B). He responded well to intravenous and oral corticosteroids for six weeks, but had a cerebellar relapse four months after his first episode. A further course of steroids was given, and MMF was started. He has been well for one year, and his MRI six months into MMF shows near complete resolution of lesions, and no new lesions (figure 2D).

Appendix e-3

figure e-1: Cytoskeleton organization in live human oligodendroglial MO3.13Ctl/MOG+ cells is intact after incubation with purified IgG from DEM patients and controls and IgG from DEM patients immunoabsorbed on HEK293MOG+ cells. F-actin (red) and β-tubulin (green) immunolabelings in human live oligodendroglial MO3.13Ctl cells incubated 45 min with 6μg of purified HC IgG or MOG antibody-positive DEM IgG (A), human live oligodendroglial MO3.13MOG+cells incubated 45 min with 6μg of purified MOG antibody-negative DEM IgG (B), and human live oligodendroglial MO3.13MOG+ cells incubated 45 min with MOG antibody-positive DEM serum immunoabsorbed on HEK293MOG+ cells (C). Representative data are shown (volume projection of entire Z-stack acquired using deconvolution microscopy). Nuclei stained with DAPI. Bar: 10μm. Dotted lines on right images represent contour of cells as determined by DIC images (not shown). (D) Immunoreactivity to MOG was assessed by flow cell-based assay. HEK293Ctl-immunoabsorbed sera from MOG antibody-positive DEM patients had high ΔMFI compared to MOG antibody-negative DEM sera, whereas HEK293MOG+-immunoabsorbed sera from MOG antibody-positive DEM patients had decreased ΔMFI similar to MOG antibody-negative DEM patients. Ab = antibody; DEM = demyelinating diseases; DIC = differential interference contrast; HC = healthy control; IgG = immunoglobulin G.

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