Pathogenic myelin-specific antibodies in multiple sclerosis target conformational proteolipid protein 1–anchored membrane domains

B cell clonal expansion and cerebrospinal fluid (CSF) oligoclonal IgG bands are established features of the immune response in multiple sclerosis (MS). Clone-specific recombinant monoclonal IgG1 Abs (rAbs) derived from MS patient CSF plasmablasts bound to conformational proteolipid protein 1 (PLP1) membrane complexes and, when injected into mouse brain with human complement, recapitulated histologic features of MS pathology: oligodendrocyte cell loss, complement deposition, and CD68+ phagocyte infiltration. Conformational PLP1 membrane epitopes were complex and governed by the local cholesterol and glycolipid microenvironment. Abs against conformational PLP1 membrane complexes targeted multiple surface epitopes, were enriched within the CSF compartment, and were detected in most MS patients, but not in inflammatory and noninflammatory neurologic controls. CSF PLP1 complex Abs provide a pathogenic autoantibody biomarker specific for MS.

b Patient-derived rAbs from CSF plasmablast clones demonstrated to bind myelin and/or PLP1-transfected cells.
c CSF was collected and analyzed following their first clinical event.Patients subsequently developed relapsing-remitting MS within a 6-18 month period.

Figure S2 .
Figure S2.Transient astrocyte loss occurs in MS rAb + HC demyelinating lesions.(A) Images show loss of AQP4 immunostaining and absence of GFAP immunostaining in areas of eGFP oligodendrocyte loss in MS#30 + HC lesion.Bar = 500 µm.(B) Images of 2B4 and MS#30 + HC ICIs into Aldhl1 transgenic Swiss Webster mice at different time points post-injection.Infiltration of CD68 + cells highlights the lesion boundaries and reveals differences in lesion severity between 2B4 and MS#30 ICIs.eGFP immunofluorescence identifies astrocyte cell bodies.Bar = 1 µm.(C) Graph shows significant loss of eGFP astrocytes between 2B4 and MS#30 lesions on day 3 post-injection (Mann-Whitney test).Significance of astrocyte recovery from day 3 to day 14 in MS#30 + HC ICIs was determined using one-way ANOVA.(D) Images of eGFP astrocyte recovery and GFAP expression in recovering day 7 and day 14 MS#30 ICIs.Dashed white lines identify corresponding areas of CD68 cell infiltraton.Bar = 200 µm.

Figure S3 .
Figure S3.MS#11 is a pathogenic antibody to PLP1-transfected cells.(A) Injection of 5 ug MS#11 rAb with 20% HC produces large areas of oligodendrocyte loss and CD68 + cell infiltration.(B) Graph compares area of oligodendrocyte loss between isotype control 2B4 and MS#11 ICIs (Mann-Whitney, **p<0.01)from a separate cohort of animals.Asterisk identifies injection site and hashed line outlines area of oligodendrocyte cell loss.(C) Image shows loss of AQP4 immunostaining in areas of eGFP+ oligodendrocyte cell loss.

Figure S5 .
Figure S5.MS#30 immunostaining co-localizes with PLP1-expression in mosaic female PLP x/- C57Bl/6 mice.Confocal images (Bar = 100 µm) show immunostaining of folia from PFA-fixed cerebellar slice culture with PLP1AA3 mAb, MS#30 rAb and anti-MBP mAb.Because the PLP1 gene is located on the X chromosome, PLP1 expression in oligodendrocytes is mosaic.Staining of white matter tracts with MS#30 rAb coincides with the pattern of PLP1 expression whereas MBP staining can be seen throughout myelin processes.

Figure S6 .
Figure S6.Oligodendrocyte developmental O1 and O4 mAbs identify cells expressing galactocerebroside and sulfatide respectively.(A) Flowchart and images of O1 and O4 mAb staining of live CHOK1 cells prior and following the individual and combined transfection of plasmid cDNA expressing galc and sulfatide biosynthetic enzymes CGT and CST.(B) Identification of CSTtransfected cells using antibodies to a FLAG epitope (red) engineered onto the C-terminus of the CST expression construct.Graphs demonstrate that positive O4 staining of CHOK1cells requires expression of both CGT and CST enzymes and that O4 staining of CST + CGT transfections segregates with CST expression.Student's t-test,**p<0.01;****p<0.0001.(C) Images show the absence of myelin-specific rAb binding to live CHOK1 cells following transfection with CGT and CST.
Figure S8.(A)Representative image of PLP1-transfected HEKE7 cells stained with MS02-19 CSF.The highlighted elements were used to calculate the normalized CSF G/R binding ratio.Bar = 10 μm.White dashed areas represent CSF IgG staining (green channel) of mAb AA3 positive (red channel) PLP1-transfected cells (white arrows).The mean green (GF) and red fluorescent (RF) staining intensities are measured from 8 or more PLP1 + cells or clusters of cells per CSF sample and each corrected for background binding to AA3 negative cells, which are highlighted in this image by the yellow dashed line and arrow.Although not readily visible in this image, live non-transfected cells are identified from parallel images in which very dim DAPI staining can be enhanced.The white arrowhead identifies a bright DAPI positive dead cell, which is omitted from all analyses.To correct for inter-experimental variability in green and red channel staining intensities, each binding assay includes a positive control staining with ON34 rAb (40 µg/ml) at Bmax.The measured G/R ratio of ON34 binding obtained for our initial experiment (ON34 rAb Day 1 ) is then divided by the binding ratio obtained for subsequent experiments (ON34 rAb Day x ) and used to compute a final normalized CSF G/R ratio as indicated in the accompanying equation.(B) Representative images of individual MS and control patient CSF or purified CSF IgG staining of PLP1-transfected HEKE7 cells.Bar = 10 μm.Images of MS04-4 and MS07-8 CSF staining are representative of weakly positive CSF and images of MS09-1 and MS93.1 represent more moderate to strong CSF positivity.

Table S1 . Clinical, MRI and CSF Features of MS Patients used for CSF monoclonal recombinant antibody production a
a Patients were not receiving immunomodulatory therapy at the time of lumbar puncture.