Neurology: Neuroimmunology & Neuroinflammation - Absence of … · tissue sections. All brainstem and subcortical WM tissue sections were immunostained for myelin (proteolipid pro-tein)

ARTICLE OPEN ACCESS

Absence of B Cells in Brainstem and WhiteMatter Lesions Associates With Less SevereDisease and Absence of Oligoclonal Bands in MSNina L Fransen MD Brigit A de Jong MD PhD Katharina Heszlig MD Tanja Kuhlmann MD PhD

Maria CJ Vincenten MSc Jorg Hamann PhD Inge Huitinga PhD and Joost Smolders MD PhD

Neurol Neuroimmunol Neuroinflamm 20218e955 doi101212NXI0000000000000955

Correspondence

Dr Smolders

jjfmsmolderserasmusmcnl

AbstractObjectiveTo determine whether B-cell presence in brainstem and white matter (WM) lesions is asso-ciated with poorer pathological and clinical characteristics in advanced MS autopsy cases

MethodsAutopsy tissue of 140 MS and 24 control cases and biopsy tissue of 24 patients with MS wereexamined for CD20+ B cells and CD138+ plasma cells The presence of these cells wascompared with pathological and clinical characteristics In corresponding CSF and plasmaimmunoglobulin (Ig) G ratio and oligoclonal band (OCB) patterns were determined In aclinical cohort of 73 patients the presence of OCBs was determined during follow-up andcompared to status at diagnosis

ResultsIn 34 of active and 71 of mixed activeinactive lesions B cells were absent which correlatedwith less pronounced meningeal B-cell infiltration (p lt 00001) The absence of B cells andplasma cells in brainstem and WM lesions was associated with a longer disease duration (p =0001) less frequent secondary progressive MS compared with relapsing and primary pro-gressive MS (p lt 00001 and p = 0046 respectively) a lower proportion of mixed activeinactive lesions (p = 001) and less often perivascular T-cell clustering (p lt 00001) Moreovera lower CSF IgG ratio (p = 0006) and more frequent absence of OCBs (p lt 00001) werenoted In a clinical cohort numbers of patients without OCBs in CSF were increased at follow-up (274)

ConclusionsThe absence of B cells is associated with a favorable clinical and pathological profile Thisfinding may reflect extremes of a continuum of genetic or environmental constitution but also aregression of WM humoral immunopathology in the natural course of advanced MS

Condashsenior authors

From the Department of Neuroimmunology (NLF MCJV JH IH JS) Netherlands Institute for Neuroscience Amsterdam The Netherlands Department of Neurology and MSCenter Amsterdam Amsterdam Neuroscience Amsterdam University Medical Centers Vrije Universiteit (BAJ) The Netherlands Institute for Neuropathology (KH TK) Uni-versity Hospital Munster Munster Germany Department of Experimental Immunology (JH) Amsterdam Infection amp Immunity Institute Amsterdam University Medical CentersUniversity of Amsterdam The Netherlands Swammerdam Institute for Life Sciences University of Amsterdam (IH) The Netherlands and MS Center ErasMS (JS) Departments ofNeurology and Immunology Erasmus Medical Center Rotterdam The Netherlands

Go to NeurologyorgNN for full disclosures Funding information is provided at the end of the article

The Article Processing Charge was funded by the authors

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CC BY-NC-ND) which permits downloadingand sharing the work provided it is properly cited The work cannot be changed in any way or used commercially without permission from the journal

Copyright copy 2021 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

MS is a heterogeneous disease differing in clinical diseasecourse1 radiologic appearance of lesions2 and response toimmunomodulatory therapies3 Of interest variability be-tween patients with MS is observed in the involvement ofhumoral immunity in the disease At the time of diagnosis10 of patients with MS show the absence of oligoclonalbands (OCBs) consisting of intrathecally produced immu-noglobulin (Ig) Gs4 The absence of OCBs is associated with adecreased number of lesions on MRI and a more benigndisease course45 Furthermore the presence of OCBs in pa-tients with clinically isolated syndrome is associated with anincreased risk for clinically definite MS and with an increasedrisk of disability progression67

In contrast to its limited presence in early MS8 advancedprogressive MS is characterized by extensive cortical de-myelination9 Active cortical demyelination is observed inconjunction with the presence of meningeal follicle-like in-flammatory structures10ndash12 The distinct zones of B cellsplasma cells and T cells resemble tertiary lymphoidstructures1213 The presence of these follicle-like structuresassociates with more severe disease reflected by an earlierdisease onset and faster accumulation of disability and earlierdeath1112 Recently Reali et al14 reported that the density ofmeningeal B cells in MS spinal cords correlates with extensiveaxonal loss and white matter (WM) lesion area but also withdensity of B cells in the WM perivascular space

Besides cortical demyelination demyelinating WM lesionsalso add up to disease severity in donors with advanced MSIn MS autopsy cases the presence of active and mixedactiveinactive lesions has been reported to be substantialand correlate with a short time to reaching expanded dis-ability status scale (EDSS) end points a shorter time todeath an unfavorable profile of risk factors for adverseoutcomes and an unfavorable profile of genetic risk factorsfor adverse outcomes1516 Furthermore we showed theseactive lesions to be populated by infiltrating T cells with adominant tissue-resident memory T (TRM)-cell fractionshowing signs of recent reactivation1718 Frischer et al19

quantified the presence of B cells in MS WM lesions andfound these to be predominantly present in perivascularcuffs and meninges and less frequently in the parenchymaThe presence of B cells was found most frequently in acutelesions in relapsing-remitting (RR) donors and less fre-quently in progressive patients19 IgG-producing cells andIgG deposits are regularly found in MS WM lesions2021

Furthermore the number of B cells reported in late MSautopsy lesions is highly variable between cases1922

The correlation of B-cell presence in WM lesions with clinicalend points and risk factors as well as with meningeal B-cellinfiltration has been limitedly explored Here we investigatedthe clinical and pathological characteristics of NetherlandsBrain Bank (NBB)MS autopsy cases in association with B-cellinfiltration of brainstem and subcortical WM lesions

MethodsDonor and Sample CharacteristicsOne hundred forty-one MS brain donors and 24 non-neurologic controls from the NBB autopsy cohort(Amsterdam The Netherlands) were included for thisanalysis of B cells and plasma cells Donors came to autopsybetween 1991 and 2015 and were diagnosed with MSaccording to the contemporary diagnostic criteria by theirtreating physicians Clinical files were collected post-mortem by the NBB By retrospective chart analysis theclinical diagnosis of MS was confirmed for all patients andthe clinical course was defined as either RR secondaryprogressive (SP) or primary progressive (PP) by a neu-rologist15 No MRI data were available None of the donorsreceived MS disease-modifying therapies in the year beforeautopsy except for 1 (B cellndashpositive) donor on fingolimodDetailed donor and tissue characteristics are described insupplementary table 1 linkslwwcomNXIA399 and treat-ment status is provided in supplementary table 2 linkslwwcomNXIA399 The pathological diagnosis of MS was con-firmed for all cases by a certified neuropathologist15 All donorswere analyzed for antindashmyelin oligodendrocyte glycoprotein(MOG) and antindashaquaporin-4 antibodies using cell-based as-says (figure e-1 linkslwwcomNXIA399)23ndash25

For the immunohistochemical part of this study 3 types oftissues were analyzed for the presence of B cells and plasma cells(1) standardly dissected tissue blocks at the level of the medullaoblongata (MO) from 140 MS autopsy cases (2) subcorticalWM lesions from 73140 MS autopsy cases (158 WM lesionswith a median 2 lesions per donor for both the donors with andwithout B cells) and 24 non-neurologic control donors and (3)earlyMS biopsyWM lesions (N= 28) from 24 patients withMSto explore how findings in postmortem autopsy samples of do-nors with advanced MS correlate with findings at the earlieststages ofMS These sections weremade available by the Institutefor Neuropathology University Hospital Munster (MunsterGermany) Additional information on the analysis and selectionof the different tissue samples is described in the supplementarymethods linkslwwcomNXIA399

GlossaryGM = gray matter IgG = immunoglobulin GMO =medulla oblongataMOG =myelin oligodendrocyte glycoproteinNBB =Netherlands Brain Bank OCB = oligoclonal band PP = primary progressive RR = relapsing-remitting SP = secondaryprogressive TRM = tissue-resident memory T (cells) WM = white matter

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 2 | March 2021 NeurologyorgNN

A CSF sample was acquired with a lumbar puncture from 73patients with MS with an average disease duration of 117 plusmn85 (mean plusmn SD) years These patients visited the MS CenterAmsterdam (Amsterdam The Netherlands) for analysis ofcognitive complaints which is a common symptom in MS26

Information on OCB pattern at the time of diagnosis wascollected by a retrospective chart analysis Patientsrsquo charac-teristics are provided in table 1

Standard Protocol Approvals Registrationsand Patient ConsentsInformed consent was given by the donors of the NBB forbrain autopsy and for the use of material and clinical data forresearch purposes NBB autopsy procedures have been ap-proved by the medical ethics committee of AmsterdamUMC location VUmc Amsterdam The Netherlands

Sampling of biopsies and CSF has been approved by themedical ethics committee of the University HospitalMunster and Amsterdam UMC location VUmcrespectively

ImmunohistochemistryImmunohistochemistry of the autopsy tissue samples wasperformed on 8-μm-thick formalin-fixed paraffin-embeddedtissue sections All brainstem and subcortical WM tissuesections were immunostained for myelin (proteolipid pro-tein) and HLA (HLA-DRDQ referred to as HLA) as pre-viously described1516 Lesions were annotated and sectionswere stained for CD20 CD13827 and CD3 as described inthe supplementary methods linkslwwcomNXIA399 ForCD138 an image of the positive control in tonsil is providedin figure e-2 linkslwwcomNXIA399

Table 1 Clinical Cohort of Patients With MS With OCB Examinations

DiagnosisCases (n())

Age at OCB(y) Sex

MS type()

Disease duration at OCB(y)

Treatment()

All MS 73 492 plusmn 100 46F27M

RR 68SP 19PP 5Various 8

117 plusmn 85 49 DMT51 none

OCB-positive 53 (726) 483 plusmn 97 32F21M



OCB-negative 20 (274) 515 plusmn 107 14F6M RR 55SP 20Various 25


All OCB-negative

At diagnosis OCB-positive 6 (30) 495 plusmn 97 5F1M BD 17RR 67SP 17


Previously elevated IgG OCBunknown

4 (20) 600 plusmn 96 2F2M CIS 25RR 50SP 25


At diagnosis OCB-negative 4 (20) 490 plusmn 106 3F1M CIS 50RR 50


Not reported 6 (30) 495 plusmn 121 4F2M RR 50SP 33NR 17


OCB-negative at diagnosis positive

Case 1 56 F RR 20 GLA

Case 2 54 F RR 8 DMF


Case 4 52 F BD 4 IFNβ


Case 6 60 M SP 16 None

Abbreviations BD = Balo disease CIS = clinically isolated syndrome DMF = dimethyl fumarate GLA = glatiramer acetate IFNβ = interferon-β IgG =immunoglobulin G NR = not reported OCB = oligoclonal band PP = primary progressive RR = relapsing-remitting SP = secondary progressive various = BDCIS and NRProvided is the mean plusmn SD

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 8 Number 2 | March 2021 3

OCB and IgG Measurement in CSF and PlasmaA selection of 16 NBB MS cases without the presence ofB cells and CD138+ plasma cells in the perivascular space andparenchyma of both MO and subcortical active WM lesionsand 16MS cases with B cells and CD138+ plasma cells at theselocations was made to analyze postmortemCSF samples Onecase was excluded due to CSF anti-MOG positivity (figuree-1 linkslwwcomNXIA399) Paired plasma samples wereavailable from 20 of these MS autopsy cases (10 with B cellsand 10 without B cells) In addition paired CSF and serumsamples of 73 patients with MS were analyzed In all samplesIgG levels were determined with nephelometry and thepresence of OCBs was analyzed with isoelectric focusingfollowed by IgG immunoblotting28

Statistical AnalysisStatistical analysis was performed using GraphPad Prism 8(811 April 2019 GraphPad San Diego CA USA) Pro-portional differences between 2 or more strata were testedwith the Fisher exact and χ2 tests respectively Brainstemlesion load and reactive site load were log transformedNormally distributed data were analyzed using a Student ttest A nonparametric Mann-Whitney U test was used whendata were not normally distributed data For disease durationand age at death a survival analysis was performed using theGehan-Breslow-Wilcoxon test

Data AvailabilityThe data that support the findings of this study are availablefrom the corresponding author on reasonable request

ResultsB Cells Are Present in Early MS Biopsy Lesionsand in Active and Mixed ActiveInactive MSAutopsy LesionsOf the NBBMS autopsy cohort we analyzed thematerial of N= 140 donors for the current study First we analyzed thepresence of B cells and CD138+ plasma cells in the MObecause this is one of the few standardly dissected regions inthe NBB MS autopsy protocol which contains both the WMand gray matter (GM) brain parenchyma and the meningesB cells were more often present in the perivascular space (p =0004) and meninges (p lt 00001) compared with the brainparenchyma (17 51 and 4 of cases respectively figure1 A and B) In the MO collection 85 sections contained MSlesions and 53 sections did not contain MS lesions B cellsand CD138+ plasma cells were found more frequently (p =0028 and p = 0038 respectively) in sections with MS lesions(21 had B cells and 19 had plasma cells) compared withnormal-appearingMO tissue sections (9 had B cells and 8had plasma cells) (figure 1 C and D)

To investigate the association with WM lesion characteristicswe scored the presence of B cells and CD138+ plasma cells insubcortical WM from 24 non-neurologic controls and 73 MS

autopsy cases containing 158 MS lesions (10 reactive 41active 66 mixed activeinactive 25 inactive and 16 remyeli-nated) Moreover we determined the presence of B cells andCD138+ plasma cells in 28 early MS biopsies from WM le-sions which were all active In the non-neurologic controlswe identified B cells (2ndash5 cells per section) in the meninges in4 (2 of 24) of the cases B cells were more frequently foundin early biopsy (93 p lt 00001) and in active (66 p lt00001) and mixed activeinactive (29 p lt 00001) autopsyMS lesions compared with control WM Notably in 34 ofthe active autopsy lesions no B cells were identified In re-active (10) inactive (8) and remyelinated lesions (6)B cells were not significantly enriched compared with controlWM (figure 1E) In control WM and meninges no CD138+

plasma cells were identified CD138+ plasma cells were foundin early biopsy lesions (56 p = 0004) and in all autopsy MSlesion subtypesmdashreactive (10 p = 0002) active (22 p lt00001) mixed activeinactive (8 p = 0007) inactive(24 p lt 00001) and remyelinated (19 p lt 00001)compared with control WM (figure 1 FndashI)

The Presence of B Cells and CD138+ PlasmaCells Is a General Donor CharacteristicWe next assessed the presence of B cells and CD138+ plasmacells within multiple locations (parenchyma perivascularspace and meninges) and tissue blocks (MO and subcorticalWM) from the same donors (figure 2 A and B) The presenceof B cells in the perivascular space was associated with thepresence of B cells in the meninges (92 and 43 in donorswith and without perivascular B cells respectively p lt 00001figure 2D) This is in accordance with Reali et al14 who alsoobserved an increased number of B cells in meninges to beassociated with an increased number of B cells in perivascularspace of MS spinal cords Furthermore the presence of B cellsin the MO was associated with the presence of B cells in thesubcortical WM (50 and 27 in donors with and withoutMO B cells respectively p = 0001 figure 2E)

Limited Presence of B Cells in MS AutopsyCases Associates With a Favorable Clinical andPathological ProfileTo assess whether the presence of B and CD138+ plasma cellsin MO and subcortical WM lesions correlates with more se-vere MS likewise earlier reported for meningeal B-cell infil-trates we compared donors with and without B cells at theselocations B cells were frequently encountered in perivascularclusters with T cells (figure 3A) Cases without B cells at theMO showed less often perivascular cuffing of T cells in theMO (11 and 35 p lt 00001 figure 3B) Cases withoutB cells in subcortical WM showed a trend for a lower numberof T cells in subcortical MS lesions (median 33 vs 83 cellsmm2 p = 006 figure 3C) and a lower overall percentage ofmixed activeinactive lesions (mean 237 vs 396 p = 001figure 3D) compared with MS donors with B cells Clinicallythey showed a higher age at death (median 690 vs 555 p =00006 figure 3E) and a less severe clinical disease coursedefined as a longer disease duration (median 310 vs 220 p =


0007 figure 3F) and they more often had a persistent re-lapsing or PP course compared with an SP course (100 and87 vs 75 p lt 00001 and p = 0046 figure 3G) There wasno difference in brainstem lesion load reactive site load

percentage of inactive remyelinated areas and incidence ofcortical GM lesions between MS cases with and withoutB cells at the investigated locations (figure e-3 linkslwwcomNXIA399)

Figure 1 B Cells Are Enriched in Biopsy Lesions and Active and Mixed ActiveInactive Lesions at Autopsy

(AB) B cells and plasma cells were enriched in the perivascular space and meninges of the MO compared with the MO parenchyma (CD) MO lesionscontained more frequently B cells and plasma cells compared with the normal-appearing medulla oblongata (NA MO) MS lesion subtypes were analyzed inthe subcortical WM (E) Early MS biopsy lesions significantly more often contained B cells compared with all autopsy lesions In active and mixed activeinactive autopsy lesions B cells were significantly more often present compared with control WM (F) Early MS biopsy lesions significantly more oftencontained plasma cells compared with autopsy lesions Plasma cells were significantly more often present in all MS lesion types compared with control WM(G) Example of an inflammatory active MS lesions of a secondary progressive MS brain donor with MS for 27 years stained for HLA (black) and proteolipidprotein (brown) Scale bar is 500 μm (HI) In the perivascular space B cells (CD20+ panel H scale bar is 50 μm) and a plasma cell (CD138+ panel I scale bar is25 μm) were present (both brown color) p lt 005 p lt 001 and p lt 00001 WM = white matter


MS Autopsy Cases With Limited Presence ofB Cells Show a Lower Intrathecal IgGProduction and Lack More Often OCBsBecause our data suggest an association between thepresence of B cells in meninges and MOsubcortical WMas well as an association with a more severe pathologicaland clinical profile we explored its relevance for intrathecalB-cell activation Because an increased intrathecal IgGproduction and OCB presence are highly correlating bio-markers of MS29 and the presence of OCBrsquos is associatedwith adverse outcomes630 we explored whether these CSFbiomarkers were associated with B-cell and CD138+

plasma-cell presence in MO and subcortical WM lesionsWe conducted an extreme-of-outcome analysis by selecting16 cases with B cells and 16 cases without B cells andCD138+ plasma cells in both MO and subcortical WM

lesions One MS case with B cells and CD138+ plasma cellswas excluded before OCB analysis because anti-MOG an-tibodies were detected in the postmortem CSF There wasno significant association of IgG index and OCB presence(figure 4A) with postmortem delay The pH of postmortemCSF showed a positive correlation with IgG index(Spearman correlation R = 0498 p = 0035) but not withOCB presence or presence of B cells (figure e-4 linkslwwcomNXIA399) Selected cases lacking B cells andCD138+ plasma cells in MO and subcortical WM lesionsshowed a lower CSF IgG level (median 004 vs 007 p =002 figure 4B) and a lower IgG CSFplasma ratio (median0003 vs 0008 p = 0007 figure 4C) indicating a lowerintrathecal IgG production compared with MS cases withB cells CSF OCBs were absent in 37 of cases withoutB cells and CD138+ plasma cells whereas all cases with

Figure 2 B-Cell and Plasma Cell Presence in the Meninges and Perivascular Space Is Consistent Within Donors

(A) In the MO of donor 1 with 27 yearsof secondary progressive MS B-celland plasma cell infiltrates were iden-tified in both the perivascular space(PVS) and themeninges (M) Scale barsare 100 μm for CD20 and 50 μm forCD138 (B) In the MO of donor 2 with aprimary progressive disease courseand a disease duration of 2 years Bcells were detected in the perivascularspace but not in themeninges and noplasma cells were identified Scalebars are 50 μm for CD20 and CD138 inthe PVS and 100 μm for CD20 in themeninges (C) In the MO of donor 3with a relapsing disease course for 38years no B cells or plasma cells wereidentified in both the meninges andthe perivascular space Scale bars areas in A (D) The absence of B cells in theperivascular space (PVS) is associatedwith the absence of B cells in the me-ninges (E) The absence of B cells in theMO is associatedwith the absence of Bcells in the subcortical white matterp lt 001 and p lt 00001 MO =medulla oblongata


B cells displayed CSF OCBs (p lt 00001 figure 4D) Thisobservation suggests that these MS cases with limitedpresence of B cells and CD138+ plasma cells in MO andsubcortical WM lesions are characterized by an overall al-tered CSF IgG clonality and lower IgG production Thisobservation is again in line with the strong correlationreported by Reali et al14 between meningeal and peri-vascular B-cell presence In addition the IgG index but notthe presence of OCBs was positively correlated with thenumber of T cells in subcortical WM (figure e-5A linkslwwcomNXIA399) Other pathological end points didnot correlate with IgG production or clonality (figure e-5BndashD linkslwwcomNXIA399)

OCBs Can Disappear Over Time in PatientsWith MSIn MS autopsy cases selected for absence of B cells and CD138+

plasma cells the prevalence of OCBs was lower than theexpected 90OCB positivity of patients withMS at diagnosis31

This difference could be explained by selection of MS donorswith an extreme profile of genetic or environmental factors16 butalso by a decline of the intrathecal humoral immune responseover time in chronic MS In our current study 2 of 6 MS caseswithout OCBs at autopsy had an elevated IgG ratio at diagnosiswithout information on OCBs 1 of 6 donors had normal di-agnostic CSF examination and no information was available forthe 3 other donors To explore whether a dynamic course of

Figure 3 MS Cases With Limited Presence of B Cells Show a Favorable pathological and Clinical Profile

(A) B cells were often encountered inperivascular clusters together withT cells Scale bars are 100 μm (B) MScases with the limited presence of Bcells showed less often perivascularclustering of CD3+ T cells (C) a lowernumber of CD3+ T cells in MS lesions(D) a lower percentage of mixed ac-tiveinactive (mAI) lesions (E) a higherage at death (F) a longer disease du-ration and (G)more often a secondaryprogressive disease course p lt 005and p lt 00001


OCB pattern throughout the disease course of MS can be aplausible explanation of our findings the presence of OCBs wasdetermined in a clinical cohort of 73 patients with MS whounderwent a lumbar puncture after an average disease durationof 117 plusmn 85 (mean plusmn SD) years In 274 (20 of 73) of thepatients withMSOCBs were absent In 6 of the 20 patients withMS without OCBs at follow-up OCBs were present at the timeof diagnosis (table 1) Although laboratory differences can beconfounders these data support the hypothesis that the contri-bution of B cells toMS pathology may decline during the courseof MS

DiscussionWe here demonstrate the absence of B cells and CD138+

plasma cells in 34 of the active WM lesions of advanced

MS cases in the NBB autopsy cohort Cases without B cellsat the MO or subcortical WM showed a more favorablepathological profile as indicated by a lower number ofT cells in MS lesions a lesser frequency of perivascularcuffing of T cells and a lower percentage of mixed activeinactive lesions Clinically they manifested with a less fre-quent SP disease course a longer disease duration and alower percentage of mixed activeinactive lesions comparedwith the MS donors with B cells in MO or subcortical WMlesions Furthermore a selected subgroup of patients withMS without WM B cells and CD138+ plasma cells had alower intrathecal IgG production and lacked more oftenunique OCBs in postmortem CSF Our findings indicatethat besides an important role of meningeal B cells in cor-tical pathology of advanced progressive MS B-cell in-filtration in WM is also a detrimental phenomenon at thelater stages of MS

Figure 4 MS Cases With Limited B Cells Show Lower Intrathecal IgG Production and More Often Lack OCBs

(A) Example of OCB patterns in pairedCSF and plasma for a donor without(donor 1) and a donor with (donor 2)CSF-unique OCBs MS cases with lim-ited presence of B cells showed (B) alower concentration of IgG in post-mortem CSF and (C) a lower CSFplasma IgG ratio suggesting a lowerintrathecal IgG production and (D)more often lacked CSF OCBs p lt 005p lt 001 and p lt 00001 IgG =immunoglobulin G OCB = oligoclonalband


In MS and also other autoimmune diseases B cells have beendescribed to play an important role in antigen presentationand cytokine production which induces the activation andproliferation of T cells32ndash35 MS cases with B cells show anincreased number of T cells in their MS lesions suggestingincreased T-cell activation We and others previously showedthat reactivated TRM cells are associated with the ongoinginflammatory lesion activity in WM lesions from advancedMS cases192236 InMS lesions these reactivated TRM cells areoften encountered in clusters in the perivascular space to-gether with B cells1737 suggesting that antigen presentationand reactivation of TRM cells induced by B cells potentiallyoccur at this location171838 This illustrates that besides IgGproduction B cells may have different functional roles in MSWM lesions

We show that CD138+ plasma cells are present more often inMS lesions compared with control and normal-appearingWM in line with earlier reports however only in a low per-centage of the MS autopsy cases2021 Of interest CD138+

plasma cells were most often present in inactive lesionscompared with the other lesion subtypes Prineas et al21

previously showed in a detailed electron microscopy study ofthe perivascular space in MS tissues that high numbers ofplasma cells are present in inactive lesion areas This suggeststhat CD138+ plasma cells play a less prominent role comparedwith B cells in the ongoing microglial activity of MS lesionsOcrelizumab and rituximab which show an effect on diseaseprogression in MS are directed against circulating CD20+

B cells but do not affect CD138+ plasma cells

A large heterogeneity in the number of B cells and the pres-ence of IgG depositions inMS lesions has been described overthe past decades In both early MS biopsies and late MSautopsies the presence39ndash41 and absence3942 of IgG depositsinMS lesions have been described Also the number of B cellsin MS autopsy lesions is highly heterogenous between MScases In 34 of the inflammatory active MS lesions in au-topsy tissue we identified no B cells and we showed that thepresence of B cells correlates between different location (MOand subcortical WM) and compartments (parenchyma per-ivascular space and meninges) in an individual donor

In a selected subgroup of MS cases without WM B cells alower intrathecal IgG production and a more frequent ab-sence of OCBs were found The presence of OCBs in 60 inthese MS cases is lower compared with clinical MS cohortswhere 90 showed OCBs at diagnosis4 Possibly we nowselected an extreme subgroup of MS cases with a geneticprofile at one side of a continuum that restricts involvementof B cells in MS lesion pathogenesis16 Alternatively becausewe identified B cells in 92 of the early MS biopsy lesionsand the MS cases with limited B-cell presence in autopsytissue had a longer disease duration and older age B-cellinvolvement in WM lesion activity might be extinguishingover time Accordingly Frischer et al19 reported highernumbers of perivascular B cells in donors with relapsing and

progressive disease compared with inactive disease Weprovided some support for this hypothesis by observing in aclinical cohort the absence of OCBs in 274 of patientsafter a disease duration of 117 plusmn 85 (mean plusmn SD) years In 6of these patients without OCBs their presence at diagnosiscould be validated In 4 of these patients the elevated IgGindex at diagnosis was validated These data require carefulinterpretation because comparison with historical data onOCB presence may be inaccurate It is not likely that treat-ment with disease-modifying therapies confounds these re-sults In clinical studies the presence of CSF OCBs was notaffected by highly efficacious therapies as fingolimod43 rit-uximab44 and alemtuzumab45 whereas treatment withnatalizumab4647 and cladribine48 was associated with re-duced OCBs Treatment with dimethyl fumarate has notbeen associated with lower CSF IgG production49 Althoughloss of CSF OCBs has been described in a cohort of in-terferon-betandash and glatiramer acetatendashtreated patients withMS50 this has not been observed in controlled studiesWhether the absence of perivascular B cells truly is a bio-marker for the regression of WM inflammatory disease ac-tivity in advanced MS remains to be determined Regardingcessation of disease-modifying therapies in advanced MSthis could be a clinically useful hypothesis to pursue

Our study has some limitations Because of the structure ofthe NBB MS tissue dissection protocol we could only in-vestigate the presence of B cells at selected locations in WMand meninges Bias in our data by sample and site selectioncannot be excluded which may be partially overcome byselecting a standardly dissected location and comparingmultiple locations within the same donor The extreme out-come analysis comparing donors with or without B cells atmultiple locations in a dichotomous approach provides arather crude estimate of biological associations than correla-tion analyses However due to limited availability of materialthis was for the current research question in this cohort themost feasible approach

In sum we here demonstrate in an advanced MS autopsycohort that the absence of B cells at the MO and subcorticalWM is associated with a favorable clinical and pathologicalprofile This finding may reflect extremes of a continuum ofgenetic or environmental constitution but also a regression ofWM humoral immunopathology in the natural course of ad-vanced MS

AcknowledgmentThe authors thank the MS brain donors for donating theirbrain and the NBB team for their excellent service Jannette SLangstraat for the measurements of IgG and OCBs andCorbert G van Eden for help with CD20 immunohisto-chemistry in the brainstem

Study FundingThis research was funded by MS Research (grant MS 14-888)and the VriendenLoterij J Smolders was supported by the


Nationaal MS Fonds (OZ2018-003) T Kuhlmann was sup-ported by the German Research Foundation (CRCTR-128Z02)

DisclosureNL Fransen reports no disclosures BA de Jong receivedlecture andor consultancy fees from Merck Serono BiogenTeva Genzyme and Novartis K Heszlig T Kuhlmann MCJVincenten and J Hamann report no disclosures I Huitingareceived lecture andor consultancy fees from Biogen andNovartis J Smolders received lecture andor consultancy feesfrom Biogen Merck Novartis and Sanofi-Genzyme Go toNeurologyorgNN for full disclosures

Publication HistoryReceived by Neurology Neuroimmunology amp NeuroinflammationJuly 4 2020 Accepted in final form November 18 2020

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Is associated with multiple sclerosis severity AJNR Am J Neuroradiol 2016371447ndash1453

3 Hegen H Auer M Deisenhammer F Predictors of response to multiple sclerosistherapeutics in individual patients Drugs 2016761421ndash1445

4 Annunziata P Giorgio A De Santi L et al Absence of cerebrospinal fluid oligoclonalbands is associated with delayed disability progression in relapsing-remitting MSpatients treated with interferon-beta J Neurol Sci 200624497ndash102

5 Zeman AZJ Kidd D McLean BN et al A study of oligoclonal band negative multiplesclerosis J Neurol Neurosurg Psychiatry 19966027ndash30

6 Tintore M Rovira A Rıo J et al Defining high medium and low impact prognosticfactors for developing multiple sclerosis Brain 20151381863ndash1874

7 Kuhle J Disanto G Dobson R et al Conversion from clinically isolated syndrome tomultiple sclerosis a large multicentre study Mult Scler 2015211013ndash1024

8 Lucchinetti CF Popescu BF Bunyan RF et al Inflammatory cortical demyelination inearly multiple sclerosis N Engl J Med 20113652188ndash2197

9 Kutzelnigg A Lucchinetti CF Stadelmann C et al Cortical demyelination and diffusewhite matter injury in multiple sclerosis Brain 20051282705ndash2712

10 Magliozzi R Howell O Vora A et al Meningeal B-cell follicles in secondary pro-gressive multiple sclerosis associate with early onset of disease and severe corticalpathology Brain 20071301089ndash1104

11 Choi SR Howell OW Carassiti D et al Meningeal inflammation plays a role in thepathology of primary progressive multiple sclerosis Brain 20121352925ndash2937

12 Howell OW Reeves CA Nicholas R et al Meningeal inflammation is widespread andlinked to cortical pathology in multiple sclerosis Brain 20111342755ndash2771

13 Serafini B Rosicarelli B Magliozzi R Stigliano E Aloisi F Detection of ectopic B-cellfollicles with germinal centers in the meninges of patients with secondary progressivemultiple sclerosis Brain Pathol 200414164ndash174

14 Reali C Magliozzi R Roncaroli F Nicholas R Howell OW Reynolds R B cell richmeningeal inflammation associates with increased spinal cord pathology in multiplesclerosisBrain Pathol 202030779ndash793

15 Luchetti S Fransen NL van Eden CG Ramaglia V Mason M Huitinga I Progressivemultiple sclerosis patients show substantial lesion activity that correlates with clinicaldisease severity and sex a retrospective autopsy cohort analysis Acta Neuropathol2018135511

16 Fransen NL Crusius JBA Smolders J et al Post‐mortem multiple sclerosis lesionpathology is influenced by single nucleotide polymorphisms Brain Pathol 202030106ndash119

17 Fransen NL Hsiao C-C van der Poel M et al Tissue-resident memory T cells invadethe brain parenchyma in multiple sclerosis white matter lesions Brain 20201431714ndash1730

18 Smolders J Heutinck KM Fransen NL et al Tissue-resident memory T cells populatethe human brain Nat Commun 201894593

19 Frischer JM Bramow S Dal-Bianco A et al The relation between inflammation andneurodegeneration in multiple sclerosis brains Brain 20091321175ndash1189

20 Esiri MM Immunoglobulin-containing cells in multiple-sclerosis plaques Lancet19772478ndash480

21 Prineas JW Wright RG Macrophages lymphocytes and plasma cells in the peri-vascular compartment in chronic multiple sclerosis Lab Invest 197838409ndash421

22 Machado-Santos J Saji E Troscher AR et al The compartmentalized inflammatoryresponse in the multiple sclerosis brain is composed of tissue-resident CD8+T lymphocytes and B cells Brain 20181412066ndash2082

23 Waters PJ Komorowski LWoodhall M et al Amulticenter comparison ofMOG-IgGcell-based assays Neurology 201992E1250ndashE1255

Appendix Authors

Name Location Contribution

Nina LFransenMD

Netherlands Institute forNeuroscienceAmsterdam TheNetherlands

Draftingrevision of themanuscript for contentincludingmedical writing forcontent major role in theacquisition of data studyconcept or design andanalysis or interpretation ofdata

Brigit A deJong MDPhD

Amsterdam UniversityMedical Centers VrijeUniversiteit AmsterdamAmsterdam TheNetherlands

Draftingrevision of themanuscript for contentincludingmedical writing forcontent major role in theacquisition of data andanalysis or interpretation ofdata

KatharinaHeszlig MD

University HospitalMunster MunsterGermany


TanjaKuhlmannMD PhD



Maria CJVincentenMSc


Major role in the acquisitionof data

JorgHamannPhD

Netherlands Institutefor NeuroscienceAmsterdam TheNetherlands AmsterdamUniversity MedicalCenters Universityof AmsterdamAmsterdam TheNetherlands

Draftingrevision of themanuscript for contentincludingmedical writing forcontent and analysis orinterpretation of data

Appendix (continued)


IngeHuitingaPhD

Netherlands Institute forNeuroscienceAmsterdam TheNetherlandsSwammerdam Institute forLife Sciences University ofAmsterdam AmsterdamThe Netherlands

Draftingrevision of themanuscript for contentincludingmedical writing forcontent study concept ordesign and analysis orinterpretation of data

JoostSmoldersMD PhD

Netherlands Institute forNeuroscienceAmsterdam TheNetherlands ErasmusMedical CenterRotterdam TheNetherlands



24 Waters P Reindl M Saiz A et al Multicentre comparison of a diagnostic assayaquaporin-4 antibodies in neuromyelitis optica J Neurol Neurosurg Psychiatry 2016871005ndash1015

25 Hoftberger R Guo Y Flanagan EP et al The pathology of central nervous systeminflammatory demyelinating disease accompanying myelin oligodendrocyte glyco-protein autoantibody Acta Neuropathol 2020139875ndash892

26 Chiaravalloti ND DeLuca J Cognitive impairment in multiple sclerosis LancetNeurol 200871139ndash1151

27 Tellier J Nutt SL Standing out from the crowd how to identify plasma cells Eur JImmunol Wiley-vch Verlag 2017471276ndash1279

28 Freedman MS Thompson EJ Deisenhammer F et al Recommended standard ofcerebrospinal fluid analysis in the diagnosis of multiple sclerosis a consensus state-ment Arch Neurol 200562865ndash870

29 Simonsen CS FlemmenHOslash Lauritzen T Berg-Hansen P Moen SM Celius EG Thediagnostic value of IgG index versus oligoclonal bands in cerebrospinal fluid of pa-tients with multiple sclerosis Mult Scler J 20206205521731990129

30 Ferreira D Voevodskaya O Imrell K et al Multiple sclerosis patients lacking oligo-clonal bands in the cerebrospinal fluid have less global and regional brain atrophyJ Neuroimmunol 2014274149ndash154

31 Calabrese M Gasperini C Tortorella C et al ldquoBetter explanationsrdquo in multiple sclerosisdiagnostic workup a 3-year longitudinal study Neurology 201992E2527ndashE2537

32 Weber MS Prodrsquohomme T Patarroyo JC et al B-cell activation influences T-cellpolarization and outcome of anti-CD20 B-cell depletion in central nervous systemautoimmunity Ann Neurol 201068369ndash383

33 Brimnes MK Hansen BE Nielsen LK Dziegiel MH Nielsen CH Uptake and pre-sentation of myelin basic protein by normal human b cells PLoS One 20149e113388

34 van Langelaar J Rijvers L Smolders J van Luijn MM B and T Cells driving multiplesclerosis identity mechanisms and potential triggers Front Immunol 202011760

35 Molnarfi N Schulze-Topphoff U Weber MS et al MHC class II-dependent B cellAPC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies J Exp Med 20132102921ndash2937

36 van Nierop GP van Luijn MM Michels SS et al Phenotypic and functional char-acterization of T cells in white matter lesions of multiple sclerosis patients ActaNeuropathol 2017134383ndash401

37 Revesz T Kidd D Thompson AJ Barnard RO McDonald WI A comparison of thepathology of primary and secondary progressive multiple sclerosis Brain 1994117759ndash765

38 Corsiero E Nerviani A Bombardieri M Pitzalis C Ectopic lymphoid structurespowerhouse of autoimmunity Front Immunol Front 20167430

39 Lucchinetti C Bruck W Parisi J Scheithauer B Rodriguez M Lassmann H Het-erogeneity of multiple sclerosis lesions implications for the pathogenesis of de-myelination Ann Neurol 200047707ndash717

40 Breij ECW Brink BP Veerhuis R et al Homogeneity of active demyelinating lesionsin established multiple sclerosis Ann Neurol 20086316ndash25

41 Barnett MH Parratt JD Cho ES Prineas JW Immunoglobulins and complement inpostmortem multiple sclerosis tissue Ann Neurol 20096532ndash46

42 Prineas JW Kwon EE Cho ES et al Immunopathology of secondary-progressivemultiple sclerosis Ann Neurol 200150646ndash657

43 Kowarik MC Pellkofer HL Cepok S et al Differential effects of fingolimod(FTY720) on immune cells in the CSF and blood of patients with MS Neurology2011761214ndash1221

44 Cross AH Stark JL Lauber J Ramsbottom MJ Lyons JA Rituximab reduces B cellsand T cells in cerebrospinal fluid of multiple sclerosis patients J Neuroimmunol 200618063ndash70

45 Hill-Cawthorne GA Button T Tuohy O et al Long term lymphocyte reconstitutionafter alemtuzumab treatment of multiple sclerosis J Neurol Neurosurg Psychiatry201283298ndash304

46 Von Glehn F Farias AS De Oliveira ACP et al Disappearance of cerebrospinal fluidoligoclonal bands after natalizumab treatment of multiple sclerosis patients MultScler 2012181038ndash1041

47 Mancuso R Franciotta D Rovaris M et al Effects of natalizumab on oligoclonal bandsin the cerebrospinal fluid of multiple sclerosis patients a longitudinal studyMult Scler2014201900ndash1903

48 Rejdak K Stelmasiak Z Grieb P Cladribine induces long lasting oligoclonal bandsdisappearance in relapsing multiple sclerosis patients 10-year observational studyMult Scler Relat Disord 201927117ndash120

49 Hoslashglund RA Polak J Vartdal F Holmoslashy T Lossius A B-cell composition in the bloodand cerebrospinal fluid of multiple sclerosis patients treated with dimethyl fumarateMult Scler Relat Disord 20182690ndash95

50 Mares J Herzig R Urbanek K et al Relapsing-remitting multiple sclerosis and oli-goclonal band pattern during disease modifying drug therapy relabujıcı-remitujıcıroztrousena skleroza a oligoklonalnı pruhy v prubehu lecby modifikujıcı prubehchoroby Cesk Slov Neurol N 2007103674ndash677


DOI 101212NXI000000000000095520218 Neurol Neuroimmunol Neuroinflamm

Nina L Fransen Brigit A de Jong Katharina Heszlig et al Disease and Absence of Oligoclonal Bands in MS

Absence of B Cells in Brainstem and White Matter Lesions Associates With Less Severe

This information is current as of January 27 2021

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This article cites 49 articles 5 of which you can access for free at

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httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosisfollowing collection(s) This article along with others on similar topics appears in the

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httpnnneurologyorgmiscaboutxhtmlpermissionsits entirety can be found online atInformation about reproducing this article in parts (figurestables) or in

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Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2021 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the AmericanPublished since April 2014 it is an open-access online-only continuous publication journal Copyright

is an official journal of the American Academy of NeurologyNeurol Neuroimmunol Neuroinflamm

MS is a heterogeneous disease differing in clinical diseasecourse1 radiologic appearance of lesions2 and response toimmunomodulatory therapies3 Of interest variability be-tween patients with MS is observed in the involvement ofhumoral immunity in the disease At the time of diagnosis10 of patients with MS show the absence of oligoclonalbands (OCBs) consisting of intrathecally produced immu-noglobulin (Ig) Gs4 The absence of OCBs is associated with adecreased number of lesions on MRI and a more benigndisease course45 Furthermore the presence of OCBs in pa-tients with clinically isolated syndrome is associated with anincreased risk for clinically definite MS and with an increasedrisk of disability progression67

In contrast to its limited presence in early MS8 advancedprogressive MS is characterized by extensive cortical de-myelination9 Active cortical demyelination is observed inconjunction with the presence of meningeal follicle-like in-flammatory structures10ndash12 The distinct zones of B cellsplasma cells and T cells resemble tertiary lymphoidstructures1213 The presence of these follicle-like structuresassociates with more severe disease reflected by an earlierdisease onset and faster accumulation of disability and earlierdeath1112 Recently Reali et al14 reported that the density ofmeningeal B cells in MS spinal cords correlates with extensiveaxonal loss and white matter (WM) lesion area but also withdensity of B cells in the WM perivascular space

Besides cortical demyelination demyelinating WM lesionsalso add up to disease severity in donors with advanced MSIn MS autopsy cases the presence of active and mixedactiveinactive lesions has been reported to be substantialand correlate with a short time to reaching expanded dis-ability status scale (EDSS) end points a shorter time todeath an unfavorable profile of risk factors for adverseoutcomes and an unfavorable profile of genetic risk factorsfor adverse outcomes1516 Furthermore we showed theseactive lesions to be populated by infiltrating T cells with adominant tissue-resident memory T (TRM)-cell fractionshowing signs of recent reactivation1718 Frischer et al19

quantified the presence of B cells in MS WM lesions andfound these to be predominantly present in perivascularcuffs and meninges and less frequently in the parenchymaThe presence of B cells was found most frequently in acutelesions in relapsing-remitting (RR) donors and less fre-quently in progressive patients19 IgG-producing cells andIgG deposits are regularly found in MS WM lesions2021

Furthermore the number of B cells reported in late MSautopsy lesions is highly variable between cases1922

The correlation of B-cell presence in WM lesions with clinicalend points and risk factors as well as with meningeal B-cellinfiltration has been limitedly explored Here we investigatedthe clinical and pathological characteristics of NetherlandsBrain Bank (NBB)MS autopsy cases in association with B-cellinfiltration of brainstem and subcortical WM lesions

MethodsDonor and Sample CharacteristicsOne hundred forty-one MS brain donors and 24 non-neurologic controls from the NBB autopsy cohort(Amsterdam The Netherlands) were included for thisanalysis of B cells and plasma cells Donors came to autopsybetween 1991 and 2015 and were diagnosed with MSaccording to the contemporary diagnostic criteria by theirtreating physicians Clinical files were collected post-mortem by the NBB By retrospective chart analysis theclinical diagnosis of MS was confirmed for all patients andthe clinical course was defined as either RR secondaryprogressive (SP) or primary progressive (PP) by a neu-rologist15 No MRI data were available None of the donorsreceived MS disease-modifying therapies in the year beforeautopsy except for 1 (B cellndashpositive) donor on fingolimodDetailed donor and tissue characteristics are described insupplementary table 1 linkslwwcomNXIA399 and treat-ment status is provided in supplementary table 2 linkslwwcomNXIA399 The pathological diagnosis of MS was con-firmed for all cases by a certified neuropathologist15 All donorswere analyzed for antindashmyelin oligodendrocyte glycoprotein(MOG) and antindashaquaporin-4 antibodies using cell-based as-says (figure e-1 linkslwwcomNXIA399)23ndash25

For the immunohistochemical part of this study 3 types oftissues were analyzed for the presence of B cells and plasma cells(1) standardly dissected tissue blocks at the level of the medullaoblongata (MO) from 140 MS autopsy cases (2) subcorticalWM lesions from 73140 MS autopsy cases (158 WM lesionswith a median 2 lesions per donor for both the donors with andwithout B cells) and 24 non-neurologic control donors and (3)earlyMS biopsyWM lesions (N= 28) from 24 patients withMSto explore how findings in postmortem autopsy samples of do-nors with advanced MS correlate with findings at the earlieststages ofMS These sections weremade available by the Institutefor Neuropathology University Hospital Munster (MunsterGermany) Additional information on the analysis and selectionof the different tissue samples is described in the supplementarymethods linkslwwcomNXIA399

GlossaryGM = gray matter IgG = immunoglobulin GMO =medulla oblongataMOG =myelin oligodendrocyte glycoproteinNBB =Netherlands Brain Bank OCB = oligoclonal band PP = primary progressive RR = relapsing-remitting SP = secondaryprogressive TRM = tissue-resident memory T (cells) WM = white matter









Age at OCB(y) Sex

MS type()


Treatment()









All OCB-negative






























































































Appendix Authors


Nina LFransenMD






KatharinaHeszlig MD



TanjaKuhlmannMD PhD






JorgHamannPhD





IngeHuitingaPhD



JoostSmoldersMD PhD












































Reprints











Age at OCB(y) Sex

MS type()


Treatment()









All OCB-negative






























































































Appendix Authors


Nina LFransenMD






KatharinaHeszlig MD



TanjaKuhlmannMD PhD






JorgHamannPhD





IngeHuitingaPhD



JoostSmoldersMD PhD












































Reprints















































































Appendix Authors


Nina LFransenMD






KatharinaHeszlig MD



TanjaKuhlmannMD PhD






JorgHamannPhD





IngeHuitingaPhD



JoostSmoldersMD PhD












































Reprints





































































Appendix Authors


Nina LFransenMD






KatharinaHeszlig MD



TanjaKuhlmannMD PhD






JorgHamannPhD





IngeHuitingaPhD



JoostSmoldersMD PhD












































Reprints
































































Appendix Authors


Nina LFransenMD






KatharinaHeszlig MD



TanjaKuhlmannMD PhD






JorgHamannPhD





IngeHuitingaPhD



JoostSmoldersMD PhD












































Reprints


























































Appendix Authors


Nina LFransenMD






KatharinaHeszlig MD



TanjaKuhlmannMD PhD






JorgHamannPhD





IngeHuitingaPhD



JoostSmoldersMD PhD












































Reprints



















































Appendix Authors


Nina LFransenMD






KatharinaHeszlig MD



TanjaKuhlmannMD PhD






JorgHamannPhD





IngeHuitingaPhD



JoostSmoldersMD PhD












































Reprints












































Appendix Authors


Nina LFransenMD






KatharinaHeszlig MD



TanjaKuhlmannMD PhD






JorgHamannPhD





IngeHuitingaPhD



JoostSmoldersMD PhD












































Reprints































Appendix Authors


Nina LFransenMD






KatharinaHeszlig MD



TanjaKuhlmannMD PhD






JorgHamannPhD





IngeHuitingaPhD



JoostSmoldersMD PhD












































Reprints












































Reprints
















Reprints




Documents

Neurology: Neuroimmunology & Neuroinflammation - Absence of … · tissue sections. All brainstem and subcortical WM tissue sections were immunostained for myelin (proteolipid pro-tein)