α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency...

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ARTICLE OPEN ACCESS α 4-integrin deciency in B cells does not aect disease in a T-cellmediated EAE disease model Rehana Z. Hussain, MSc, Petra D. Cravens, PhD, William A. Miller-Little, BA, Richard Doelger, BS, MSc, Valerie Granados, PhD, Emily Herndon, MD, Darin T. Okuda, MD, Todd N. Eagar, PhD, and Olaf St¨ uve, MD, PhD Neurol Neuroimmunol Neuroinamm 2019;6:e563. doi:10.1212/NXI.0000000000000563 Correspondence Dr. St¨ uve [email protected] Abstract Objective The goal of this study was to investigate the role of CD 19 + B cells within the brain and spinal cord during CNS autoimmunity in a peptide-induced, primarily T-cellmediated experimental autoimmune encephalomyelitis (EAE) model of MS. We hypothesized that CD19 + B cells outside the CNS drive inammation in EAE. Methods We generated CD19.Cre +/- α4-integrin /mice. EAE was induced by active immunization with myelin oligodendrocyte glycoprotein peptide (MOG p35-55 ). Multiparameter ow cytometry was used to phenotype leukocyte subsets in primary and secondary lymphoid organs and the CNS. Serum cytokine levels and Ig levels were assessed by bead array. B-cell adoptive transfer was used to determine the compartment-specic pathogenic role of antigen-specic and nonantigen-specic B cells. Results A genetic ablation of α4-integrin in CD19 +/- B cells signicantly reduced the number of CD19 + B cells in the CNS but does not aect EAE disease activity in active MOG p35-55 -induced disease. The composition of B-cell subsets in the brain, primary lymphoid organs, and secondary lymphoid organs of CD19.Cre +/- α4-integrin /mice was unchanged during MOG p35-55 - induced EAE. Adoptive transfer of puried CD19 + B cells from CD19.Cre +/- α4-integrin /mice or C57BL/6 wild-type (WT) control mice immunized with recombinant rMOG 1-125 or ovalbumin 323-339 into MOG p35-55 -immunized CD19.Cre +/- α4-integrin /mice caused worse clinical EAE than was observed in MOG p35-55 -immunized C57BL/6 WT control mice that did not receive adoptively transferred CD19 + B cells. Conclusions Observations made in CD19.Cre +/- α4-integrin /mice in active MOG p35-55 -induced EAE suggest a compartment-specic pathogenic role of CD19 + B cells mostly outside of the CNS that is not necessarily antigen specic. From the Department of Neurology and Neurotherapeutics (R.Z.H., P.C.C., W.A.M.-L., R.D., V.G., D.T.O., O.S.) and Department of Pathology (E.H.), University of Texas Southwestern Medical Center, Dallas; Department of Pathology and Genomic Medicine (T.N.E.), Houston Methodist Hospital; Neurology Section (O.S.), VA North Texas Health Care System, Medical Service; and Department of Neurology (O.S.), Klinikum Rechts der Isar, Technische Universit¨ at M¨ unchen, Germany. Funding information and disclosures are provided at the end of the article. Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/NN. The Article Processing Charge was funded by United States Department of Veterans Affairs I01BX001674. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and 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 © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1

Transcript of α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency...

Page 1: α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency in B cells does not affect disease in a T-cell–mediated EAE disease model Rehana

ARTICLE OPEN ACCESS

α4-integrin deficiency in B cells does not affectdisease in a T-cellndashmediated EAE disease modelRehana Z Hussain MSc Petra D Cravens PhD William A Miller-Little BA Richard Doelger BS MSc

Valerie Granados PhD Emily HerndonMD Darin T OkudaMD ToddN Eagar PhD andOlaf StuveMD PhD

Neurol Neuroimmunol Neuroinflamm 20196e563 doi101212NXI0000000000000563

Correspondence

Dr Stuve

olafstuveutsouthwesternedu

AbstractObjectiveThe goal of this study was to investigate the role of CD 19+ B cells within the brain and spinalcord during CNS autoimmunity in a peptide-induced primarily T-cellndashmediated experimentalautoimmune encephalomyelitis (EAE) model of MS We hypothesized that CD19+ B cellsoutside the CNS drive inflammation in EAE

MethodsWe generated CD19Cre+minus α4-integrinflflmice EAEwas induced by active immunization withmyelin oligodendrocyte glycoprotein peptide (MOGp35-55) Multiparameter flow cytometrywas used to phenotype leukocyte subsets in primary and secondary lymphoid organs and theCNS Serum cytokine levels and Ig levels were assessed by bead array B-cell adoptive transferwas used to determine the compartment-specific pathogenic role of antigen-specific andnonndashantigen-specific B cells

ResultsA genetic ablation of α4-integrin in CD19+minus B cells significantly reduced the number of CD19+

B cells in the CNS but does not affect EAE disease activity in activeMOGp35-55-induced diseaseThe composition of B-cell subsets in the brain primary lymphoid organs and secondarylymphoid organs of CD19Cre+minus α4-integrinflfl mice was unchanged during MOGp35-55-induced EAE Adoptive transfer of purified CD19+ B cells from CD19Cre+minus α4-integrinflfl

mice or C57BL6 wild-type (WT) control mice immunized with recombinant rMOG1-125 orovalbumin323-339 into MOGp35-55-immunized CD19Cre+minus α4-integrinflfl mice caused worseclinical EAE than was observed in MOGp35-55-immunized C57BL6 WT control mice that didnot receive adoptively transferred CD19+ B cells

ConclusionsObservations made in CD19Cre+minus α4-integrinflfl mice in active MOGp35-55-induced EAEsuggest a compartment-specific pathogenic role of CD19+ B cells mostly outside of the CNSthat is not necessarily antigen specific

From the Department of Neurology and Neurotherapeutics (RZH PCC WAM-L RD VG DTO OS) and Department of Pathology (EH) University of Texas SouthwesternMedical Center Dallas Department of Pathology and Genomic Medicine (TNE) Houston Methodist Hospital Neurology Section (OS) VA North Texas Health Care System MedicalService and Department of Neurology (OS) Klinikum Rechts der Isar Technische Universitat Munchen Germany

Funding information and disclosures are provided at the end of the article Full disclosure form information provided by the authors is available with the full text of this article atNeurologyorgNN

The Article Processing Charge was funded by United States Department of Veterans Affairs I01BX001674

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 2019 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Recent clinical trials with B-cellndashdepleting anti-CD20 thera-peutic monoclonal antibodies illustrated a pathogenic role forB lymphocytes in MS1ndash4 Whether B-cell depletion outside ofthe CNS is sufficient to provide a detectable benefit in MS orwhether a reduction in the number of B lymphocytes withinthe CNS compartment is required to diminish inflammationremains incompletely understood

In 1992 it was determined that the binding of leukocytes toinflamed CNS venules was inhibited by antibodies against α4-integrin5 Natalizumab a humanized recombinant mono-clonal antibody was the first approved α4-integrin antagonistfor treatment of relapsing forms ofMS6 Natalizumab is highlyeffective in decreasing the number of CD19+ B cells in CSF7

The goal of this study was to investigate the role of α4-integrinablation in CD19+ B cells in a peptide-induced primarilyT-cellndashmediated experimental autoimmune encephalomyelitis(EAE)model and to identify compartment-specific contributionsof B cells to disease initiation and perpetuation A T-cellndashmediated EAEmodel was chosen to reflect the role of α4-integrinin B cells in patients with MS as closely as possible GeneticallyMS is most strongly associated with human leukcoyte antigen-DRB1150189 an association that implies a pathogenicinvolvement of an antigen-specific CD4+ T cell in MS

Flow cytometry was used to phenotype leukocyte subsets inlymphoid organs and the CNS Serum cytokine levels andimmunoglobulin (Ig) levels were assessed by ELISA B-celladoptive transfer was used to determine the compartment-specific pathogenic role of antigen-specific B cells

MethodsGeneration of CD19Cre+2 α4-integrinndashdeficient miceBecause α4-integrin is an absolute requirement for normalorgan development α4-integrinndashdeficient (αminusminus) mice areembryonic lethal10 Thus it is not possible to conduct EAEexperiments in animals that are completely devoid of α4-integrin To examine how the deficiency of α4-integrin affectsthe migration of dendritic cells and B cells into the CNS andT-cell reactivation and retention in the CNS we used cre-loxPndashmediated recombination11 to create B-cell lineagendashspecific α4-integrin gene knockout mice Specifically wecrossed female mice that are homozygous for the α4-integrinndashfloxed allele (α4ff)12 with commercially availableCD19Cre+ males for the ablation of α4-integrin in B cellsInsertion of cre disrupts the CD19 coding sequence leading to

a CD19 deficiency and a concomitant reduction in germinalcenters (GCs) in homozygous animals ConsequentlyCD19Cre++ mice behave functionally very similarly to B-cellndashdeficient mice CD19Cre++ mice on the C57BL6 back-ground were used to generate CD19Cre+minus α4-integrinflflmicethat appear developmentally normal and fertile C57BL6 micewere purchased from (The Jackson Laboratories Bar HarborMN) α4-integrinflfl mice were used as controls Male and fe-male mice were used for experiments We observed no differ-ences regarding disease scores cellular composition or any ofthe biochemical and cellular outcomes between the 2 sexes

PeptidesMouse myelin oligodendrocyte glycoprotein peptide(MOGp)35-55 (MEVGWYRSPFSRVVHLYRNGK) and oval-bumin (OVA)323-339 (ISQAVHAAHAEINEAGR) were syn-thesized by solid-phase Fmoc chemistry by QCB Inc(Hopkinton MA) and CS Bio (Menlo Park CA) RecombinantrMOG1-125 was as donation ofDrHans-Christian vonBudingenat the University of California San Francisco (UCSF)

Experimental autoimmune encephalomyelitisTo induce active EAE experimental mice were immunizedsubcutaneously with myelin MOGp35-55 (200 μg100 μLmouse) emulsified in an equal volume of complete Freund ad-juvant containing 4 mgmL H37Ra Mycobacterium tuberculosis(Difco BD Franklin Lakes NJ) in each flank as described13

For B-cell adoptive transfer spleens of donor mice immunizedwith MOG1-125 or OVA323-339 were removed at day 12 andsingle-cell suspensions were prepared as previously described14

The Miltenyi kit 130-090-862 was used to purify a total of 10 times106 CD19+ donor B cells (Miltenyi Biotec San Diego CA)Briefly highly pure resting B cells were isolated by magneticlabeling and depleted of CD43-expressing B cells (activatedB cells plasma cells [PCs] andCD5+B-1a cells) and nonndashB cellsPurified cells were subsequently transferred IV into recipientCD19cre+minus α4-integrinflfl mice that were then immediatelyimmunized with MOGp35-55 For all experiments individualanimals were observed and scored as described13

Isolation of lymph node cells and splenocytesLymph node (LN) cells and splenocytes were isolated bypressing through a 70-μMnylonmesh cell strainer as described13

Percoll PLUS density gradientIn all experiments in which tissue is referred to as CNS andnot specifically as brains or spinal cords CNS leukocytes wereisolated by Percoll PLUS (GE Healthcare Bio-SciencesPittsburgh PA) gradient as previously described13

GlossaryBM = bone marrow DC = dendritic cell EAE = experimental autoimmune encephalomyelitis GC = germinal center IFN =interferon gamma IL = interleukin LN = lymph nodeMGZ = marginal zoneMOG = myelin oligodendrocyte glycoproteinOVA = ovalbumin PB = plasma blast PC = plasma cell WT = wild type

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

Enzymatic CNS digestionsFor some experiments brains and spinal cords were dissoci-ated enzymatically as described13

Flow cytometryTo determine the absolute number of B cells and T cells indifferent compartments during EAE multiparameter flowcytometry was used Cells from bone marrows (BMs)spleens LNs brains and spinal cords were brought intosingle-cell suspension as described13 Cells were stained for 30minutes at 4degC with the following antibodies CD45 PE-Cyannin-7 (30-F11 eBioscience San Diego CA) CD3 AlexaFlour 700 (17A2 eBioscience) CD19 PE-Texas Red (6D5Invitrogen Waltham MA) CD19 PE (1D3 BD BioscienceSan Jose CA) CD49d FITC (PS2 Santa Cruz Bio-technology Dallas TX) CD4 APC (RM4-5 BD Bioscience)CD45R B220-APC (RA3-6B2 BioLegend San Diego CA)PE PE Dazzle 594 PE-Texas Red AF700 Pac OrangePerCP CD138 BV421(281-2 BioLegend) CD11b (APCAPC-Cy-7 PE PerCPCy55 V450 B700 (M170 BioL-egend) CD23 PE-Cy7 (B3B4 BioLegend) CD5 PE-Cy5(53-73 BioLegend) CD1d PE (1B1 BioLegend) GL-7FITC (GL7 BioLegend) and CD21CD35 BV510 (7G6BD Bioscience San Jose CA) Isotype-matched mAbs wereused to set the gates Next 30000ndash300000 gated events wereacquired on an FACSAria II flow cytometer (BD Bio-sciences) equipped with Diva acquisition software (BDBiosciences) FlowJo (BD Biosciences) software was alsoused for some data analysis

Intracellular cytokine stainingPhorbol 12-myristate 13-acetate (500 ng) ionomycin (500 ng)and GolgiPlug (1 μL) (BD Biosciences) were added to 1 times 106

cells and incubated for 3 hours at 37degC Cells were then washedin phosphate-buffered saline and stained following the extra-cellular staining protocol for CD45-Alexa Fluor 700 After ex-tracellular staining CD19+ cells were purified as described andfixed using Fixation Buffer (BioLegend) for 15 minutes in thedark at room temperature Cells were then washed with Per-meabilization Buffer (BioLegend) twice and then stained usinginterferon gamma (IFNγ)-PE-Cy7 (BD Biosciences) in-terleukin (IL)-17A-FITC (BioLegend) tumor necrosis factorα-PE (eBioscience) and IL-6-APC (BioLegend) Cells were thenwashed and acquired and analyzed by flow cytometry

ELISASerum samples were collected by submandibular bleeding atdays 13 19 and 29 during early active EAE maximum EAEdisease activity and during chronic EAE Quantitative ELISAfor IL-17 IL-10 IL-4 IL-5 and IFNγ was performed usingpaired mAb specific for corresponding cytokines as per themanufacturerrsquos recommendations (BD Biosciences or RampDSystems) IgM and IgG serum levels were also determined byELISA The results of ELISA assays are expressed as an averageof triplicate wells plusmn SD The EPOCH (BioTek Winooski VT)ELISA plate reader and software were used for data analysis(Molecular Devices Corporation Sunnyvale CA)

T-cell proliferation assayTo determine the capability of B cells to serve as APC to CD4+

T cells flow cytometric proliferation assays using the greenfluorescent dye carboxyfluorescein succinimidyl ester or V450(BD Biosciences) were performed according to publishedmethods15 On day 15 after active induction of EAE spleens andLNs were harvested and processed into single-cell suspensionSplenocytes and lymphocytes were stained with carboxy-fluorescein succinimidyl ester and plated at 1 times 106 cells per wellThe cells were then restimulated with 10 μgmL of MOGp35-55

and incubated at 37degC for 5 days Con A at a concentration of 2mgmL and media were used as controls On day 6 the cellswere collected blockedwith Fc block antibody stainedwithAPCCD4 antibody and acquired and analyzed by flow cytometry

HistologyFollowing fixation in 10 buffered formalin LNs were pro-cessed and embedded in paraffin blocks Four-micrometersections were cut mounted on Fisherbrand Superfrost Plusglass slides (Fisher Scientific Pittsburgh PA) and stainedwith hematoxylin amp eosin (Fisher Scientific) or anti-CD19

Statistical analysisFor parametric tests data were assessed for normality using theKolmogorov-Smirnov test Normally distributed values werecompared using the unpaired 2-sided Student t test Correla-tions between continuous and categorical variables wereassessed using the Mann-Whitney U-test All statistical testswere 2 sided and p lt 005 indicated significance All analyseswere performed with Prism 7 (Graphpad La Jolla CA)

Standard protocol approvals and registrationsAll experimental animals were maintained in a specificpathogen-free facility at the University of Texas (UT) South-western Medical Center All protocols involving mice handlingwere approved by the UT Southwestern animal care facility

Data availabilityData not shown will be shared by request

ResultsThe percentage of CD49d+ CD19+ B cells isdiminished in primary and secondarylymphoid organs in naive CD19Cre+2 α4-integrinflfl miceTo characterize CD19Cre+minus α4-integrinflfl mice the ex-pression of α4-integrin (CD49d) was determined by multi-parameter flow cytometry The percentage of CD49d+

CD19+ B cells in the BM LNs spleens and Peyer patches issignificantly diminished in naive CD19Cre+minus α4-integrinflfl

mice compared with C57BL6 wild-type (WT) controlmice (figure 1A) Cre recombination efficiency is in-complete16 and determined mostly by the nucleotidesequence in the spacer region of the lox site Also there isa negative correlation between the Crelox recombinationefficiency and the length of DNA between the 2 lox sites As

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 3

stated above other investigators found a deletion efficiencybetween 75 and 80 in BM-derived prendashB cells and ap-proximately 90 in splenic B cells17

CD19Cre+2 α4-integrinflfl mice immunizedwith MOGp35-55 display a regular clinicalEAE courseTo determine the effect of α4-integrin expression of B cells ina T-cellndashmediated active model of EAE CD19Cre+minus α4-integrinflfl mice and C57BL6 control mice were immunizedwithMOGp35-55 No significant differences regarding the EAEdisease incidence severity or phenotype were observed be-tween the mouse strains (figure 1B and table) α4-integrinflfl

mice were also used as controls and showed similar diseaseactivity (data not shown)

α4-integrin expressionon splenic B cells affectsantigen-specific CD4+ T-cell proliferationIt was previously demonstrated that α4-integrin can participatein costimulation of CD4+ T cells1819 Yet other investigatorsshowed that anti-CD49d plus anti-CD3anti-CD28ndashcoatedpolystyrene beads induced significantly greater T-cell pro-liferation than anti-CD3anti-CD28 polystyrene beads alone20

To test the effect of α4-integrin ablation on the capability ofCD19+ B cells to present antigen to antigen-reactive CD4+

T cells flow cytometric proliferation assays were performedCD4+ T-cell recall responses to MOGp35-55 in spleens weresignificantly diminished in CD19Cre+minus α4-integrinflfl mice(figure 1C) However in LNs we did not observe any differ-ence in CD4+ T-cell proliferation between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice (figure 1D)

Figure 1 CD19Cre+minus α4-integrinflfl mice are fully susceptible to actively MOGp35-55-induced experimental autoimmuneencephalomyelitis

(A) The percentage of CD49d+ CD19+ B cells in thebone marrow lymph nodes spleens and Peyerpatches is significantly diminished in naiveCD19Cre+minus α4-integrinflfl mice compared withC57BL6 control mice Cells were immunophe-notyped by multiparameter flow cytometry (B)CD19Cre+minus α4-integrinflfl mice behave similar toC57BL6 wild-type (WT) mice regarding the dis-ease incidence onset and severity of active ex-perimental autoimmune encephalomyelitis (EAE)α4-integrinflfl mice were used as controls andshowed similar disease activity (data not shown)Active EAE was induced in CD19Cre+minus α4-integ-rinflfl and C57BL6 age-matched control mice bysubcutaneous immunizations with MOGp35-55 inincomplete Freund adjuvant (IFA) containing 4mgmL mycobacteria Mice received in-traperitoneal injection of 200 μL of pertussis toxin(Ptx) at 200 ngmL on days 0 and 2 Mice wereobserved daily and EAE severity was scored usinga 5-point scale CD4+ T-cell recall responses toMOGp35-55 in (C) spleens were significantly di-minished in CD19Cre+minus α4-integrinflfl mice but(D) indistinguishable to those in C57BL6 WTmicein lymphnodes Cell proliferationwas determinedby a flow cytometric proliferation assay using thegreen fluorescent dye CFSE or V450 (E) At maxi-mum disease activity (days 13ndash15) the percent-age of CD3+ T cells in the CNS was similar inCD19Cre+minus α4-integrinflfl and C57BL6 WT miceIn contrast the percentage of CD19+ B cells in theCNS was significantly diminished in CD19Cre+minus

α4-integrinflfl mice (F) The percentage of α4-integrinndashpositive (CD49d+) CD19+ B cells in theCNS during maximum EAE disease activity wascomparable between both mouse strains Lym-phocytes were immunophenotyped by multipa-rameter flow cytometry The numberappearance and architecture of germinal centersin lymph nodes of (G and H) C57BL6 WT controlmice were comparable to those in (I and J)CD19Cre+minus α4-integrinflfl mice Panels G and Iwere stained with hematoxylin amp eosin andpanels H and J were stained with anti-CD19Magnification for GndashJ is times4 p lt 005 p lt 001p lt 0001 p lt 00001 CFSE = carboxy-fluorescein succinimidyl ester

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In both mouse strains T-cell proliferation was substantial Ourobservations appear to differ from those of other investigatorswho showed that B cells do not presentMOGp35-55 to T cells21

The Methods section of that article states that ldquohellipB cells weremagnetically activated cell sorting (MACS Miltenyi BiotecBergisch Gladbach Germany)-separated from lymph nodes orspleensrdquo Thus a differential capability of B cells isolated fromeither LNs or spleens on MOG peptide presentation may nothave been fully tested

The number of B cells in the CNS during activeMOGp35-55-induced EAE is diminished inCD19Cre+2 α4-integrinflfl miceTo examine the effect of α4-integrin deletion in CD19+ B cellson the ability of B lymphocytes to enter the CNS cells wereisolated from brains and spinal cords by Percoll gradient andanalyzed by multiparameter flow cytometry As MOGp35-55-induced EAE is a T-cellndashmediated form of EAE the number ofCD3+ T cells was also assessed by the same method At maxi-mum disease activity (days 13ndash15) the percentage of CD3+

T cells in the CNS was similar in CD19Cre+minus α4-integrinflfl

mice and C57Bl6 WT mice (figure 1E) In contrast the per-centage of CD19+ B cells in the CNS was significantly reducedin CD19Cre+minus α4-integrinflfl mice (figure 1E) These obser-vations suggest that antigen-specific B cells within the CNS playa minor role in initiating and perpetuating EAE given that thesemice showed the same disease susceptibility and disease courseas C57Bl6 WT mice Immunohistochemical studies to de-termine a differential anatomic distribution of B cells in theCNSbetween CD19Cre+minus α4-integrinflfl mice and C57Bl6 WTmice were not performed as the absolute number of B cells inCD19Cre+minus α4-integrinflfl mice was very low

CD19+ B cells from CD19Cre+2 α4-integrinflfl

mice use CD49d to gain access to the CNSduring active MOGp35-55-induced EAETo determine whether CD19+ B cells from CD19Cre+minus α4-integrinflfl mice that gain access to the CNS during active EAEuse α4-integrin the percentage of α4-integrinndashpositive (CD49d+)CD19+ B cells was determined by multiparameter flowcytometry and found to be comparable between CD19Cre+minus

α4-integrinflfl mice and C57Bl6 WT controls (figure 1F)

The number and architecture of GCs in LNs ofCD19Cre+2 α4-integrinflfl mice are normalCD19Cremice express cre under the transcriptional control ofthe B lineagendashrestricted CD19 gene17 Insertion of cre disruptsthe CD19 coding sequence leading to a CD19 deficiency anda concomitant reduction in GCs in CD19Cre++ mice At day15 during the acute phase of MOGp35-55-induced EAE thenumber and architecture of GCs in LNs of CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice were com-parable by hematoxylin amp eosin (HampE) staining (figure 1 Gand H) and anti-CD19 staining (figure 1 I and J)

B cells of CD19Cre+2 α4-integrinflfl mice havenormal cytokine profiles during activeMOGp35-

55-induced EAETo test whether the absence of α4-integrin in CD19+ B cellsaffects their differentiation during inflammatory conditionsexperimental animals were killed at day 13 during the acutephase of MOGp35-55-induced EAE and B cells in disease-relevant compartments were immunophenotyped by multi-parameter flow cytometry and intracellular cytokine stainingCompared with B cells from C57BL6 WT control mice thepercentage of CD19+ B cells expressing IFNγ IL-6 IL-10 orIL-17 in CD19Cre+minus α4-integrinflfl mice was similar in theBM (figure 2A) spleen (figure 2B) LNs (figure 2C) andbrain (figure 2D) of CD19Cre+minus α4-integrinflfl mice

Outside the CNS B cells of CD19Cre+2 α4-integrinflfl mice express normal maturationand activation markers during active MOGp35-

55-induced EAEAs stated above α4-integrin is considered a costimulatorymolecule through which CD19+ B cells are capable ofinteracting with CD4+ T cells To further elucidate theeffect of α4-integrin deficiency on CD19+ B cells on theirmaturation and activation in secondary lymphoid organsexperimental animals were killed at day 13 during theacute phase of MOGp35-55-induced EAE and B cells inspleens and LNs were immunophenotyped by multipa-rameter flow cytometry The percentages of CD19+ B220+

B cells expressing IgD IgM IgG and major histocom-patibility complex (MHC) II in LNs (figure 2E) andspleen (figure 2F) were comparable in CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice

CNS B cells from CD19Cre+2 α4-integrinflfl

mice do not upregulate activation markersduring active MOGp35-55-induced EAEThe absolute number of CD19+ B cells is diminished in theCNS of CD19Cre+minus α4-integrinflfl mice compared withC57BL6 WT control mice during acute MOGp35-55-in-duced EAE (figure 1E) To evaluate the activation state ofthese cells CNS B cells were isolated and assessed by mul-tiparameter flow cytometry during active EAE at day 13 afteractive induction or EAE with MOGp35-55 The surface ex-pression of MHC II was similar in CNS-derived B cells fromCD19Cre+minus α4-integrinflfl and C57BL6 WT controls

Table Disease incidence mean maximum disease scoreand mean day of active MOGp35-55-induced EAEdisease onset in CD19+minus α4-integrin mice andC57BL6 control mice

Group IncidenceMean maximumdisease score

Mean day ofdisease onset

C57BL6 7280 289 1057 plusmn 518

CD19+2 α4-integrinflfl

6281 245 926 plusmn 607

Abbreviations EAE = experimental autoimmune encephalomyelitis MOG =myelin oligodendrocyte glycoproteinResults of 15 experiments are shown

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 5

(figure 2G) Also the expression of the costimulator mole-cules CD80 and CD86 and the first apoptosis signal receptorCD95 was comparable in brain-derived B cells fromCD19Cre+minus α4-integrinflfl mice and C57BL6 WT con-trols (figure 2H)

The composition of B-cell subsets in the brainof CD19Cre+2 α4-integrinflfl mice isunchanged during MOGp35-55-induced EAETo determine whether the deletion of α4-integrin on CD19+

B cells affects B-cell subsets differentially in their capability toenter the CNS the percentage of innate and adaptiveB-lymphocyte subsets including CD11b+CD23minusB220minus B1B cells CD11b+CD23minusB220lominusCD5minus B1b B cellsCD11b+CD23minusB220lominusCD5+ B1a B cellsCD19+CD5+CD1dhi B10 B cells CD19+CD23+CD5minus

naive follicular B cells B220+CD138minusCD95+GL7+ GCsB cells B220+CD1d+CD5minusCD23minusCD21+ marginal zone

(MGZ) B cells CD19lowndashCD138+B220lowndash plasma blast(PB) and CD19minusCD138+ PCs was assessed by multipa-rameter flow cytometry during acute MOGp35-55-inducedEAE at day 13 during early active EAE (figure 2I) day 19during maximum EAE disease activity (figure 2J) and day 29during chronic EAE (figure 2K) after active induction orEAE with MOGp35-55 and was found to be similar inCD19Cre+minus α4-integrinflfl mice and C57BL6 WT con-trols at all time points A gating strategy used to identifysome of the B-cell subsets is shown in figure 3

The composition of B-cell subsets in primaryand secondary lymphoid organs of CD19Cre+2

α4-integrinflfl mice is similar to those of WTcontrol mice during MOGp35-55-induced EAETo assess the effect of α4-integrin ablation on CD19+ B cellson B-cell phenotypes in lymphoid organs innate and adaptiveB-lymphocyte subsets were characterized by flow cytometry

Figure 2 CD19+ B cells in the CNS of CD19Cre+minus α4-integrinflfl mice are phenotypically similar to wild-type (WT) B cellsduring myelin oligodendrocyte glycoprotein peptide (MOGp35-55)-induced experimental autoimmuneencephalomyelitis

Experimental animals were killed at day 13 during the acute phase of MOGp35-55-induced experimental autoimmune encephalomyelitis (EAE) and B cells indisease-relevant compartmentswere immunophenotyped bymultiparameter flow cytometry and intracellular cytokine staining Comparedwith B cells fromC57BL6 WT control mice the percentage of CD19+ B cells expressing interferon gamma (IFNγ) interleukin (IL)-6 IL-10 or IL-17 in CD19Cre+minus α4-integrinflfl

mice was similar in the (A) bone marrow (BM) (B) spleen (C) lymph node (LNs) and (D) brain of CD19Cre+minus α4-integrinflfl mice The percentages of CD19+

B220+ B cells expressing IgD IgM IgG and MHC II in (E) LNs and (F) spleen were comparable in both mouse strains (G) In the CNS the surface expression ofMHC II was similar in CD19Cre+minus α4-integrinflfl mice and controls (H) In the brain the expression of the costimulator molecules CD80 and CD86 and the firstapoptosis signal receptor CD95 was indistinguishable between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT controls The percentage ofCD11b+CD23B220lo B1 B cells CD11b+CD23B220lominusCD5+ B1a B cells CD11b+CD23B220lominusCD5minus B1b B cells B220+CD5+CD1dhi B10 B cells CD19+CD23+CD5minus

naive follicular B cells (FB) B220+CD138minusCD95+GL7+ germinal center (GC) B cells B220+CD1d+CD5minusCD23minusCD21+marginal zone (MGZ) B cells CD138+B220lowndash

plasma blast (PB) and B220minusCD138+ plasma cells (PC) was similar in both mouse strains at (I) day 13 during early active EAE (J) day 19 during maximum EAEdisease activity and (K) day 29 during chronic EAE Lymphocytes were immunophenotyped by multiparameter flow cytometry A gating strategy used toidentify some of the B-cell subsets is shown in Figure 3 p lt 001

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

We were unable to detect a change in composition ofCD19+CD11b+CD23minusB220minus B1 B cellsCD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cellsCD19+CD5+CD1dhi B10 B cellsCD19+B220+CD1d+CD5minusCD23minusCD21+ MGZ B cells andCD19lowndashCD138+B220lowndash PB CD19Cre+minus α4-integrinflfl

mice and C57BL6 WT controls at day 13 during early activeEAE in the BM (figure 4A) LNs (Figure 4B) and spleen(figure 4C)

Serum soluble inflammatory markers areunchanged in CD19Cre+2 α4-integrinflfl miceduring different stages of MOGp35-55-induced EAEDeletion of α4-integrin on CD19+ B cells diminishes theability of CD19+ B cells to enter the CNS during acuteMOGp35-55-induced EAE (figure 1E) It is conceivable thatinflammatory B cells accumulate in peripheral blood alteringthe inflammatory milieu in that compartment This wasobserved in patients withMS treated with natalizumab22 Wefound that the serum expression of the cytokines IL-17A(figure 5A) IL-10 (Figure 5B) IL-4 (Figure 5C) IL-5

(figure 5D) and IFNγ (figure 5E) as measured by ELISA(ELISA) during acute and chronic MOGp35-55-induced EAEon days 13 19 and 29 was comparable betweenCD19Cre+minus α4-integrinflflmice and C57BL6 WT controlmice

Serum Ig levels are unchanged in CD19Cre+2

α4-integrinflfl mice during different stages ofMOGp35-55-induced EAETo further investigate whether the sequestration of CD19+

B cells through deletion of α4-integrin affects their acti-vation proliferation and antibody secretion IgM and IgGlevels in serum were determined by ELISA Serum levels ofIgM (figure 5F) and IgG (figure 5G) as measured byELISA were also similar between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice on days13 19 and 29 of MOGp35-55-induced EAE Given that theabsolute levels of IgM and IgG were similar between themouse strains and that other investigators previouslyshowed that B cells from mice on the C57BL6 back-ground do not readily recognize MOGp35-55

21

we did not test the serum levels of MOGp35-55-specific IgMor IgG

Figure 3 Immunophenotyping of murine B cells

B-lymphocyte subsets were immunophenotyped by multiparameter flow cytometry A gating strategy that was used to identify some of the B-cell sub-populations is shown (A) First cellular area height and width measurements were obtained in a channel with linear scale to gate on singlets and excludedoublets (B) Next gates were set to include CD45+ leukocytes and exclude FSClow cell debris out (C) CD138 and B220 were used to gate on plasmablasts(CD138+B220lowndash) plasma cells (CD138+B220ndash) and B cells (B220+) Panels DndashF show the gating for (E) B220+CD1d+CD5minusCD23minusCD21+IgM+ marginal zone Bcells (E) B220+CD1d+CD5minusCD23minusCD21minusIgM+ transitional 1 (T1) B cells and (F) B220+CD1d+CD5minusCD23+CD21+IgM+ T2 B cells Panels GndashI show gating for B1 B-cell populations and the identification of (H) CD11b+CD23minusB220lominusCD5minus B1b B cells and (I) CD11b+CD23minusB220lominusCD5+ B1a B cells Panels J and K show theidentification of B220+CD138minusCD95+GL7+ germinal center B cells Panels LndashO illustrate the gating for B220+CD19+CD5minusGL7minusCD23+CD21loint naive follicular Bcells FSC = forward side scatter

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 7

Activated peripheral B cells contribute todisease severity in MOGp35-55-induced EAETo determine the compartment-specific pathogenic role ofB cells in the induction and perpetuation of the EAE animalmodel CD19Cre+minus α4-integrinflfl mice were immunizedwith MOGp35-55 As demonstrated B cells in these mice havea diminished capacity to enter the CNS (figure 1E) Adoptivetransfer of purified CD19+ B cells from CD19Cre+minus α4-integrinflfl mice and C57BL6 WT mice immunized withrMOG1-125 or the control antigen OVA323-339 worsened the

clinical course of EAE in recipient mice immunized withMOGp35-55 (figure 5H) although these changes were notsignificant

DiscussionThere is accumulating evidence to suggest that B cells andmyelin-specific antibodies play crucial roles in the pathogen-esis of MS Whether B-cell depletion mediates its beneficial

Figure 4 CD19+ B cells in primary and secondary lymphoid organs of CD19Cre+minus α4-integrinflfl mice are phenotypicallysimilar to wild-type B cells during MOGp35-55-induced experimental autoimmune encephalomyelitis

(A) In the bonemarrow (B) lymph nodes and (C) spleen the percentage of CD19+CD11b+CD23minusB220minus B1 B cells CD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cells CD19+CD5+CD1dhi B10 B cells CD19+B220+CD1d+CD5minusCD23minusCD21+ marginal zone (MGZ) B cells andCD19lowndashCD138+B220lowndash plasma blast (PB) was similar in CD19Cre+minus α4-integrinflfl mice and C57BL6 WT controls at day 13 during early activeEAE Lymphocytes were immunophenotyped by multiparameter flow cytometry

Figure 5ActivatedB cells outside of theCNSdrive disease activity inmyelin oligodendrocyte glycoprotein peptide (MOGp35-

55)-induced experimental autoimmune encephalomyelitis (EAE) in an antigen no-specific manner

The serum expression of the cytokines(A) IL-17A (B) IL-10 (C) IL-4 (D) IL-5 and(E) IFNγ as measured by ELISA duringacute and chronic MOGp35-55-inducedEAE on days 13 19 and 29 was com-parable between CD19Cre+minus α4-integ-rinflfl mice and C57BL6 wild-type (WT)control mice Serum levels of (F) IgMand (G) IgG measured by ELSA at thesame time points were also similar be-tween mouse strains (H) Adoptivetransfer of purified CD19+ B cells fromCD19Cre+minus α4-integrinflfl mice orC57BL6 WT control mice immunizedwith recombinant (r)MOG1-125 or oval-bumin (OVA)323-339 into CD19+minus α4-integrinflfl mice that were then imme-diately immunized with MOGp35-55

caused worse clinical EAE than wasobserved in MOGp35-55-immunizedC57BL6 WT control mice (ldquoC57BL6rdquo)that did not receive adoptively trans-ferred CD19+ B cells

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

effects in patients with MS through B cells in the periphery inthe CNS or both is currently not completely understood

Other investigators investigated the role of α4-integrin deficiencyin CD19+ B cells in EAE23 These investigators showed thatB-cell very late activation antigen-4ndashdeficient mice developedmore severe clinical disease than control mice after active in-duction of EAE with MOGp35-55 and that that this clinical effectis due to the requirement of regulatory B cells (Bregs) for α4-integrin to enter the CNS Bregs were defined as CD19+IL-10+

or CD1dhiCD5+ B-cell subsets Another group of investigatorsalso testedCD19Cre Itga4flflmice inMOGp35-55-induced activeEAE24 CD19Cre Itga4flfl mice developed more severe EAEthan controlmice and these investigators demonstrated a criticalrole for α4-integrin on the generation of Bregs in peripheralimmune organs

Our own results diverge from these reports in that we couldnot detect a difference in active EAE disease severity betweenCD19Cre+minus α4-integrinflfl mice and C57BL6 WT controlmice These experiments were conducted 15 times (table)and we are confident in our findings Differences in EAEdisease severity and phenotype can be affected by conditionsunder which mouse colonies are maintained as well as otherfactors that may affect the overall inflammatory milieu of ex-perimental conditions This may explain the differences inresults It is also conceivable that different mice were used bythe other investigators for their experiments The inves-tigators of the 2 previous studies state that they usedCD19Creα4flfl mice23 or that they used CD19Cre Itga4flfl

mice25 It is not stated whether mice were heterozygous orhomozygous for CD19cre or a mixture of both As stated inthe Methods section CD19Cre++ mice behave functionallyvery similarly to B-cellndashdeficient mice which develop moresevere EAE when immunized with MOGp35-55

26

We found one potentially meaningful biological difference be-tween CD19Cre+minus α4-integrinflfl mice and C57BL6 controlmice There was decreased MOGp35-55-specific CD4+ T-cellproliferation in spleens which is an expected result given thatα4-integrin can participate in costimulation of CD4+T cells1819

The observation that CD19+ B cells of CD19Cre+minus α4-integrinflfl mice did deplete α4-integrin and that they weresignificantly less capable of entering the CNS after mice wereimmunized with MOGp35-55 suggests 3 interpretations (1)Antigen-activated B cells contribute to disease burden in EAEin a seemingly dose-dependent manner (2) they do not ab-solutely require recognition of a CNS autoantigen or a mimicthereof to do so and (3) they do not require full α4-integrinndashmediated access to the CNS to exacerbate diseaseThe first interpretation is supported by work of other inves-tigators who have previously demonstrated that B-cellndashdeficient C57BL6 mice are resistant to active rMOG-inducedEAE26 and that the administration of a B-cellndashdepletingmonoclonal antibody prevents or reverses established rMOG-induced EAE in C57BL6 mice21 Weber and colleagues21

further showed that B-cell depletion results in a reduction ofMOGp35-55-specific Th1 and Th17 cells Our own data suggestthat the differentiation ofMOGp35-55-specific T cells to becomeencephalitogenic does not absolutely require the presentationof autoantigen but is likely driven by other B-cell factors in-cluding soluble inflammatory mediators The third in-terpretation of this experiment is supported by ourdemonstration that B cells from CD19Cre+minus α4-integrinflfl

mice are significantly impaired in their ability to enter the CNS(figure 1E) Finally it should be emphasized that our findingsdo not conclusively rule out a role of antigen-specific B cells inthe CNS on the course of CNS autoimmunity as thresholdeffects may be difficult to detect with the EAE experiments thatwere conducted

Our data suggest that the beneficial effects of anti-CD20therapies in patients with MS are mediated through theireffects on peripheral B cells We also conclude that benefitsobserved in patients with MS during anti-α4-integrin therapyare likely not predominantly due to its effect of suppressingthe migration of B cells into the CNS The interpretation ofour findings should be limited to early forms of MS as theEAE model that was used and the observation period that wasused do not provide insight into late or progressive forms ofCNS autoimmunity Magliozzi et al27 were the first inves-tigators to show that some patients with secondary-progressive MS develop lymphoid tissue in the meningesthat resemble B-cell follicles in secondary lymphoid organsB cells in these structures may be susceptible to anti-CD20therapies with monoclonal antibodies or small molecules andit is conceivable that their depletion may benefit patientsafflicted with that MS phenotype

AcknowledgmentThe authors thank Dr Thalia Papayannopoulou at theUniversity of Washington for providing them with α4-integrinfloxflox mice Dr Stuve was funded by a Merit Reviewgrant (federal award document number [FAIN]I01BX001674) from the United States (US) Departmentof Veterans Affairs Biomedical Laboratory Research andDevelopment

Study fundingNo targeted funding reported

DisclosureRZ Hussain PC Cravens WA Miller-Little R Doelger VGrandos and E Herndon report no disclosures DT Okudareceived advisory and consulting fees from Celgene Gen-entech Genzyme EMD Serono and Novartis and researchsupport from Biogen served on the scientific advisory boardof Osmotica and served on the speakersrsquo bureau of AcordaGenzyme and Teva TN Eagar serves as section editor ofArchives of Pathology and received research support from theNIH O Stuve served on the scientific advisory boards of TGTherapeutics and Genentech-Roche served on the editorialboard of Therapeutic Advances in Neurological Disorders

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 9

consulted for Celgene EMD Serono and Novartis andreceived research support from Sanofi and GenzymeDisclosures available NeurologyorgNN

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationNovember 2 2018 Accepted in final form February 1 2019

References1 Hauser SL Waubant E Arnold DL et al B-cell depletion with rituximab in relapsing-

remitting multiple sclerosis N Engl J Med 2008358676ndash6882 Hawker K OrsquoConnor P Freedman MS et al Rituximab in patients with primary

progressive multiple sclerosis results of a randomized double-blind placebo-controlled multicenter trial Ann Neurol 200966460ndash471

3 Hauser SL Bar-Or A Comi G et al Ocrelizumab versus interferon beta-1a in re-lapsing multiple sclerosis N Engl J Med 2017376221ndash234

4 Montalban X Hauser SL Kappos L et al Ocrelizumab versus placebo in primaryprogressive multiple sclerosis N Engl J Med 2017376209ndash220

5 Yednock TA Cannon C Fritz LC Sanchez-Madrid F Steinman L Karin N Pre-vention of experimental autoimmune encephalomyelitis by antibodies against alpha 4beta 1 integrin Nature 199235663ndash66

6 Frohman EM Racke MK Raine CS Multiple sclerosismdashthe plaque and its patho-genesis N Engl J Med 2006354942ndash955

7 Stuve O Marra CM Jerome KR et al Immune surveillance in multiple sclerosispatients treated with natalizumab Ann Neurol 200659743ndash747

8 Haines JL Ter Minassian M Bazyk A et al A complete genomic screen for multiplesclerosis underscores a role for the major histocompatability complex The MultipleSclerosis Genetics Group Nat Genet 199613469ndash471

9 Sawcer S Jones HB Feakes R et al A genome screen in multiple sclerosis revealssusceptibility loci on chromosome 6p21 and 17q22 Nat Genet 199613464ndash468

10 Yang JT Rayburn H Hynes RO Cell adhesion events mediated by alpha 4 integrinsare essential in placental and cardiac development Development 1995121549ndash560

11 Sternberg N Hamilton D Bacteriophage P1 site-specific recombination I Re-combination between loxP sites J Mol Biol 1981150467ndash486

12 Scott LM Priestley GV Papayannopoulou T Deletion of alpha4 integrins from adulthematopoietic cells reveals roles in homeostasis regeneration and homing Mol CellBiol 2003239349ndash9360

13 Hussain RZ Miller-Little WA Doelger R et al Defining standard enzymatic disso-ciation methods for individual brains and spinal cords in EAE Neurol NeuroimmunolNeuroinflamm 20185e437 doi 101212NXI0000000000000437

14 Cravens PD Hussain RZ Zacharias TE et al Lymph node-derived donor encepha-litogenic CD4+ T cells in C57BL6 mice adoptive transfer experimental autoimmuneencephalomyelitis highly express GM-CSF and T-bet J Neuroinflammation 2011873

15 Karandikar NJ Crawford MP Yan X et al Glatiramer acetate (Copaxone) therapyinduces CD8(+) T cell responses in patients with multiple sclerosis J Clin Invest2002109641ndash649

16 Gu H Marth JD Orban PC Mossmann H Rajewsky K Deletion of a DNA poly-merase beta gene segment in T cells using cell type-specific gene targeting Science1994265103ndash106

17 Rickert RC Roes J Rajewsky K B lymphocyte-specific Cre-mediated mutagenesis inmice Nucleic Acids Res 1997251317ndash1318

18 Davis LS Oppenheimer-Marks N Bednarczyk JL McIntyre BW Lipsky PE Fibro-nectin promotes proliferation of naive and memory T cells by signaling through boththe VLA-4 and VLA-5 integrin molecules J Immunol 1990145785ndash793

19 Shimizu Y van Seventer GA Horgan KJ Shaw S Costimulation of proliferativeresponses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 withfibronectin or VLA-6 with laminin J Immunol 199014559ndash67

20 Theien BE Vanderlugt CL Eagar TN et al Discordant effects of anti-VLA-4 treat-ment before and after onset of relapsing experimental autoimmune encephalomyelitisJ Clin Invest 2001107995ndash1006

21 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

22 Krumbholz M Meinl I Kumpfel T Hohlfeld R Meinl E Natalizumab dispropor-tionately increases circulating pre-B and B cells in multiple sclerosis Neurology 2008711350ndash1354

23 Lehmann-Horn K Sagan SA Winger RC et al CNS accumulation of regulatoryB cells is VLA-4-dependent Neurol Neuroimmunol Neuroinflamm 20163e212 doi101212NXI0000000000000212

24 Glatigny S Wagner CA Bettelli E Correction cutting edge integrin alpha4 is re-quired for regulatory B cell control of experimental autoimmune encephalomyelitisJ Immunol 20171983756

25 Glatigny S Duhen R Oukka M Bettelli E Cutting edge loss of alpha4 integrinexpression differentially affects the homing of Th1 and Th17 cells J Immunol 20111876176ndash6179

26 Lyons JA SanM HappMP Cross AH B cells are critical to induction of experimentalallergic encephalomyelitis by protein but not by a short encephalitogenic peptide EurJ Immunol 1999293432ndash3439

27 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

Appendix Authors

Name Location Role Contribution

Rehana ZHussainMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

Petra CCravensPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

William AMiller-Little BA

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

RichardDoelgerMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

ValerieGranadosPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the data andanalyzed the data

EmilyHerndonMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Darin TOkudaMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Todd NEagar PhD

Houston MethodistHospital HoustonTX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

OlafStuve MDPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

DOI 101212NXI000000000000056320196 Neurol Neuroimmunol Neuroinflamm

Rehana Z Hussain Petra D Cravens William A Miller-Little et al disease model

mediated EAEminus4-integrin deficiency in B cells does not affect disease in a T-cellα

This information is current as of April 16 2019

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

ServicesUpdated Information amp

httpnnneurologyorgcontent64e563fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent64e563fullhtmlref-list-1

This article cites 27 articles 7 of which you can access for free at

Citations httpnnneurologyorgcontent64e563fullhtmlotherarticles

This article has been cited by 1 HighWire-hosted articles

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httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

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httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the

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nextAn erratum has been published regarding this article Please see

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Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

CORRECTION

α4-integrin deficiency in B cells does not affect disease in aT-cellndashmediated EAE disease modelNeurol Neuroimmunol Neuroinflamm 20196e585 doi101212NXI0000000000000585

In the article ldquoα4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAEdisease model by Hussain et al1 first published online April 16 2019 funding for the papershould have been listed in the Study Funding section The authors regret the error

Reference1 Hussain RZ Cravens PD Miller-Little WA et al α4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAE

disease model Neurol Neuroimmunol Neuroinflamm 20196e563

Copyright copy 2019 American Academy of Neurology 1

Copyright copy 2019 American Academy of Neurology Unauthorized reproduction of this article is prohibited

Page 2: α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency in B cells does not affect disease in a T-cell–mediated EAE disease model Rehana

Recent clinical trials with B-cellndashdepleting anti-CD20 thera-peutic monoclonal antibodies illustrated a pathogenic role forB lymphocytes in MS1ndash4 Whether B-cell depletion outside ofthe CNS is sufficient to provide a detectable benefit in MS orwhether a reduction in the number of B lymphocytes withinthe CNS compartment is required to diminish inflammationremains incompletely understood

In 1992 it was determined that the binding of leukocytes toinflamed CNS venules was inhibited by antibodies against α4-integrin5 Natalizumab a humanized recombinant mono-clonal antibody was the first approved α4-integrin antagonistfor treatment of relapsing forms ofMS6 Natalizumab is highlyeffective in decreasing the number of CD19+ B cells in CSF7

The goal of this study was to investigate the role of α4-integrinablation in CD19+ B cells in a peptide-induced primarilyT-cellndashmediated experimental autoimmune encephalomyelitis(EAE)model and to identify compartment-specific contributionsof B cells to disease initiation and perpetuation A T-cellndashmediated EAEmodel was chosen to reflect the role of α4-integrinin B cells in patients with MS as closely as possible GeneticallyMS is most strongly associated with human leukcoyte antigen-DRB1150189 an association that implies a pathogenicinvolvement of an antigen-specific CD4+ T cell in MS

Flow cytometry was used to phenotype leukocyte subsets inlymphoid organs and the CNS Serum cytokine levels andimmunoglobulin (Ig) levels were assessed by ELISA B-celladoptive transfer was used to determine the compartment-specific pathogenic role of antigen-specific B cells

MethodsGeneration of CD19Cre+2 α4-integrinndashdeficient miceBecause α4-integrin is an absolute requirement for normalorgan development α4-integrinndashdeficient (αminusminus) mice areembryonic lethal10 Thus it is not possible to conduct EAEexperiments in animals that are completely devoid of α4-integrin To examine how the deficiency of α4-integrin affectsthe migration of dendritic cells and B cells into the CNS andT-cell reactivation and retention in the CNS we used cre-loxPndashmediated recombination11 to create B-cell lineagendashspecific α4-integrin gene knockout mice Specifically wecrossed female mice that are homozygous for the α4-integrinndashfloxed allele (α4ff)12 with commercially availableCD19Cre+ males for the ablation of α4-integrin in B cellsInsertion of cre disrupts the CD19 coding sequence leading to

a CD19 deficiency and a concomitant reduction in germinalcenters (GCs) in homozygous animals ConsequentlyCD19Cre++ mice behave functionally very similarly to B-cellndashdeficient mice CD19Cre++ mice on the C57BL6 back-ground were used to generate CD19Cre+minus α4-integrinflflmicethat appear developmentally normal and fertile C57BL6 micewere purchased from (The Jackson Laboratories Bar HarborMN) α4-integrinflfl mice were used as controls Male and fe-male mice were used for experiments We observed no differ-ences regarding disease scores cellular composition or any ofthe biochemical and cellular outcomes between the 2 sexes

PeptidesMouse myelin oligodendrocyte glycoprotein peptide(MOGp)35-55 (MEVGWYRSPFSRVVHLYRNGK) and oval-bumin (OVA)323-339 (ISQAVHAAHAEINEAGR) were syn-thesized by solid-phase Fmoc chemistry by QCB Inc(Hopkinton MA) and CS Bio (Menlo Park CA) RecombinantrMOG1-125 was as donation ofDrHans-Christian vonBudingenat the University of California San Francisco (UCSF)

Experimental autoimmune encephalomyelitisTo induce active EAE experimental mice were immunizedsubcutaneously with myelin MOGp35-55 (200 μg100 μLmouse) emulsified in an equal volume of complete Freund ad-juvant containing 4 mgmL H37Ra Mycobacterium tuberculosis(Difco BD Franklin Lakes NJ) in each flank as described13

For B-cell adoptive transfer spleens of donor mice immunizedwith MOG1-125 or OVA323-339 were removed at day 12 andsingle-cell suspensions were prepared as previously described14

The Miltenyi kit 130-090-862 was used to purify a total of 10 times106 CD19+ donor B cells (Miltenyi Biotec San Diego CA)Briefly highly pure resting B cells were isolated by magneticlabeling and depleted of CD43-expressing B cells (activatedB cells plasma cells [PCs] andCD5+B-1a cells) and nonndashB cellsPurified cells were subsequently transferred IV into recipientCD19cre+minus α4-integrinflfl mice that were then immediatelyimmunized with MOGp35-55 For all experiments individualanimals were observed and scored as described13

Isolation of lymph node cells and splenocytesLymph node (LN) cells and splenocytes were isolated bypressing through a 70-μMnylonmesh cell strainer as described13

Percoll PLUS density gradientIn all experiments in which tissue is referred to as CNS andnot specifically as brains or spinal cords CNS leukocytes wereisolated by Percoll PLUS (GE Healthcare Bio-SciencesPittsburgh PA) gradient as previously described13

GlossaryBM = bone marrow DC = dendritic cell EAE = experimental autoimmune encephalomyelitis GC = germinal center IFN =interferon gamma IL = interleukin LN = lymph nodeMGZ = marginal zoneMOG = myelin oligodendrocyte glycoproteinOVA = ovalbumin PB = plasma blast PC = plasma cell WT = wild type

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

Enzymatic CNS digestionsFor some experiments brains and spinal cords were dissoci-ated enzymatically as described13

Flow cytometryTo determine the absolute number of B cells and T cells indifferent compartments during EAE multiparameter flowcytometry was used Cells from bone marrows (BMs)spleens LNs brains and spinal cords were brought intosingle-cell suspension as described13 Cells were stained for 30minutes at 4degC with the following antibodies CD45 PE-Cyannin-7 (30-F11 eBioscience San Diego CA) CD3 AlexaFlour 700 (17A2 eBioscience) CD19 PE-Texas Red (6D5Invitrogen Waltham MA) CD19 PE (1D3 BD BioscienceSan Jose CA) CD49d FITC (PS2 Santa Cruz Bio-technology Dallas TX) CD4 APC (RM4-5 BD Bioscience)CD45R B220-APC (RA3-6B2 BioLegend San Diego CA)PE PE Dazzle 594 PE-Texas Red AF700 Pac OrangePerCP CD138 BV421(281-2 BioLegend) CD11b (APCAPC-Cy-7 PE PerCPCy55 V450 B700 (M170 BioL-egend) CD23 PE-Cy7 (B3B4 BioLegend) CD5 PE-Cy5(53-73 BioLegend) CD1d PE (1B1 BioLegend) GL-7FITC (GL7 BioLegend) and CD21CD35 BV510 (7G6BD Bioscience San Jose CA) Isotype-matched mAbs wereused to set the gates Next 30000ndash300000 gated events wereacquired on an FACSAria II flow cytometer (BD Bio-sciences) equipped with Diva acquisition software (BDBiosciences) FlowJo (BD Biosciences) software was alsoused for some data analysis

Intracellular cytokine stainingPhorbol 12-myristate 13-acetate (500 ng) ionomycin (500 ng)and GolgiPlug (1 μL) (BD Biosciences) were added to 1 times 106

cells and incubated for 3 hours at 37degC Cells were then washedin phosphate-buffered saline and stained following the extra-cellular staining protocol for CD45-Alexa Fluor 700 After ex-tracellular staining CD19+ cells were purified as described andfixed using Fixation Buffer (BioLegend) for 15 minutes in thedark at room temperature Cells were then washed with Per-meabilization Buffer (BioLegend) twice and then stained usinginterferon gamma (IFNγ)-PE-Cy7 (BD Biosciences) in-terleukin (IL)-17A-FITC (BioLegend) tumor necrosis factorα-PE (eBioscience) and IL-6-APC (BioLegend) Cells were thenwashed and acquired and analyzed by flow cytometry

ELISASerum samples were collected by submandibular bleeding atdays 13 19 and 29 during early active EAE maximum EAEdisease activity and during chronic EAE Quantitative ELISAfor IL-17 IL-10 IL-4 IL-5 and IFNγ was performed usingpaired mAb specific for corresponding cytokines as per themanufacturerrsquos recommendations (BD Biosciences or RampDSystems) IgM and IgG serum levels were also determined byELISA The results of ELISA assays are expressed as an averageof triplicate wells plusmn SD The EPOCH (BioTek Winooski VT)ELISA plate reader and software were used for data analysis(Molecular Devices Corporation Sunnyvale CA)

T-cell proliferation assayTo determine the capability of B cells to serve as APC to CD4+

T cells flow cytometric proliferation assays using the greenfluorescent dye carboxyfluorescein succinimidyl ester or V450(BD Biosciences) were performed according to publishedmethods15 On day 15 after active induction of EAE spleens andLNs were harvested and processed into single-cell suspensionSplenocytes and lymphocytes were stained with carboxy-fluorescein succinimidyl ester and plated at 1 times 106 cells per wellThe cells were then restimulated with 10 μgmL of MOGp35-55

and incubated at 37degC for 5 days Con A at a concentration of 2mgmL and media were used as controls On day 6 the cellswere collected blockedwith Fc block antibody stainedwithAPCCD4 antibody and acquired and analyzed by flow cytometry

HistologyFollowing fixation in 10 buffered formalin LNs were pro-cessed and embedded in paraffin blocks Four-micrometersections were cut mounted on Fisherbrand Superfrost Plusglass slides (Fisher Scientific Pittsburgh PA) and stainedwith hematoxylin amp eosin (Fisher Scientific) or anti-CD19

Statistical analysisFor parametric tests data were assessed for normality using theKolmogorov-Smirnov test Normally distributed values werecompared using the unpaired 2-sided Student t test Correla-tions between continuous and categorical variables wereassessed using the Mann-Whitney U-test All statistical testswere 2 sided and p lt 005 indicated significance All analyseswere performed with Prism 7 (Graphpad La Jolla CA)

Standard protocol approvals and registrationsAll experimental animals were maintained in a specificpathogen-free facility at the University of Texas (UT) South-western Medical Center All protocols involving mice handlingwere approved by the UT Southwestern animal care facility

Data availabilityData not shown will be shared by request

ResultsThe percentage of CD49d+ CD19+ B cells isdiminished in primary and secondarylymphoid organs in naive CD19Cre+2 α4-integrinflfl miceTo characterize CD19Cre+minus α4-integrinflfl mice the ex-pression of α4-integrin (CD49d) was determined by multi-parameter flow cytometry The percentage of CD49d+

CD19+ B cells in the BM LNs spleens and Peyer patches issignificantly diminished in naive CD19Cre+minus α4-integrinflfl

mice compared with C57BL6 wild-type (WT) controlmice (figure 1A) Cre recombination efficiency is in-complete16 and determined mostly by the nucleotidesequence in the spacer region of the lox site Also there isa negative correlation between the Crelox recombinationefficiency and the length of DNA between the 2 lox sites As

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 3

stated above other investigators found a deletion efficiencybetween 75 and 80 in BM-derived prendashB cells and ap-proximately 90 in splenic B cells17

CD19Cre+2 α4-integrinflfl mice immunizedwith MOGp35-55 display a regular clinicalEAE courseTo determine the effect of α4-integrin expression of B cells ina T-cellndashmediated active model of EAE CD19Cre+minus α4-integrinflfl mice and C57BL6 control mice were immunizedwithMOGp35-55 No significant differences regarding the EAEdisease incidence severity or phenotype were observed be-tween the mouse strains (figure 1B and table) α4-integrinflfl

mice were also used as controls and showed similar diseaseactivity (data not shown)

α4-integrin expressionon splenic B cells affectsantigen-specific CD4+ T-cell proliferationIt was previously demonstrated that α4-integrin can participatein costimulation of CD4+ T cells1819 Yet other investigatorsshowed that anti-CD49d plus anti-CD3anti-CD28ndashcoatedpolystyrene beads induced significantly greater T-cell pro-liferation than anti-CD3anti-CD28 polystyrene beads alone20

To test the effect of α4-integrin ablation on the capability ofCD19+ B cells to present antigen to antigen-reactive CD4+

T cells flow cytometric proliferation assays were performedCD4+ T-cell recall responses to MOGp35-55 in spleens weresignificantly diminished in CD19Cre+minus α4-integrinflfl mice(figure 1C) However in LNs we did not observe any differ-ence in CD4+ T-cell proliferation between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice (figure 1D)

Figure 1 CD19Cre+minus α4-integrinflfl mice are fully susceptible to actively MOGp35-55-induced experimental autoimmuneencephalomyelitis

(A) The percentage of CD49d+ CD19+ B cells in thebone marrow lymph nodes spleens and Peyerpatches is significantly diminished in naiveCD19Cre+minus α4-integrinflfl mice compared withC57BL6 control mice Cells were immunophe-notyped by multiparameter flow cytometry (B)CD19Cre+minus α4-integrinflfl mice behave similar toC57BL6 wild-type (WT) mice regarding the dis-ease incidence onset and severity of active ex-perimental autoimmune encephalomyelitis (EAE)α4-integrinflfl mice were used as controls andshowed similar disease activity (data not shown)Active EAE was induced in CD19Cre+minus α4-integ-rinflfl and C57BL6 age-matched control mice bysubcutaneous immunizations with MOGp35-55 inincomplete Freund adjuvant (IFA) containing 4mgmL mycobacteria Mice received in-traperitoneal injection of 200 μL of pertussis toxin(Ptx) at 200 ngmL on days 0 and 2 Mice wereobserved daily and EAE severity was scored usinga 5-point scale CD4+ T-cell recall responses toMOGp35-55 in (C) spleens were significantly di-minished in CD19Cre+minus α4-integrinflfl mice but(D) indistinguishable to those in C57BL6 WTmicein lymphnodes Cell proliferationwas determinedby a flow cytometric proliferation assay using thegreen fluorescent dye CFSE or V450 (E) At maxi-mum disease activity (days 13ndash15) the percent-age of CD3+ T cells in the CNS was similar inCD19Cre+minus α4-integrinflfl and C57BL6 WT miceIn contrast the percentage of CD19+ B cells in theCNS was significantly diminished in CD19Cre+minus

α4-integrinflfl mice (F) The percentage of α4-integrinndashpositive (CD49d+) CD19+ B cells in theCNS during maximum EAE disease activity wascomparable between both mouse strains Lym-phocytes were immunophenotyped by multipa-rameter flow cytometry The numberappearance and architecture of germinal centersin lymph nodes of (G and H) C57BL6 WT controlmice were comparable to those in (I and J)CD19Cre+minus α4-integrinflfl mice Panels G and Iwere stained with hematoxylin amp eosin andpanels H and J were stained with anti-CD19Magnification for GndashJ is times4 p lt 005 p lt 001p lt 0001 p lt 00001 CFSE = carboxy-fluorescein succinimidyl ester

4 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

In both mouse strains T-cell proliferation was substantial Ourobservations appear to differ from those of other investigatorswho showed that B cells do not presentMOGp35-55 to T cells21

The Methods section of that article states that ldquohellipB cells weremagnetically activated cell sorting (MACS Miltenyi BiotecBergisch Gladbach Germany)-separated from lymph nodes orspleensrdquo Thus a differential capability of B cells isolated fromeither LNs or spleens on MOG peptide presentation may nothave been fully tested

The number of B cells in the CNS during activeMOGp35-55-induced EAE is diminished inCD19Cre+2 α4-integrinflfl miceTo examine the effect of α4-integrin deletion in CD19+ B cellson the ability of B lymphocytes to enter the CNS cells wereisolated from brains and spinal cords by Percoll gradient andanalyzed by multiparameter flow cytometry As MOGp35-55-induced EAE is a T-cellndashmediated form of EAE the number ofCD3+ T cells was also assessed by the same method At maxi-mum disease activity (days 13ndash15) the percentage of CD3+

T cells in the CNS was similar in CD19Cre+minus α4-integrinflfl

mice and C57Bl6 WT mice (figure 1E) In contrast the per-centage of CD19+ B cells in the CNS was significantly reducedin CD19Cre+minus α4-integrinflfl mice (figure 1E) These obser-vations suggest that antigen-specific B cells within the CNS playa minor role in initiating and perpetuating EAE given that thesemice showed the same disease susceptibility and disease courseas C57Bl6 WT mice Immunohistochemical studies to de-termine a differential anatomic distribution of B cells in theCNSbetween CD19Cre+minus α4-integrinflfl mice and C57Bl6 WTmice were not performed as the absolute number of B cells inCD19Cre+minus α4-integrinflfl mice was very low

CD19+ B cells from CD19Cre+2 α4-integrinflfl

mice use CD49d to gain access to the CNSduring active MOGp35-55-induced EAETo determine whether CD19+ B cells from CD19Cre+minus α4-integrinflfl mice that gain access to the CNS during active EAEuse α4-integrin the percentage of α4-integrinndashpositive (CD49d+)CD19+ B cells was determined by multiparameter flowcytometry and found to be comparable between CD19Cre+minus

α4-integrinflfl mice and C57Bl6 WT controls (figure 1F)

The number and architecture of GCs in LNs ofCD19Cre+2 α4-integrinflfl mice are normalCD19Cremice express cre under the transcriptional control ofthe B lineagendashrestricted CD19 gene17 Insertion of cre disruptsthe CD19 coding sequence leading to a CD19 deficiency anda concomitant reduction in GCs in CD19Cre++ mice At day15 during the acute phase of MOGp35-55-induced EAE thenumber and architecture of GCs in LNs of CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice were com-parable by hematoxylin amp eosin (HampE) staining (figure 1 Gand H) and anti-CD19 staining (figure 1 I and J)

B cells of CD19Cre+2 α4-integrinflfl mice havenormal cytokine profiles during activeMOGp35-

55-induced EAETo test whether the absence of α4-integrin in CD19+ B cellsaffects their differentiation during inflammatory conditionsexperimental animals were killed at day 13 during the acutephase of MOGp35-55-induced EAE and B cells in disease-relevant compartments were immunophenotyped by multi-parameter flow cytometry and intracellular cytokine stainingCompared with B cells from C57BL6 WT control mice thepercentage of CD19+ B cells expressing IFNγ IL-6 IL-10 orIL-17 in CD19Cre+minus α4-integrinflfl mice was similar in theBM (figure 2A) spleen (figure 2B) LNs (figure 2C) andbrain (figure 2D) of CD19Cre+minus α4-integrinflfl mice

Outside the CNS B cells of CD19Cre+2 α4-integrinflfl mice express normal maturationand activation markers during active MOGp35-

55-induced EAEAs stated above α4-integrin is considered a costimulatorymolecule through which CD19+ B cells are capable ofinteracting with CD4+ T cells To further elucidate theeffect of α4-integrin deficiency on CD19+ B cells on theirmaturation and activation in secondary lymphoid organsexperimental animals were killed at day 13 during theacute phase of MOGp35-55-induced EAE and B cells inspleens and LNs were immunophenotyped by multipa-rameter flow cytometry The percentages of CD19+ B220+

B cells expressing IgD IgM IgG and major histocom-patibility complex (MHC) II in LNs (figure 2E) andspleen (figure 2F) were comparable in CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice

CNS B cells from CD19Cre+2 α4-integrinflfl

mice do not upregulate activation markersduring active MOGp35-55-induced EAEThe absolute number of CD19+ B cells is diminished in theCNS of CD19Cre+minus α4-integrinflfl mice compared withC57BL6 WT control mice during acute MOGp35-55-in-duced EAE (figure 1E) To evaluate the activation state ofthese cells CNS B cells were isolated and assessed by mul-tiparameter flow cytometry during active EAE at day 13 afteractive induction or EAE with MOGp35-55 The surface ex-pression of MHC II was similar in CNS-derived B cells fromCD19Cre+minus α4-integrinflfl and C57BL6 WT controls

Table Disease incidence mean maximum disease scoreand mean day of active MOGp35-55-induced EAEdisease onset in CD19+minus α4-integrin mice andC57BL6 control mice

Group IncidenceMean maximumdisease score

Mean day ofdisease onset

C57BL6 7280 289 1057 plusmn 518

CD19+2 α4-integrinflfl

6281 245 926 plusmn 607

Abbreviations EAE = experimental autoimmune encephalomyelitis MOG =myelin oligodendrocyte glycoproteinResults of 15 experiments are shown

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 5

(figure 2G) Also the expression of the costimulator mole-cules CD80 and CD86 and the first apoptosis signal receptorCD95 was comparable in brain-derived B cells fromCD19Cre+minus α4-integrinflfl mice and C57BL6 WT con-trols (figure 2H)

The composition of B-cell subsets in the brainof CD19Cre+2 α4-integrinflfl mice isunchanged during MOGp35-55-induced EAETo determine whether the deletion of α4-integrin on CD19+

B cells affects B-cell subsets differentially in their capability toenter the CNS the percentage of innate and adaptiveB-lymphocyte subsets including CD11b+CD23minusB220minus B1B cells CD11b+CD23minusB220lominusCD5minus B1b B cellsCD11b+CD23minusB220lominusCD5+ B1a B cellsCD19+CD5+CD1dhi B10 B cells CD19+CD23+CD5minus

naive follicular B cells B220+CD138minusCD95+GL7+ GCsB cells B220+CD1d+CD5minusCD23minusCD21+ marginal zone

(MGZ) B cells CD19lowndashCD138+B220lowndash plasma blast(PB) and CD19minusCD138+ PCs was assessed by multipa-rameter flow cytometry during acute MOGp35-55-inducedEAE at day 13 during early active EAE (figure 2I) day 19during maximum EAE disease activity (figure 2J) and day 29during chronic EAE (figure 2K) after active induction orEAE with MOGp35-55 and was found to be similar inCD19Cre+minus α4-integrinflfl mice and C57BL6 WT con-trols at all time points A gating strategy used to identifysome of the B-cell subsets is shown in figure 3

The composition of B-cell subsets in primaryand secondary lymphoid organs of CD19Cre+2

α4-integrinflfl mice is similar to those of WTcontrol mice during MOGp35-55-induced EAETo assess the effect of α4-integrin ablation on CD19+ B cellson B-cell phenotypes in lymphoid organs innate and adaptiveB-lymphocyte subsets were characterized by flow cytometry

Figure 2 CD19+ B cells in the CNS of CD19Cre+minus α4-integrinflfl mice are phenotypically similar to wild-type (WT) B cellsduring myelin oligodendrocyte glycoprotein peptide (MOGp35-55)-induced experimental autoimmuneencephalomyelitis

Experimental animals were killed at day 13 during the acute phase of MOGp35-55-induced experimental autoimmune encephalomyelitis (EAE) and B cells indisease-relevant compartmentswere immunophenotyped bymultiparameter flow cytometry and intracellular cytokine staining Comparedwith B cells fromC57BL6 WT control mice the percentage of CD19+ B cells expressing interferon gamma (IFNγ) interleukin (IL)-6 IL-10 or IL-17 in CD19Cre+minus α4-integrinflfl

mice was similar in the (A) bone marrow (BM) (B) spleen (C) lymph node (LNs) and (D) brain of CD19Cre+minus α4-integrinflfl mice The percentages of CD19+

B220+ B cells expressing IgD IgM IgG and MHC II in (E) LNs and (F) spleen were comparable in both mouse strains (G) In the CNS the surface expression ofMHC II was similar in CD19Cre+minus α4-integrinflfl mice and controls (H) In the brain the expression of the costimulator molecules CD80 and CD86 and the firstapoptosis signal receptor CD95 was indistinguishable between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT controls The percentage ofCD11b+CD23B220lo B1 B cells CD11b+CD23B220lominusCD5+ B1a B cells CD11b+CD23B220lominusCD5minus B1b B cells B220+CD5+CD1dhi B10 B cells CD19+CD23+CD5minus

naive follicular B cells (FB) B220+CD138minusCD95+GL7+ germinal center (GC) B cells B220+CD1d+CD5minusCD23minusCD21+marginal zone (MGZ) B cells CD138+B220lowndash

plasma blast (PB) and B220minusCD138+ plasma cells (PC) was similar in both mouse strains at (I) day 13 during early active EAE (J) day 19 during maximum EAEdisease activity and (K) day 29 during chronic EAE Lymphocytes were immunophenotyped by multiparameter flow cytometry A gating strategy used toidentify some of the B-cell subsets is shown in Figure 3 p lt 001

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

We were unable to detect a change in composition ofCD19+CD11b+CD23minusB220minus B1 B cellsCD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cellsCD19+CD5+CD1dhi B10 B cellsCD19+B220+CD1d+CD5minusCD23minusCD21+ MGZ B cells andCD19lowndashCD138+B220lowndash PB CD19Cre+minus α4-integrinflfl

mice and C57BL6 WT controls at day 13 during early activeEAE in the BM (figure 4A) LNs (Figure 4B) and spleen(figure 4C)

Serum soluble inflammatory markers areunchanged in CD19Cre+2 α4-integrinflfl miceduring different stages of MOGp35-55-induced EAEDeletion of α4-integrin on CD19+ B cells diminishes theability of CD19+ B cells to enter the CNS during acuteMOGp35-55-induced EAE (figure 1E) It is conceivable thatinflammatory B cells accumulate in peripheral blood alteringthe inflammatory milieu in that compartment This wasobserved in patients withMS treated with natalizumab22 Wefound that the serum expression of the cytokines IL-17A(figure 5A) IL-10 (Figure 5B) IL-4 (Figure 5C) IL-5

(figure 5D) and IFNγ (figure 5E) as measured by ELISA(ELISA) during acute and chronic MOGp35-55-induced EAEon days 13 19 and 29 was comparable betweenCD19Cre+minus α4-integrinflflmice and C57BL6 WT controlmice

Serum Ig levels are unchanged in CD19Cre+2

α4-integrinflfl mice during different stages ofMOGp35-55-induced EAETo further investigate whether the sequestration of CD19+

B cells through deletion of α4-integrin affects their acti-vation proliferation and antibody secretion IgM and IgGlevels in serum were determined by ELISA Serum levels ofIgM (figure 5F) and IgG (figure 5G) as measured byELISA were also similar between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice on days13 19 and 29 of MOGp35-55-induced EAE Given that theabsolute levels of IgM and IgG were similar between themouse strains and that other investigators previouslyshowed that B cells from mice on the C57BL6 back-ground do not readily recognize MOGp35-55

21

we did not test the serum levels of MOGp35-55-specific IgMor IgG

Figure 3 Immunophenotyping of murine B cells

B-lymphocyte subsets were immunophenotyped by multiparameter flow cytometry A gating strategy that was used to identify some of the B-cell sub-populations is shown (A) First cellular area height and width measurements were obtained in a channel with linear scale to gate on singlets and excludedoublets (B) Next gates were set to include CD45+ leukocytes and exclude FSClow cell debris out (C) CD138 and B220 were used to gate on plasmablasts(CD138+B220lowndash) plasma cells (CD138+B220ndash) and B cells (B220+) Panels DndashF show the gating for (E) B220+CD1d+CD5minusCD23minusCD21+IgM+ marginal zone Bcells (E) B220+CD1d+CD5minusCD23minusCD21minusIgM+ transitional 1 (T1) B cells and (F) B220+CD1d+CD5minusCD23+CD21+IgM+ T2 B cells Panels GndashI show gating for B1 B-cell populations and the identification of (H) CD11b+CD23minusB220lominusCD5minus B1b B cells and (I) CD11b+CD23minusB220lominusCD5+ B1a B cells Panels J and K show theidentification of B220+CD138minusCD95+GL7+ germinal center B cells Panels LndashO illustrate the gating for B220+CD19+CD5minusGL7minusCD23+CD21loint naive follicular Bcells FSC = forward side scatter

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 7

Activated peripheral B cells contribute todisease severity in MOGp35-55-induced EAETo determine the compartment-specific pathogenic role ofB cells in the induction and perpetuation of the EAE animalmodel CD19Cre+minus α4-integrinflfl mice were immunizedwith MOGp35-55 As demonstrated B cells in these mice havea diminished capacity to enter the CNS (figure 1E) Adoptivetransfer of purified CD19+ B cells from CD19Cre+minus α4-integrinflfl mice and C57BL6 WT mice immunized withrMOG1-125 or the control antigen OVA323-339 worsened the

clinical course of EAE in recipient mice immunized withMOGp35-55 (figure 5H) although these changes were notsignificant

DiscussionThere is accumulating evidence to suggest that B cells andmyelin-specific antibodies play crucial roles in the pathogen-esis of MS Whether B-cell depletion mediates its beneficial

Figure 4 CD19+ B cells in primary and secondary lymphoid organs of CD19Cre+minus α4-integrinflfl mice are phenotypicallysimilar to wild-type B cells during MOGp35-55-induced experimental autoimmune encephalomyelitis

(A) In the bonemarrow (B) lymph nodes and (C) spleen the percentage of CD19+CD11b+CD23minusB220minus B1 B cells CD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cells CD19+CD5+CD1dhi B10 B cells CD19+B220+CD1d+CD5minusCD23minusCD21+ marginal zone (MGZ) B cells andCD19lowndashCD138+B220lowndash plasma blast (PB) was similar in CD19Cre+minus α4-integrinflfl mice and C57BL6 WT controls at day 13 during early activeEAE Lymphocytes were immunophenotyped by multiparameter flow cytometry

Figure 5ActivatedB cells outside of theCNSdrive disease activity inmyelin oligodendrocyte glycoprotein peptide (MOGp35-

55)-induced experimental autoimmune encephalomyelitis (EAE) in an antigen no-specific manner

The serum expression of the cytokines(A) IL-17A (B) IL-10 (C) IL-4 (D) IL-5 and(E) IFNγ as measured by ELISA duringacute and chronic MOGp35-55-inducedEAE on days 13 19 and 29 was com-parable between CD19Cre+minus α4-integ-rinflfl mice and C57BL6 wild-type (WT)control mice Serum levels of (F) IgMand (G) IgG measured by ELSA at thesame time points were also similar be-tween mouse strains (H) Adoptivetransfer of purified CD19+ B cells fromCD19Cre+minus α4-integrinflfl mice orC57BL6 WT control mice immunizedwith recombinant (r)MOG1-125 or oval-bumin (OVA)323-339 into CD19+minus α4-integrinflfl mice that were then imme-diately immunized with MOGp35-55

caused worse clinical EAE than wasobserved in MOGp35-55-immunizedC57BL6 WT control mice (ldquoC57BL6rdquo)that did not receive adoptively trans-ferred CD19+ B cells

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

effects in patients with MS through B cells in the periphery inthe CNS or both is currently not completely understood

Other investigators investigated the role of α4-integrin deficiencyin CD19+ B cells in EAE23 These investigators showed thatB-cell very late activation antigen-4ndashdeficient mice developedmore severe clinical disease than control mice after active in-duction of EAE with MOGp35-55 and that that this clinical effectis due to the requirement of regulatory B cells (Bregs) for α4-integrin to enter the CNS Bregs were defined as CD19+IL-10+

or CD1dhiCD5+ B-cell subsets Another group of investigatorsalso testedCD19Cre Itga4flflmice inMOGp35-55-induced activeEAE24 CD19Cre Itga4flfl mice developed more severe EAEthan controlmice and these investigators demonstrated a criticalrole for α4-integrin on the generation of Bregs in peripheralimmune organs

Our own results diverge from these reports in that we couldnot detect a difference in active EAE disease severity betweenCD19Cre+minus α4-integrinflfl mice and C57BL6 WT controlmice These experiments were conducted 15 times (table)and we are confident in our findings Differences in EAEdisease severity and phenotype can be affected by conditionsunder which mouse colonies are maintained as well as otherfactors that may affect the overall inflammatory milieu of ex-perimental conditions This may explain the differences inresults It is also conceivable that different mice were used bythe other investigators for their experiments The inves-tigators of the 2 previous studies state that they usedCD19Creα4flfl mice23 or that they used CD19Cre Itga4flfl

mice25 It is not stated whether mice were heterozygous orhomozygous for CD19cre or a mixture of both As stated inthe Methods section CD19Cre++ mice behave functionallyvery similarly to B-cellndashdeficient mice which develop moresevere EAE when immunized with MOGp35-55

26

We found one potentially meaningful biological difference be-tween CD19Cre+minus α4-integrinflfl mice and C57BL6 controlmice There was decreased MOGp35-55-specific CD4+ T-cellproliferation in spleens which is an expected result given thatα4-integrin can participate in costimulation of CD4+T cells1819

The observation that CD19+ B cells of CD19Cre+minus α4-integrinflfl mice did deplete α4-integrin and that they weresignificantly less capable of entering the CNS after mice wereimmunized with MOGp35-55 suggests 3 interpretations (1)Antigen-activated B cells contribute to disease burden in EAEin a seemingly dose-dependent manner (2) they do not ab-solutely require recognition of a CNS autoantigen or a mimicthereof to do so and (3) they do not require full α4-integrinndashmediated access to the CNS to exacerbate diseaseThe first interpretation is supported by work of other inves-tigators who have previously demonstrated that B-cellndashdeficient C57BL6 mice are resistant to active rMOG-inducedEAE26 and that the administration of a B-cellndashdepletingmonoclonal antibody prevents or reverses established rMOG-induced EAE in C57BL6 mice21 Weber and colleagues21

further showed that B-cell depletion results in a reduction ofMOGp35-55-specific Th1 and Th17 cells Our own data suggestthat the differentiation ofMOGp35-55-specific T cells to becomeencephalitogenic does not absolutely require the presentationof autoantigen but is likely driven by other B-cell factors in-cluding soluble inflammatory mediators The third in-terpretation of this experiment is supported by ourdemonstration that B cells from CD19Cre+minus α4-integrinflfl

mice are significantly impaired in their ability to enter the CNS(figure 1E) Finally it should be emphasized that our findingsdo not conclusively rule out a role of antigen-specific B cells inthe CNS on the course of CNS autoimmunity as thresholdeffects may be difficult to detect with the EAE experiments thatwere conducted

Our data suggest that the beneficial effects of anti-CD20therapies in patients with MS are mediated through theireffects on peripheral B cells We also conclude that benefitsobserved in patients with MS during anti-α4-integrin therapyare likely not predominantly due to its effect of suppressingthe migration of B cells into the CNS The interpretation ofour findings should be limited to early forms of MS as theEAE model that was used and the observation period that wasused do not provide insight into late or progressive forms ofCNS autoimmunity Magliozzi et al27 were the first inves-tigators to show that some patients with secondary-progressive MS develop lymphoid tissue in the meningesthat resemble B-cell follicles in secondary lymphoid organsB cells in these structures may be susceptible to anti-CD20therapies with monoclonal antibodies or small molecules andit is conceivable that their depletion may benefit patientsafflicted with that MS phenotype

AcknowledgmentThe authors thank Dr Thalia Papayannopoulou at theUniversity of Washington for providing them with α4-integrinfloxflox mice Dr Stuve was funded by a Merit Reviewgrant (federal award document number [FAIN]I01BX001674) from the United States (US) Departmentof Veterans Affairs Biomedical Laboratory Research andDevelopment

Study fundingNo targeted funding reported

DisclosureRZ Hussain PC Cravens WA Miller-Little R Doelger VGrandos and E Herndon report no disclosures DT Okudareceived advisory and consulting fees from Celgene Gen-entech Genzyme EMD Serono and Novartis and researchsupport from Biogen served on the scientific advisory boardof Osmotica and served on the speakersrsquo bureau of AcordaGenzyme and Teva TN Eagar serves as section editor ofArchives of Pathology and received research support from theNIH O Stuve served on the scientific advisory boards of TGTherapeutics and Genentech-Roche served on the editorialboard of Therapeutic Advances in Neurological Disorders

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 9

consulted for Celgene EMD Serono and Novartis andreceived research support from Sanofi and GenzymeDisclosures available NeurologyorgNN

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationNovember 2 2018 Accepted in final form February 1 2019

References1 Hauser SL Waubant E Arnold DL et al B-cell depletion with rituximab in relapsing-

remitting multiple sclerosis N Engl J Med 2008358676ndash6882 Hawker K OrsquoConnor P Freedman MS et al Rituximab in patients with primary

progressive multiple sclerosis results of a randomized double-blind placebo-controlled multicenter trial Ann Neurol 200966460ndash471

3 Hauser SL Bar-Or A Comi G et al Ocrelizumab versus interferon beta-1a in re-lapsing multiple sclerosis N Engl J Med 2017376221ndash234

4 Montalban X Hauser SL Kappos L et al Ocrelizumab versus placebo in primaryprogressive multiple sclerosis N Engl J Med 2017376209ndash220

5 Yednock TA Cannon C Fritz LC Sanchez-Madrid F Steinman L Karin N Pre-vention of experimental autoimmune encephalomyelitis by antibodies against alpha 4beta 1 integrin Nature 199235663ndash66

6 Frohman EM Racke MK Raine CS Multiple sclerosismdashthe plaque and its patho-genesis N Engl J Med 2006354942ndash955

7 Stuve O Marra CM Jerome KR et al Immune surveillance in multiple sclerosispatients treated with natalizumab Ann Neurol 200659743ndash747

8 Haines JL Ter Minassian M Bazyk A et al A complete genomic screen for multiplesclerosis underscores a role for the major histocompatability complex The MultipleSclerosis Genetics Group Nat Genet 199613469ndash471

9 Sawcer S Jones HB Feakes R et al A genome screen in multiple sclerosis revealssusceptibility loci on chromosome 6p21 and 17q22 Nat Genet 199613464ndash468

10 Yang JT Rayburn H Hynes RO Cell adhesion events mediated by alpha 4 integrinsare essential in placental and cardiac development Development 1995121549ndash560

11 Sternberg N Hamilton D Bacteriophage P1 site-specific recombination I Re-combination between loxP sites J Mol Biol 1981150467ndash486

12 Scott LM Priestley GV Papayannopoulou T Deletion of alpha4 integrins from adulthematopoietic cells reveals roles in homeostasis regeneration and homing Mol CellBiol 2003239349ndash9360

13 Hussain RZ Miller-Little WA Doelger R et al Defining standard enzymatic disso-ciation methods for individual brains and spinal cords in EAE Neurol NeuroimmunolNeuroinflamm 20185e437 doi 101212NXI0000000000000437

14 Cravens PD Hussain RZ Zacharias TE et al Lymph node-derived donor encepha-litogenic CD4+ T cells in C57BL6 mice adoptive transfer experimental autoimmuneencephalomyelitis highly express GM-CSF and T-bet J Neuroinflammation 2011873

15 Karandikar NJ Crawford MP Yan X et al Glatiramer acetate (Copaxone) therapyinduces CD8(+) T cell responses in patients with multiple sclerosis J Clin Invest2002109641ndash649

16 Gu H Marth JD Orban PC Mossmann H Rajewsky K Deletion of a DNA poly-merase beta gene segment in T cells using cell type-specific gene targeting Science1994265103ndash106

17 Rickert RC Roes J Rajewsky K B lymphocyte-specific Cre-mediated mutagenesis inmice Nucleic Acids Res 1997251317ndash1318

18 Davis LS Oppenheimer-Marks N Bednarczyk JL McIntyre BW Lipsky PE Fibro-nectin promotes proliferation of naive and memory T cells by signaling through boththe VLA-4 and VLA-5 integrin molecules J Immunol 1990145785ndash793

19 Shimizu Y van Seventer GA Horgan KJ Shaw S Costimulation of proliferativeresponses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 withfibronectin or VLA-6 with laminin J Immunol 199014559ndash67

20 Theien BE Vanderlugt CL Eagar TN et al Discordant effects of anti-VLA-4 treat-ment before and after onset of relapsing experimental autoimmune encephalomyelitisJ Clin Invest 2001107995ndash1006

21 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

22 Krumbholz M Meinl I Kumpfel T Hohlfeld R Meinl E Natalizumab dispropor-tionately increases circulating pre-B and B cells in multiple sclerosis Neurology 2008711350ndash1354

23 Lehmann-Horn K Sagan SA Winger RC et al CNS accumulation of regulatoryB cells is VLA-4-dependent Neurol Neuroimmunol Neuroinflamm 20163e212 doi101212NXI0000000000000212

24 Glatigny S Wagner CA Bettelli E Correction cutting edge integrin alpha4 is re-quired for regulatory B cell control of experimental autoimmune encephalomyelitisJ Immunol 20171983756

25 Glatigny S Duhen R Oukka M Bettelli E Cutting edge loss of alpha4 integrinexpression differentially affects the homing of Th1 and Th17 cells J Immunol 20111876176ndash6179

26 Lyons JA SanM HappMP Cross AH B cells are critical to induction of experimentalallergic encephalomyelitis by protein but not by a short encephalitogenic peptide EurJ Immunol 1999293432ndash3439

27 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

Appendix Authors

Name Location Role Contribution

Rehana ZHussainMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

Petra CCravensPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

William AMiller-Little BA

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

RichardDoelgerMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

ValerieGranadosPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the data andanalyzed the data

EmilyHerndonMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Darin TOkudaMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Todd NEagar PhD

Houston MethodistHospital HoustonTX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

OlafStuve MDPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

DOI 101212NXI000000000000056320196 Neurol Neuroimmunol Neuroinflamm

Rehana Z Hussain Petra D Cravens William A Miller-Little et al disease model

mediated EAEminus4-integrin deficiency in B cells does not affect disease in a T-cellα

This information is current as of April 16 2019

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

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Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

CORRECTION

α4-integrin deficiency in B cells does not affect disease in aT-cellndashmediated EAE disease modelNeurol Neuroimmunol Neuroinflamm 20196e585 doi101212NXI0000000000000585

In the article ldquoα4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAEdisease model by Hussain et al1 first published online April 16 2019 funding for the papershould have been listed in the Study Funding section The authors regret the error

Reference1 Hussain RZ Cravens PD Miller-Little WA et al α4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAE

disease model Neurol Neuroimmunol Neuroinflamm 20196e563

Copyright copy 2019 American Academy of Neurology 1

Copyright copy 2019 American Academy of Neurology Unauthorized reproduction of this article is prohibited

Page 3: α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency in B cells does not affect disease in a T-cell–mediated EAE disease model Rehana

Enzymatic CNS digestionsFor some experiments brains and spinal cords were dissoci-ated enzymatically as described13

Flow cytometryTo determine the absolute number of B cells and T cells indifferent compartments during EAE multiparameter flowcytometry was used Cells from bone marrows (BMs)spleens LNs brains and spinal cords were brought intosingle-cell suspension as described13 Cells were stained for 30minutes at 4degC with the following antibodies CD45 PE-Cyannin-7 (30-F11 eBioscience San Diego CA) CD3 AlexaFlour 700 (17A2 eBioscience) CD19 PE-Texas Red (6D5Invitrogen Waltham MA) CD19 PE (1D3 BD BioscienceSan Jose CA) CD49d FITC (PS2 Santa Cruz Bio-technology Dallas TX) CD4 APC (RM4-5 BD Bioscience)CD45R B220-APC (RA3-6B2 BioLegend San Diego CA)PE PE Dazzle 594 PE-Texas Red AF700 Pac OrangePerCP CD138 BV421(281-2 BioLegend) CD11b (APCAPC-Cy-7 PE PerCPCy55 V450 B700 (M170 BioL-egend) CD23 PE-Cy7 (B3B4 BioLegend) CD5 PE-Cy5(53-73 BioLegend) CD1d PE (1B1 BioLegend) GL-7FITC (GL7 BioLegend) and CD21CD35 BV510 (7G6BD Bioscience San Jose CA) Isotype-matched mAbs wereused to set the gates Next 30000ndash300000 gated events wereacquired on an FACSAria II flow cytometer (BD Bio-sciences) equipped with Diva acquisition software (BDBiosciences) FlowJo (BD Biosciences) software was alsoused for some data analysis

Intracellular cytokine stainingPhorbol 12-myristate 13-acetate (500 ng) ionomycin (500 ng)and GolgiPlug (1 μL) (BD Biosciences) were added to 1 times 106

cells and incubated for 3 hours at 37degC Cells were then washedin phosphate-buffered saline and stained following the extra-cellular staining protocol for CD45-Alexa Fluor 700 After ex-tracellular staining CD19+ cells were purified as described andfixed using Fixation Buffer (BioLegend) for 15 minutes in thedark at room temperature Cells were then washed with Per-meabilization Buffer (BioLegend) twice and then stained usinginterferon gamma (IFNγ)-PE-Cy7 (BD Biosciences) in-terleukin (IL)-17A-FITC (BioLegend) tumor necrosis factorα-PE (eBioscience) and IL-6-APC (BioLegend) Cells were thenwashed and acquired and analyzed by flow cytometry

ELISASerum samples were collected by submandibular bleeding atdays 13 19 and 29 during early active EAE maximum EAEdisease activity and during chronic EAE Quantitative ELISAfor IL-17 IL-10 IL-4 IL-5 and IFNγ was performed usingpaired mAb specific for corresponding cytokines as per themanufacturerrsquos recommendations (BD Biosciences or RampDSystems) IgM and IgG serum levels were also determined byELISA The results of ELISA assays are expressed as an averageof triplicate wells plusmn SD The EPOCH (BioTek Winooski VT)ELISA plate reader and software were used for data analysis(Molecular Devices Corporation Sunnyvale CA)

T-cell proliferation assayTo determine the capability of B cells to serve as APC to CD4+

T cells flow cytometric proliferation assays using the greenfluorescent dye carboxyfluorescein succinimidyl ester or V450(BD Biosciences) were performed according to publishedmethods15 On day 15 after active induction of EAE spleens andLNs were harvested and processed into single-cell suspensionSplenocytes and lymphocytes were stained with carboxy-fluorescein succinimidyl ester and plated at 1 times 106 cells per wellThe cells were then restimulated with 10 μgmL of MOGp35-55

and incubated at 37degC for 5 days Con A at a concentration of 2mgmL and media were used as controls On day 6 the cellswere collected blockedwith Fc block antibody stainedwithAPCCD4 antibody and acquired and analyzed by flow cytometry

HistologyFollowing fixation in 10 buffered formalin LNs were pro-cessed and embedded in paraffin blocks Four-micrometersections were cut mounted on Fisherbrand Superfrost Plusglass slides (Fisher Scientific Pittsburgh PA) and stainedwith hematoxylin amp eosin (Fisher Scientific) or anti-CD19

Statistical analysisFor parametric tests data were assessed for normality using theKolmogorov-Smirnov test Normally distributed values werecompared using the unpaired 2-sided Student t test Correla-tions between continuous and categorical variables wereassessed using the Mann-Whitney U-test All statistical testswere 2 sided and p lt 005 indicated significance All analyseswere performed with Prism 7 (Graphpad La Jolla CA)

Standard protocol approvals and registrationsAll experimental animals were maintained in a specificpathogen-free facility at the University of Texas (UT) South-western Medical Center All protocols involving mice handlingwere approved by the UT Southwestern animal care facility

Data availabilityData not shown will be shared by request

ResultsThe percentage of CD49d+ CD19+ B cells isdiminished in primary and secondarylymphoid organs in naive CD19Cre+2 α4-integrinflfl miceTo characterize CD19Cre+minus α4-integrinflfl mice the ex-pression of α4-integrin (CD49d) was determined by multi-parameter flow cytometry The percentage of CD49d+

CD19+ B cells in the BM LNs spleens and Peyer patches issignificantly diminished in naive CD19Cre+minus α4-integrinflfl

mice compared with C57BL6 wild-type (WT) controlmice (figure 1A) Cre recombination efficiency is in-complete16 and determined mostly by the nucleotidesequence in the spacer region of the lox site Also there isa negative correlation between the Crelox recombinationefficiency and the length of DNA between the 2 lox sites As

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 3

stated above other investigators found a deletion efficiencybetween 75 and 80 in BM-derived prendashB cells and ap-proximately 90 in splenic B cells17

CD19Cre+2 α4-integrinflfl mice immunizedwith MOGp35-55 display a regular clinicalEAE courseTo determine the effect of α4-integrin expression of B cells ina T-cellndashmediated active model of EAE CD19Cre+minus α4-integrinflfl mice and C57BL6 control mice were immunizedwithMOGp35-55 No significant differences regarding the EAEdisease incidence severity or phenotype were observed be-tween the mouse strains (figure 1B and table) α4-integrinflfl

mice were also used as controls and showed similar diseaseactivity (data not shown)

α4-integrin expressionon splenic B cells affectsantigen-specific CD4+ T-cell proliferationIt was previously demonstrated that α4-integrin can participatein costimulation of CD4+ T cells1819 Yet other investigatorsshowed that anti-CD49d plus anti-CD3anti-CD28ndashcoatedpolystyrene beads induced significantly greater T-cell pro-liferation than anti-CD3anti-CD28 polystyrene beads alone20

To test the effect of α4-integrin ablation on the capability ofCD19+ B cells to present antigen to antigen-reactive CD4+

T cells flow cytometric proliferation assays were performedCD4+ T-cell recall responses to MOGp35-55 in spleens weresignificantly diminished in CD19Cre+minus α4-integrinflfl mice(figure 1C) However in LNs we did not observe any differ-ence in CD4+ T-cell proliferation between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice (figure 1D)

Figure 1 CD19Cre+minus α4-integrinflfl mice are fully susceptible to actively MOGp35-55-induced experimental autoimmuneencephalomyelitis

(A) The percentage of CD49d+ CD19+ B cells in thebone marrow lymph nodes spleens and Peyerpatches is significantly diminished in naiveCD19Cre+minus α4-integrinflfl mice compared withC57BL6 control mice Cells were immunophe-notyped by multiparameter flow cytometry (B)CD19Cre+minus α4-integrinflfl mice behave similar toC57BL6 wild-type (WT) mice regarding the dis-ease incidence onset and severity of active ex-perimental autoimmune encephalomyelitis (EAE)α4-integrinflfl mice were used as controls andshowed similar disease activity (data not shown)Active EAE was induced in CD19Cre+minus α4-integ-rinflfl and C57BL6 age-matched control mice bysubcutaneous immunizations with MOGp35-55 inincomplete Freund adjuvant (IFA) containing 4mgmL mycobacteria Mice received in-traperitoneal injection of 200 μL of pertussis toxin(Ptx) at 200 ngmL on days 0 and 2 Mice wereobserved daily and EAE severity was scored usinga 5-point scale CD4+ T-cell recall responses toMOGp35-55 in (C) spleens were significantly di-minished in CD19Cre+minus α4-integrinflfl mice but(D) indistinguishable to those in C57BL6 WTmicein lymphnodes Cell proliferationwas determinedby a flow cytometric proliferation assay using thegreen fluorescent dye CFSE or V450 (E) At maxi-mum disease activity (days 13ndash15) the percent-age of CD3+ T cells in the CNS was similar inCD19Cre+minus α4-integrinflfl and C57BL6 WT miceIn contrast the percentage of CD19+ B cells in theCNS was significantly diminished in CD19Cre+minus

α4-integrinflfl mice (F) The percentage of α4-integrinndashpositive (CD49d+) CD19+ B cells in theCNS during maximum EAE disease activity wascomparable between both mouse strains Lym-phocytes were immunophenotyped by multipa-rameter flow cytometry The numberappearance and architecture of germinal centersin lymph nodes of (G and H) C57BL6 WT controlmice were comparable to those in (I and J)CD19Cre+minus α4-integrinflfl mice Panels G and Iwere stained with hematoxylin amp eosin andpanels H and J were stained with anti-CD19Magnification for GndashJ is times4 p lt 005 p lt 001p lt 0001 p lt 00001 CFSE = carboxy-fluorescein succinimidyl ester

4 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

In both mouse strains T-cell proliferation was substantial Ourobservations appear to differ from those of other investigatorswho showed that B cells do not presentMOGp35-55 to T cells21

The Methods section of that article states that ldquohellipB cells weremagnetically activated cell sorting (MACS Miltenyi BiotecBergisch Gladbach Germany)-separated from lymph nodes orspleensrdquo Thus a differential capability of B cells isolated fromeither LNs or spleens on MOG peptide presentation may nothave been fully tested

The number of B cells in the CNS during activeMOGp35-55-induced EAE is diminished inCD19Cre+2 α4-integrinflfl miceTo examine the effect of α4-integrin deletion in CD19+ B cellson the ability of B lymphocytes to enter the CNS cells wereisolated from brains and spinal cords by Percoll gradient andanalyzed by multiparameter flow cytometry As MOGp35-55-induced EAE is a T-cellndashmediated form of EAE the number ofCD3+ T cells was also assessed by the same method At maxi-mum disease activity (days 13ndash15) the percentage of CD3+

T cells in the CNS was similar in CD19Cre+minus α4-integrinflfl

mice and C57Bl6 WT mice (figure 1E) In contrast the per-centage of CD19+ B cells in the CNS was significantly reducedin CD19Cre+minus α4-integrinflfl mice (figure 1E) These obser-vations suggest that antigen-specific B cells within the CNS playa minor role in initiating and perpetuating EAE given that thesemice showed the same disease susceptibility and disease courseas C57Bl6 WT mice Immunohistochemical studies to de-termine a differential anatomic distribution of B cells in theCNSbetween CD19Cre+minus α4-integrinflfl mice and C57Bl6 WTmice were not performed as the absolute number of B cells inCD19Cre+minus α4-integrinflfl mice was very low

CD19+ B cells from CD19Cre+2 α4-integrinflfl

mice use CD49d to gain access to the CNSduring active MOGp35-55-induced EAETo determine whether CD19+ B cells from CD19Cre+minus α4-integrinflfl mice that gain access to the CNS during active EAEuse α4-integrin the percentage of α4-integrinndashpositive (CD49d+)CD19+ B cells was determined by multiparameter flowcytometry and found to be comparable between CD19Cre+minus

α4-integrinflfl mice and C57Bl6 WT controls (figure 1F)

The number and architecture of GCs in LNs ofCD19Cre+2 α4-integrinflfl mice are normalCD19Cremice express cre under the transcriptional control ofthe B lineagendashrestricted CD19 gene17 Insertion of cre disruptsthe CD19 coding sequence leading to a CD19 deficiency anda concomitant reduction in GCs in CD19Cre++ mice At day15 during the acute phase of MOGp35-55-induced EAE thenumber and architecture of GCs in LNs of CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice were com-parable by hematoxylin amp eosin (HampE) staining (figure 1 Gand H) and anti-CD19 staining (figure 1 I and J)

B cells of CD19Cre+2 α4-integrinflfl mice havenormal cytokine profiles during activeMOGp35-

55-induced EAETo test whether the absence of α4-integrin in CD19+ B cellsaffects their differentiation during inflammatory conditionsexperimental animals were killed at day 13 during the acutephase of MOGp35-55-induced EAE and B cells in disease-relevant compartments were immunophenotyped by multi-parameter flow cytometry and intracellular cytokine stainingCompared with B cells from C57BL6 WT control mice thepercentage of CD19+ B cells expressing IFNγ IL-6 IL-10 orIL-17 in CD19Cre+minus α4-integrinflfl mice was similar in theBM (figure 2A) spleen (figure 2B) LNs (figure 2C) andbrain (figure 2D) of CD19Cre+minus α4-integrinflfl mice

Outside the CNS B cells of CD19Cre+2 α4-integrinflfl mice express normal maturationand activation markers during active MOGp35-

55-induced EAEAs stated above α4-integrin is considered a costimulatorymolecule through which CD19+ B cells are capable ofinteracting with CD4+ T cells To further elucidate theeffect of α4-integrin deficiency on CD19+ B cells on theirmaturation and activation in secondary lymphoid organsexperimental animals were killed at day 13 during theacute phase of MOGp35-55-induced EAE and B cells inspleens and LNs were immunophenotyped by multipa-rameter flow cytometry The percentages of CD19+ B220+

B cells expressing IgD IgM IgG and major histocom-patibility complex (MHC) II in LNs (figure 2E) andspleen (figure 2F) were comparable in CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice

CNS B cells from CD19Cre+2 α4-integrinflfl

mice do not upregulate activation markersduring active MOGp35-55-induced EAEThe absolute number of CD19+ B cells is diminished in theCNS of CD19Cre+minus α4-integrinflfl mice compared withC57BL6 WT control mice during acute MOGp35-55-in-duced EAE (figure 1E) To evaluate the activation state ofthese cells CNS B cells were isolated and assessed by mul-tiparameter flow cytometry during active EAE at day 13 afteractive induction or EAE with MOGp35-55 The surface ex-pression of MHC II was similar in CNS-derived B cells fromCD19Cre+minus α4-integrinflfl and C57BL6 WT controls

Table Disease incidence mean maximum disease scoreand mean day of active MOGp35-55-induced EAEdisease onset in CD19+minus α4-integrin mice andC57BL6 control mice

Group IncidenceMean maximumdisease score

Mean day ofdisease onset

C57BL6 7280 289 1057 plusmn 518

CD19+2 α4-integrinflfl

6281 245 926 plusmn 607

Abbreviations EAE = experimental autoimmune encephalomyelitis MOG =myelin oligodendrocyte glycoproteinResults of 15 experiments are shown

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 5

(figure 2G) Also the expression of the costimulator mole-cules CD80 and CD86 and the first apoptosis signal receptorCD95 was comparable in brain-derived B cells fromCD19Cre+minus α4-integrinflfl mice and C57BL6 WT con-trols (figure 2H)

The composition of B-cell subsets in the brainof CD19Cre+2 α4-integrinflfl mice isunchanged during MOGp35-55-induced EAETo determine whether the deletion of α4-integrin on CD19+

B cells affects B-cell subsets differentially in their capability toenter the CNS the percentage of innate and adaptiveB-lymphocyte subsets including CD11b+CD23minusB220minus B1B cells CD11b+CD23minusB220lominusCD5minus B1b B cellsCD11b+CD23minusB220lominusCD5+ B1a B cellsCD19+CD5+CD1dhi B10 B cells CD19+CD23+CD5minus

naive follicular B cells B220+CD138minusCD95+GL7+ GCsB cells B220+CD1d+CD5minusCD23minusCD21+ marginal zone

(MGZ) B cells CD19lowndashCD138+B220lowndash plasma blast(PB) and CD19minusCD138+ PCs was assessed by multipa-rameter flow cytometry during acute MOGp35-55-inducedEAE at day 13 during early active EAE (figure 2I) day 19during maximum EAE disease activity (figure 2J) and day 29during chronic EAE (figure 2K) after active induction orEAE with MOGp35-55 and was found to be similar inCD19Cre+minus α4-integrinflfl mice and C57BL6 WT con-trols at all time points A gating strategy used to identifysome of the B-cell subsets is shown in figure 3

The composition of B-cell subsets in primaryand secondary lymphoid organs of CD19Cre+2

α4-integrinflfl mice is similar to those of WTcontrol mice during MOGp35-55-induced EAETo assess the effect of α4-integrin ablation on CD19+ B cellson B-cell phenotypes in lymphoid organs innate and adaptiveB-lymphocyte subsets were characterized by flow cytometry

Figure 2 CD19+ B cells in the CNS of CD19Cre+minus α4-integrinflfl mice are phenotypically similar to wild-type (WT) B cellsduring myelin oligodendrocyte glycoprotein peptide (MOGp35-55)-induced experimental autoimmuneencephalomyelitis

Experimental animals were killed at day 13 during the acute phase of MOGp35-55-induced experimental autoimmune encephalomyelitis (EAE) and B cells indisease-relevant compartmentswere immunophenotyped bymultiparameter flow cytometry and intracellular cytokine staining Comparedwith B cells fromC57BL6 WT control mice the percentage of CD19+ B cells expressing interferon gamma (IFNγ) interleukin (IL)-6 IL-10 or IL-17 in CD19Cre+minus α4-integrinflfl

mice was similar in the (A) bone marrow (BM) (B) spleen (C) lymph node (LNs) and (D) brain of CD19Cre+minus α4-integrinflfl mice The percentages of CD19+

B220+ B cells expressing IgD IgM IgG and MHC II in (E) LNs and (F) spleen were comparable in both mouse strains (G) In the CNS the surface expression ofMHC II was similar in CD19Cre+minus α4-integrinflfl mice and controls (H) In the brain the expression of the costimulator molecules CD80 and CD86 and the firstapoptosis signal receptor CD95 was indistinguishable between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT controls The percentage ofCD11b+CD23B220lo B1 B cells CD11b+CD23B220lominusCD5+ B1a B cells CD11b+CD23B220lominusCD5minus B1b B cells B220+CD5+CD1dhi B10 B cells CD19+CD23+CD5minus

naive follicular B cells (FB) B220+CD138minusCD95+GL7+ germinal center (GC) B cells B220+CD1d+CD5minusCD23minusCD21+marginal zone (MGZ) B cells CD138+B220lowndash

plasma blast (PB) and B220minusCD138+ plasma cells (PC) was similar in both mouse strains at (I) day 13 during early active EAE (J) day 19 during maximum EAEdisease activity and (K) day 29 during chronic EAE Lymphocytes were immunophenotyped by multiparameter flow cytometry A gating strategy used toidentify some of the B-cell subsets is shown in Figure 3 p lt 001

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

We were unable to detect a change in composition ofCD19+CD11b+CD23minusB220minus B1 B cellsCD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cellsCD19+CD5+CD1dhi B10 B cellsCD19+B220+CD1d+CD5minusCD23minusCD21+ MGZ B cells andCD19lowndashCD138+B220lowndash PB CD19Cre+minus α4-integrinflfl

mice and C57BL6 WT controls at day 13 during early activeEAE in the BM (figure 4A) LNs (Figure 4B) and spleen(figure 4C)

Serum soluble inflammatory markers areunchanged in CD19Cre+2 α4-integrinflfl miceduring different stages of MOGp35-55-induced EAEDeletion of α4-integrin on CD19+ B cells diminishes theability of CD19+ B cells to enter the CNS during acuteMOGp35-55-induced EAE (figure 1E) It is conceivable thatinflammatory B cells accumulate in peripheral blood alteringthe inflammatory milieu in that compartment This wasobserved in patients withMS treated with natalizumab22 Wefound that the serum expression of the cytokines IL-17A(figure 5A) IL-10 (Figure 5B) IL-4 (Figure 5C) IL-5

(figure 5D) and IFNγ (figure 5E) as measured by ELISA(ELISA) during acute and chronic MOGp35-55-induced EAEon days 13 19 and 29 was comparable betweenCD19Cre+minus α4-integrinflflmice and C57BL6 WT controlmice

Serum Ig levels are unchanged in CD19Cre+2

α4-integrinflfl mice during different stages ofMOGp35-55-induced EAETo further investigate whether the sequestration of CD19+

B cells through deletion of α4-integrin affects their acti-vation proliferation and antibody secretion IgM and IgGlevels in serum were determined by ELISA Serum levels ofIgM (figure 5F) and IgG (figure 5G) as measured byELISA were also similar between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice on days13 19 and 29 of MOGp35-55-induced EAE Given that theabsolute levels of IgM and IgG were similar between themouse strains and that other investigators previouslyshowed that B cells from mice on the C57BL6 back-ground do not readily recognize MOGp35-55

21

we did not test the serum levels of MOGp35-55-specific IgMor IgG

Figure 3 Immunophenotyping of murine B cells

B-lymphocyte subsets were immunophenotyped by multiparameter flow cytometry A gating strategy that was used to identify some of the B-cell sub-populations is shown (A) First cellular area height and width measurements were obtained in a channel with linear scale to gate on singlets and excludedoublets (B) Next gates were set to include CD45+ leukocytes and exclude FSClow cell debris out (C) CD138 and B220 were used to gate on plasmablasts(CD138+B220lowndash) plasma cells (CD138+B220ndash) and B cells (B220+) Panels DndashF show the gating for (E) B220+CD1d+CD5minusCD23minusCD21+IgM+ marginal zone Bcells (E) B220+CD1d+CD5minusCD23minusCD21minusIgM+ transitional 1 (T1) B cells and (F) B220+CD1d+CD5minusCD23+CD21+IgM+ T2 B cells Panels GndashI show gating for B1 B-cell populations and the identification of (H) CD11b+CD23minusB220lominusCD5minus B1b B cells and (I) CD11b+CD23minusB220lominusCD5+ B1a B cells Panels J and K show theidentification of B220+CD138minusCD95+GL7+ germinal center B cells Panels LndashO illustrate the gating for B220+CD19+CD5minusGL7minusCD23+CD21loint naive follicular Bcells FSC = forward side scatter

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 7

Activated peripheral B cells contribute todisease severity in MOGp35-55-induced EAETo determine the compartment-specific pathogenic role ofB cells in the induction and perpetuation of the EAE animalmodel CD19Cre+minus α4-integrinflfl mice were immunizedwith MOGp35-55 As demonstrated B cells in these mice havea diminished capacity to enter the CNS (figure 1E) Adoptivetransfer of purified CD19+ B cells from CD19Cre+minus α4-integrinflfl mice and C57BL6 WT mice immunized withrMOG1-125 or the control antigen OVA323-339 worsened the

clinical course of EAE in recipient mice immunized withMOGp35-55 (figure 5H) although these changes were notsignificant

DiscussionThere is accumulating evidence to suggest that B cells andmyelin-specific antibodies play crucial roles in the pathogen-esis of MS Whether B-cell depletion mediates its beneficial

Figure 4 CD19+ B cells in primary and secondary lymphoid organs of CD19Cre+minus α4-integrinflfl mice are phenotypicallysimilar to wild-type B cells during MOGp35-55-induced experimental autoimmune encephalomyelitis

(A) In the bonemarrow (B) lymph nodes and (C) spleen the percentage of CD19+CD11b+CD23minusB220minus B1 B cells CD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cells CD19+CD5+CD1dhi B10 B cells CD19+B220+CD1d+CD5minusCD23minusCD21+ marginal zone (MGZ) B cells andCD19lowndashCD138+B220lowndash plasma blast (PB) was similar in CD19Cre+minus α4-integrinflfl mice and C57BL6 WT controls at day 13 during early activeEAE Lymphocytes were immunophenotyped by multiparameter flow cytometry

Figure 5ActivatedB cells outside of theCNSdrive disease activity inmyelin oligodendrocyte glycoprotein peptide (MOGp35-

55)-induced experimental autoimmune encephalomyelitis (EAE) in an antigen no-specific manner

The serum expression of the cytokines(A) IL-17A (B) IL-10 (C) IL-4 (D) IL-5 and(E) IFNγ as measured by ELISA duringacute and chronic MOGp35-55-inducedEAE on days 13 19 and 29 was com-parable between CD19Cre+minus α4-integ-rinflfl mice and C57BL6 wild-type (WT)control mice Serum levels of (F) IgMand (G) IgG measured by ELSA at thesame time points were also similar be-tween mouse strains (H) Adoptivetransfer of purified CD19+ B cells fromCD19Cre+minus α4-integrinflfl mice orC57BL6 WT control mice immunizedwith recombinant (r)MOG1-125 or oval-bumin (OVA)323-339 into CD19+minus α4-integrinflfl mice that were then imme-diately immunized with MOGp35-55

caused worse clinical EAE than wasobserved in MOGp35-55-immunizedC57BL6 WT control mice (ldquoC57BL6rdquo)that did not receive adoptively trans-ferred CD19+ B cells

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

effects in patients with MS through B cells in the periphery inthe CNS or both is currently not completely understood

Other investigators investigated the role of α4-integrin deficiencyin CD19+ B cells in EAE23 These investigators showed thatB-cell very late activation antigen-4ndashdeficient mice developedmore severe clinical disease than control mice after active in-duction of EAE with MOGp35-55 and that that this clinical effectis due to the requirement of regulatory B cells (Bregs) for α4-integrin to enter the CNS Bregs were defined as CD19+IL-10+

or CD1dhiCD5+ B-cell subsets Another group of investigatorsalso testedCD19Cre Itga4flflmice inMOGp35-55-induced activeEAE24 CD19Cre Itga4flfl mice developed more severe EAEthan controlmice and these investigators demonstrated a criticalrole for α4-integrin on the generation of Bregs in peripheralimmune organs

Our own results diverge from these reports in that we couldnot detect a difference in active EAE disease severity betweenCD19Cre+minus α4-integrinflfl mice and C57BL6 WT controlmice These experiments were conducted 15 times (table)and we are confident in our findings Differences in EAEdisease severity and phenotype can be affected by conditionsunder which mouse colonies are maintained as well as otherfactors that may affect the overall inflammatory milieu of ex-perimental conditions This may explain the differences inresults It is also conceivable that different mice were used bythe other investigators for their experiments The inves-tigators of the 2 previous studies state that they usedCD19Creα4flfl mice23 or that they used CD19Cre Itga4flfl

mice25 It is not stated whether mice were heterozygous orhomozygous for CD19cre or a mixture of both As stated inthe Methods section CD19Cre++ mice behave functionallyvery similarly to B-cellndashdeficient mice which develop moresevere EAE when immunized with MOGp35-55

26

We found one potentially meaningful biological difference be-tween CD19Cre+minus α4-integrinflfl mice and C57BL6 controlmice There was decreased MOGp35-55-specific CD4+ T-cellproliferation in spleens which is an expected result given thatα4-integrin can participate in costimulation of CD4+T cells1819

The observation that CD19+ B cells of CD19Cre+minus α4-integrinflfl mice did deplete α4-integrin and that they weresignificantly less capable of entering the CNS after mice wereimmunized with MOGp35-55 suggests 3 interpretations (1)Antigen-activated B cells contribute to disease burden in EAEin a seemingly dose-dependent manner (2) they do not ab-solutely require recognition of a CNS autoantigen or a mimicthereof to do so and (3) they do not require full α4-integrinndashmediated access to the CNS to exacerbate diseaseThe first interpretation is supported by work of other inves-tigators who have previously demonstrated that B-cellndashdeficient C57BL6 mice are resistant to active rMOG-inducedEAE26 and that the administration of a B-cellndashdepletingmonoclonal antibody prevents or reverses established rMOG-induced EAE in C57BL6 mice21 Weber and colleagues21

further showed that B-cell depletion results in a reduction ofMOGp35-55-specific Th1 and Th17 cells Our own data suggestthat the differentiation ofMOGp35-55-specific T cells to becomeencephalitogenic does not absolutely require the presentationof autoantigen but is likely driven by other B-cell factors in-cluding soluble inflammatory mediators The third in-terpretation of this experiment is supported by ourdemonstration that B cells from CD19Cre+minus α4-integrinflfl

mice are significantly impaired in their ability to enter the CNS(figure 1E) Finally it should be emphasized that our findingsdo not conclusively rule out a role of antigen-specific B cells inthe CNS on the course of CNS autoimmunity as thresholdeffects may be difficult to detect with the EAE experiments thatwere conducted

Our data suggest that the beneficial effects of anti-CD20therapies in patients with MS are mediated through theireffects on peripheral B cells We also conclude that benefitsobserved in patients with MS during anti-α4-integrin therapyare likely not predominantly due to its effect of suppressingthe migration of B cells into the CNS The interpretation ofour findings should be limited to early forms of MS as theEAE model that was used and the observation period that wasused do not provide insight into late or progressive forms ofCNS autoimmunity Magliozzi et al27 were the first inves-tigators to show that some patients with secondary-progressive MS develop lymphoid tissue in the meningesthat resemble B-cell follicles in secondary lymphoid organsB cells in these structures may be susceptible to anti-CD20therapies with monoclonal antibodies or small molecules andit is conceivable that their depletion may benefit patientsafflicted with that MS phenotype

AcknowledgmentThe authors thank Dr Thalia Papayannopoulou at theUniversity of Washington for providing them with α4-integrinfloxflox mice Dr Stuve was funded by a Merit Reviewgrant (federal award document number [FAIN]I01BX001674) from the United States (US) Departmentof Veterans Affairs Biomedical Laboratory Research andDevelopment

Study fundingNo targeted funding reported

DisclosureRZ Hussain PC Cravens WA Miller-Little R Doelger VGrandos and E Herndon report no disclosures DT Okudareceived advisory and consulting fees from Celgene Gen-entech Genzyme EMD Serono and Novartis and researchsupport from Biogen served on the scientific advisory boardof Osmotica and served on the speakersrsquo bureau of AcordaGenzyme and Teva TN Eagar serves as section editor ofArchives of Pathology and received research support from theNIH O Stuve served on the scientific advisory boards of TGTherapeutics and Genentech-Roche served on the editorialboard of Therapeutic Advances in Neurological Disorders

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 9

consulted for Celgene EMD Serono and Novartis andreceived research support from Sanofi and GenzymeDisclosures available NeurologyorgNN

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationNovember 2 2018 Accepted in final form February 1 2019

References1 Hauser SL Waubant E Arnold DL et al B-cell depletion with rituximab in relapsing-

remitting multiple sclerosis N Engl J Med 2008358676ndash6882 Hawker K OrsquoConnor P Freedman MS et al Rituximab in patients with primary

progressive multiple sclerosis results of a randomized double-blind placebo-controlled multicenter trial Ann Neurol 200966460ndash471

3 Hauser SL Bar-Or A Comi G et al Ocrelizumab versus interferon beta-1a in re-lapsing multiple sclerosis N Engl J Med 2017376221ndash234

4 Montalban X Hauser SL Kappos L et al Ocrelizumab versus placebo in primaryprogressive multiple sclerosis N Engl J Med 2017376209ndash220

5 Yednock TA Cannon C Fritz LC Sanchez-Madrid F Steinman L Karin N Pre-vention of experimental autoimmune encephalomyelitis by antibodies against alpha 4beta 1 integrin Nature 199235663ndash66

6 Frohman EM Racke MK Raine CS Multiple sclerosismdashthe plaque and its patho-genesis N Engl J Med 2006354942ndash955

7 Stuve O Marra CM Jerome KR et al Immune surveillance in multiple sclerosispatients treated with natalizumab Ann Neurol 200659743ndash747

8 Haines JL Ter Minassian M Bazyk A et al A complete genomic screen for multiplesclerosis underscores a role for the major histocompatability complex The MultipleSclerosis Genetics Group Nat Genet 199613469ndash471

9 Sawcer S Jones HB Feakes R et al A genome screen in multiple sclerosis revealssusceptibility loci on chromosome 6p21 and 17q22 Nat Genet 199613464ndash468

10 Yang JT Rayburn H Hynes RO Cell adhesion events mediated by alpha 4 integrinsare essential in placental and cardiac development Development 1995121549ndash560

11 Sternberg N Hamilton D Bacteriophage P1 site-specific recombination I Re-combination between loxP sites J Mol Biol 1981150467ndash486

12 Scott LM Priestley GV Papayannopoulou T Deletion of alpha4 integrins from adulthematopoietic cells reveals roles in homeostasis regeneration and homing Mol CellBiol 2003239349ndash9360

13 Hussain RZ Miller-Little WA Doelger R et al Defining standard enzymatic disso-ciation methods for individual brains and spinal cords in EAE Neurol NeuroimmunolNeuroinflamm 20185e437 doi 101212NXI0000000000000437

14 Cravens PD Hussain RZ Zacharias TE et al Lymph node-derived donor encepha-litogenic CD4+ T cells in C57BL6 mice adoptive transfer experimental autoimmuneencephalomyelitis highly express GM-CSF and T-bet J Neuroinflammation 2011873

15 Karandikar NJ Crawford MP Yan X et al Glatiramer acetate (Copaxone) therapyinduces CD8(+) T cell responses in patients with multiple sclerosis J Clin Invest2002109641ndash649

16 Gu H Marth JD Orban PC Mossmann H Rajewsky K Deletion of a DNA poly-merase beta gene segment in T cells using cell type-specific gene targeting Science1994265103ndash106

17 Rickert RC Roes J Rajewsky K B lymphocyte-specific Cre-mediated mutagenesis inmice Nucleic Acids Res 1997251317ndash1318

18 Davis LS Oppenheimer-Marks N Bednarczyk JL McIntyre BW Lipsky PE Fibro-nectin promotes proliferation of naive and memory T cells by signaling through boththe VLA-4 and VLA-5 integrin molecules J Immunol 1990145785ndash793

19 Shimizu Y van Seventer GA Horgan KJ Shaw S Costimulation of proliferativeresponses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 withfibronectin or VLA-6 with laminin J Immunol 199014559ndash67

20 Theien BE Vanderlugt CL Eagar TN et al Discordant effects of anti-VLA-4 treat-ment before and after onset of relapsing experimental autoimmune encephalomyelitisJ Clin Invest 2001107995ndash1006

21 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

22 Krumbholz M Meinl I Kumpfel T Hohlfeld R Meinl E Natalizumab dispropor-tionately increases circulating pre-B and B cells in multiple sclerosis Neurology 2008711350ndash1354

23 Lehmann-Horn K Sagan SA Winger RC et al CNS accumulation of regulatoryB cells is VLA-4-dependent Neurol Neuroimmunol Neuroinflamm 20163e212 doi101212NXI0000000000000212

24 Glatigny S Wagner CA Bettelli E Correction cutting edge integrin alpha4 is re-quired for regulatory B cell control of experimental autoimmune encephalomyelitisJ Immunol 20171983756

25 Glatigny S Duhen R Oukka M Bettelli E Cutting edge loss of alpha4 integrinexpression differentially affects the homing of Th1 and Th17 cells J Immunol 20111876176ndash6179

26 Lyons JA SanM HappMP Cross AH B cells are critical to induction of experimentalallergic encephalomyelitis by protein but not by a short encephalitogenic peptide EurJ Immunol 1999293432ndash3439

27 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

Appendix Authors

Name Location Role Contribution

Rehana ZHussainMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

Petra CCravensPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

William AMiller-Little BA

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

RichardDoelgerMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

ValerieGranadosPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the data andanalyzed the data

EmilyHerndonMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Darin TOkudaMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Todd NEagar PhD

Houston MethodistHospital HoustonTX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

OlafStuve MDPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

DOI 101212NXI000000000000056320196 Neurol Neuroimmunol Neuroinflamm

Rehana Z Hussain Petra D Cravens William A Miller-Little et al disease model

mediated EAEminus4-integrin deficiency in B cells does not affect disease in a T-cellα

This information is current as of April 16 2019

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

ServicesUpdated Information amp

httpnnneurologyorgcontent64e563fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent64e563fullhtmlref-list-1

This article cites 27 articles 7 of which you can access for free at

Citations httpnnneurologyorgcontent64e563fullhtmlotherarticles

This article has been cited by 1 HighWire-hosted articles

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseases

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httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the

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nextAn erratum has been published regarding this article Please see

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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 2019 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

CORRECTION

α4-integrin deficiency in B cells does not affect disease in aT-cellndashmediated EAE disease modelNeurol Neuroimmunol Neuroinflamm 20196e585 doi101212NXI0000000000000585

In the article ldquoα4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAEdisease model by Hussain et al1 first published online April 16 2019 funding for the papershould have been listed in the Study Funding section The authors regret the error

Reference1 Hussain RZ Cravens PD Miller-Little WA et al α4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAE

disease model Neurol Neuroimmunol Neuroinflamm 20196e563

Copyright copy 2019 American Academy of Neurology 1

Copyright copy 2019 American Academy of Neurology Unauthorized reproduction of this article is prohibited

Page 4: α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency in B cells does not affect disease in a T-cell–mediated EAE disease model Rehana

stated above other investigators found a deletion efficiencybetween 75 and 80 in BM-derived prendashB cells and ap-proximately 90 in splenic B cells17

CD19Cre+2 α4-integrinflfl mice immunizedwith MOGp35-55 display a regular clinicalEAE courseTo determine the effect of α4-integrin expression of B cells ina T-cellndashmediated active model of EAE CD19Cre+minus α4-integrinflfl mice and C57BL6 control mice were immunizedwithMOGp35-55 No significant differences regarding the EAEdisease incidence severity or phenotype were observed be-tween the mouse strains (figure 1B and table) α4-integrinflfl

mice were also used as controls and showed similar diseaseactivity (data not shown)

α4-integrin expressionon splenic B cells affectsantigen-specific CD4+ T-cell proliferationIt was previously demonstrated that α4-integrin can participatein costimulation of CD4+ T cells1819 Yet other investigatorsshowed that anti-CD49d plus anti-CD3anti-CD28ndashcoatedpolystyrene beads induced significantly greater T-cell pro-liferation than anti-CD3anti-CD28 polystyrene beads alone20

To test the effect of α4-integrin ablation on the capability ofCD19+ B cells to present antigen to antigen-reactive CD4+

T cells flow cytometric proliferation assays were performedCD4+ T-cell recall responses to MOGp35-55 in spleens weresignificantly diminished in CD19Cre+minus α4-integrinflfl mice(figure 1C) However in LNs we did not observe any differ-ence in CD4+ T-cell proliferation between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice (figure 1D)

Figure 1 CD19Cre+minus α4-integrinflfl mice are fully susceptible to actively MOGp35-55-induced experimental autoimmuneencephalomyelitis

(A) The percentage of CD49d+ CD19+ B cells in thebone marrow lymph nodes spleens and Peyerpatches is significantly diminished in naiveCD19Cre+minus α4-integrinflfl mice compared withC57BL6 control mice Cells were immunophe-notyped by multiparameter flow cytometry (B)CD19Cre+minus α4-integrinflfl mice behave similar toC57BL6 wild-type (WT) mice regarding the dis-ease incidence onset and severity of active ex-perimental autoimmune encephalomyelitis (EAE)α4-integrinflfl mice were used as controls andshowed similar disease activity (data not shown)Active EAE was induced in CD19Cre+minus α4-integ-rinflfl and C57BL6 age-matched control mice bysubcutaneous immunizations with MOGp35-55 inincomplete Freund adjuvant (IFA) containing 4mgmL mycobacteria Mice received in-traperitoneal injection of 200 μL of pertussis toxin(Ptx) at 200 ngmL on days 0 and 2 Mice wereobserved daily and EAE severity was scored usinga 5-point scale CD4+ T-cell recall responses toMOGp35-55 in (C) spleens were significantly di-minished in CD19Cre+minus α4-integrinflfl mice but(D) indistinguishable to those in C57BL6 WTmicein lymphnodes Cell proliferationwas determinedby a flow cytometric proliferation assay using thegreen fluorescent dye CFSE or V450 (E) At maxi-mum disease activity (days 13ndash15) the percent-age of CD3+ T cells in the CNS was similar inCD19Cre+minus α4-integrinflfl and C57BL6 WT miceIn contrast the percentage of CD19+ B cells in theCNS was significantly diminished in CD19Cre+minus

α4-integrinflfl mice (F) The percentage of α4-integrinndashpositive (CD49d+) CD19+ B cells in theCNS during maximum EAE disease activity wascomparable between both mouse strains Lym-phocytes were immunophenotyped by multipa-rameter flow cytometry The numberappearance and architecture of germinal centersin lymph nodes of (G and H) C57BL6 WT controlmice were comparable to those in (I and J)CD19Cre+minus α4-integrinflfl mice Panels G and Iwere stained with hematoxylin amp eosin andpanels H and J were stained with anti-CD19Magnification for GndashJ is times4 p lt 005 p lt 001p lt 0001 p lt 00001 CFSE = carboxy-fluorescein succinimidyl ester

4 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

In both mouse strains T-cell proliferation was substantial Ourobservations appear to differ from those of other investigatorswho showed that B cells do not presentMOGp35-55 to T cells21

The Methods section of that article states that ldquohellipB cells weremagnetically activated cell sorting (MACS Miltenyi BiotecBergisch Gladbach Germany)-separated from lymph nodes orspleensrdquo Thus a differential capability of B cells isolated fromeither LNs or spleens on MOG peptide presentation may nothave been fully tested

The number of B cells in the CNS during activeMOGp35-55-induced EAE is diminished inCD19Cre+2 α4-integrinflfl miceTo examine the effect of α4-integrin deletion in CD19+ B cellson the ability of B lymphocytes to enter the CNS cells wereisolated from brains and spinal cords by Percoll gradient andanalyzed by multiparameter flow cytometry As MOGp35-55-induced EAE is a T-cellndashmediated form of EAE the number ofCD3+ T cells was also assessed by the same method At maxi-mum disease activity (days 13ndash15) the percentage of CD3+

T cells in the CNS was similar in CD19Cre+minus α4-integrinflfl

mice and C57Bl6 WT mice (figure 1E) In contrast the per-centage of CD19+ B cells in the CNS was significantly reducedin CD19Cre+minus α4-integrinflfl mice (figure 1E) These obser-vations suggest that antigen-specific B cells within the CNS playa minor role in initiating and perpetuating EAE given that thesemice showed the same disease susceptibility and disease courseas C57Bl6 WT mice Immunohistochemical studies to de-termine a differential anatomic distribution of B cells in theCNSbetween CD19Cre+minus α4-integrinflfl mice and C57Bl6 WTmice were not performed as the absolute number of B cells inCD19Cre+minus α4-integrinflfl mice was very low

CD19+ B cells from CD19Cre+2 α4-integrinflfl

mice use CD49d to gain access to the CNSduring active MOGp35-55-induced EAETo determine whether CD19+ B cells from CD19Cre+minus α4-integrinflfl mice that gain access to the CNS during active EAEuse α4-integrin the percentage of α4-integrinndashpositive (CD49d+)CD19+ B cells was determined by multiparameter flowcytometry and found to be comparable between CD19Cre+minus

α4-integrinflfl mice and C57Bl6 WT controls (figure 1F)

The number and architecture of GCs in LNs ofCD19Cre+2 α4-integrinflfl mice are normalCD19Cremice express cre under the transcriptional control ofthe B lineagendashrestricted CD19 gene17 Insertion of cre disruptsthe CD19 coding sequence leading to a CD19 deficiency anda concomitant reduction in GCs in CD19Cre++ mice At day15 during the acute phase of MOGp35-55-induced EAE thenumber and architecture of GCs in LNs of CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice were com-parable by hematoxylin amp eosin (HampE) staining (figure 1 Gand H) and anti-CD19 staining (figure 1 I and J)

B cells of CD19Cre+2 α4-integrinflfl mice havenormal cytokine profiles during activeMOGp35-

55-induced EAETo test whether the absence of α4-integrin in CD19+ B cellsaffects their differentiation during inflammatory conditionsexperimental animals were killed at day 13 during the acutephase of MOGp35-55-induced EAE and B cells in disease-relevant compartments were immunophenotyped by multi-parameter flow cytometry and intracellular cytokine stainingCompared with B cells from C57BL6 WT control mice thepercentage of CD19+ B cells expressing IFNγ IL-6 IL-10 orIL-17 in CD19Cre+minus α4-integrinflfl mice was similar in theBM (figure 2A) spleen (figure 2B) LNs (figure 2C) andbrain (figure 2D) of CD19Cre+minus α4-integrinflfl mice

Outside the CNS B cells of CD19Cre+2 α4-integrinflfl mice express normal maturationand activation markers during active MOGp35-

55-induced EAEAs stated above α4-integrin is considered a costimulatorymolecule through which CD19+ B cells are capable ofinteracting with CD4+ T cells To further elucidate theeffect of α4-integrin deficiency on CD19+ B cells on theirmaturation and activation in secondary lymphoid organsexperimental animals were killed at day 13 during theacute phase of MOGp35-55-induced EAE and B cells inspleens and LNs were immunophenotyped by multipa-rameter flow cytometry The percentages of CD19+ B220+

B cells expressing IgD IgM IgG and major histocom-patibility complex (MHC) II in LNs (figure 2E) andspleen (figure 2F) were comparable in CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice

CNS B cells from CD19Cre+2 α4-integrinflfl

mice do not upregulate activation markersduring active MOGp35-55-induced EAEThe absolute number of CD19+ B cells is diminished in theCNS of CD19Cre+minus α4-integrinflfl mice compared withC57BL6 WT control mice during acute MOGp35-55-in-duced EAE (figure 1E) To evaluate the activation state ofthese cells CNS B cells were isolated and assessed by mul-tiparameter flow cytometry during active EAE at day 13 afteractive induction or EAE with MOGp35-55 The surface ex-pression of MHC II was similar in CNS-derived B cells fromCD19Cre+minus α4-integrinflfl and C57BL6 WT controls

Table Disease incidence mean maximum disease scoreand mean day of active MOGp35-55-induced EAEdisease onset in CD19+minus α4-integrin mice andC57BL6 control mice

Group IncidenceMean maximumdisease score

Mean day ofdisease onset

C57BL6 7280 289 1057 plusmn 518

CD19+2 α4-integrinflfl

6281 245 926 plusmn 607

Abbreviations EAE = experimental autoimmune encephalomyelitis MOG =myelin oligodendrocyte glycoproteinResults of 15 experiments are shown

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 5

(figure 2G) Also the expression of the costimulator mole-cules CD80 and CD86 and the first apoptosis signal receptorCD95 was comparable in brain-derived B cells fromCD19Cre+minus α4-integrinflfl mice and C57BL6 WT con-trols (figure 2H)

The composition of B-cell subsets in the brainof CD19Cre+2 α4-integrinflfl mice isunchanged during MOGp35-55-induced EAETo determine whether the deletion of α4-integrin on CD19+

B cells affects B-cell subsets differentially in their capability toenter the CNS the percentage of innate and adaptiveB-lymphocyte subsets including CD11b+CD23minusB220minus B1B cells CD11b+CD23minusB220lominusCD5minus B1b B cellsCD11b+CD23minusB220lominusCD5+ B1a B cellsCD19+CD5+CD1dhi B10 B cells CD19+CD23+CD5minus

naive follicular B cells B220+CD138minusCD95+GL7+ GCsB cells B220+CD1d+CD5minusCD23minusCD21+ marginal zone

(MGZ) B cells CD19lowndashCD138+B220lowndash plasma blast(PB) and CD19minusCD138+ PCs was assessed by multipa-rameter flow cytometry during acute MOGp35-55-inducedEAE at day 13 during early active EAE (figure 2I) day 19during maximum EAE disease activity (figure 2J) and day 29during chronic EAE (figure 2K) after active induction orEAE with MOGp35-55 and was found to be similar inCD19Cre+minus α4-integrinflfl mice and C57BL6 WT con-trols at all time points A gating strategy used to identifysome of the B-cell subsets is shown in figure 3

The composition of B-cell subsets in primaryand secondary lymphoid organs of CD19Cre+2

α4-integrinflfl mice is similar to those of WTcontrol mice during MOGp35-55-induced EAETo assess the effect of α4-integrin ablation on CD19+ B cellson B-cell phenotypes in lymphoid organs innate and adaptiveB-lymphocyte subsets were characterized by flow cytometry

Figure 2 CD19+ B cells in the CNS of CD19Cre+minus α4-integrinflfl mice are phenotypically similar to wild-type (WT) B cellsduring myelin oligodendrocyte glycoprotein peptide (MOGp35-55)-induced experimental autoimmuneencephalomyelitis

Experimental animals were killed at day 13 during the acute phase of MOGp35-55-induced experimental autoimmune encephalomyelitis (EAE) and B cells indisease-relevant compartmentswere immunophenotyped bymultiparameter flow cytometry and intracellular cytokine staining Comparedwith B cells fromC57BL6 WT control mice the percentage of CD19+ B cells expressing interferon gamma (IFNγ) interleukin (IL)-6 IL-10 or IL-17 in CD19Cre+minus α4-integrinflfl

mice was similar in the (A) bone marrow (BM) (B) spleen (C) lymph node (LNs) and (D) brain of CD19Cre+minus α4-integrinflfl mice The percentages of CD19+

B220+ B cells expressing IgD IgM IgG and MHC II in (E) LNs and (F) spleen were comparable in both mouse strains (G) In the CNS the surface expression ofMHC II was similar in CD19Cre+minus α4-integrinflfl mice and controls (H) In the brain the expression of the costimulator molecules CD80 and CD86 and the firstapoptosis signal receptor CD95 was indistinguishable between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT controls The percentage ofCD11b+CD23B220lo B1 B cells CD11b+CD23B220lominusCD5+ B1a B cells CD11b+CD23B220lominusCD5minus B1b B cells B220+CD5+CD1dhi B10 B cells CD19+CD23+CD5minus

naive follicular B cells (FB) B220+CD138minusCD95+GL7+ germinal center (GC) B cells B220+CD1d+CD5minusCD23minusCD21+marginal zone (MGZ) B cells CD138+B220lowndash

plasma blast (PB) and B220minusCD138+ plasma cells (PC) was similar in both mouse strains at (I) day 13 during early active EAE (J) day 19 during maximum EAEdisease activity and (K) day 29 during chronic EAE Lymphocytes were immunophenotyped by multiparameter flow cytometry A gating strategy used toidentify some of the B-cell subsets is shown in Figure 3 p lt 001

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

We were unable to detect a change in composition ofCD19+CD11b+CD23minusB220minus B1 B cellsCD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cellsCD19+CD5+CD1dhi B10 B cellsCD19+B220+CD1d+CD5minusCD23minusCD21+ MGZ B cells andCD19lowndashCD138+B220lowndash PB CD19Cre+minus α4-integrinflfl

mice and C57BL6 WT controls at day 13 during early activeEAE in the BM (figure 4A) LNs (Figure 4B) and spleen(figure 4C)

Serum soluble inflammatory markers areunchanged in CD19Cre+2 α4-integrinflfl miceduring different stages of MOGp35-55-induced EAEDeletion of α4-integrin on CD19+ B cells diminishes theability of CD19+ B cells to enter the CNS during acuteMOGp35-55-induced EAE (figure 1E) It is conceivable thatinflammatory B cells accumulate in peripheral blood alteringthe inflammatory milieu in that compartment This wasobserved in patients withMS treated with natalizumab22 Wefound that the serum expression of the cytokines IL-17A(figure 5A) IL-10 (Figure 5B) IL-4 (Figure 5C) IL-5

(figure 5D) and IFNγ (figure 5E) as measured by ELISA(ELISA) during acute and chronic MOGp35-55-induced EAEon days 13 19 and 29 was comparable betweenCD19Cre+minus α4-integrinflflmice and C57BL6 WT controlmice

Serum Ig levels are unchanged in CD19Cre+2

α4-integrinflfl mice during different stages ofMOGp35-55-induced EAETo further investigate whether the sequestration of CD19+

B cells through deletion of α4-integrin affects their acti-vation proliferation and antibody secretion IgM and IgGlevels in serum were determined by ELISA Serum levels ofIgM (figure 5F) and IgG (figure 5G) as measured byELISA were also similar between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice on days13 19 and 29 of MOGp35-55-induced EAE Given that theabsolute levels of IgM and IgG were similar between themouse strains and that other investigators previouslyshowed that B cells from mice on the C57BL6 back-ground do not readily recognize MOGp35-55

21

we did not test the serum levels of MOGp35-55-specific IgMor IgG

Figure 3 Immunophenotyping of murine B cells

B-lymphocyte subsets were immunophenotyped by multiparameter flow cytometry A gating strategy that was used to identify some of the B-cell sub-populations is shown (A) First cellular area height and width measurements were obtained in a channel with linear scale to gate on singlets and excludedoublets (B) Next gates were set to include CD45+ leukocytes and exclude FSClow cell debris out (C) CD138 and B220 were used to gate on plasmablasts(CD138+B220lowndash) plasma cells (CD138+B220ndash) and B cells (B220+) Panels DndashF show the gating for (E) B220+CD1d+CD5minusCD23minusCD21+IgM+ marginal zone Bcells (E) B220+CD1d+CD5minusCD23minusCD21minusIgM+ transitional 1 (T1) B cells and (F) B220+CD1d+CD5minusCD23+CD21+IgM+ T2 B cells Panels GndashI show gating for B1 B-cell populations and the identification of (H) CD11b+CD23minusB220lominusCD5minus B1b B cells and (I) CD11b+CD23minusB220lominusCD5+ B1a B cells Panels J and K show theidentification of B220+CD138minusCD95+GL7+ germinal center B cells Panels LndashO illustrate the gating for B220+CD19+CD5minusGL7minusCD23+CD21loint naive follicular Bcells FSC = forward side scatter

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 7

Activated peripheral B cells contribute todisease severity in MOGp35-55-induced EAETo determine the compartment-specific pathogenic role ofB cells in the induction and perpetuation of the EAE animalmodel CD19Cre+minus α4-integrinflfl mice were immunizedwith MOGp35-55 As demonstrated B cells in these mice havea diminished capacity to enter the CNS (figure 1E) Adoptivetransfer of purified CD19+ B cells from CD19Cre+minus α4-integrinflfl mice and C57BL6 WT mice immunized withrMOG1-125 or the control antigen OVA323-339 worsened the

clinical course of EAE in recipient mice immunized withMOGp35-55 (figure 5H) although these changes were notsignificant

DiscussionThere is accumulating evidence to suggest that B cells andmyelin-specific antibodies play crucial roles in the pathogen-esis of MS Whether B-cell depletion mediates its beneficial

Figure 4 CD19+ B cells in primary and secondary lymphoid organs of CD19Cre+minus α4-integrinflfl mice are phenotypicallysimilar to wild-type B cells during MOGp35-55-induced experimental autoimmune encephalomyelitis

(A) In the bonemarrow (B) lymph nodes and (C) spleen the percentage of CD19+CD11b+CD23minusB220minus B1 B cells CD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cells CD19+CD5+CD1dhi B10 B cells CD19+B220+CD1d+CD5minusCD23minusCD21+ marginal zone (MGZ) B cells andCD19lowndashCD138+B220lowndash plasma blast (PB) was similar in CD19Cre+minus α4-integrinflfl mice and C57BL6 WT controls at day 13 during early activeEAE Lymphocytes were immunophenotyped by multiparameter flow cytometry

Figure 5ActivatedB cells outside of theCNSdrive disease activity inmyelin oligodendrocyte glycoprotein peptide (MOGp35-

55)-induced experimental autoimmune encephalomyelitis (EAE) in an antigen no-specific manner

The serum expression of the cytokines(A) IL-17A (B) IL-10 (C) IL-4 (D) IL-5 and(E) IFNγ as measured by ELISA duringacute and chronic MOGp35-55-inducedEAE on days 13 19 and 29 was com-parable between CD19Cre+minus α4-integ-rinflfl mice and C57BL6 wild-type (WT)control mice Serum levels of (F) IgMand (G) IgG measured by ELSA at thesame time points were also similar be-tween mouse strains (H) Adoptivetransfer of purified CD19+ B cells fromCD19Cre+minus α4-integrinflfl mice orC57BL6 WT control mice immunizedwith recombinant (r)MOG1-125 or oval-bumin (OVA)323-339 into CD19+minus α4-integrinflfl mice that were then imme-diately immunized with MOGp35-55

caused worse clinical EAE than wasobserved in MOGp35-55-immunizedC57BL6 WT control mice (ldquoC57BL6rdquo)that did not receive adoptively trans-ferred CD19+ B cells

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

effects in patients with MS through B cells in the periphery inthe CNS or both is currently not completely understood

Other investigators investigated the role of α4-integrin deficiencyin CD19+ B cells in EAE23 These investigators showed thatB-cell very late activation antigen-4ndashdeficient mice developedmore severe clinical disease than control mice after active in-duction of EAE with MOGp35-55 and that that this clinical effectis due to the requirement of regulatory B cells (Bregs) for α4-integrin to enter the CNS Bregs were defined as CD19+IL-10+

or CD1dhiCD5+ B-cell subsets Another group of investigatorsalso testedCD19Cre Itga4flflmice inMOGp35-55-induced activeEAE24 CD19Cre Itga4flfl mice developed more severe EAEthan controlmice and these investigators demonstrated a criticalrole for α4-integrin on the generation of Bregs in peripheralimmune organs

Our own results diverge from these reports in that we couldnot detect a difference in active EAE disease severity betweenCD19Cre+minus α4-integrinflfl mice and C57BL6 WT controlmice These experiments were conducted 15 times (table)and we are confident in our findings Differences in EAEdisease severity and phenotype can be affected by conditionsunder which mouse colonies are maintained as well as otherfactors that may affect the overall inflammatory milieu of ex-perimental conditions This may explain the differences inresults It is also conceivable that different mice were used bythe other investigators for their experiments The inves-tigators of the 2 previous studies state that they usedCD19Creα4flfl mice23 or that they used CD19Cre Itga4flfl

mice25 It is not stated whether mice were heterozygous orhomozygous for CD19cre or a mixture of both As stated inthe Methods section CD19Cre++ mice behave functionallyvery similarly to B-cellndashdeficient mice which develop moresevere EAE when immunized with MOGp35-55

26

We found one potentially meaningful biological difference be-tween CD19Cre+minus α4-integrinflfl mice and C57BL6 controlmice There was decreased MOGp35-55-specific CD4+ T-cellproliferation in spleens which is an expected result given thatα4-integrin can participate in costimulation of CD4+T cells1819

The observation that CD19+ B cells of CD19Cre+minus α4-integrinflfl mice did deplete α4-integrin and that they weresignificantly less capable of entering the CNS after mice wereimmunized with MOGp35-55 suggests 3 interpretations (1)Antigen-activated B cells contribute to disease burden in EAEin a seemingly dose-dependent manner (2) they do not ab-solutely require recognition of a CNS autoantigen or a mimicthereof to do so and (3) they do not require full α4-integrinndashmediated access to the CNS to exacerbate diseaseThe first interpretation is supported by work of other inves-tigators who have previously demonstrated that B-cellndashdeficient C57BL6 mice are resistant to active rMOG-inducedEAE26 and that the administration of a B-cellndashdepletingmonoclonal antibody prevents or reverses established rMOG-induced EAE in C57BL6 mice21 Weber and colleagues21

further showed that B-cell depletion results in a reduction ofMOGp35-55-specific Th1 and Th17 cells Our own data suggestthat the differentiation ofMOGp35-55-specific T cells to becomeencephalitogenic does not absolutely require the presentationof autoantigen but is likely driven by other B-cell factors in-cluding soluble inflammatory mediators The third in-terpretation of this experiment is supported by ourdemonstration that B cells from CD19Cre+minus α4-integrinflfl

mice are significantly impaired in their ability to enter the CNS(figure 1E) Finally it should be emphasized that our findingsdo not conclusively rule out a role of antigen-specific B cells inthe CNS on the course of CNS autoimmunity as thresholdeffects may be difficult to detect with the EAE experiments thatwere conducted

Our data suggest that the beneficial effects of anti-CD20therapies in patients with MS are mediated through theireffects on peripheral B cells We also conclude that benefitsobserved in patients with MS during anti-α4-integrin therapyare likely not predominantly due to its effect of suppressingthe migration of B cells into the CNS The interpretation ofour findings should be limited to early forms of MS as theEAE model that was used and the observation period that wasused do not provide insight into late or progressive forms ofCNS autoimmunity Magliozzi et al27 were the first inves-tigators to show that some patients with secondary-progressive MS develop lymphoid tissue in the meningesthat resemble B-cell follicles in secondary lymphoid organsB cells in these structures may be susceptible to anti-CD20therapies with monoclonal antibodies or small molecules andit is conceivable that their depletion may benefit patientsafflicted with that MS phenotype

AcknowledgmentThe authors thank Dr Thalia Papayannopoulou at theUniversity of Washington for providing them with α4-integrinfloxflox mice Dr Stuve was funded by a Merit Reviewgrant (federal award document number [FAIN]I01BX001674) from the United States (US) Departmentof Veterans Affairs Biomedical Laboratory Research andDevelopment

Study fundingNo targeted funding reported

DisclosureRZ Hussain PC Cravens WA Miller-Little R Doelger VGrandos and E Herndon report no disclosures DT Okudareceived advisory and consulting fees from Celgene Gen-entech Genzyme EMD Serono and Novartis and researchsupport from Biogen served on the scientific advisory boardof Osmotica and served on the speakersrsquo bureau of AcordaGenzyme and Teva TN Eagar serves as section editor ofArchives of Pathology and received research support from theNIH O Stuve served on the scientific advisory boards of TGTherapeutics and Genentech-Roche served on the editorialboard of Therapeutic Advances in Neurological Disorders

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 9

consulted for Celgene EMD Serono and Novartis andreceived research support from Sanofi and GenzymeDisclosures available NeurologyorgNN

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationNovember 2 2018 Accepted in final form February 1 2019

References1 Hauser SL Waubant E Arnold DL et al B-cell depletion with rituximab in relapsing-

remitting multiple sclerosis N Engl J Med 2008358676ndash6882 Hawker K OrsquoConnor P Freedman MS et al Rituximab in patients with primary

progressive multiple sclerosis results of a randomized double-blind placebo-controlled multicenter trial Ann Neurol 200966460ndash471

3 Hauser SL Bar-Or A Comi G et al Ocrelizumab versus interferon beta-1a in re-lapsing multiple sclerosis N Engl J Med 2017376221ndash234

4 Montalban X Hauser SL Kappos L et al Ocrelizumab versus placebo in primaryprogressive multiple sclerosis N Engl J Med 2017376209ndash220

5 Yednock TA Cannon C Fritz LC Sanchez-Madrid F Steinman L Karin N Pre-vention of experimental autoimmune encephalomyelitis by antibodies against alpha 4beta 1 integrin Nature 199235663ndash66

6 Frohman EM Racke MK Raine CS Multiple sclerosismdashthe plaque and its patho-genesis N Engl J Med 2006354942ndash955

7 Stuve O Marra CM Jerome KR et al Immune surveillance in multiple sclerosispatients treated with natalizumab Ann Neurol 200659743ndash747

8 Haines JL Ter Minassian M Bazyk A et al A complete genomic screen for multiplesclerosis underscores a role for the major histocompatability complex The MultipleSclerosis Genetics Group Nat Genet 199613469ndash471

9 Sawcer S Jones HB Feakes R et al A genome screen in multiple sclerosis revealssusceptibility loci on chromosome 6p21 and 17q22 Nat Genet 199613464ndash468

10 Yang JT Rayburn H Hynes RO Cell adhesion events mediated by alpha 4 integrinsare essential in placental and cardiac development Development 1995121549ndash560

11 Sternberg N Hamilton D Bacteriophage P1 site-specific recombination I Re-combination between loxP sites J Mol Biol 1981150467ndash486

12 Scott LM Priestley GV Papayannopoulou T Deletion of alpha4 integrins from adulthematopoietic cells reveals roles in homeostasis regeneration and homing Mol CellBiol 2003239349ndash9360

13 Hussain RZ Miller-Little WA Doelger R et al Defining standard enzymatic disso-ciation methods for individual brains and spinal cords in EAE Neurol NeuroimmunolNeuroinflamm 20185e437 doi 101212NXI0000000000000437

14 Cravens PD Hussain RZ Zacharias TE et al Lymph node-derived donor encepha-litogenic CD4+ T cells in C57BL6 mice adoptive transfer experimental autoimmuneencephalomyelitis highly express GM-CSF and T-bet J Neuroinflammation 2011873

15 Karandikar NJ Crawford MP Yan X et al Glatiramer acetate (Copaxone) therapyinduces CD8(+) T cell responses in patients with multiple sclerosis J Clin Invest2002109641ndash649

16 Gu H Marth JD Orban PC Mossmann H Rajewsky K Deletion of a DNA poly-merase beta gene segment in T cells using cell type-specific gene targeting Science1994265103ndash106

17 Rickert RC Roes J Rajewsky K B lymphocyte-specific Cre-mediated mutagenesis inmice Nucleic Acids Res 1997251317ndash1318

18 Davis LS Oppenheimer-Marks N Bednarczyk JL McIntyre BW Lipsky PE Fibro-nectin promotes proliferation of naive and memory T cells by signaling through boththe VLA-4 and VLA-5 integrin molecules J Immunol 1990145785ndash793

19 Shimizu Y van Seventer GA Horgan KJ Shaw S Costimulation of proliferativeresponses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 withfibronectin or VLA-6 with laminin J Immunol 199014559ndash67

20 Theien BE Vanderlugt CL Eagar TN et al Discordant effects of anti-VLA-4 treat-ment before and after onset of relapsing experimental autoimmune encephalomyelitisJ Clin Invest 2001107995ndash1006

21 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

22 Krumbholz M Meinl I Kumpfel T Hohlfeld R Meinl E Natalizumab dispropor-tionately increases circulating pre-B and B cells in multiple sclerosis Neurology 2008711350ndash1354

23 Lehmann-Horn K Sagan SA Winger RC et al CNS accumulation of regulatoryB cells is VLA-4-dependent Neurol Neuroimmunol Neuroinflamm 20163e212 doi101212NXI0000000000000212

24 Glatigny S Wagner CA Bettelli E Correction cutting edge integrin alpha4 is re-quired for regulatory B cell control of experimental autoimmune encephalomyelitisJ Immunol 20171983756

25 Glatigny S Duhen R Oukka M Bettelli E Cutting edge loss of alpha4 integrinexpression differentially affects the homing of Th1 and Th17 cells J Immunol 20111876176ndash6179

26 Lyons JA SanM HappMP Cross AH B cells are critical to induction of experimentalallergic encephalomyelitis by protein but not by a short encephalitogenic peptide EurJ Immunol 1999293432ndash3439

27 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

Appendix Authors

Name Location Role Contribution

Rehana ZHussainMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

Petra CCravensPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

William AMiller-Little BA

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

RichardDoelgerMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

ValerieGranadosPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the data andanalyzed the data

EmilyHerndonMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Darin TOkudaMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Todd NEagar PhD

Houston MethodistHospital HoustonTX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

OlafStuve MDPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

DOI 101212NXI000000000000056320196 Neurol Neuroimmunol Neuroinflamm

Rehana Z Hussain Petra D Cravens William A Miller-Little et al disease model

mediated EAEminus4-integrin deficiency in B cells does not affect disease in a T-cellα

This information is current as of April 16 2019

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

ServicesUpdated Information amp

httpnnneurologyorgcontent64e563fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent64e563fullhtmlref-list-1

This article cites 27 articles 7 of which you can access for free at

Citations httpnnneurologyorgcontent64e563fullhtmlotherarticles

This article has been cited by 1 HighWire-hosted articles

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseases

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httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the

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Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

CORRECTION

α4-integrin deficiency in B cells does not affect disease in aT-cellndashmediated EAE disease modelNeurol Neuroimmunol Neuroinflamm 20196e585 doi101212NXI0000000000000585

In the article ldquoα4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAEdisease model by Hussain et al1 first published online April 16 2019 funding for the papershould have been listed in the Study Funding section The authors regret the error

Reference1 Hussain RZ Cravens PD Miller-Little WA et al α4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAE

disease model Neurol Neuroimmunol Neuroinflamm 20196e563

Copyright copy 2019 American Academy of Neurology 1

Copyright copy 2019 American Academy of Neurology Unauthorized reproduction of this article is prohibited

Page 5: α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency in B cells does not affect disease in a T-cell–mediated EAE disease model Rehana

In both mouse strains T-cell proliferation was substantial Ourobservations appear to differ from those of other investigatorswho showed that B cells do not presentMOGp35-55 to T cells21

The Methods section of that article states that ldquohellipB cells weremagnetically activated cell sorting (MACS Miltenyi BiotecBergisch Gladbach Germany)-separated from lymph nodes orspleensrdquo Thus a differential capability of B cells isolated fromeither LNs or spleens on MOG peptide presentation may nothave been fully tested

The number of B cells in the CNS during activeMOGp35-55-induced EAE is diminished inCD19Cre+2 α4-integrinflfl miceTo examine the effect of α4-integrin deletion in CD19+ B cellson the ability of B lymphocytes to enter the CNS cells wereisolated from brains and spinal cords by Percoll gradient andanalyzed by multiparameter flow cytometry As MOGp35-55-induced EAE is a T-cellndashmediated form of EAE the number ofCD3+ T cells was also assessed by the same method At maxi-mum disease activity (days 13ndash15) the percentage of CD3+

T cells in the CNS was similar in CD19Cre+minus α4-integrinflfl

mice and C57Bl6 WT mice (figure 1E) In contrast the per-centage of CD19+ B cells in the CNS was significantly reducedin CD19Cre+minus α4-integrinflfl mice (figure 1E) These obser-vations suggest that antigen-specific B cells within the CNS playa minor role in initiating and perpetuating EAE given that thesemice showed the same disease susceptibility and disease courseas C57Bl6 WT mice Immunohistochemical studies to de-termine a differential anatomic distribution of B cells in theCNSbetween CD19Cre+minus α4-integrinflfl mice and C57Bl6 WTmice were not performed as the absolute number of B cells inCD19Cre+minus α4-integrinflfl mice was very low

CD19+ B cells from CD19Cre+2 α4-integrinflfl

mice use CD49d to gain access to the CNSduring active MOGp35-55-induced EAETo determine whether CD19+ B cells from CD19Cre+minus α4-integrinflfl mice that gain access to the CNS during active EAEuse α4-integrin the percentage of α4-integrinndashpositive (CD49d+)CD19+ B cells was determined by multiparameter flowcytometry and found to be comparable between CD19Cre+minus

α4-integrinflfl mice and C57Bl6 WT controls (figure 1F)

The number and architecture of GCs in LNs ofCD19Cre+2 α4-integrinflfl mice are normalCD19Cremice express cre under the transcriptional control ofthe B lineagendashrestricted CD19 gene17 Insertion of cre disruptsthe CD19 coding sequence leading to a CD19 deficiency anda concomitant reduction in GCs in CD19Cre++ mice At day15 during the acute phase of MOGp35-55-induced EAE thenumber and architecture of GCs in LNs of CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice were com-parable by hematoxylin amp eosin (HampE) staining (figure 1 Gand H) and anti-CD19 staining (figure 1 I and J)

B cells of CD19Cre+2 α4-integrinflfl mice havenormal cytokine profiles during activeMOGp35-

55-induced EAETo test whether the absence of α4-integrin in CD19+ B cellsaffects their differentiation during inflammatory conditionsexperimental animals were killed at day 13 during the acutephase of MOGp35-55-induced EAE and B cells in disease-relevant compartments were immunophenotyped by multi-parameter flow cytometry and intracellular cytokine stainingCompared with B cells from C57BL6 WT control mice thepercentage of CD19+ B cells expressing IFNγ IL-6 IL-10 orIL-17 in CD19Cre+minus α4-integrinflfl mice was similar in theBM (figure 2A) spleen (figure 2B) LNs (figure 2C) andbrain (figure 2D) of CD19Cre+minus α4-integrinflfl mice

Outside the CNS B cells of CD19Cre+2 α4-integrinflfl mice express normal maturationand activation markers during active MOGp35-

55-induced EAEAs stated above α4-integrin is considered a costimulatorymolecule through which CD19+ B cells are capable ofinteracting with CD4+ T cells To further elucidate theeffect of α4-integrin deficiency on CD19+ B cells on theirmaturation and activation in secondary lymphoid organsexperimental animals were killed at day 13 during theacute phase of MOGp35-55-induced EAE and B cells inspleens and LNs were immunophenotyped by multipa-rameter flow cytometry The percentages of CD19+ B220+

B cells expressing IgD IgM IgG and major histocom-patibility complex (MHC) II in LNs (figure 2E) andspleen (figure 2F) were comparable in CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice

CNS B cells from CD19Cre+2 α4-integrinflfl

mice do not upregulate activation markersduring active MOGp35-55-induced EAEThe absolute number of CD19+ B cells is diminished in theCNS of CD19Cre+minus α4-integrinflfl mice compared withC57BL6 WT control mice during acute MOGp35-55-in-duced EAE (figure 1E) To evaluate the activation state ofthese cells CNS B cells were isolated and assessed by mul-tiparameter flow cytometry during active EAE at day 13 afteractive induction or EAE with MOGp35-55 The surface ex-pression of MHC II was similar in CNS-derived B cells fromCD19Cre+minus α4-integrinflfl and C57BL6 WT controls

Table Disease incidence mean maximum disease scoreand mean day of active MOGp35-55-induced EAEdisease onset in CD19+minus α4-integrin mice andC57BL6 control mice

Group IncidenceMean maximumdisease score

Mean day ofdisease onset

C57BL6 7280 289 1057 plusmn 518

CD19+2 α4-integrinflfl

6281 245 926 plusmn 607

Abbreviations EAE = experimental autoimmune encephalomyelitis MOG =myelin oligodendrocyte glycoproteinResults of 15 experiments are shown

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 5

(figure 2G) Also the expression of the costimulator mole-cules CD80 and CD86 and the first apoptosis signal receptorCD95 was comparable in brain-derived B cells fromCD19Cre+minus α4-integrinflfl mice and C57BL6 WT con-trols (figure 2H)

The composition of B-cell subsets in the brainof CD19Cre+2 α4-integrinflfl mice isunchanged during MOGp35-55-induced EAETo determine whether the deletion of α4-integrin on CD19+

B cells affects B-cell subsets differentially in their capability toenter the CNS the percentage of innate and adaptiveB-lymphocyte subsets including CD11b+CD23minusB220minus B1B cells CD11b+CD23minusB220lominusCD5minus B1b B cellsCD11b+CD23minusB220lominusCD5+ B1a B cellsCD19+CD5+CD1dhi B10 B cells CD19+CD23+CD5minus

naive follicular B cells B220+CD138minusCD95+GL7+ GCsB cells B220+CD1d+CD5minusCD23minusCD21+ marginal zone

(MGZ) B cells CD19lowndashCD138+B220lowndash plasma blast(PB) and CD19minusCD138+ PCs was assessed by multipa-rameter flow cytometry during acute MOGp35-55-inducedEAE at day 13 during early active EAE (figure 2I) day 19during maximum EAE disease activity (figure 2J) and day 29during chronic EAE (figure 2K) after active induction orEAE with MOGp35-55 and was found to be similar inCD19Cre+minus α4-integrinflfl mice and C57BL6 WT con-trols at all time points A gating strategy used to identifysome of the B-cell subsets is shown in figure 3

The composition of B-cell subsets in primaryand secondary lymphoid organs of CD19Cre+2

α4-integrinflfl mice is similar to those of WTcontrol mice during MOGp35-55-induced EAETo assess the effect of α4-integrin ablation on CD19+ B cellson B-cell phenotypes in lymphoid organs innate and adaptiveB-lymphocyte subsets were characterized by flow cytometry

Figure 2 CD19+ B cells in the CNS of CD19Cre+minus α4-integrinflfl mice are phenotypically similar to wild-type (WT) B cellsduring myelin oligodendrocyte glycoprotein peptide (MOGp35-55)-induced experimental autoimmuneencephalomyelitis

Experimental animals were killed at day 13 during the acute phase of MOGp35-55-induced experimental autoimmune encephalomyelitis (EAE) and B cells indisease-relevant compartmentswere immunophenotyped bymultiparameter flow cytometry and intracellular cytokine staining Comparedwith B cells fromC57BL6 WT control mice the percentage of CD19+ B cells expressing interferon gamma (IFNγ) interleukin (IL)-6 IL-10 or IL-17 in CD19Cre+minus α4-integrinflfl

mice was similar in the (A) bone marrow (BM) (B) spleen (C) lymph node (LNs) and (D) brain of CD19Cre+minus α4-integrinflfl mice The percentages of CD19+

B220+ B cells expressing IgD IgM IgG and MHC II in (E) LNs and (F) spleen were comparable in both mouse strains (G) In the CNS the surface expression ofMHC II was similar in CD19Cre+minus α4-integrinflfl mice and controls (H) In the brain the expression of the costimulator molecules CD80 and CD86 and the firstapoptosis signal receptor CD95 was indistinguishable between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT controls The percentage ofCD11b+CD23B220lo B1 B cells CD11b+CD23B220lominusCD5+ B1a B cells CD11b+CD23B220lominusCD5minus B1b B cells B220+CD5+CD1dhi B10 B cells CD19+CD23+CD5minus

naive follicular B cells (FB) B220+CD138minusCD95+GL7+ germinal center (GC) B cells B220+CD1d+CD5minusCD23minusCD21+marginal zone (MGZ) B cells CD138+B220lowndash

plasma blast (PB) and B220minusCD138+ plasma cells (PC) was similar in both mouse strains at (I) day 13 during early active EAE (J) day 19 during maximum EAEdisease activity and (K) day 29 during chronic EAE Lymphocytes were immunophenotyped by multiparameter flow cytometry A gating strategy used toidentify some of the B-cell subsets is shown in Figure 3 p lt 001

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

We were unable to detect a change in composition ofCD19+CD11b+CD23minusB220minus B1 B cellsCD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cellsCD19+CD5+CD1dhi B10 B cellsCD19+B220+CD1d+CD5minusCD23minusCD21+ MGZ B cells andCD19lowndashCD138+B220lowndash PB CD19Cre+minus α4-integrinflfl

mice and C57BL6 WT controls at day 13 during early activeEAE in the BM (figure 4A) LNs (Figure 4B) and spleen(figure 4C)

Serum soluble inflammatory markers areunchanged in CD19Cre+2 α4-integrinflfl miceduring different stages of MOGp35-55-induced EAEDeletion of α4-integrin on CD19+ B cells diminishes theability of CD19+ B cells to enter the CNS during acuteMOGp35-55-induced EAE (figure 1E) It is conceivable thatinflammatory B cells accumulate in peripheral blood alteringthe inflammatory milieu in that compartment This wasobserved in patients withMS treated with natalizumab22 Wefound that the serum expression of the cytokines IL-17A(figure 5A) IL-10 (Figure 5B) IL-4 (Figure 5C) IL-5

(figure 5D) and IFNγ (figure 5E) as measured by ELISA(ELISA) during acute and chronic MOGp35-55-induced EAEon days 13 19 and 29 was comparable betweenCD19Cre+minus α4-integrinflflmice and C57BL6 WT controlmice

Serum Ig levels are unchanged in CD19Cre+2

α4-integrinflfl mice during different stages ofMOGp35-55-induced EAETo further investigate whether the sequestration of CD19+

B cells through deletion of α4-integrin affects their acti-vation proliferation and antibody secretion IgM and IgGlevels in serum were determined by ELISA Serum levels ofIgM (figure 5F) and IgG (figure 5G) as measured byELISA were also similar between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice on days13 19 and 29 of MOGp35-55-induced EAE Given that theabsolute levels of IgM and IgG were similar between themouse strains and that other investigators previouslyshowed that B cells from mice on the C57BL6 back-ground do not readily recognize MOGp35-55

21

we did not test the serum levels of MOGp35-55-specific IgMor IgG

Figure 3 Immunophenotyping of murine B cells

B-lymphocyte subsets were immunophenotyped by multiparameter flow cytometry A gating strategy that was used to identify some of the B-cell sub-populations is shown (A) First cellular area height and width measurements were obtained in a channel with linear scale to gate on singlets and excludedoublets (B) Next gates were set to include CD45+ leukocytes and exclude FSClow cell debris out (C) CD138 and B220 were used to gate on plasmablasts(CD138+B220lowndash) plasma cells (CD138+B220ndash) and B cells (B220+) Panels DndashF show the gating for (E) B220+CD1d+CD5minusCD23minusCD21+IgM+ marginal zone Bcells (E) B220+CD1d+CD5minusCD23minusCD21minusIgM+ transitional 1 (T1) B cells and (F) B220+CD1d+CD5minusCD23+CD21+IgM+ T2 B cells Panels GndashI show gating for B1 B-cell populations and the identification of (H) CD11b+CD23minusB220lominusCD5minus B1b B cells and (I) CD11b+CD23minusB220lominusCD5+ B1a B cells Panels J and K show theidentification of B220+CD138minusCD95+GL7+ germinal center B cells Panels LndashO illustrate the gating for B220+CD19+CD5minusGL7minusCD23+CD21loint naive follicular Bcells FSC = forward side scatter

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 7

Activated peripheral B cells contribute todisease severity in MOGp35-55-induced EAETo determine the compartment-specific pathogenic role ofB cells in the induction and perpetuation of the EAE animalmodel CD19Cre+minus α4-integrinflfl mice were immunizedwith MOGp35-55 As demonstrated B cells in these mice havea diminished capacity to enter the CNS (figure 1E) Adoptivetransfer of purified CD19+ B cells from CD19Cre+minus α4-integrinflfl mice and C57BL6 WT mice immunized withrMOG1-125 or the control antigen OVA323-339 worsened the

clinical course of EAE in recipient mice immunized withMOGp35-55 (figure 5H) although these changes were notsignificant

DiscussionThere is accumulating evidence to suggest that B cells andmyelin-specific antibodies play crucial roles in the pathogen-esis of MS Whether B-cell depletion mediates its beneficial

Figure 4 CD19+ B cells in primary and secondary lymphoid organs of CD19Cre+minus α4-integrinflfl mice are phenotypicallysimilar to wild-type B cells during MOGp35-55-induced experimental autoimmune encephalomyelitis

(A) In the bonemarrow (B) lymph nodes and (C) spleen the percentage of CD19+CD11b+CD23minusB220minus B1 B cells CD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cells CD19+CD5+CD1dhi B10 B cells CD19+B220+CD1d+CD5minusCD23minusCD21+ marginal zone (MGZ) B cells andCD19lowndashCD138+B220lowndash plasma blast (PB) was similar in CD19Cre+minus α4-integrinflfl mice and C57BL6 WT controls at day 13 during early activeEAE Lymphocytes were immunophenotyped by multiparameter flow cytometry

Figure 5ActivatedB cells outside of theCNSdrive disease activity inmyelin oligodendrocyte glycoprotein peptide (MOGp35-

55)-induced experimental autoimmune encephalomyelitis (EAE) in an antigen no-specific manner

The serum expression of the cytokines(A) IL-17A (B) IL-10 (C) IL-4 (D) IL-5 and(E) IFNγ as measured by ELISA duringacute and chronic MOGp35-55-inducedEAE on days 13 19 and 29 was com-parable between CD19Cre+minus α4-integ-rinflfl mice and C57BL6 wild-type (WT)control mice Serum levels of (F) IgMand (G) IgG measured by ELSA at thesame time points were also similar be-tween mouse strains (H) Adoptivetransfer of purified CD19+ B cells fromCD19Cre+minus α4-integrinflfl mice orC57BL6 WT control mice immunizedwith recombinant (r)MOG1-125 or oval-bumin (OVA)323-339 into CD19+minus α4-integrinflfl mice that were then imme-diately immunized with MOGp35-55

caused worse clinical EAE than wasobserved in MOGp35-55-immunizedC57BL6 WT control mice (ldquoC57BL6rdquo)that did not receive adoptively trans-ferred CD19+ B cells

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

effects in patients with MS through B cells in the periphery inthe CNS or both is currently not completely understood

Other investigators investigated the role of α4-integrin deficiencyin CD19+ B cells in EAE23 These investigators showed thatB-cell very late activation antigen-4ndashdeficient mice developedmore severe clinical disease than control mice after active in-duction of EAE with MOGp35-55 and that that this clinical effectis due to the requirement of regulatory B cells (Bregs) for α4-integrin to enter the CNS Bregs were defined as CD19+IL-10+

or CD1dhiCD5+ B-cell subsets Another group of investigatorsalso testedCD19Cre Itga4flflmice inMOGp35-55-induced activeEAE24 CD19Cre Itga4flfl mice developed more severe EAEthan controlmice and these investigators demonstrated a criticalrole for α4-integrin on the generation of Bregs in peripheralimmune organs

Our own results diverge from these reports in that we couldnot detect a difference in active EAE disease severity betweenCD19Cre+minus α4-integrinflfl mice and C57BL6 WT controlmice These experiments were conducted 15 times (table)and we are confident in our findings Differences in EAEdisease severity and phenotype can be affected by conditionsunder which mouse colonies are maintained as well as otherfactors that may affect the overall inflammatory milieu of ex-perimental conditions This may explain the differences inresults It is also conceivable that different mice were used bythe other investigators for their experiments The inves-tigators of the 2 previous studies state that they usedCD19Creα4flfl mice23 or that they used CD19Cre Itga4flfl

mice25 It is not stated whether mice were heterozygous orhomozygous for CD19cre or a mixture of both As stated inthe Methods section CD19Cre++ mice behave functionallyvery similarly to B-cellndashdeficient mice which develop moresevere EAE when immunized with MOGp35-55

26

We found one potentially meaningful biological difference be-tween CD19Cre+minus α4-integrinflfl mice and C57BL6 controlmice There was decreased MOGp35-55-specific CD4+ T-cellproliferation in spleens which is an expected result given thatα4-integrin can participate in costimulation of CD4+T cells1819

The observation that CD19+ B cells of CD19Cre+minus α4-integrinflfl mice did deplete α4-integrin and that they weresignificantly less capable of entering the CNS after mice wereimmunized with MOGp35-55 suggests 3 interpretations (1)Antigen-activated B cells contribute to disease burden in EAEin a seemingly dose-dependent manner (2) they do not ab-solutely require recognition of a CNS autoantigen or a mimicthereof to do so and (3) they do not require full α4-integrinndashmediated access to the CNS to exacerbate diseaseThe first interpretation is supported by work of other inves-tigators who have previously demonstrated that B-cellndashdeficient C57BL6 mice are resistant to active rMOG-inducedEAE26 and that the administration of a B-cellndashdepletingmonoclonal antibody prevents or reverses established rMOG-induced EAE in C57BL6 mice21 Weber and colleagues21

further showed that B-cell depletion results in a reduction ofMOGp35-55-specific Th1 and Th17 cells Our own data suggestthat the differentiation ofMOGp35-55-specific T cells to becomeencephalitogenic does not absolutely require the presentationof autoantigen but is likely driven by other B-cell factors in-cluding soluble inflammatory mediators The third in-terpretation of this experiment is supported by ourdemonstration that B cells from CD19Cre+minus α4-integrinflfl

mice are significantly impaired in their ability to enter the CNS(figure 1E) Finally it should be emphasized that our findingsdo not conclusively rule out a role of antigen-specific B cells inthe CNS on the course of CNS autoimmunity as thresholdeffects may be difficult to detect with the EAE experiments thatwere conducted

Our data suggest that the beneficial effects of anti-CD20therapies in patients with MS are mediated through theireffects on peripheral B cells We also conclude that benefitsobserved in patients with MS during anti-α4-integrin therapyare likely not predominantly due to its effect of suppressingthe migration of B cells into the CNS The interpretation ofour findings should be limited to early forms of MS as theEAE model that was used and the observation period that wasused do not provide insight into late or progressive forms ofCNS autoimmunity Magliozzi et al27 were the first inves-tigators to show that some patients with secondary-progressive MS develop lymphoid tissue in the meningesthat resemble B-cell follicles in secondary lymphoid organsB cells in these structures may be susceptible to anti-CD20therapies with monoclonal antibodies or small molecules andit is conceivable that their depletion may benefit patientsafflicted with that MS phenotype

AcknowledgmentThe authors thank Dr Thalia Papayannopoulou at theUniversity of Washington for providing them with α4-integrinfloxflox mice Dr Stuve was funded by a Merit Reviewgrant (federal award document number [FAIN]I01BX001674) from the United States (US) Departmentof Veterans Affairs Biomedical Laboratory Research andDevelopment

Study fundingNo targeted funding reported

DisclosureRZ Hussain PC Cravens WA Miller-Little R Doelger VGrandos and E Herndon report no disclosures DT Okudareceived advisory and consulting fees from Celgene Gen-entech Genzyme EMD Serono and Novartis and researchsupport from Biogen served on the scientific advisory boardof Osmotica and served on the speakersrsquo bureau of AcordaGenzyme and Teva TN Eagar serves as section editor ofArchives of Pathology and received research support from theNIH O Stuve served on the scientific advisory boards of TGTherapeutics and Genentech-Roche served on the editorialboard of Therapeutic Advances in Neurological Disorders

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 9

consulted for Celgene EMD Serono and Novartis andreceived research support from Sanofi and GenzymeDisclosures available NeurologyorgNN

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationNovember 2 2018 Accepted in final form February 1 2019

References1 Hauser SL Waubant E Arnold DL et al B-cell depletion with rituximab in relapsing-

remitting multiple sclerosis N Engl J Med 2008358676ndash6882 Hawker K OrsquoConnor P Freedman MS et al Rituximab in patients with primary

progressive multiple sclerosis results of a randomized double-blind placebo-controlled multicenter trial Ann Neurol 200966460ndash471

3 Hauser SL Bar-Or A Comi G et al Ocrelizumab versus interferon beta-1a in re-lapsing multiple sclerosis N Engl J Med 2017376221ndash234

4 Montalban X Hauser SL Kappos L et al Ocrelizumab versus placebo in primaryprogressive multiple sclerosis N Engl J Med 2017376209ndash220

5 Yednock TA Cannon C Fritz LC Sanchez-Madrid F Steinman L Karin N Pre-vention of experimental autoimmune encephalomyelitis by antibodies against alpha 4beta 1 integrin Nature 199235663ndash66

6 Frohman EM Racke MK Raine CS Multiple sclerosismdashthe plaque and its patho-genesis N Engl J Med 2006354942ndash955

7 Stuve O Marra CM Jerome KR et al Immune surveillance in multiple sclerosispatients treated with natalizumab Ann Neurol 200659743ndash747

8 Haines JL Ter Minassian M Bazyk A et al A complete genomic screen for multiplesclerosis underscores a role for the major histocompatability complex The MultipleSclerosis Genetics Group Nat Genet 199613469ndash471

9 Sawcer S Jones HB Feakes R et al A genome screen in multiple sclerosis revealssusceptibility loci on chromosome 6p21 and 17q22 Nat Genet 199613464ndash468

10 Yang JT Rayburn H Hynes RO Cell adhesion events mediated by alpha 4 integrinsare essential in placental and cardiac development Development 1995121549ndash560

11 Sternberg N Hamilton D Bacteriophage P1 site-specific recombination I Re-combination between loxP sites J Mol Biol 1981150467ndash486

12 Scott LM Priestley GV Papayannopoulou T Deletion of alpha4 integrins from adulthematopoietic cells reveals roles in homeostasis regeneration and homing Mol CellBiol 2003239349ndash9360

13 Hussain RZ Miller-Little WA Doelger R et al Defining standard enzymatic disso-ciation methods for individual brains and spinal cords in EAE Neurol NeuroimmunolNeuroinflamm 20185e437 doi 101212NXI0000000000000437

14 Cravens PD Hussain RZ Zacharias TE et al Lymph node-derived donor encepha-litogenic CD4+ T cells in C57BL6 mice adoptive transfer experimental autoimmuneencephalomyelitis highly express GM-CSF and T-bet J Neuroinflammation 2011873

15 Karandikar NJ Crawford MP Yan X et al Glatiramer acetate (Copaxone) therapyinduces CD8(+) T cell responses in patients with multiple sclerosis J Clin Invest2002109641ndash649

16 Gu H Marth JD Orban PC Mossmann H Rajewsky K Deletion of a DNA poly-merase beta gene segment in T cells using cell type-specific gene targeting Science1994265103ndash106

17 Rickert RC Roes J Rajewsky K B lymphocyte-specific Cre-mediated mutagenesis inmice Nucleic Acids Res 1997251317ndash1318

18 Davis LS Oppenheimer-Marks N Bednarczyk JL McIntyre BW Lipsky PE Fibro-nectin promotes proliferation of naive and memory T cells by signaling through boththe VLA-4 and VLA-5 integrin molecules J Immunol 1990145785ndash793

19 Shimizu Y van Seventer GA Horgan KJ Shaw S Costimulation of proliferativeresponses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 withfibronectin or VLA-6 with laminin J Immunol 199014559ndash67

20 Theien BE Vanderlugt CL Eagar TN et al Discordant effects of anti-VLA-4 treat-ment before and after onset of relapsing experimental autoimmune encephalomyelitisJ Clin Invest 2001107995ndash1006

21 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

22 Krumbholz M Meinl I Kumpfel T Hohlfeld R Meinl E Natalizumab dispropor-tionately increases circulating pre-B and B cells in multiple sclerosis Neurology 2008711350ndash1354

23 Lehmann-Horn K Sagan SA Winger RC et al CNS accumulation of regulatoryB cells is VLA-4-dependent Neurol Neuroimmunol Neuroinflamm 20163e212 doi101212NXI0000000000000212

24 Glatigny S Wagner CA Bettelli E Correction cutting edge integrin alpha4 is re-quired for regulatory B cell control of experimental autoimmune encephalomyelitisJ Immunol 20171983756

25 Glatigny S Duhen R Oukka M Bettelli E Cutting edge loss of alpha4 integrinexpression differentially affects the homing of Th1 and Th17 cells J Immunol 20111876176ndash6179

26 Lyons JA SanM HappMP Cross AH B cells are critical to induction of experimentalallergic encephalomyelitis by protein but not by a short encephalitogenic peptide EurJ Immunol 1999293432ndash3439

27 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

Appendix Authors

Name Location Role Contribution

Rehana ZHussainMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

Petra CCravensPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

William AMiller-Little BA

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

RichardDoelgerMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

ValerieGranadosPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the data andanalyzed the data

EmilyHerndonMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Darin TOkudaMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Todd NEagar PhD

Houston MethodistHospital HoustonTX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

OlafStuve MDPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

DOI 101212NXI000000000000056320196 Neurol Neuroimmunol Neuroinflamm

Rehana Z Hussain Petra D Cravens William A Miller-Little et al disease model

mediated EAEminus4-integrin deficiency in B cells does not affect disease in a T-cellα

This information is current as of April 16 2019

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

ServicesUpdated Information amp

httpnnneurologyorgcontent64e563fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent64e563fullhtmlref-list-1

This article cites 27 articles 7 of which you can access for free at

Citations httpnnneurologyorgcontent64e563fullhtmlotherarticles

This article has been cited by 1 HighWire-hosted articles

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httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

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Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

CORRECTION

α4-integrin deficiency in B cells does not affect disease in aT-cellndashmediated EAE disease modelNeurol Neuroimmunol Neuroinflamm 20196e585 doi101212NXI0000000000000585

In the article ldquoα4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAEdisease model by Hussain et al1 first published online April 16 2019 funding for the papershould have been listed in the Study Funding section The authors regret the error

Reference1 Hussain RZ Cravens PD Miller-Little WA et al α4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAE

disease model Neurol Neuroimmunol Neuroinflamm 20196e563

Copyright copy 2019 American Academy of Neurology 1

Copyright copy 2019 American Academy of Neurology Unauthorized reproduction of this article is prohibited

Page 6: α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency in B cells does not affect disease in a T-cell–mediated EAE disease model Rehana

(figure 2G) Also the expression of the costimulator mole-cules CD80 and CD86 and the first apoptosis signal receptorCD95 was comparable in brain-derived B cells fromCD19Cre+minus α4-integrinflfl mice and C57BL6 WT con-trols (figure 2H)

The composition of B-cell subsets in the brainof CD19Cre+2 α4-integrinflfl mice isunchanged during MOGp35-55-induced EAETo determine whether the deletion of α4-integrin on CD19+

B cells affects B-cell subsets differentially in their capability toenter the CNS the percentage of innate and adaptiveB-lymphocyte subsets including CD11b+CD23minusB220minus B1B cells CD11b+CD23minusB220lominusCD5minus B1b B cellsCD11b+CD23minusB220lominusCD5+ B1a B cellsCD19+CD5+CD1dhi B10 B cells CD19+CD23+CD5minus

naive follicular B cells B220+CD138minusCD95+GL7+ GCsB cells B220+CD1d+CD5minusCD23minusCD21+ marginal zone

(MGZ) B cells CD19lowndashCD138+B220lowndash plasma blast(PB) and CD19minusCD138+ PCs was assessed by multipa-rameter flow cytometry during acute MOGp35-55-inducedEAE at day 13 during early active EAE (figure 2I) day 19during maximum EAE disease activity (figure 2J) and day 29during chronic EAE (figure 2K) after active induction orEAE with MOGp35-55 and was found to be similar inCD19Cre+minus α4-integrinflfl mice and C57BL6 WT con-trols at all time points A gating strategy used to identifysome of the B-cell subsets is shown in figure 3

The composition of B-cell subsets in primaryand secondary lymphoid organs of CD19Cre+2

α4-integrinflfl mice is similar to those of WTcontrol mice during MOGp35-55-induced EAETo assess the effect of α4-integrin ablation on CD19+ B cellson B-cell phenotypes in lymphoid organs innate and adaptiveB-lymphocyte subsets were characterized by flow cytometry

Figure 2 CD19+ B cells in the CNS of CD19Cre+minus α4-integrinflfl mice are phenotypically similar to wild-type (WT) B cellsduring myelin oligodendrocyte glycoprotein peptide (MOGp35-55)-induced experimental autoimmuneencephalomyelitis

Experimental animals were killed at day 13 during the acute phase of MOGp35-55-induced experimental autoimmune encephalomyelitis (EAE) and B cells indisease-relevant compartmentswere immunophenotyped bymultiparameter flow cytometry and intracellular cytokine staining Comparedwith B cells fromC57BL6 WT control mice the percentage of CD19+ B cells expressing interferon gamma (IFNγ) interleukin (IL)-6 IL-10 or IL-17 in CD19Cre+minus α4-integrinflfl

mice was similar in the (A) bone marrow (BM) (B) spleen (C) lymph node (LNs) and (D) brain of CD19Cre+minus α4-integrinflfl mice The percentages of CD19+

B220+ B cells expressing IgD IgM IgG and MHC II in (E) LNs and (F) spleen were comparable in both mouse strains (G) In the CNS the surface expression ofMHC II was similar in CD19Cre+minus α4-integrinflfl mice and controls (H) In the brain the expression of the costimulator molecules CD80 and CD86 and the firstapoptosis signal receptor CD95 was indistinguishable between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT controls The percentage ofCD11b+CD23B220lo B1 B cells CD11b+CD23B220lominusCD5+ B1a B cells CD11b+CD23B220lominusCD5minus B1b B cells B220+CD5+CD1dhi B10 B cells CD19+CD23+CD5minus

naive follicular B cells (FB) B220+CD138minusCD95+GL7+ germinal center (GC) B cells B220+CD1d+CD5minusCD23minusCD21+marginal zone (MGZ) B cells CD138+B220lowndash

plasma blast (PB) and B220minusCD138+ plasma cells (PC) was similar in both mouse strains at (I) day 13 during early active EAE (J) day 19 during maximum EAEdisease activity and (K) day 29 during chronic EAE Lymphocytes were immunophenotyped by multiparameter flow cytometry A gating strategy used toidentify some of the B-cell subsets is shown in Figure 3 p lt 001

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

We were unable to detect a change in composition ofCD19+CD11b+CD23minusB220minus B1 B cellsCD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cellsCD19+CD5+CD1dhi B10 B cellsCD19+B220+CD1d+CD5minusCD23minusCD21+ MGZ B cells andCD19lowndashCD138+B220lowndash PB CD19Cre+minus α4-integrinflfl

mice and C57BL6 WT controls at day 13 during early activeEAE in the BM (figure 4A) LNs (Figure 4B) and spleen(figure 4C)

Serum soluble inflammatory markers areunchanged in CD19Cre+2 α4-integrinflfl miceduring different stages of MOGp35-55-induced EAEDeletion of α4-integrin on CD19+ B cells diminishes theability of CD19+ B cells to enter the CNS during acuteMOGp35-55-induced EAE (figure 1E) It is conceivable thatinflammatory B cells accumulate in peripheral blood alteringthe inflammatory milieu in that compartment This wasobserved in patients withMS treated with natalizumab22 Wefound that the serum expression of the cytokines IL-17A(figure 5A) IL-10 (Figure 5B) IL-4 (Figure 5C) IL-5

(figure 5D) and IFNγ (figure 5E) as measured by ELISA(ELISA) during acute and chronic MOGp35-55-induced EAEon days 13 19 and 29 was comparable betweenCD19Cre+minus α4-integrinflflmice and C57BL6 WT controlmice

Serum Ig levels are unchanged in CD19Cre+2

α4-integrinflfl mice during different stages ofMOGp35-55-induced EAETo further investigate whether the sequestration of CD19+

B cells through deletion of α4-integrin affects their acti-vation proliferation and antibody secretion IgM and IgGlevels in serum were determined by ELISA Serum levels ofIgM (figure 5F) and IgG (figure 5G) as measured byELISA were also similar between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice on days13 19 and 29 of MOGp35-55-induced EAE Given that theabsolute levels of IgM and IgG were similar between themouse strains and that other investigators previouslyshowed that B cells from mice on the C57BL6 back-ground do not readily recognize MOGp35-55

21

we did not test the serum levels of MOGp35-55-specific IgMor IgG

Figure 3 Immunophenotyping of murine B cells

B-lymphocyte subsets were immunophenotyped by multiparameter flow cytometry A gating strategy that was used to identify some of the B-cell sub-populations is shown (A) First cellular area height and width measurements were obtained in a channel with linear scale to gate on singlets and excludedoublets (B) Next gates were set to include CD45+ leukocytes and exclude FSClow cell debris out (C) CD138 and B220 were used to gate on plasmablasts(CD138+B220lowndash) plasma cells (CD138+B220ndash) and B cells (B220+) Panels DndashF show the gating for (E) B220+CD1d+CD5minusCD23minusCD21+IgM+ marginal zone Bcells (E) B220+CD1d+CD5minusCD23minusCD21minusIgM+ transitional 1 (T1) B cells and (F) B220+CD1d+CD5minusCD23+CD21+IgM+ T2 B cells Panels GndashI show gating for B1 B-cell populations and the identification of (H) CD11b+CD23minusB220lominusCD5minus B1b B cells and (I) CD11b+CD23minusB220lominusCD5+ B1a B cells Panels J and K show theidentification of B220+CD138minusCD95+GL7+ germinal center B cells Panels LndashO illustrate the gating for B220+CD19+CD5minusGL7minusCD23+CD21loint naive follicular Bcells FSC = forward side scatter

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 7

Activated peripheral B cells contribute todisease severity in MOGp35-55-induced EAETo determine the compartment-specific pathogenic role ofB cells in the induction and perpetuation of the EAE animalmodel CD19Cre+minus α4-integrinflfl mice were immunizedwith MOGp35-55 As demonstrated B cells in these mice havea diminished capacity to enter the CNS (figure 1E) Adoptivetransfer of purified CD19+ B cells from CD19Cre+minus α4-integrinflfl mice and C57BL6 WT mice immunized withrMOG1-125 or the control antigen OVA323-339 worsened the

clinical course of EAE in recipient mice immunized withMOGp35-55 (figure 5H) although these changes were notsignificant

DiscussionThere is accumulating evidence to suggest that B cells andmyelin-specific antibodies play crucial roles in the pathogen-esis of MS Whether B-cell depletion mediates its beneficial

Figure 4 CD19+ B cells in primary and secondary lymphoid organs of CD19Cre+minus α4-integrinflfl mice are phenotypicallysimilar to wild-type B cells during MOGp35-55-induced experimental autoimmune encephalomyelitis

(A) In the bonemarrow (B) lymph nodes and (C) spleen the percentage of CD19+CD11b+CD23minusB220minus B1 B cells CD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cells CD19+CD5+CD1dhi B10 B cells CD19+B220+CD1d+CD5minusCD23minusCD21+ marginal zone (MGZ) B cells andCD19lowndashCD138+B220lowndash plasma blast (PB) was similar in CD19Cre+minus α4-integrinflfl mice and C57BL6 WT controls at day 13 during early activeEAE Lymphocytes were immunophenotyped by multiparameter flow cytometry

Figure 5ActivatedB cells outside of theCNSdrive disease activity inmyelin oligodendrocyte glycoprotein peptide (MOGp35-

55)-induced experimental autoimmune encephalomyelitis (EAE) in an antigen no-specific manner

The serum expression of the cytokines(A) IL-17A (B) IL-10 (C) IL-4 (D) IL-5 and(E) IFNγ as measured by ELISA duringacute and chronic MOGp35-55-inducedEAE on days 13 19 and 29 was com-parable between CD19Cre+minus α4-integ-rinflfl mice and C57BL6 wild-type (WT)control mice Serum levels of (F) IgMand (G) IgG measured by ELSA at thesame time points were also similar be-tween mouse strains (H) Adoptivetransfer of purified CD19+ B cells fromCD19Cre+minus α4-integrinflfl mice orC57BL6 WT control mice immunizedwith recombinant (r)MOG1-125 or oval-bumin (OVA)323-339 into CD19+minus α4-integrinflfl mice that were then imme-diately immunized with MOGp35-55

caused worse clinical EAE than wasobserved in MOGp35-55-immunizedC57BL6 WT control mice (ldquoC57BL6rdquo)that did not receive adoptively trans-ferred CD19+ B cells

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

effects in patients with MS through B cells in the periphery inthe CNS or both is currently not completely understood

Other investigators investigated the role of α4-integrin deficiencyin CD19+ B cells in EAE23 These investigators showed thatB-cell very late activation antigen-4ndashdeficient mice developedmore severe clinical disease than control mice after active in-duction of EAE with MOGp35-55 and that that this clinical effectis due to the requirement of regulatory B cells (Bregs) for α4-integrin to enter the CNS Bregs were defined as CD19+IL-10+

or CD1dhiCD5+ B-cell subsets Another group of investigatorsalso testedCD19Cre Itga4flflmice inMOGp35-55-induced activeEAE24 CD19Cre Itga4flfl mice developed more severe EAEthan controlmice and these investigators demonstrated a criticalrole for α4-integrin on the generation of Bregs in peripheralimmune organs

Our own results diverge from these reports in that we couldnot detect a difference in active EAE disease severity betweenCD19Cre+minus α4-integrinflfl mice and C57BL6 WT controlmice These experiments were conducted 15 times (table)and we are confident in our findings Differences in EAEdisease severity and phenotype can be affected by conditionsunder which mouse colonies are maintained as well as otherfactors that may affect the overall inflammatory milieu of ex-perimental conditions This may explain the differences inresults It is also conceivable that different mice were used bythe other investigators for their experiments The inves-tigators of the 2 previous studies state that they usedCD19Creα4flfl mice23 or that they used CD19Cre Itga4flfl

mice25 It is not stated whether mice were heterozygous orhomozygous for CD19cre or a mixture of both As stated inthe Methods section CD19Cre++ mice behave functionallyvery similarly to B-cellndashdeficient mice which develop moresevere EAE when immunized with MOGp35-55

26

We found one potentially meaningful biological difference be-tween CD19Cre+minus α4-integrinflfl mice and C57BL6 controlmice There was decreased MOGp35-55-specific CD4+ T-cellproliferation in spleens which is an expected result given thatα4-integrin can participate in costimulation of CD4+T cells1819

The observation that CD19+ B cells of CD19Cre+minus α4-integrinflfl mice did deplete α4-integrin and that they weresignificantly less capable of entering the CNS after mice wereimmunized with MOGp35-55 suggests 3 interpretations (1)Antigen-activated B cells contribute to disease burden in EAEin a seemingly dose-dependent manner (2) they do not ab-solutely require recognition of a CNS autoantigen or a mimicthereof to do so and (3) they do not require full α4-integrinndashmediated access to the CNS to exacerbate diseaseThe first interpretation is supported by work of other inves-tigators who have previously demonstrated that B-cellndashdeficient C57BL6 mice are resistant to active rMOG-inducedEAE26 and that the administration of a B-cellndashdepletingmonoclonal antibody prevents or reverses established rMOG-induced EAE in C57BL6 mice21 Weber and colleagues21

further showed that B-cell depletion results in a reduction ofMOGp35-55-specific Th1 and Th17 cells Our own data suggestthat the differentiation ofMOGp35-55-specific T cells to becomeencephalitogenic does not absolutely require the presentationof autoantigen but is likely driven by other B-cell factors in-cluding soluble inflammatory mediators The third in-terpretation of this experiment is supported by ourdemonstration that B cells from CD19Cre+minus α4-integrinflfl

mice are significantly impaired in their ability to enter the CNS(figure 1E) Finally it should be emphasized that our findingsdo not conclusively rule out a role of antigen-specific B cells inthe CNS on the course of CNS autoimmunity as thresholdeffects may be difficult to detect with the EAE experiments thatwere conducted

Our data suggest that the beneficial effects of anti-CD20therapies in patients with MS are mediated through theireffects on peripheral B cells We also conclude that benefitsobserved in patients with MS during anti-α4-integrin therapyare likely not predominantly due to its effect of suppressingthe migration of B cells into the CNS The interpretation ofour findings should be limited to early forms of MS as theEAE model that was used and the observation period that wasused do not provide insight into late or progressive forms ofCNS autoimmunity Magliozzi et al27 were the first inves-tigators to show that some patients with secondary-progressive MS develop lymphoid tissue in the meningesthat resemble B-cell follicles in secondary lymphoid organsB cells in these structures may be susceptible to anti-CD20therapies with monoclonal antibodies or small molecules andit is conceivable that their depletion may benefit patientsafflicted with that MS phenotype

AcknowledgmentThe authors thank Dr Thalia Papayannopoulou at theUniversity of Washington for providing them with α4-integrinfloxflox mice Dr Stuve was funded by a Merit Reviewgrant (federal award document number [FAIN]I01BX001674) from the United States (US) Departmentof Veterans Affairs Biomedical Laboratory Research andDevelopment

Study fundingNo targeted funding reported

DisclosureRZ Hussain PC Cravens WA Miller-Little R Doelger VGrandos and E Herndon report no disclosures DT Okudareceived advisory and consulting fees from Celgene Gen-entech Genzyme EMD Serono and Novartis and researchsupport from Biogen served on the scientific advisory boardof Osmotica and served on the speakersrsquo bureau of AcordaGenzyme and Teva TN Eagar serves as section editor ofArchives of Pathology and received research support from theNIH O Stuve served on the scientific advisory boards of TGTherapeutics and Genentech-Roche served on the editorialboard of Therapeutic Advances in Neurological Disorders

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 9

consulted for Celgene EMD Serono and Novartis andreceived research support from Sanofi and GenzymeDisclosures available NeurologyorgNN

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationNovember 2 2018 Accepted in final form February 1 2019

References1 Hauser SL Waubant E Arnold DL et al B-cell depletion with rituximab in relapsing-

remitting multiple sclerosis N Engl J Med 2008358676ndash6882 Hawker K OrsquoConnor P Freedman MS et al Rituximab in patients with primary

progressive multiple sclerosis results of a randomized double-blind placebo-controlled multicenter trial Ann Neurol 200966460ndash471

3 Hauser SL Bar-Or A Comi G et al Ocrelizumab versus interferon beta-1a in re-lapsing multiple sclerosis N Engl J Med 2017376221ndash234

4 Montalban X Hauser SL Kappos L et al Ocrelizumab versus placebo in primaryprogressive multiple sclerosis N Engl J Med 2017376209ndash220

5 Yednock TA Cannon C Fritz LC Sanchez-Madrid F Steinman L Karin N Pre-vention of experimental autoimmune encephalomyelitis by antibodies against alpha 4beta 1 integrin Nature 199235663ndash66

6 Frohman EM Racke MK Raine CS Multiple sclerosismdashthe plaque and its patho-genesis N Engl J Med 2006354942ndash955

7 Stuve O Marra CM Jerome KR et al Immune surveillance in multiple sclerosispatients treated with natalizumab Ann Neurol 200659743ndash747

8 Haines JL Ter Minassian M Bazyk A et al A complete genomic screen for multiplesclerosis underscores a role for the major histocompatability complex The MultipleSclerosis Genetics Group Nat Genet 199613469ndash471

9 Sawcer S Jones HB Feakes R et al A genome screen in multiple sclerosis revealssusceptibility loci on chromosome 6p21 and 17q22 Nat Genet 199613464ndash468

10 Yang JT Rayburn H Hynes RO Cell adhesion events mediated by alpha 4 integrinsare essential in placental and cardiac development Development 1995121549ndash560

11 Sternberg N Hamilton D Bacteriophage P1 site-specific recombination I Re-combination between loxP sites J Mol Biol 1981150467ndash486

12 Scott LM Priestley GV Papayannopoulou T Deletion of alpha4 integrins from adulthematopoietic cells reveals roles in homeostasis regeneration and homing Mol CellBiol 2003239349ndash9360

13 Hussain RZ Miller-Little WA Doelger R et al Defining standard enzymatic disso-ciation methods for individual brains and spinal cords in EAE Neurol NeuroimmunolNeuroinflamm 20185e437 doi 101212NXI0000000000000437

14 Cravens PD Hussain RZ Zacharias TE et al Lymph node-derived donor encepha-litogenic CD4+ T cells in C57BL6 mice adoptive transfer experimental autoimmuneencephalomyelitis highly express GM-CSF and T-bet J Neuroinflammation 2011873

15 Karandikar NJ Crawford MP Yan X et al Glatiramer acetate (Copaxone) therapyinduces CD8(+) T cell responses in patients with multiple sclerosis J Clin Invest2002109641ndash649

16 Gu H Marth JD Orban PC Mossmann H Rajewsky K Deletion of a DNA poly-merase beta gene segment in T cells using cell type-specific gene targeting Science1994265103ndash106

17 Rickert RC Roes J Rajewsky K B lymphocyte-specific Cre-mediated mutagenesis inmice Nucleic Acids Res 1997251317ndash1318

18 Davis LS Oppenheimer-Marks N Bednarczyk JL McIntyre BW Lipsky PE Fibro-nectin promotes proliferation of naive and memory T cells by signaling through boththe VLA-4 and VLA-5 integrin molecules J Immunol 1990145785ndash793

19 Shimizu Y van Seventer GA Horgan KJ Shaw S Costimulation of proliferativeresponses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 withfibronectin or VLA-6 with laminin J Immunol 199014559ndash67

20 Theien BE Vanderlugt CL Eagar TN et al Discordant effects of anti-VLA-4 treat-ment before and after onset of relapsing experimental autoimmune encephalomyelitisJ Clin Invest 2001107995ndash1006

21 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

22 Krumbholz M Meinl I Kumpfel T Hohlfeld R Meinl E Natalizumab dispropor-tionately increases circulating pre-B and B cells in multiple sclerosis Neurology 2008711350ndash1354

23 Lehmann-Horn K Sagan SA Winger RC et al CNS accumulation of regulatoryB cells is VLA-4-dependent Neurol Neuroimmunol Neuroinflamm 20163e212 doi101212NXI0000000000000212

24 Glatigny S Wagner CA Bettelli E Correction cutting edge integrin alpha4 is re-quired for regulatory B cell control of experimental autoimmune encephalomyelitisJ Immunol 20171983756

25 Glatigny S Duhen R Oukka M Bettelli E Cutting edge loss of alpha4 integrinexpression differentially affects the homing of Th1 and Th17 cells J Immunol 20111876176ndash6179

26 Lyons JA SanM HappMP Cross AH B cells are critical to induction of experimentalallergic encephalomyelitis by protein but not by a short encephalitogenic peptide EurJ Immunol 1999293432ndash3439

27 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

Appendix Authors

Name Location Role Contribution

Rehana ZHussainMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

Petra CCravensPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

William AMiller-Little BA

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

RichardDoelgerMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

ValerieGranadosPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the data andanalyzed the data

EmilyHerndonMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Darin TOkudaMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Todd NEagar PhD

Houston MethodistHospital HoustonTX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

OlafStuve MDPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

DOI 101212NXI000000000000056320196 Neurol Neuroimmunol Neuroinflamm

Rehana Z Hussain Petra D Cravens William A Miller-Little et al disease model

mediated EAEminus4-integrin deficiency in B cells does not affect disease in a T-cellα

This information is current as of April 16 2019

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

ServicesUpdated Information amp

httpnnneurologyorgcontent64e563fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent64e563fullhtmlref-list-1

This article cites 27 articles 7 of which you can access for free at

Citations httpnnneurologyorgcontent64e563fullhtmlotherarticles

This article has been cited by 1 HighWire-hosted articles

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseases

httpnnneurologyorgcgicollectionall_immunologyAll Immunology

httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the

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nextAn erratum has been published regarding this article Please see

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

Reprints

httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

CORRECTION

α4-integrin deficiency in B cells does not affect disease in aT-cellndashmediated EAE disease modelNeurol Neuroimmunol Neuroinflamm 20196e585 doi101212NXI0000000000000585

In the article ldquoα4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAEdisease model by Hussain et al1 first published online April 16 2019 funding for the papershould have been listed in the Study Funding section The authors regret the error

Reference1 Hussain RZ Cravens PD Miller-Little WA et al α4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAE

disease model Neurol Neuroimmunol Neuroinflamm 20196e563

Copyright copy 2019 American Academy of Neurology 1

Copyright copy 2019 American Academy of Neurology Unauthorized reproduction of this article is prohibited

Page 7: α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency in B cells does not affect disease in a T-cell–mediated EAE disease model Rehana

We were unable to detect a change in composition ofCD19+CD11b+CD23minusB220minus B1 B cellsCD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cellsCD19+CD5+CD1dhi B10 B cellsCD19+B220+CD1d+CD5minusCD23minusCD21+ MGZ B cells andCD19lowndashCD138+B220lowndash PB CD19Cre+minus α4-integrinflfl

mice and C57BL6 WT controls at day 13 during early activeEAE in the BM (figure 4A) LNs (Figure 4B) and spleen(figure 4C)

Serum soluble inflammatory markers areunchanged in CD19Cre+2 α4-integrinflfl miceduring different stages of MOGp35-55-induced EAEDeletion of α4-integrin on CD19+ B cells diminishes theability of CD19+ B cells to enter the CNS during acuteMOGp35-55-induced EAE (figure 1E) It is conceivable thatinflammatory B cells accumulate in peripheral blood alteringthe inflammatory milieu in that compartment This wasobserved in patients withMS treated with natalizumab22 Wefound that the serum expression of the cytokines IL-17A(figure 5A) IL-10 (Figure 5B) IL-4 (Figure 5C) IL-5

(figure 5D) and IFNγ (figure 5E) as measured by ELISA(ELISA) during acute and chronic MOGp35-55-induced EAEon days 13 19 and 29 was comparable betweenCD19Cre+minus α4-integrinflflmice and C57BL6 WT controlmice

Serum Ig levels are unchanged in CD19Cre+2

α4-integrinflfl mice during different stages ofMOGp35-55-induced EAETo further investigate whether the sequestration of CD19+

B cells through deletion of α4-integrin affects their acti-vation proliferation and antibody secretion IgM and IgGlevels in serum were determined by ELISA Serum levels ofIgM (figure 5F) and IgG (figure 5G) as measured byELISA were also similar between CD19Cre+minus α4-integrinflfl mice and C57BL6 WT control mice on days13 19 and 29 of MOGp35-55-induced EAE Given that theabsolute levels of IgM and IgG were similar between themouse strains and that other investigators previouslyshowed that B cells from mice on the C57BL6 back-ground do not readily recognize MOGp35-55

21

we did not test the serum levels of MOGp35-55-specific IgMor IgG

Figure 3 Immunophenotyping of murine B cells

B-lymphocyte subsets were immunophenotyped by multiparameter flow cytometry A gating strategy that was used to identify some of the B-cell sub-populations is shown (A) First cellular area height and width measurements were obtained in a channel with linear scale to gate on singlets and excludedoublets (B) Next gates were set to include CD45+ leukocytes and exclude FSClow cell debris out (C) CD138 and B220 were used to gate on plasmablasts(CD138+B220lowndash) plasma cells (CD138+B220ndash) and B cells (B220+) Panels DndashF show the gating for (E) B220+CD1d+CD5minusCD23minusCD21+IgM+ marginal zone Bcells (E) B220+CD1d+CD5minusCD23minusCD21minusIgM+ transitional 1 (T1) B cells and (F) B220+CD1d+CD5minusCD23+CD21+IgM+ T2 B cells Panels GndashI show gating for B1 B-cell populations and the identification of (H) CD11b+CD23minusB220lominusCD5minus B1b B cells and (I) CD11b+CD23minusB220lominusCD5+ B1a B cells Panels J and K show theidentification of B220+CD138minusCD95+GL7+ germinal center B cells Panels LndashO illustrate the gating for B220+CD19+CD5minusGL7minusCD23+CD21loint naive follicular Bcells FSC = forward side scatter

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 7

Activated peripheral B cells contribute todisease severity in MOGp35-55-induced EAETo determine the compartment-specific pathogenic role ofB cells in the induction and perpetuation of the EAE animalmodel CD19Cre+minus α4-integrinflfl mice were immunizedwith MOGp35-55 As demonstrated B cells in these mice havea diminished capacity to enter the CNS (figure 1E) Adoptivetransfer of purified CD19+ B cells from CD19Cre+minus α4-integrinflfl mice and C57BL6 WT mice immunized withrMOG1-125 or the control antigen OVA323-339 worsened the

clinical course of EAE in recipient mice immunized withMOGp35-55 (figure 5H) although these changes were notsignificant

DiscussionThere is accumulating evidence to suggest that B cells andmyelin-specific antibodies play crucial roles in the pathogen-esis of MS Whether B-cell depletion mediates its beneficial

Figure 4 CD19+ B cells in primary and secondary lymphoid organs of CD19Cre+minus α4-integrinflfl mice are phenotypicallysimilar to wild-type B cells during MOGp35-55-induced experimental autoimmune encephalomyelitis

(A) In the bonemarrow (B) lymph nodes and (C) spleen the percentage of CD19+CD11b+CD23minusB220minus B1 B cells CD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cells CD19+CD5+CD1dhi B10 B cells CD19+B220+CD1d+CD5minusCD23minusCD21+ marginal zone (MGZ) B cells andCD19lowndashCD138+B220lowndash plasma blast (PB) was similar in CD19Cre+minus α4-integrinflfl mice and C57BL6 WT controls at day 13 during early activeEAE Lymphocytes were immunophenotyped by multiparameter flow cytometry

Figure 5ActivatedB cells outside of theCNSdrive disease activity inmyelin oligodendrocyte glycoprotein peptide (MOGp35-

55)-induced experimental autoimmune encephalomyelitis (EAE) in an antigen no-specific manner

The serum expression of the cytokines(A) IL-17A (B) IL-10 (C) IL-4 (D) IL-5 and(E) IFNγ as measured by ELISA duringacute and chronic MOGp35-55-inducedEAE on days 13 19 and 29 was com-parable between CD19Cre+minus α4-integ-rinflfl mice and C57BL6 wild-type (WT)control mice Serum levels of (F) IgMand (G) IgG measured by ELSA at thesame time points were also similar be-tween mouse strains (H) Adoptivetransfer of purified CD19+ B cells fromCD19Cre+minus α4-integrinflfl mice orC57BL6 WT control mice immunizedwith recombinant (r)MOG1-125 or oval-bumin (OVA)323-339 into CD19+minus α4-integrinflfl mice that were then imme-diately immunized with MOGp35-55

caused worse clinical EAE than wasobserved in MOGp35-55-immunizedC57BL6 WT control mice (ldquoC57BL6rdquo)that did not receive adoptively trans-ferred CD19+ B cells

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

effects in patients with MS through B cells in the periphery inthe CNS or both is currently not completely understood

Other investigators investigated the role of α4-integrin deficiencyin CD19+ B cells in EAE23 These investigators showed thatB-cell very late activation antigen-4ndashdeficient mice developedmore severe clinical disease than control mice after active in-duction of EAE with MOGp35-55 and that that this clinical effectis due to the requirement of regulatory B cells (Bregs) for α4-integrin to enter the CNS Bregs were defined as CD19+IL-10+

or CD1dhiCD5+ B-cell subsets Another group of investigatorsalso testedCD19Cre Itga4flflmice inMOGp35-55-induced activeEAE24 CD19Cre Itga4flfl mice developed more severe EAEthan controlmice and these investigators demonstrated a criticalrole for α4-integrin on the generation of Bregs in peripheralimmune organs

Our own results diverge from these reports in that we couldnot detect a difference in active EAE disease severity betweenCD19Cre+minus α4-integrinflfl mice and C57BL6 WT controlmice These experiments were conducted 15 times (table)and we are confident in our findings Differences in EAEdisease severity and phenotype can be affected by conditionsunder which mouse colonies are maintained as well as otherfactors that may affect the overall inflammatory milieu of ex-perimental conditions This may explain the differences inresults It is also conceivable that different mice were used bythe other investigators for their experiments The inves-tigators of the 2 previous studies state that they usedCD19Creα4flfl mice23 or that they used CD19Cre Itga4flfl

mice25 It is not stated whether mice were heterozygous orhomozygous for CD19cre or a mixture of both As stated inthe Methods section CD19Cre++ mice behave functionallyvery similarly to B-cellndashdeficient mice which develop moresevere EAE when immunized with MOGp35-55

26

We found one potentially meaningful biological difference be-tween CD19Cre+minus α4-integrinflfl mice and C57BL6 controlmice There was decreased MOGp35-55-specific CD4+ T-cellproliferation in spleens which is an expected result given thatα4-integrin can participate in costimulation of CD4+T cells1819

The observation that CD19+ B cells of CD19Cre+minus α4-integrinflfl mice did deplete α4-integrin and that they weresignificantly less capable of entering the CNS after mice wereimmunized with MOGp35-55 suggests 3 interpretations (1)Antigen-activated B cells contribute to disease burden in EAEin a seemingly dose-dependent manner (2) they do not ab-solutely require recognition of a CNS autoantigen or a mimicthereof to do so and (3) they do not require full α4-integrinndashmediated access to the CNS to exacerbate diseaseThe first interpretation is supported by work of other inves-tigators who have previously demonstrated that B-cellndashdeficient C57BL6 mice are resistant to active rMOG-inducedEAE26 and that the administration of a B-cellndashdepletingmonoclonal antibody prevents or reverses established rMOG-induced EAE in C57BL6 mice21 Weber and colleagues21

further showed that B-cell depletion results in a reduction ofMOGp35-55-specific Th1 and Th17 cells Our own data suggestthat the differentiation ofMOGp35-55-specific T cells to becomeencephalitogenic does not absolutely require the presentationof autoantigen but is likely driven by other B-cell factors in-cluding soluble inflammatory mediators The third in-terpretation of this experiment is supported by ourdemonstration that B cells from CD19Cre+minus α4-integrinflfl

mice are significantly impaired in their ability to enter the CNS(figure 1E) Finally it should be emphasized that our findingsdo not conclusively rule out a role of antigen-specific B cells inthe CNS on the course of CNS autoimmunity as thresholdeffects may be difficult to detect with the EAE experiments thatwere conducted

Our data suggest that the beneficial effects of anti-CD20therapies in patients with MS are mediated through theireffects on peripheral B cells We also conclude that benefitsobserved in patients with MS during anti-α4-integrin therapyare likely not predominantly due to its effect of suppressingthe migration of B cells into the CNS The interpretation ofour findings should be limited to early forms of MS as theEAE model that was used and the observation period that wasused do not provide insight into late or progressive forms ofCNS autoimmunity Magliozzi et al27 were the first inves-tigators to show that some patients with secondary-progressive MS develop lymphoid tissue in the meningesthat resemble B-cell follicles in secondary lymphoid organsB cells in these structures may be susceptible to anti-CD20therapies with monoclonal antibodies or small molecules andit is conceivable that their depletion may benefit patientsafflicted with that MS phenotype

AcknowledgmentThe authors thank Dr Thalia Papayannopoulou at theUniversity of Washington for providing them with α4-integrinfloxflox mice Dr Stuve was funded by a Merit Reviewgrant (federal award document number [FAIN]I01BX001674) from the United States (US) Departmentof Veterans Affairs Biomedical Laboratory Research andDevelopment

Study fundingNo targeted funding reported

DisclosureRZ Hussain PC Cravens WA Miller-Little R Doelger VGrandos and E Herndon report no disclosures DT Okudareceived advisory and consulting fees from Celgene Gen-entech Genzyme EMD Serono and Novartis and researchsupport from Biogen served on the scientific advisory boardof Osmotica and served on the speakersrsquo bureau of AcordaGenzyme and Teva TN Eagar serves as section editor ofArchives of Pathology and received research support from theNIH O Stuve served on the scientific advisory boards of TGTherapeutics and Genentech-Roche served on the editorialboard of Therapeutic Advances in Neurological Disorders

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 9

consulted for Celgene EMD Serono and Novartis andreceived research support from Sanofi and GenzymeDisclosures available NeurologyorgNN

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationNovember 2 2018 Accepted in final form February 1 2019

References1 Hauser SL Waubant E Arnold DL et al B-cell depletion with rituximab in relapsing-

remitting multiple sclerosis N Engl J Med 2008358676ndash6882 Hawker K OrsquoConnor P Freedman MS et al Rituximab in patients with primary

progressive multiple sclerosis results of a randomized double-blind placebo-controlled multicenter trial Ann Neurol 200966460ndash471

3 Hauser SL Bar-Or A Comi G et al Ocrelizumab versus interferon beta-1a in re-lapsing multiple sclerosis N Engl J Med 2017376221ndash234

4 Montalban X Hauser SL Kappos L et al Ocrelizumab versus placebo in primaryprogressive multiple sclerosis N Engl J Med 2017376209ndash220

5 Yednock TA Cannon C Fritz LC Sanchez-Madrid F Steinman L Karin N Pre-vention of experimental autoimmune encephalomyelitis by antibodies against alpha 4beta 1 integrin Nature 199235663ndash66

6 Frohman EM Racke MK Raine CS Multiple sclerosismdashthe plaque and its patho-genesis N Engl J Med 2006354942ndash955

7 Stuve O Marra CM Jerome KR et al Immune surveillance in multiple sclerosispatients treated with natalizumab Ann Neurol 200659743ndash747

8 Haines JL Ter Minassian M Bazyk A et al A complete genomic screen for multiplesclerosis underscores a role for the major histocompatability complex The MultipleSclerosis Genetics Group Nat Genet 199613469ndash471

9 Sawcer S Jones HB Feakes R et al A genome screen in multiple sclerosis revealssusceptibility loci on chromosome 6p21 and 17q22 Nat Genet 199613464ndash468

10 Yang JT Rayburn H Hynes RO Cell adhesion events mediated by alpha 4 integrinsare essential in placental and cardiac development Development 1995121549ndash560

11 Sternberg N Hamilton D Bacteriophage P1 site-specific recombination I Re-combination between loxP sites J Mol Biol 1981150467ndash486

12 Scott LM Priestley GV Papayannopoulou T Deletion of alpha4 integrins from adulthematopoietic cells reveals roles in homeostasis regeneration and homing Mol CellBiol 2003239349ndash9360

13 Hussain RZ Miller-Little WA Doelger R et al Defining standard enzymatic disso-ciation methods for individual brains and spinal cords in EAE Neurol NeuroimmunolNeuroinflamm 20185e437 doi 101212NXI0000000000000437

14 Cravens PD Hussain RZ Zacharias TE et al Lymph node-derived donor encepha-litogenic CD4+ T cells in C57BL6 mice adoptive transfer experimental autoimmuneencephalomyelitis highly express GM-CSF and T-bet J Neuroinflammation 2011873

15 Karandikar NJ Crawford MP Yan X et al Glatiramer acetate (Copaxone) therapyinduces CD8(+) T cell responses in patients with multiple sclerosis J Clin Invest2002109641ndash649

16 Gu H Marth JD Orban PC Mossmann H Rajewsky K Deletion of a DNA poly-merase beta gene segment in T cells using cell type-specific gene targeting Science1994265103ndash106

17 Rickert RC Roes J Rajewsky K B lymphocyte-specific Cre-mediated mutagenesis inmice Nucleic Acids Res 1997251317ndash1318

18 Davis LS Oppenheimer-Marks N Bednarczyk JL McIntyre BW Lipsky PE Fibro-nectin promotes proliferation of naive and memory T cells by signaling through boththe VLA-4 and VLA-5 integrin molecules J Immunol 1990145785ndash793

19 Shimizu Y van Seventer GA Horgan KJ Shaw S Costimulation of proliferativeresponses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 withfibronectin or VLA-6 with laminin J Immunol 199014559ndash67

20 Theien BE Vanderlugt CL Eagar TN et al Discordant effects of anti-VLA-4 treat-ment before and after onset of relapsing experimental autoimmune encephalomyelitisJ Clin Invest 2001107995ndash1006

21 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

22 Krumbholz M Meinl I Kumpfel T Hohlfeld R Meinl E Natalizumab dispropor-tionately increases circulating pre-B and B cells in multiple sclerosis Neurology 2008711350ndash1354

23 Lehmann-Horn K Sagan SA Winger RC et al CNS accumulation of regulatoryB cells is VLA-4-dependent Neurol Neuroimmunol Neuroinflamm 20163e212 doi101212NXI0000000000000212

24 Glatigny S Wagner CA Bettelli E Correction cutting edge integrin alpha4 is re-quired for regulatory B cell control of experimental autoimmune encephalomyelitisJ Immunol 20171983756

25 Glatigny S Duhen R Oukka M Bettelli E Cutting edge loss of alpha4 integrinexpression differentially affects the homing of Th1 and Th17 cells J Immunol 20111876176ndash6179

26 Lyons JA SanM HappMP Cross AH B cells are critical to induction of experimentalallergic encephalomyelitis by protein but not by a short encephalitogenic peptide EurJ Immunol 1999293432ndash3439

27 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

Appendix Authors

Name Location Role Contribution

Rehana ZHussainMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

Petra CCravensPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

William AMiller-Little BA

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

RichardDoelgerMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

ValerieGranadosPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the data andanalyzed the data

EmilyHerndonMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Darin TOkudaMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Todd NEagar PhD

Houston MethodistHospital HoustonTX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

OlafStuve MDPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

DOI 101212NXI000000000000056320196 Neurol Neuroimmunol Neuroinflamm

Rehana Z Hussain Petra D Cravens William A Miller-Little et al disease model

mediated EAEminus4-integrin deficiency in B cells does not affect disease in a T-cellα

This information is current as of April 16 2019

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

ServicesUpdated Information amp

httpnnneurologyorgcontent64e563fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent64e563fullhtmlref-list-1

This article cites 27 articles 7 of which you can access for free at

Citations httpnnneurologyorgcontent64e563fullhtmlotherarticles

This article has been cited by 1 HighWire-hosted articles

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseases

httpnnneurologyorgcgicollectionall_immunologyAll Immunology

httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the

Errata

content64e585fullpdf or page

nextAn erratum has been published regarding this article Please see

Permissions amp Licensing

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

Reprints

httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

CORRECTION

α4-integrin deficiency in B cells does not affect disease in aT-cellndashmediated EAE disease modelNeurol Neuroimmunol Neuroinflamm 20196e585 doi101212NXI0000000000000585

In the article ldquoα4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAEdisease model by Hussain et al1 first published online April 16 2019 funding for the papershould have been listed in the Study Funding section The authors regret the error

Reference1 Hussain RZ Cravens PD Miller-Little WA et al α4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAE

disease model Neurol Neuroimmunol Neuroinflamm 20196e563

Copyright copy 2019 American Academy of Neurology 1

Copyright copy 2019 American Academy of Neurology Unauthorized reproduction of this article is prohibited

Page 8: α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency in B cells does not affect disease in a T-cell–mediated EAE disease model Rehana

Activated peripheral B cells contribute todisease severity in MOGp35-55-induced EAETo determine the compartment-specific pathogenic role ofB cells in the induction and perpetuation of the EAE animalmodel CD19Cre+minus α4-integrinflfl mice were immunizedwith MOGp35-55 As demonstrated B cells in these mice havea diminished capacity to enter the CNS (figure 1E) Adoptivetransfer of purified CD19+ B cells from CD19Cre+minus α4-integrinflfl mice and C57BL6 WT mice immunized withrMOG1-125 or the control antigen OVA323-339 worsened the

clinical course of EAE in recipient mice immunized withMOGp35-55 (figure 5H) although these changes were notsignificant

DiscussionThere is accumulating evidence to suggest that B cells andmyelin-specific antibodies play crucial roles in the pathogen-esis of MS Whether B-cell depletion mediates its beneficial

Figure 4 CD19+ B cells in primary and secondary lymphoid organs of CD19Cre+minus α4-integrinflfl mice are phenotypicallysimilar to wild-type B cells during MOGp35-55-induced experimental autoimmune encephalomyelitis

(A) In the bonemarrow (B) lymph nodes and (C) spleen the percentage of CD19+CD11b+CD23minusB220minus B1 B cells CD19+CD11b+CD23minusB220minusCD5+ B1a B cellsCD19+CD11b+CD23minusB220minusCD5minus B1b B cells CD19+CD5+CD1dhi B10 B cells CD19+B220+CD1d+CD5minusCD23minusCD21+ marginal zone (MGZ) B cells andCD19lowndashCD138+B220lowndash plasma blast (PB) was similar in CD19Cre+minus α4-integrinflfl mice and C57BL6 WT controls at day 13 during early activeEAE Lymphocytes were immunophenotyped by multiparameter flow cytometry

Figure 5ActivatedB cells outside of theCNSdrive disease activity inmyelin oligodendrocyte glycoprotein peptide (MOGp35-

55)-induced experimental autoimmune encephalomyelitis (EAE) in an antigen no-specific manner

The serum expression of the cytokines(A) IL-17A (B) IL-10 (C) IL-4 (D) IL-5 and(E) IFNγ as measured by ELISA duringacute and chronic MOGp35-55-inducedEAE on days 13 19 and 29 was com-parable between CD19Cre+minus α4-integ-rinflfl mice and C57BL6 wild-type (WT)control mice Serum levels of (F) IgMand (G) IgG measured by ELSA at thesame time points were also similar be-tween mouse strains (H) Adoptivetransfer of purified CD19+ B cells fromCD19Cre+minus α4-integrinflfl mice orC57BL6 WT control mice immunizedwith recombinant (r)MOG1-125 or oval-bumin (OVA)323-339 into CD19+minus α4-integrinflfl mice that were then imme-diately immunized with MOGp35-55

caused worse clinical EAE than wasobserved in MOGp35-55-immunizedC57BL6 WT control mice (ldquoC57BL6rdquo)that did not receive adoptively trans-ferred CD19+ B cells

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

effects in patients with MS through B cells in the periphery inthe CNS or both is currently not completely understood

Other investigators investigated the role of α4-integrin deficiencyin CD19+ B cells in EAE23 These investigators showed thatB-cell very late activation antigen-4ndashdeficient mice developedmore severe clinical disease than control mice after active in-duction of EAE with MOGp35-55 and that that this clinical effectis due to the requirement of regulatory B cells (Bregs) for α4-integrin to enter the CNS Bregs were defined as CD19+IL-10+

or CD1dhiCD5+ B-cell subsets Another group of investigatorsalso testedCD19Cre Itga4flflmice inMOGp35-55-induced activeEAE24 CD19Cre Itga4flfl mice developed more severe EAEthan controlmice and these investigators demonstrated a criticalrole for α4-integrin on the generation of Bregs in peripheralimmune organs

Our own results diverge from these reports in that we couldnot detect a difference in active EAE disease severity betweenCD19Cre+minus α4-integrinflfl mice and C57BL6 WT controlmice These experiments were conducted 15 times (table)and we are confident in our findings Differences in EAEdisease severity and phenotype can be affected by conditionsunder which mouse colonies are maintained as well as otherfactors that may affect the overall inflammatory milieu of ex-perimental conditions This may explain the differences inresults It is also conceivable that different mice were used bythe other investigators for their experiments The inves-tigators of the 2 previous studies state that they usedCD19Creα4flfl mice23 or that they used CD19Cre Itga4flfl

mice25 It is not stated whether mice were heterozygous orhomozygous for CD19cre or a mixture of both As stated inthe Methods section CD19Cre++ mice behave functionallyvery similarly to B-cellndashdeficient mice which develop moresevere EAE when immunized with MOGp35-55

26

We found one potentially meaningful biological difference be-tween CD19Cre+minus α4-integrinflfl mice and C57BL6 controlmice There was decreased MOGp35-55-specific CD4+ T-cellproliferation in spleens which is an expected result given thatα4-integrin can participate in costimulation of CD4+T cells1819

The observation that CD19+ B cells of CD19Cre+minus α4-integrinflfl mice did deplete α4-integrin and that they weresignificantly less capable of entering the CNS after mice wereimmunized with MOGp35-55 suggests 3 interpretations (1)Antigen-activated B cells contribute to disease burden in EAEin a seemingly dose-dependent manner (2) they do not ab-solutely require recognition of a CNS autoantigen or a mimicthereof to do so and (3) they do not require full α4-integrinndashmediated access to the CNS to exacerbate diseaseThe first interpretation is supported by work of other inves-tigators who have previously demonstrated that B-cellndashdeficient C57BL6 mice are resistant to active rMOG-inducedEAE26 and that the administration of a B-cellndashdepletingmonoclonal antibody prevents or reverses established rMOG-induced EAE in C57BL6 mice21 Weber and colleagues21

further showed that B-cell depletion results in a reduction ofMOGp35-55-specific Th1 and Th17 cells Our own data suggestthat the differentiation ofMOGp35-55-specific T cells to becomeencephalitogenic does not absolutely require the presentationof autoantigen but is likely driven by other B-cell factors in-cluding soluble inflammatory mediators The third in-terpretation of this experiment is supported by ourdemonstration that B cells from CD19Cre+minus α4-integrinflfl

mice are significantly impaired in their ability to enter the CNS(figure 1E) Finally it should be emphasized that our findingsdo not conclusively rule out a role of antigen-specific B cells inthe CNS on the course of CNS autoimmunity as thresholdeffects may be difficult to detect with the EAE experiments thatwere conducted

Our data suggest that the beneficial effects of anti-CD20therapies in patients with MS are mediated through theireffects on peripheral B cells We also conclude that benefitsobserved in patients with MS during anti-α4-integrin therapyare likely not predominantly due to its effect of suppressingthe migration of B cells into the CNS The interpretation ofour findings should be limited to early forms of MS as theEAE model that was used and the observation period that wasused do not provide insight into late or progressive forms ofCNS autoimmunity Magliozzi et al27 were the first inves-tigators to show that some patients with secondary-progressive MS develop lymphoid tissue in the meningesthat resemble B-cell follicles in secondary lymphoid organsB cells in these structures may be susceptible to anti-CD20therapies with monoclonal antibodies or small molecules andit is conceivable that their depletion may benefit patientsafflicted with that MS phenotype

AcknowledgmentThe authors thank Dr Thalia Papayannopoulou at theUniversity of Washington for providing them with α4-integrinfloxflox mice Dr Stuve was funded by a Merit Reviewgrant (federal award document number [FAIN]I01BX001674) from the United States (US) Departmentof Veterans Affairs Biomedical Laboratory Research andDevelopment

Study fundingNo targeted funding reported

DisclosureRZ Hussain PC Cravens WA Miller-Little R Doelger VGrandos and E Herndon report no disclosures DT Okudareceived advisory and consulting fees from Celgene Gen-entech Genzyme EMD Serono and Novartis and researchsupport from Biogen served on the scientific advisory boardof Osmotica and served on the speakersrsquo bureau of AcordaGenzyme and Teva TN Eagar serves as section editor ofArchives of Pathology and received research support from theNIH O Stuve served on the scientific advisory boards of TGTherapeutics and Genentech-Roche served on the editorialboard of Therapeutic Advances in Neurological Disorders

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 9

consulted for Celgene EMD Serono and Novartis andreceived research support from Sanofi and GenzymeDisclosures available NeurologyorgNN

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationNovember 2 2018 Accepted in final form February 1 2019

References1 Hauser SL Waubant E Arnold DL et al B-cell depletion with rituximab in relapsing-

remitting multiple sclerosis N Engl J Med 2008358676ndash6882 Hawker K OrsquoConnor P Freedman MS et al Rituximab in patients with primary

progressive multiple sclerosis results of a randomized double-blind placebo-controlled multicenter trial Ann Neurol 200966460ndash471

3 Hauser SL Bar-Or A Comi G et al Ocrelizumab versus interferon beta-1a in re-lapsing multiple sclerosis N Engl J Med 2017376221ndash234

4 Montalban X Hauser SL Kappos L et al Ocrelizumab versus placebo in primaryprogressive multiple sclerosis N Engl J Med 2017376209ndash220

5 Yednock TA Cannon C Fritz LC Sanchez-Madrid F Steinman L Karin N Pre-vention of experimental autoimmune encephalomyelitis by antibodies against alpha 4beta 1 integrin Nature 199235663ndash66

6 Frohman EM Racke MK Raine CS Multiple sclerosismdashthe plaque and its patho-genesis N Engl J Med 2006354942ndash955

7 Stuve O Marra CM Jerome KR et al Immune surveillance in multiple sclerosispatients treated with natalizumab Ann Neurol 200659743ndash747

8 Haines JL Ter Minassian M Bazyk A et al A complete genomic screen for multiplesclerosis underscores a role for the major histocompatability complex The MultipleSclerosis Genetics Group Nat Genet 199613469ndash471

9 Sawcer S Jones HB Feakes R et al A genome screen in multiple sclerosis revealssusceptibility loci on chromosome 6p21 and 17q22 Nat Genet 199613464ndash468

10 Yang JT Rayburn H Hynes RO Cell adhesion events mediated by alpha 4 integrinsare essential in placental and cardiac development Development 1995121549ndash560

11 Sternberg N Hamilton D Bacteriophage P1 site-specific recombination I Re-combination between loxP sites J Mol Biol 1981150467ndash486

12 Scott LM Priestley GV Papayannopoulou T Deletion of alpha4 integrins from adulthematopoietic cells reveals roles in homeostasis regeneration and homing Mol CellBiol 2003239349ndash9360

13 Hussain RZ Miller-Little WA Doelger R et al Defining standard enzymatic disso-ciation methods for individual brains and spinal cords in EAE Neurol NeuroimmunolNeuroinflamm 20185e437 doi 101212NXI0000000000000437

14 Cravens PD Hussain RZ Zacharias TE et al Lymph node-derived donor encepha-litogenic CD4+ T cells in C57BL6 mice adoptive transfer experimental autoimmuneencephalomyelitis highly express GM-CSF and T-bet J Neuroinflammation 2011873

15 Karandikar NJ Crawford MP Yan X et al Glatiramer acetate (Copaxone) therapyinduces CD8(+) T cell responses in patients with multiple sclerosis J Clin Invest2002109641ndash649

16 Gu H Marth JD Orban PC Mossmann H Rajewsky K Deletion of a DNA poly-merase beta gene segment in T cells using cell type-specific gene targeting Science1994265103ndash106

17 Rickert RC Roes J Rajewsky K B lymphocyte-specific Cre-mediated mutagenesis inmice Nucleic Acids Res 1997251317ndash1318

18 Davis LS Oppenheimer-Marks N Bednarczyk JL McIntyre BW Lipsky PE Fibro-nectin promotes proliferation of naive and memory T cells by signaling through boththe VLA-4 and VLA-5 integrin molecules J Immunol 1990145785ndash793

19 Shimizu Y van Seventer GA Horgan KJ Shaw S Costimulation of proliferativeresponses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 withfibronectin or VLA-6 with laminin J Immunol 199014559ndash67

20 Theien BE Vanderlugt CL Eagar TN et al Discordant effects of anti-VLA-4 treat-ment before and after onset of relapsing experimental autoimmune encephalomyelitisJ Clin Invest 2001107995ndash1006

21 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

22 Krumbholz M Meinl I Kumpfel T Hohlfeld R Meinl E Natalizumab dispropor-tionately increases circulating pre-B and B cells in multiple sclerosis Neurology 2008711350ndash1354

23 Lehmann-Horn K Sagan SA Winger RC et al CNS accumulation of regulatoryB cells is VLA-4-dependent Neurol Neuroimmunol Neuroinflamm 20163e212 doi101212NXI0000000000000212

24 Glatigny S Wagner CA Bettelli E Correction cutting edge integrin alpha4 is re-quired for regulatory B cell control of experimental autoimmune encephalomyelitisJ Immunol 20171983756

25 Glatigny S Duhen R Oukka M Bettelli E Cutting edge loss of alpha4 integrinexpression differentially affects the homing of Th1 and Th17 cells J Immunol 20111876176ndash6179

26 Lyons JA SanM HappMP Cross AH B cells are critical to induction of experimentalallergic encephalomyelitis by protein but not by a short encephalitogenic peptide EurJ Immunol 1999293432ndash3439

27 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

Appendix Authors

Name Location Role Contribution

Rehana ZHussainMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

Petra CCravensPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

William AMiller-Little BA

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

RichardDoelgerMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

ValerieGranadosPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the data andanalyzed the data

EmilyHerndonMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Darin TOkudaMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Todd NEagar PhD

Houston MethodistHospital HoustonTX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

OlafStuve MDPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

DOI 101212NXI000000000000056320196 Neurol Neuroimmunol Neuroinflamm

Rehana Z Hussain Petra D Cravens William A Miller-Little et al disease model

mediated EAEminus4-integrin deficiency in B cells does not affect disease in a T-cellα

This information is current as of April 16 2019

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

ServicesUpdated Information amp

httpnnneurologyorgcontent64e563fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent64e563fullhtmlref-list-1

This article cites 27 articles 7 of which you can access for free at

Citations httpnnneurologyorgcontent64e563fullhtmlotherarticles

This article has been cited by 1 HighWire-hosted articles

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseases

httpnnneurologyorgcgicollectionall_immunologyAll Immunology

httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the

Errata

content64e585fullpdf or page

nextAn erratum has been published regarding this article Please see

Permissions amp Licensing

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

Reprints

httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

CORRECTION

α4-integrin deficiency in B cells does not affect disease in aT-cellndashmediated EAE disease modelNeurol Neuroimmunol Neuroinflamm 20196e585 doi101212NXI0000000000000585

In the article ldquoα4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAEdisease model by Hussain et al1 first published online April 16 2019 funding for the papershould have been listed in the Study Funding section The authors regret the error

Reference1 Hussain RZ Cravens PD Miller-Little WA et al α4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAE

disease model Neurol Neuroimmunol Neuroinflamm 20196e563

Copyright copy 2019 American Academy of Neurology 1

Copyright copy 2019 American Academy of Neurology Unauthorized reproduction of this article is prohibited

Page 9: α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency in B cells does not affect disease in a T-cell–mediated EAE disease model Rehana

effects in patients with MS through B cells in the periphery inthe CNS or both is currently not completely understood

Other investigators investigated the role of α4-integrin deficiencyin CD19+ B cells in EAE23 These investigators showed thatB-cell very late activation antigen-4ndashdeficient mice developedmore severe clinical disease than control mice after active in-duction of EAE with MOGp35-55 and that that this clinical effectis due to the requirement of regulatory B cells (Bregs) for α4-integrin to enter the CNS Bregs were defined as CD19+IL-10+

or CD1dhiCD5+ B-cell subsets Another group of investigatorsalso testedCD19Cre Itga4flflmice inMOGp35-55-induced activeEAE24 CD19Cre Itga4flfl mice developed more severe EAEthan controlmice and these investigators demonstrated a criticalrole for α4-integrin on the generation of Bregs in peripheralimmune organs

Our own results diverge from these reports in that we couldnot detect a difference in active EAE disease severity betweenCD19Cre+minus α4-integrinflfl mice and C57BL6 WT controlmice These experiments were conducted 15 times (table)and we are confident in our findings Differences in EAEdisease severity and phenotype can be affected by conditionsunder which mouse colonies are maintained as well as otherfactors that may affect the overall inflammatory milieu of ex-perimental conditions This may explain the differences inresults It is also conceivable that different mice were used bythe other investigators for their experiments The inves-tigators of the 2 previous studies state that they usedCD19Creα4flfl mice23 or that they used CD19Cre Itga4flfl

mice25 It is not stated whether mice were heterozygous orhomozygous for CD19cre or a mixture of both As stated inthe Methods section CD19Cre++ mice behave functionallyvery similarly to B-cellndashdeficient mice which develop moresevere EAE when immunized with MOGp35-55

26

We found one potentially meaningful biological difference be-tween CD19Cre+minus α4-integrinflfl mice and C57BL6 controlmice There was decreased MOGp35-55-specific CD4+ T-cellproliferation in spleens which is an expected result given thatα4-integrin can participate in costimulation of CD4+T cells1819

The observation that CD19+ B cells of CD19Cre+minus α4-integrinflfl mice did deplete α4-integrin and that they weresignificantly less capable of entering the CNS after mice wereimmunized with MOGp35-55 suggests 3 interpretations (1)Antigen-activated B cells contribute to disease burden in EAEin a seemingly dose-dependent manner (2) they do not ab-solutely require recognition of a CNS autoantigen or a mimicthereof to do so and (3) they do not require full α4-integrinndashmediated access to the CNS to exacerbate diseaseThe first interpretation is supported by work of other inves-tigators who have previously demonstrated that B-cellndashdeficient C57BL6 mice are resistant to active rMOG-inducedEAE26 and that the administration of a B-cellndashdepletingmonoclonal antibody prevents or reverses established rMOG-induced EAE in C57BL6 mice21 Weber and colleagues21

further showed that B-cell depletion results in a reduction ofMOGp35-55-specific Th1 and Th17 cells Our own data suggestthat the differentiation ofMOGp35-55-specific T cells to becomeencephalitogenic does not absolutely require the presentationof autoantigen but is likely driven by other B-cell factors in-cluding soluble inflammatory mediators The third in-terpretation of this experiment is supported by ourdemonstration that B cells from CD19Cre+minus α4-integrinflfl

mice are significantly impaired in their ability to enter the CNS(figure 1E) Finally it should be emphasized that our findingsdo not conclusively rule out a role of antigen-specific B cells inthe CNS on the course of CNS autoimmunity as thresholdeffects may be difficult to detect with the EAE experiments thatwere conducted

Our data suggest that the beneficial effects of anti-CD20therapies in patients with MS are mediated through theireffects on peripheral B cells We also conclude that benefitsobserved in patients with MS during anti-α4-integrin therapyare likely not predominantly due to its effect of suppressingthe migration of B cells into the CNS The interpretation ofour findings should be limited to early forms of MS as theEAE model that was used and the observation period that wasused do not provide insight into late or progressive forms ofCNS autoimmunity Magliozzi et al27 were the first inves-tigators to show that some patients with secondary-progressive MS develop lymphoid tissue in the meningesthat resemble B-cell follicles in secondary lymphoid organsB cells in these structures may be susceptible to anti-CD20therapies with monoclonal antibodies or small molecules andit is conceivable that their depletion may benefit patientsafflicted with that MS phenotype

AcknowledgmentThe authors thank Dr Thalia Papayannopoulou at theUniversity of Washington for providing them with α4-integrinfloxflox mice Dr Stuve was funded by a Merit Reviewgrant (federal award document number [FAIN]I01BX001674) from the United States (US) Departmentof Veterans Affairs Biomedical Laboratory Research andDevelopment

Study fundingNo targeted funding reported

DisclosureRZ Hussain PC Cravens WA Miller-Little R Doelger VGrandos and E Herndon report no disclosures DT Okudareceived advisory and consulting fees from Celgene Gen-entech Genzyme EMD Serono and Novartis and researchsupport from Biogen served on the scientific advisory boardof Osmotica and served on the speakersrsquo bureau of AcordaGenzyme and Teva TN Eagar serves as section editor ofArchives of Pathology and received research support from theNIH O Stuve served on the scientific advisory boards of TGTherapeutics and Genentech-Roche served on the editorialboard of Therapeutic Advances in Neurological Disorders

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 9

consulted for Celgene EMD Serono and Novartis andreceived research support from Sanofi and GenzymeDisclosures available NeurologyorgNN

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationNovember 2 2018 Accepted in final form February 1 2019

References1 Hauser SL Waubant E Arnold DL et al B-cell depletion with rituximab in relapsing-

remitting multiple sclerosis N Engl J Med 2008358676ndash6882 Hawker K OrsquoConnor P Freedman MS et al Rituximab in patients with primary

progressive multiple sclerosis results of a randomized double-blind placebo-controlled multicenter trial Ann Neurol 200966460ndash471

3 Hauser SL Bar-Or A Comi G et al Ocrelizumab versus interferon beta-1a in re-lapsing multiple sclerosis N Engl J Med 2017376221ndash234

4 Montalban X Hauser SL Kappos L et al Ocrelizumab versus placebo in primaryprogressive multiple sclerosis N Engl J Med 2017376209ndash220

5 Yednock TA Cannon C Fritz LC Sanchez-Madrid F Steinman L Karin N Pre-vention of experimental autoimmune encephalomyelitis by antibodies against alpha 4beta 1 integrin Nature 199235663ndash66

6 Frohman EM Racke MK Raine CS Multiple sclerosismdashthe plaque and its patho-genesis N Engl J Med 2006354942ndash955

7 Stuve O Marra CM Jerome KR et al Immune surveillance in multiple sclerosispatients treated with natalizumab Ann Neurol 200659743ndash747

8 Haines JL Ter Minassian M Bazyk A et al A complete genomic screen for multiplesclerosis underscores a role for the major histocompatability complex The MultipleSclerosis Genetics Group Nat Genet 199613469ndash471

9 Sawcer S Jones HB Feakes R et al A genome screen in multiple sclerosis revealssusceptibility loci on chromosome 6p21 and 17q22 Nat Genet 199613464ndash468

10 Yang JT Rayburn H Hynes RO Cell adhesion events mediated by alpha 4 integrinsare essential in placental and cardiac development Development 1995121549ndash560

11 Sternberg N Hamilton D Bacteriophage P1 site-specific recombination I Re-combination between loxP sites J Mol Biol 1981150467ndash486

12 Scott LM Priestley GV Papayannopoulou T Deletion of alpha4 integrins from adulthematopoietic cells reveals roles in homeostasis regeneration and homing Mol CellBiol 2003239349ndash9360

13 Hussain RZ Miller-Little WA Doelger R et al Defining standard enzymatic disso-ciation methods for individual brains and spinal cords in EAE Neurol NeuroimmunolNeuroinflamm 20185e437 doi 101212NXI0000000000000437

14 Cravens PD Hussain RZ Zacharias TE et al Lymph node-derived donor encepha-litogenic CD4+ T cells in C57BL6 mice adoptive transfer experimental autoimmuneencephalomyelitis highly express GM-CSF and T-bet J Neuroinflammation 2011873

15 Karandikar NJ Crawford MP Yan X et al Glatiramer acetate (Copaxone) therapyinduces CD8(+) T cell responses in patients with multiple sclerosis J Clin Invest2002109641ndash649

16 Gu H Marth JD Orban PC Mossmann H Rajewsky K Deletion of a DNA poly-merase beta gene segment in T cells using cell type-specific gene targeting Science1994265103ndash106

17 Rickert RC Roes J Rajewsky K B lymphocyte-specific Cre-mediated mutagenesis inmice Nucleic Acids Res 1997251317ndash1318

18 Davis LS Oppenheimer-Marks N Bednarczyk JL McIntyre BW Lipsky PE Fibro-nectin promotes proliferation of naive and memory T cells by signaling through boththe VLA-4 and VLA-5 integrin molecules J Immunol 1990145785ndash793

19 Shimizu Y van Seventer GA Horgan KJ Shaw S Costimulation of proliferativeresponses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 withfibronectin or VLA-6 with laminin J Immunol 199014559ndash67

20 Theien BE Vanderlugt CL Eagar TN et al Discordant effects of anti-VLA-4 treat-ment before and after onset of relapsing experimental autoimmune encephalomyelitisJ Clin Invest 2001107995ndash1006

21 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

22 Krumbholz M Meinl I Kumpfel T Hohlfeld R Meinl E Natalizumab dispropor-tionately increases circulating pre-B and B cells in multiple sclerosis Neurology 2008711350ndash1354

23 Lehmann-Horn K Sagan SA Winger RC et al CNS accumulation of regulatoryB cells is VLA-4-dependent Neurol Neuroimmunol Neuroinflamm 20163e212 doi101212NXI0000000000000212

24 Glatigny S Wagner CA Bettelli E Correction cutting edge integrin alpha4 is re-quired for regulatory B cell control of experimental autoimmune encephalomyelitisJ Immunol 20171983756

25 Glatigny S Duhen R Oukka M Bettelli E Cutting edge loss of alpha4 integrinexpression differentially affects the homing of Th1 and Th17 cells J Immunol 20111876176ndash6179

26 Lyons JA SanM HappMP Cross AH B cells are critical to induction of experimentalallergic encephalomyelitis by protein but not by a short encephalitogenic peptide EurJ Immunol 1999293432ndash3439

27 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

Appendix Authors

Name Location Role Contribution

Rehana ZHussainMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

Petra CCravensPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

William AMiller-Little BA

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

RichardDoelgerMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

ValerieGranadosPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the data andanalyzed the data

EmilyHerndonMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Darin TOkudaMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Todd NEagar PhD

Houston MethodistHospital HoustonTX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

OlafStuve MDPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

DOI 101212NXI000000000000056320196 Neurol Neuroimmunol Neuroinflamm

Rehana Z Hussain Petra D Cravens William A Miller-Little et al disease model

mediated EAEminus4-integrin deficiency in B cells does not affect disease in a T-cellα

This information is current as of April 16 2019

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

ServicesUpdated Information amp

httpnnneurologyorgcontent64e563fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent64e563fullhtmlref-list-1

This article cites 27 articles 7 of which you can access for free at

Citations httpnnneurologyorgcontent64e563fullhtmlotherarticles

This article has been cited by 1 HighWire-hosted articles

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseases

httpnnneurologyorgcgicollectionall_immunologyAll Immunology

httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the

Errata

content64e585fullpdf or page

nextAn erratum has been published regarding this article Please see

Permissions amp Licensing

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

Reprints

httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

CORRECTION

α4-integrin deficiency in B cells does not affect disease in aT-cellndashmediated EAE disease modelNeurol Neuroimmunol Neuroinflamm 20196e585 doi101212NXI0000000000000585

In the article ldquoα4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAEdisease model by Hussain et al1 first published online April 16 2019 funding for the papershould have been listed in the Study Funding section The authors regret the error

Reference1 Hussain RZ Cravens PD Miller-Little WA et al α4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAE

disease model Neurol Neuroimmunol Neuroinflamm 20196e563

Copyright copy 2019 American Academy of Neurology 1

Copyright copy 2019 American Academy of Neurology Unauthorized reproduction of this article is prohibited

Page 10: α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency in B cells does not affect disease in a T-cell–mediated EAE disease model Rehana

consulted for Celgene EMD Serono and Novartis andreceived research support from Sanofi and GenzymeDisclosures available NeurologyorgNN

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationNovember 2 2018 Accepted in final form February 1 2019

References1 Hauser SL Waubant E Arnold DL et al B-cell depletion with rituximab in relapsing-

remitting multiple sclerosis N Engl J Med 2008358676ndash6882 Hawker K OrsquoConnor P Freedman MS et al Rituximab in patients with primary

progressive multiple sclerosis results of a randomized double-blind placebo-controlled multicenter trial Ann Neurol 200966460ndash471

3 Hauser SL Bar-Or A Comi G et al Ocrelizumab versus interferon beta-1a in re-lapsing multiple sclerosis N Engl J Med 2017376221ndash234

4 Montalban X Hauser SL Kappos L et al Ocrelizumab versus placebo in primaryprogressive multiple sclerosis N Engl J Med 2017376209ndash220

5 Yednock TA Cannon C Fritz LC Sanchez-Madrid F Steinman L Karin N Pre-vention of experimental autoimmune encephalomyelitis by antibodies against alpha 4beta 1 integrin Nature 199235663ndash66

6 Frohman EM Racke MK Raine CS Multiple sclerosismdashthe plaque and its patho-genesis N Engl J Med 2006354942ndash955

7 Stuve O Marra CM Jerome KR et al Immune surveillance in multiple sclerosispatients treated with natalizumab Ann Neurol 200659743ndash747

8 Haines JL Ter Minassian M Bazyk A et al A complete genomic screen for multiplesclerosis underscores a role for the major histocompatability complex The MultipleSclerosis Genetics Group Nat Genet 199613469ndash471

9 Sawcer S Jones HB Feakes R et al A genome screen in multiple sclerosis revealssusceptibility loci on chromosome 6p21 and 17q22 Nat Genet 199613464ndash468

10 Yang JT Rayburn H Hynes RO Cell adhesion events mediated by alpha 4 integrinsare essential in placental and cardiac development Development 1995121549ndash560

11 Sternberg N Hamilton D Bacteriophage P1 site-specific recombination I Re-combination between loxP sites J Mol Biol 1981150467ndash486

12 Scott LM Priestley GV Papayannopoulou T Deletion of alpha4 integrins from adulthematopoietic cells reveals roles in homeostasis regeneration and homing Mol CellBiol 2003239349ndash9360

13 Hussain RZ Miller-Little WA Doelger R et al Defining standard enzymatic disso-ciation methods for individual brains and spinal cords in EAE Neurol NeuroimmunolNeuroinflamm 20185e437 doi 101212NXI0000000000000437

14 Cravens PD Hussain RZ Zacharias TE et al Lymph node-derived donor encepha-litogenic CD4+ T cells in C57BL6 mice adoptive transfer experimental autoimmuneencephalomyelitis highly express GM-CSF and T-bet J Neuroinflammation 2011873

15 Karandikar NJ Crawford MP Yan X et al Glatiramer acetate (Copaxone) therapyinduces CD8(+) T cell responses in patients with multiple sclerosis J Clin Invest2002109641ndash649

16 Gu H Marth JD Orban PC Mossmann H Rajewsky K Deletion of a DNA poly-merase beta gene segment in T cells using cell type-specific gene targeting Science1994265103ndash106

17 Rickert RC Roes J Rajewsky K B lymphocyte-specific Cre-mediated mutagenesis inmice Nucleic Acids Res 1997251317ndash1318

18 Davis LS Oppenheimer-Marks N Bednarczyk JL McIntyre BW Lipsky PE Fibro-nectin promotes proliferation of naive and memory T cells by signaling through boththe VLA-4 and VLA-5 integrin molecules J Immunol 1990145785ndash793

19 Shimizu Y van Seventer GA Horgan KJ Shaw S Costimulation of proliferativeresponses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 withfibronectin or VLA-6 with laminin J Immunol 199014559ndash67

20 Theien BE Vanderlugt CL Eagar TN et al Discordant effects of anti-VLA-4 treat-ment before and after onset of relapsing experimental autoimmune encephalomyelitisJ Clin Invest 2001107995ndash1006

21 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

22 Krumbholz M Meinl I Kumpfel T Hohlfeld R Meinl E Natalizumab dispropor-tionately increases circulating pre-B and B cells in multiple sclerosis Neurology 2008711350ndash1354

23 Lehmann-Horn K Sagan SA Winger RC et al CNS accumulation of regulatoryB cells is VLA-4-dependent Neurol Neuroimmunol Neuroinflamm 20163e212 doi101212NXI0000000000000212

24 Glatigny S Wagner CA Bettelli E Correction cutting edge integrin alpha4 is re-quired for regulatory B cell control of experimental autoimmune encephalomyelitisJ Immunol 20171983756

25 Glatigny S Duhen R Oukka M Bettelli E Cutting edge loss of alpha4 integrinexpression differentially affects the homing of Th1 and Th17 cells J Immunol 20111876176ndash6179

26 Lyons JA SanM HappMP Cross AH B cells are critical to induction of experimentalallergic encephalomyelitis by protein but not by a short encephalitogenic peptide EurJ Immunol 1999293432ndash3439

27 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

Appendix Authors

Name Location Role Contribution

Rehana ZHussainMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

Petra CCravensPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

William AMiller-Little BA

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

RichardDoelgerMSc

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the dataanalyzed the data andrevised the manuscript

ValerieGranadosPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Acquired the data andanalyzed the data

EmilyHerndonMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Darin TOkudaMD

University of TexasSouthwesternMedical CenterDallas TX

Author Interpreted the data andrevised the manuscriptfor intellectual content

Todd NEagar PhD

Houston MethodistHospital HoustonTX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

OlafStuve MDPhD

University of TexasSouthwesternMedical CenterDallas TX

Author Designed andconceptualized thestudy analyzed the dataand drafted themanuscript forintellectual content

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 6 Number 4 | July 2019 NeurologyorgNN

DOI 101212NXI000000000000056320196 Neurol Neuroimmunol Neuroinflamm

Rehana Z Hussain Petra D Cravens William A Miller-Little et al disease model

mediated EAEminus4-integrin deficiency in B cells does not affect disease in a T-cellα

This information is current as of April 16 2019

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

ServicesUpdated Information amp

httpnnneurologyorgcontent64e563fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent64e563fullhtmlref-list-1

This article cites 27 articles 7 of which you can access for free at

Citations httpnnneurologyorgcontent64e563fullhtmlotherarticles

This article has been cited by 1 HighWire-hosted articles

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseases

httpnnneurologyorgcgicollectionall_immunologyAll Immunology

httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the

Errata

content64e585fullpdf or page

nextAn erratum has been published regarding this article Please see

Permissions amp Licensing

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

Reprints

httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

CORRECTION

α4-integrin deficiency in B cells does not affect disease in aT-cellndashmediated EAE disease modelNeurol Neuroimmunol Neuroinflamm 20196e585 doi101212NXI0000000000000585

In the article ldquoα4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAEdisease model by Hussain et al1 first published online April 16 2019 funding for the papershould have been listed in the Study Funding section The authors regret the error

Reference1 Hussain RZ Cravens PD Miller-Little WA et al α4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAE

disease model Neurol Neuroimmunol Neuroinflamm 20196e563

Copyright copy 2019 American Academy of Neurology 1

Copyright copy 2019 American Academy of Neurology Unauthorized reproduction of this article is prohibited

Page 11: α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency in B cells does not affect disease in a T-cell–mediated EAE disease model Rehana

DOI 101212NXI000000000000056320196 Neurol Neuroimmunol Neuroinflamm

Rehana Z Hussain Petra D Cravens William A Miller-Little et al disease model

mediated EAEminus4-integrin deficiency in B cells does not affect disease in a T-cellα

This information is current as of April 16 2019

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

ServicesUpdated Information amp

httpnnneurologyorgcontent64e563fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent64e563fullhtmlref-list-1

This article cites 27 articles 7 of which you can access for free at

Citations httpnnneurologyorgcontent64e563fullhtmlotherarticles

This article has been cited by 1 HighWire-hosted articles

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseases

httpnnneurologyorgcgicollectionall_immunologyAll Immunology

httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the

Errata

content64e585fullpdf or page

nextAn erratum has been published regarding this article Please see

Permissions amp Licensing

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

Reprints

httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

CORRECTION

α4-integrin deficiency in B cells does not affect disease in aT-cellndashmediated EAE disease modelNeurol Neuroimmunol Neuroinflamm 20196e585 doi101212NXI0000000000000585

In the article ldquoα4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAEdisease model by Hussain et al1 first published online April 16 2019 funding for the papershould have been listed in the Study Funding section The authors regret the error

Reference1 Hussain RZ Cravens PD Miller-Little WA et al α4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAE

disease model Neurol Neuroimmunol Neuroinflamm 20196e563

Copyright copy 2019 American Academy of Neurology 1

Copyright copy 2019 American Academy of Neurology Unauthorized reproduction of this article is prohibited

Page 12: α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency in B cells does not affect disease in a T-cell–mediated EAE disease model Rehana

ServicesUpdated Information amp

httpnnneurologyorgcontent64e563fullhtmlincluding high resolution figures can be found at

References httpnnneurologyorgcontent64e563fullhtmlref-list-1

This article cites 27 articles 7 of which you can access for free at

Citations httpnnneurologyorgcontent64e563fullhtmlotherarticles

This article has been cited by 1 HighWire-hosted articles

Subspecialty Collections

httpnnneurologyorgcgicollectionmultiple_sclerosisMultiple sclerosis

httpnnneurologyorgcgicollectionautoimmune_diseasesAutoimmune diseases

httpnnneurologyorgcgicollectionall_immunologyAll Immunology

httpnnneurologyorgcgicollectionall_demyelinating_disease_cnsAll Demyelinating disease (CNS)following collection(s) This article along with others on similar topics appears in the

Errata

content64e585fullpdf or page

nextAn erratum has been published regarding this article Please see

Permissions amp Licensing

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

Reprints

httpnnneurologyorgmiscaddirxhtmlreprintsusInformation about ordering reprints can be found online

Academy of Neurology All rights reserved Online ISSN 2332-7812Copyright copy 2019 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

CORRECTION

α4-integrin deficiency in B cells does not affect disease in aT-cellndashmediated EAE disease modelNeurol Neuroimmunol Neuroinflamm 20196e585 doi101212NXI0000000000000585

In the article ldquoα4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAEdisease model by Hussain et al1 first published online April 16 2019 funding for the papershould have been listed in the Study Funding section The authors regret the error

Reference1 Hussain RZ Cravens PD Miller-Little WA et al α4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAE

disease model Neurol Neuroimmunol Neuroinflamm 20196e563

Copyright copy 2019 American Academy of Neurology 1

Copyright copy 2019 American Academy of Neurology Unauthorized reproduction of this article is prohibited

Page 13: α4-integrin deficiency in B cells does not affect …ARTICLE OPEN ACCESS α4-integrin deficiency in B cells does not affect disease in a T-cell–mediated EAE disease model Rehana

CORRECTION

α4-integrin deficiency in B cells does not affect disease in aT-cellndashmediated EAE disease modelNeurol Neuroimmunol Neuroinflamm 20196e585 doi101212NXI0000000000000585

In the article ldquoα4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAEdisease model by Hussain et al1 first published online April 16 2019 funding for the papershould have been listed in the Study Funding section The authors regret the error

Reference1 Hussain RZ Cravens PD Miller-Little WA et al α4-integrin deficiency in B cells does not affect disease in a T-cellndashmediated EAE

disease model Neurol Neuroimmunol Neuroinflamm 20196e563

Copyright copy 2019 American Academy of Neurology 1

Copyright copy 2019 American Academy of Neurology Unauthorized reproduction of this article is prohibited