ARTICLE OPEN ACCESS

Glatiramer acetate immune modulates B-cellantigen presentation in treatment of MSDarius Hausler PhD Zivar Hajiyeva MD Jan W Traub MD Scott S Zamvil MD PhD Patrice H Lalive MD

Wolfgang Bruck MD and Martin S Weber MD

Neurol Neuroimmunol Neuroinflamm 20207e698 doi101212NXI0000000000000698

Correspondence

Dr Weber

martinweber

meduni-goettingende

AbstractObjectiveWe examined the effect of glatiramer acetate (GA) on B-cell maturation differentiation andantigen presentation in MS and experimental autoimmune encephalomyelitis (EAE)

MethodsA cross-sectional study of blood samples from 20 GA-treated and 18 untreated patients withMS was performed by flow cytometry 6 GA-treated patients with MS were analyzed longi-tudinally GA-mediated effects on B-cell antigen-presenting function were investigated in EAEor alternatively B cells were treated with GA in vitro using vehicle as a control

ResultsIn MS GA diminished transitional B-cell and plasmablast frequency downregulated CD69CD25 and CD95 expression and decreased TNF-α production whereas IL-10 secretion andMHC Class II expression were increased In EAE we observed an equivalent dampening ofproinflammatory B-cell properties and an enhanced expression of MHC Class II When usedas antigen-presenting cells for activation of naive T cells GA-treated B cells promoteddevelopment of regulatory T cells whereas proinflammatory T-cell differentiation wasdiminished

ConclusionsGA immune modulates B-cell function in EAE and MS and efficiently interferes with patho-genic B cellndashT cell interaction

These authors contributed equally to this work

From the Institute of Neuropathology (DH JWT WB MSW) University Medical Center Department of Neurology (ZH JWT MSW) University Medical Center GottingenGermany Department of Neurology (SSZ) University of California San Francisco Division of Neurology (PHL) Department of Neurosciences Hospital and University of Genevaand Department of Pathology and Immunology (PHL) Faculty of Medicine Geneva Switzerland

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

The Article Processing Charge was funded by the University Medical Center Gottingen

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 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Glatiramer acetate (GA) a synthetic random basic copolymercomposed of glutamic acid lysine tyrosine and alanine iswidely used in the treatment of MS1 GA has been shown toreduce relapse rates and progression of neurologic disability2

The precise mechanism of action by which GA mediates thisbenefit is still not fully understood Studies showed a prefer-ential differentiation of CD4+ T cells into T helper (Th)-2cells34 downregulation of Th17 cell differentiation5 increasedfrequency and function of CD4+CD25+FoxP3+ regulatory T(Treg) cells67 and modulation of CD8+ T cells8 MoreoverGA was found to promote M2 monocyte differentiation79 andto reduce activation and proinflammatory cytokine secretion inmonocytes910 and plasmacytoid dendritic cells11

Several lines of evidence highlight essential roles of B cells inthe pathogenesis of MS1213 This is broadly supported by thebeneficial effect of B cellndashdepleting therapies both inrelapsing-remitting (RR)MS1415 and primary progressiveMS1617 Some studies have also shown immunomodulatoryproperties of GA on B cells including reduction in thenumber of circulating B cells and a shift from a proin-flammatory to an anti-inflammatory B-cell phenotype18ndash20

The functional consequences of these phenotypical changesyet remained to be elucidated Accordingly we here focusedon the question to what extent GA-mediated effects on B cellscan change their ability to act as antigen-presenting cells(APCs) for the activation of T cells an assumed key process inthe development and propagation of MS

MethodsHumanBlood samples were collected from patients with relapsing-remitting MS at the Clinical MS Center of the Uni-versitatsmedizin Gottingen (UMG) in Germany between2015 and 2018 The diagnosis of RRMS was based on theMcDonald criteria Twenty patients with MS naive to ap-proved disease-modifying therapy were treated with GA forge1 month Eighteen untreated patients with MS served ascontrols Six GA-treated patients were analyzed longitudi-nally having had blood samples taken at 2 different timepoints with an interval of at least 3 months Demographic anddisease-related information is summarized in the table

MiceSix- to ten-week-old female wild-type (WT) C57BL6 micewere purchased from Charles River MOG p35-55 TCRtransgenic 2D2 mice were kindly provided by Dr Kuchroo(Boston MA)

Ethical approvalsEthical approvals for all human samples used were given bythe ethical review committee of the UMG (approval number27414) All animal experiments were performed in accor-dance with the guidelines of the Central Department forAnimal Experiments UMG and approved by the Office forConsumer Protection and Food Safety of the State of LowerSaxony (protocol number 339-42502-04-172615)

EAE induction and scoringFemale WT mice were immunized with 50 μg MOG pep-tide35-55 MEVGWYRSPFSRVVHLYRNGK emulsified incomplete Freundrsquos adjuvant followed by intraperitonealinjections of 100 ng of Bordetella pertussis toxin at the day ofimmunization and 2 days thereafter Experimental autoim-mune encephalomyelitis (EAE) severity was assessed dailyand scored on a scale from 0 to 5 scale as described21

GA treatmentGA was provided by Teva Pharmaceutical Industries Micereceived daily SC injections of 150 μg GA suspended in 01mL phosphate-buffered saline (PBS) or PBS alone

Detection of anti-GA antibodiesNinety-six-well plates were coated with 10 μgmL GA in PBSovernight Thereafter diluted serum samples were incubatedfor 2 hours After washing plate-bound antibodies were de-tected with horseradish peroxidasendashconjugated anti-mouseIgG directed against the Fc part of the bound antibodies Ab-sorbance wasmeasured at 450 nmwith subtraction of a 540-nmreference wavelength on the iMark Microplate Reader

Isolation of human and murine leukocytesHuman immune cell counts were determined in our hospitalrsquosroutine laboratory Human peripheral blood mononuclear cells(PBMCs) were isolated after Ficoll gradient centrifugation andcryopreserved at minus80deg Splenic B cells were purified by negativemagnetic activated cell sorting (MACS) separation usinga mouse lineage panel Splenic T cells were isolated by negativeMACS separation using a mouse pan T-cell isolation kit II

Flow cytometryPregating and gating strategy for humanB-cell subsets and surfacemolecule expression was done as described in figure e-1 (linkslwwcomNXIA218) Fc receptors were blocked using humanTruStain FcX Dead cells were stained with a fixable viability kitHuman B-cell differentiation was determined using CD19(HIB19)CD20 (L27)CD24 (ML5) CD27 (O323) andCD38(HIT2) B-cell activation was evaluated by CD25 (BC96) CD40(5C3) CD69 (FN50) CD80 (L3074) CD86 (FUN-1) CD95

GlossaryAPC = antigen-presenting cell EAE = experimental autoimmune encephalomyelitis GA = glatiramer acetate NMO =neuromyelitis optica PBMC = peripheral blood mononuclear cell PBS = phosphate-buffered saline RRMS = relapsing-remitting MS WT = wild type

2 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN

(DX2) andmajor histocompatibility complex (MHC) II (Tu36)after 2 μgmL CpG stimulation for 20 hours T cells and mon-ocytes were determined using CD4 (RPA-T4) CD8 (HIT-8a)and CD14 (M5E2) respectively To investigate B-cell cytokineproduction cell suspensions were stimulated with 1 μgmLCpG500 ngmL ionomycin and 20 ngmL phorbol 12-myristate 13-acetate in the presence of 1 μLmL brefeldin A for 22 hoursfollowed by cell fixationpermeabilization and intracellularstaining for IL-6 (MQ2-13A5) IL-10 (JES3-19F1) and TNF-α(MAb11) For murine experiments Fc receptors were blockedusing monoclonal antibody specific for CD16CD32 (93) Deadcells were stained with a fixable viability kit Splenic B-cellactivationdifferentiation was determined using CD19 (6D5)CD25 (PC61) CD69 (H12F3) CD40 (323) CD80 (16-10A1) CD86 (GL-1) and MHCII (AF6-1201) after 1 μgmLCpG stimulation for 20 hours To investigate B-cell cytokineproduction cell suspensions were stimulated with 1 μgmL CpGin the presence of 1 μLmL brefeldin A for 6 hours followed bycell fixationpermeabilization and intracellular staining for IL-10(JES5-16E3) and IL-6 (MP5-20F3) Treg cell differentiation wasevaluated by CD4 (GK15) CD25 (PC61) and by intracellularstaining for FoxP3 (FJK-16s) after fixation and permeabilizationusing the fixationpermeabilization kit To investigate Th1 andTh17 cell differentiation cell suspensions were stimulatedwith 50ngmL phorbol 12-myristate 13-acetate and 05 μgmL ion-omycin in the presence of 1 μLmL brefeldin A for 6 hoursfollowed by a CD4 (GK15) staining Cytokine production wasanalyzed by intracellular staining for IFN-γ (XMG12) and IL-17A (TC11-18H10) Samples were acquired on a BD LSRFor-tessa All data evaluation was performed using FlowJo software

ELISAProduction of cytokines was measured using ELISA MAXStandard Set kits Absorbance was measured at 450 nm withsubtraction of a 540-nm reference wavelength on the iMarkMicroplate Reader

T-cell proliferation assayMACS-purified splenic B cells were cocultured with MACS-purified MOG-specific CFSE-stained (CFSE Cell Division

Tracker Kit) splenic T cells from 2D2 mice and wererestimulated with MOG peptide35-55 After 72 hours T-cellproliferation was evaluated by flow cytometry

Statistical analysisStatistical analysis was performed using the software Graph-Pad Prism 501 and 601 Human data sets were tested forGauss distribution using the DrsquoAgostino-Pearson omnibusnormality test Shapiro-Wilk normality test and Kolmogorov-Smirnov normality test For the comparison of 2 cross-sectional cohorts with Gauss distribution an unpaired and forlongitudinal samples a paired t test was used respectively Ifthe data were not Gauss distributed a Mann-Whitney U testwas applied in the cross-sectional analysis and the Wilcoxonmatched-pairs signed-rank test was used for the longitudinaldata Clinical scores and T-cell proliferation are depicted asmean plusmn SEM and were analyzed by the Mann-WhitneyU testGA antibody titers are shown as median and were analyzedusing the Student t test All other data are shown as medianand the statistical comparison was made using the Mann-Whitney U test A value of p lt 005 was considered significantand is shown by 1 asterisk Two asterisks and 3 asterisksindicate significances of p lt 001 and p lt 0001 respectively

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

ResultsIn the present study we compared 20 GA-treated patientswith MS with 18 untreated MS controls (table) The meanGA therapy duration was 59 months and ranged from 1 to145 months Six GA-treated patients with MS were analyzedlongitudinally at 2 different time points with an interval of gt3months Patients were naive to any approved disease-modifying therapy and had not received steroids for at least3 months before GA treatment

GA alters PBMC compositionAt first we determined the impact of GA on PBMC com-position GA treatment resulted in a trend toward decreasedabsolute leukocyte and neutrophil numbers whereas mono-cyte numbers became significantly elevated (figure 1 A andB) Frequency analysis of the cross-sectional and longitudinalstudy showed a tendency for decreased CD4+ T cells andsignificantly diminished CD19+ B cells respectively (figure 1CndashF) This GA treatment effect did not correlate the re-spective treatment duration ranging between 1 and 145months (figure e-2A linkslwwcomNXIA218)

GA decreases B-cell activation differentiationand proinflammatory cytokine productionwhereas IL-10 secretion and MHC Class IIexpression are increasedTo assess whether GA has an effect on B-cell activationdifferentiation and cytokine production we analyzed

Table Patient characteristics

UntreatedGA treated(GA1)

GA treated(GA2)

No of patients 18 20 6

Age (y) (mean plusmn SD) 324 plusmn 97 420 plusmn 94 426 plusmn 98

Female sex () 777 545 500

EDSS score (mean plusmn SD) 21 plusmn 17 20 plusmn 14 24 plusmn 13

Disease duration (y)(mean plusmn SD)

47 plusmn 62 74 plusmn 61 98 plusmn 5

GA since (mo) (mean plusmn SD) mdash 59 plusmn 41 65 plusmn 56

Abbreviation GA = glatiramer acetate

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 3

peripheral blood B cells (figure 2 AndashI figure e-2 B and ClinkslwwcomNXIA218) GA therapy was associated witha reduction in plasmablasts both in the cross-sectional andlongitudinal analyses of blood samples (figure 2 A and B) Inaddition frequency of immature transitional B cells wasdecreased in the longitudinal study (figure 2B) and corre-lated with GA treatment duration (figure 2C) GA longitu-dinally downregulated CD25 CD69 and CD95 expressionon B cells whereas MHC Class II expression was upregu-lated as compared to untreated MS controls (figure 2 DndashG)Other molecules involved in antigen presentation such asCD40 CD80 and CD86 showed no difference (figure 2 Fand G) IL-6 production was not altered by GA treatmentboth in the cross-sectional and longitudinal studies whereas

GA increased anti-inflammatory IL-10 and decreasedproinflammatory TNF-α cytokine production in the longi-tudinal analysis however no correlation with longer GAtreatment duration was found (figure 2 H and I figure e-2BlinkslwwcomNXIA218)

GA upregulates MHC Class II B-cell expressionindependent of EAETo identify whether the observed reduction on B-cell acti-vation TNF-α production and the increase in IL-10 secre-tion and MHC Class II expression in patients with MS isa result of GA treatment or a concomitant disease-relatedeffect we initially administered daily subcutaneous GA tonaive unimmunized wild-type mice (figure 3A) GA had no

Figure 1 GA treatment alters the composition of leukocytes in patients with MS

Peripheral blood samples were taken from control (n = 18) and glatiramer acetatendashtreated (GA n = 20) patients with MS (A) Leukocyte counts and (B)neutrophil lymphocytemonocyte eosinophil and basophil counts weremeasured in routine clinical laboratory blood counts if available (p lt 005 unpairedt test) Next peripheral bloodmononuclear cells (PBMCs) were isolated from the samples (C) Cell frequencies of CD4+ T cells (TC) CD8+ TC CD14+monocytes(Mo) and CD19+ B cells (BC) were determined using flow cytometry (ns unpaired t test) (D) CD4+ CD8+ TC CD14+ Mo and BC of patients with MS at 2 timepoints during GA medication line connects an individual patient (n = 5 p lt 005 Wilcoxon matched-pairs signed-rank test) (E and F) Fold changes of thehorizontal and longitudinal cell frequency changes GA = glatiramer acetate

4 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN

Figure 2 GA therapy changes the phenotype of human B cells in patients with MS

Humanperipheral bloodmononuclear cells (PBMCs) were isolated fromglatiramer acetate (GA n = 20) or non-GA (control n = 18) treated patients withMS Inaddition 6 patients were analyzed longitudinally on GA treatment Red circles represent GA treatment squares control treatment (A) Mean frequency plusmn SEMof B-cell subpopulations defined as follows transitional B cells (CD24high CD38high transitional) mature B cells (CD24var CD38lowmature) antigen-activated Bcells (CD27+ ag-activated) memory B cells (CD27var CD38minus memory) and plasmablasts (CD20minus CD27+ CD38+ p lt 005 unpaired t test) (B) B-cell subsetfrequencies of patients with MS at 2 time points during GA therapy line connects an individual patient (n = 6 p lt 005 Wilcoxon matched-pairs signed-ranktest) (C) The individual patientsrsquo frequencies of BC subsets were correlated with the duration of GA treatment (p lt 005 linear regression) (D) MFI plusmn SEM ofactivation molecules expressed on B cells (ns unpaired t test) (E) B-cell activation marker expression of patients with MS at 2 time points during GA therapyline connects an individual patient (n = 6 p lt 005 Wilcoxon matched-pairs signed-rank test) (F) Mean MFI of molecules involved in antigen presentationexpressed on B cells (p lt 005 unpaired t test) (G) Expression of molecules involved in antigen presentation of patients with MS at 2 time points during GAmedication line connects an individual patient (ns Wilcoxon matched-pairs signed-rank test) (H) Shown is the frequency of positive cells regarding therespective cytokine (tumor necrosis factor [TNF] interleukin [IL]-6 and IL-10mean plusmn SEM ns unpaired t test) (I) TNF IL-6 and IL-10-positive B cells of patientswith MS at 2 time points during GA medication line connects an individual patient (p lt 005 Wilcoxon matched-pairs signed-rank test) GA = glatirameracetate

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 5

impact on B-cell activation or cytokine production howeverMHC Class II was significantly upregulated after ex vivostimulation (figure 3 BndashH)

GA downregulates B-cell activation andameliorates clinical severity of active EAETo investigate the effect of GA on B-cell phenotype andfunction during pathologic conditions mice receiveda daily subcutaneous GA injection starting 7 days beforeimmunization (figure 4A) GA ameliorated EAE (figure4B) which was associated with a production of antibodies

against GA (figure 4C) a decrease in expression of the earlyactivation marker CD69 on B cells and diminished secre-tion of IL-6 whereas the expression of costimulatorymolecule CD86 and MHC Class II was upregulated(figure 4D)

GA increases B-cell antigen-presentingcapacity resulting in regulatoryT-cell inductionTo elucidate whether our findings on B-cell properties havemechanistic consequences on antigen-presenting function

Figure 3 GA upregulates MHC Class II expression on B cells

(A) Naivemice received a daily SC injection of 150μgGA Onday 10 post-treatment onset splenic B cells were isolated and analyzed (B and C) for expression ofactivation markers (DndashF) costimulatory molecules and (G) the antigen-presenting molecule MHC Class II as well as (H) for secretion of cytokines Data areshown as median n = 4 p lt 005 Mann-Whitney U test GA = glatiramer acetate

6 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN

Figure 4 GA prevents B-cell activation and ameliorates clinical severity of active EAE

(A) GA therapy was performed by a daily SC injection of 150 μg starting 7 days before MOG peptide35-55 immunization Serum and splenic B cells wereisolated on day 23 post-immunization (B) Mean group EAE severity is given as mean plusmn SEM disease incidence is indicated in brackets n = 15 p lt005 Mann-Whitney U test (C) GA antibody titers were measured at 450 nm (data given as median n = 3ndash4 p lt 0001 Student t test) (D) B-cellactivation expression of molecules involved in antigen presentation and cytokine secretion were analyzed by FACS (data given as median n = 5 p lt005 p lt 001 Mann-Whitney U test) (E) B cells were cocultured with CFSE-labeled myelin-specific (2D2) naive T cells in the presence of 5 25 or100 μgmL MOG peptide35-55 T-cell proliferation was evaluated by CFSE dilution and stratified by division frequency as follows few divisions (1ndash2black) intermediate divisions (3 medium gray) and many divisions (ge4 light gray) T-cell divisions are shown as mean plusmn SEM n = 5 p lt 005 Mann-Whitney U test Differentiation of myelin-specific naive T cells into (F) Treg cells (CD25+FoxP3+CD4+) or (G) Th1- (IFN-γ+CD4+) and Th17 cells (IL-17+CD4+) was analyzed by FACS (data given as median n = 5) EAE = experimental autoimmune encephalomyelitis GA = glatiramer acetate

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 7

B cells were isolated 30 days post-GA treatment onset and23 days post-EAE induction and cocultured with MOG-specific (2D2) T cells in the presence of increasing MOGpeptide35-55 concentrations (figure 4A) As shown in figure4E B cells purified from GA-treated mice triggered a sig-nificantly higher proliferation of myelin-specific T cellsImportantly this related to an expansion of Treg cells(figure 4F) whereas Th1- and Th17 cell frequenciesremained unaffected (figure 4G) Based on these findingswe next assessed the direct effect of GA exposure on B-cellAPC function in vitro Purified naive B cells were pre-incubated with GA following coculture with myelin-specificT cells in the presence of MOG peptide35-55 (figure 5A)GA pre-incubation resulted in a B-cell stimulatory ef-fect (figure e-3 linkslwwcomNXIA218) which wasaccompanied by enhanced capacity to generate Tregcells paralleling our ex vivo findings on GA treatment(figure 5 BndashE)

DiscussionGA has been shown to reduce the relapse rate and pro-gression of neurologic disability in MS2 Past studiesdemonstrated anti-inflammatory properties of GA onT cells468 and myeloid cells91022 First lines of evidenceindicate an immunomodulatory effect on B cells18ndash20 al-though it remained unclear whether this may affect theability of B cells to act as APCs In this article we in-vestigated the phenotype and APC function of B cells in MSand its murine model on treatment with GA We founddecreased frequencies of immature (transitional) B cellsand plasmablasts in GA-treated patients with MS A re-duction in circulating CD19+ B cells in GA-treated patientswith RRMS has been also described previously23 whichcould reflect diminished B-cell survival factors such asBAFF and APRIL after GA therapy as it was observed inEAE20 In this regard of interest may be that we founda correlation between high baseline B-cell frequencies an

Figure 5 GA-treated B cells preferentially generate T regs whereas development of proinflammatory T cells is diminished

(A) Naive B cells purified from WT mice were in-cubatedwith 50μgmLGA or vehicle at 37degC for 3hours After washing B cells were coculturedwith CFSE-labeled myelin-specific (2D2) naiveT cells in the presence of 5 25 or 100 μgmLMOG peptide35-55 (B) T-cell proliferation wasevaluated by CFSE dilution and stratified by di-vision frequency as follows few divisions (1ndash2black) intermediate divisions (3 medium gray)and many divisions (ge4 light gray) T-cell divi-sions are shown as mean plusmn SEM n = 4 p lt 005Mann-Whitney U test Differentiation ofmyelin-specific naive T cells into (C) Treg cells(CD25+FoxP3+CD4+) or (D) Th1- (IFN-γ+CD4+) and(E) Th17 cells (IL-17+CD4+) was analyzed by FACS(data given as median n = 4 p lt 005 Mann-Whitney U test) GA = glatiramer acetate

8 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN

active disease course and a poor GA treatment response(figure e-2C linkslwwcomNXIA218) possibly sug-gesting that patients with MS with increased peripheralblood B-cell numbers might not properly respond to GAtherapy

By longitudinally analyzing the GA effect on B-cell pheno-type we observed a downregulation of the activation markerCD69 CD95 and CD25 and a decrease in TNF-α pro-duction and an increase in IL-10 secretion which wassupported by a recent study showing a shift toward anti-inflammatory cytokine production by B cells on GAtherapy19 Of interest we found a modest but significantupregulation of MHC Class II expression on B cells in GA-treated patients with MS B cells are thought to act as APCsfor presentation of GA to T cells24 Direct binding of GA tomultiple murine and human MHC Class II epitopes2526 hasbeen shown raising the question whether our observationmight have consequences in terms of B-cell APC functionTo address this pivotal issue we first administered GA tonaive WTmice to rule out a disease-related effect and indeednoticed an upregulation of MHC Class II expression onB cells without any effect on other markers of activationDuring pathologic conditions following EAE induction GAtreatment decreased clinical severity B-cell activation andproinflammatory cytokine production whereas the cos-timulatory molecule CD86 and MHC Class II were againupregulated To further elucidate the observed B-cell im-mune modulation with focus on B-cell antigen presentationwe used a coculture in which purified B cells from GA-treated mice or alternatively naive B cells following GApreincubation in vitro were used as APCs to activate naivemyelin-specific T cells GA-treated B cells triggered a signif-icantly higher proliferation of naive myelin-specific T cellscomposed of increased CD4+CD25+FoxP3+ Treg cells AsTGF-szlig is associated with the development of Treg cells wealso measured TGF-szlig production by B cells in our modelhowever at no detectable levels This mechanistic observa-tion which is well supported by earlier reports on an ex-pansion of Treg cells on GA treatment in MS6 and indicatesthat GA centrally interferes with pathogenic B cellndashT cellinteraction in development and propagation of CNS de-myelinating disease

Our findings indicate common features to IFN-β which alsohave been shown to exert immunomodulatory properties onB cells by abrogating proinflammatory and by fostering anti-inflammatory cytokine production27 However IFN-β isthought to primarily downregulate costimulatory moleculesand MHC-Class II27ndash29 our findings suggest the modulationof B-cell antigen presentation by GA as a key role for B cellndashfostered Treg cell development

AntindashCD20-mediated B-cell depletion has been shown tobe a very efficient therapy in MS14ndash17 however treatmentcessation may lead to a recovery of highly differentiatedpathogenic B cells30 and long-term treatment may lower

immunoglobulin production possibly raising the risk ofinfections over time31 Our data support the concept thatGA could act as a suitable maintenance therapy after ces-sation of anti-CD20 treatment by fostering regulatoryproperties in repopulating B cells The first trial in humansprovided inconclusive results32 Although the beneficial ef-fect by GA as maintenance therapy showed superior efficacythan GA therapy alone this benefit seemed to wane withinthe study period More trials are needed as that study waslimited due to a small number of patients and the lack ofa control group receiving no maintenance therapy after rit-uximab cessation

Moreover GA could also have beneficial effects in otherB cellndashmediated diseases such as neuromyelitis optica(NMO) Although aquaporin-4 antibody (AQP4-IgG)-sero-positive patients showed inefficient results3334 first lines ofevidence indicate that patients with AQP4-IgGndashseronegativeNMO may respond to GA therapy333536

In conclusion our data indicate that the pleotropic immu-nomodulatory effect of GA includes B cells and B-cell antigenpresentation resulting in a normalization of MS-specificpathogenic B-cell differentiation and in an expansion of Tregcells These novel findings may complement other establishedeffects of GA in MS may pioneer its preferential use afterB-cell depletion and may lastly be of clinical relevance inother B cellndashdriven CNS autoimmune diseases

AcknowledgmentThe authors thank Katja Grondey and Julian Koch forexcellent technical support

Study fundingD Hausler is supported by the Startprogramm of the UMGJ W Traub is supported by the VorSPrUNG program of theUMG S S Zamvil is supported by research grants from theUSNIH (1 RO1NS092835-01 1 R01 AI131624-01A1 1 R21NS108159-01 and 1 R21AI142186-01A1) the US NationalMultiple Sclerosis Society (1 RG1701-26628) the Weill In-stitute and the Maisin Foundation PH Lalive is supportedby the Swiss National Science Foundation (SNSF_ 310030_176078) MS Weber receives research support from theNational Multiple Sclerosis Society (NMSS PP 1660) theDeutsche Forschungsgemeinschaft (DFG WE 35475-1)from Novartis Teva Biogen Idec Roche Merck and theProFutura Programm of the UMG

DisclosureD Hausler Z Hajiyeva JW Traub SS Zamvil PH Laliveand W Bruck report no disclosures MS Weber is serving asan editor for PLoS One Go to NeurologyorgNN for fulldisclosures

Publication historyReceived by Neurology Neuroimmunology amp NeuroinflammationNovember 13 2019 Accepted in final form January 31 2020

NeurologyorgNN Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 9

References1 Weber MS Menge T Lehmann-Horn K et al Current treatment strategies for multiple

sclerosismdashefficacy versus neurological adverse effects Curr PharmDes 201218209ndash2192 Johnson KP Brooks BR Cohen JA et al Copolymer 1 reduces relapse rate and

improves disability in relapsing-remitting multiple sclerosis results of a phase IIImulticenter double-blind placebo-controlled trial The Copolymer 1 Multiple Scle-rosis Study Group Neurology 1995451268ndash1276

3 Duda PW Schmied MC Cook SL Krieger JI Hafler DA Glatiramer acetate(Copaxone) induces degenerate Th2-polarized immune responses in patients withmultiple sclerosis J Clin Invest 2000105967ndash976

4 Neuhaus O Farina C Yassouridis A et al Multiple sclerosis comparison ofcopolymer-1- reactive T cell lines from treated and untreated subjects reveals cytokineshift from T helper 1 to T helper 2 cells Proc Natl Acad Sci USA 2000977452ndash7457

5 Aharoni R Eilam R Stock A et al Glatiramer acetate reduces Th-17 inflammationand induces regulatory T-cells in the CNS of mice with relapsing-remitting or chronicEAE J Neuroimmunol 2010225100ndash111

6 Hong J Li N Zhang X Zheng B Zhang JZ Induction of CD4+CD25+ regulatoryT cells by copolymer-I through activation of transcription factor Foxp3 Proc NatlAcad Sci USA 20051026449ndash6454

7 Weber MS Prodrsquohomme T Youssef S et al Type II monocytes modulate T cell-mediated central nervous system autoimmune disease Nat Med 200713935ndash943

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

9 Weber MS Starck MWagenpfeil S Meinl E Hohlfeld R Farina C Multiple sclerosisglatiramer acetate inhibits monocyte reactivity in vitro and in vivo Brain 20041271370ndash1378

10 Kim HJ Ifergan I Antel JP et al Type 2 monocyte and microglia differentiationmediated by glatiramer acetate therapy in patients with multiple sclerosis J Immunol20041727144ndash7153

11 StasiolekM Bayas A KruseN et al Impairedmaturation and altered regulatory functionof plasmacytoid dendritic cells in multiple sclerosis Brain 20061291293ndash1305

12 Weber MS Hemmer B Cooperation of B cells and T cells in the pathogenesis ofmultiple sclerosis Results Probl Cell Differ 201051115ndash126

13 Kinzel S Weber MS B cell-directed therapeutics in multiple sclerosis rationale andclinical evidence CNS Drugs 2016301137ndash1148

14 Hauser SL Waubant E Arnold DL et al B-cell depletion with rituximab in relapsing-remitting multiple sclerosis N Engl J Med 2008358676ndash688

15 Kappos L Li D Calabresi PA et al Ocrelizumab in relapsing-remitting multiplesclerosis a phase 2 randomised placebo-controlled multicentre trial Lancet 20113781779ndash1787

16 Hawker K OrsquoConnor P Freedman MS et al Rituximab in patients with primaryprogressive multiple sclerosis results of a randomized double-blind placebo-controlled multicenter trial Ann Neurol 200966460ndash471

17 Montalban X Belachew S Wolinsky JS Ocrelizumab in primary progressive andrelapsing multiple sclerosis N Engl J Med 20173761694

18 Kala M Rhodes SN Piao WH Shi FD Campagnolo DI Vollmer TL B cells fromglatiramer acetate-treated mice suppress experimental autoimmune encephalomy-elitis Exp Neurol 2010221136ndash145

19 Ireland SJ Guzman AA OrsquoBrien DE et al The effect of glatiramer acetate therapy onfunctional properties of B cells from patients with relapsing-remitting multiple scle-rosis JAMA Neurol 2014711421ndash1428

20 Begum-Haque S Sharma A Christy M et al Increased expression of B cell-associatedregulatory cytokines by glatiramer acetate in mice with experimental autoimmuneencephalomyelitis J Neuroimmunol 201021947ndash53

21 Hausler D Torke S Peelen E et al High dose vitamin D exacerbates central nervoussystem autoimmunity by raising T-cell excitatory calcium Brain 20191422737ndash2755

22 Vieira PL Heystek HC Wormmeester J Wierenga EA Kapsenberg ML Glatirameracetate (copolymer-1 copaxone) promotes Th2 cell development and increased IL-10 production through modulation of dendritic cells J Immunol 20031704483ndash4488

23 Carrieri PB Carbone F Perna F et al Longitudinal assessment of immuno-metabolicparameters in multiple sclerosis patients during treatment with glatiramer acetateMetabolism 2015641112ndash1121

24 Jackson LJ Selva S Niedzielko T Vollmer T B cell receptor recognition of glatirameracetate is required for efficacy through antigen presentation and cytokine productionJ Clin Cell Immunol 20145185

25 Fridkis-Hareli M Teitelbaum D Gurevich E et al Direct binding of myelin basicprotein and synthetic copolymer 1 to class II major histocompatibility complexmolecules on living antigen-presenting cellsmdashspecificity and promiscuity Proc NatlAcad Sci USA 1994914872ndash4876

26 Fridkis-Hareli M Strominger JL Promiscuous binding of synthetic copolymer 1 topurified HLA-DR molecules J Immunol 19981604386ndash4397

27 Ramgolam VS Sha Y Marcus KL et al B cells as a therapeutic target for IFN-beta inrelapsing-remitting multiple sclerosis J Immunol 20111864518ndash4526

28 Niino M Hirotani M Miyazaki Y Sasaki H Memory and naive B-cell subsets inpatients with multiple sclerosis Neurosci Lett 200946474ndash78

29 Jiang H Milo R Swoveland P Johnson KP Panitch H Dhib-Jalbut S Interferon beta-1b reduces interferon gamma-induced antigen-presenting capacity of human glial andB cells J Neuroimmunol 19956117ndash25

30 Hausler D Hausser-Kinzel S Feldmann L et al Functional characterization ofreappearing B cells after anti-CD20 treatment of CNS autoimmune disease Proc NatlAcad Sci USA 20181159773ndash9778

31 Marcinno A Marnetto F Valentino P et al Rituximab-induced hypo-gammaglobulinemia in patients with neuromyelitis optica spectrum disorders NeurolNeuroimmunol Neuroinflamm 20185e498 doi101212NXI0000000000000498

32 Honce JM Nair KV Sillau S et al Rituximab vs placebo induction prior to glatirameracetate monotherapy in multiple sclerosis Neurology 201992e723ndashe732

33 Ayzenberg I Schollhammer J Hoepner R et al Efficacy of glatiramer acetate inneuromyelitis optica spectrum disorder a multicenter retrospective study J Neurol2016263575ndash582

34 Stellmann JP Krumbholz M Friede T et al Immunotherapies in neuromyelitis opticaspectrum disorder efficacy and predictors of response J Neurol Neurosurg Psychiatry201788639ndash647

35 Bergamaschi R Glatiramer acetate treatment in Devicrsquos neuromyelitis optica Brain2003126(pt 6)1E author reply 1E-a

36 Gartzen K Limmroth V Putzki N Relapsing neuromyelitis optica responsive toglatiramer acetate treatment Eur J Neurol 200714e12ndashe13

Appendix Authors

Name Location Contribution

DariusHauslerPhD

UMG Performed mouse experimentsand analyzed the data preparedthe figures and wrote themanuscript

ZivarHajiyevaMD

UMG Performed human experimentsand analyzed the data and wrotethe manuscript

Jan WTraub MD

UMG Prepared the figures and reviewingand editing

Scott SZamvil MDPhD

University ofCalifornia SanFrancisco

Reviewing and editing

Patrice HLalive MD

University ofGeneva

Reviewing and editing

WolfgangBruck MD

UMG Reviewing and editing

Martin SWeber MD

UMG Supervised the research and wrotethe manuscript

10 Neurology Neuroimmunology amp Neuroinflammation | Volume 7 Number 3 | May 2020 NeurologyorgNN

DOI 101212NXI000000000000069820207 Neurol Neuroimmunol Neuroinflamm

Darius Haumlusler Zivar Hajiyeva Jan W Traub et al Glatiramer acetate immune modulates B-cell antigen presentation in treatment of MS

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