Dimethyl Fumarate Treatment Mediates an Anti-Inflammatory Shift in B Cell Subsets of Patients with Multiple Sclerosis

This information is current as Rui Li, Ayman Rezk, Mathab Ghadiri, Felix Luessi, Frauke of September 29, 2021. Zipp, Hulun Li, Paul S. Giacomini, Jack Antel and Amit Bar-Or J Immunol 2017; 198:691-698; Prepublished online 14 December 2016;

doi: 10.4049/jimmunol.1601649 Downloaded from http://www.jimmunol.org/content/198/2/691

Supplementary http://www.jimmunol.org/content/suppl/2016/12/14/jimmunol.160164 Material 9.DCSupplemental http://www.jimmunol.org/ References This article cites 32 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/198/2/691.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts Errata An erratum has been published regarding this article. Please see next page or: /content/202/7/2172.full.pdf

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Dimethyl Fumarate Treatment Mediates an Anti-Inflammatory Shift in B Cell Subsets of Patients with Multiple Sclerosis

Rui Li,* Ayman Rezk,* Mathab Ghadiri,* Felix Luessi,*,† Frauke Zipp,† Hulun Li,‡ Paul S. Giacomini,* Jack Antel,* and Amit Bar-Or*

The therapeutic mode of action of dimethyl fumarate (DMF), approved for treating patients with relapsing-remitting multiple sclerosis, is not fully understood. Recently, we and others demonstrated that Ab-independent functions of distinct B cell subsets are important in mediating multiple sclerosis (MS) relapsing disease activity. Our objective was to test whether and how DMF influences both the phenotype and functional responses of disease-implicated B cell subsets in patients with MS. High-quality PBMC

were obtained from relapsing-remitting MS patients prior to and serially after initiation of DMF treatment. Multiparametric flow Downloaded from cytometry was used to monitor the phenotype and functional response-profiles of distinct B cell subsets. Total B cell counts decreased following DMF treatment, largely reflecting losses of circulating mature/differentiated (but not of immature transitional) B cells. Within the mature B cell pool, DMF had a greater impact on memory than naive B cells. In keeping with these in vivo effects, DMF treatment in vitro remarkably diminished mature (but not transitional B cell) survival, mediated by inducing apoptotic cell death. Although DMF treatment (both in vivo and in vitro) minimally impacted B cell IL-10 expression, it strongly reduced B cell ex- pression of GM-CSF, IL-6, and TNF-a, resulting in a significant anti-inflammatory shift of B cell response profiles. The DMF- http://www.jimmunol.org/ mediated decrease in B cell proinflammatory cytokine responses was further associated with reduced phosphorylation of STAT5/6 and NF-kB in surviving B cells. Together, these data implicate novel mechanisms by which DMF may modulate MS disease activity through shifting the balance between pro- and anti-inflammatory B cell responses. The Journal of Immunology, 2017, 198: 691–698.

ultiple sclerosis (MS) is a chronic inflammatory demy- tively little is known about the impact of DMF on B cell subset elinating disease of the CNS (1). An imbalance between responses, which are now strongly implicated in relapsing MS

M proinflammatory immune effectors and anti-inflammatory disease activity based on the clinical success of B cell depleting by guest on September 29, 2021 immune regulators has been implicated in MS disease pathogenesis, therapy. with a traditional focus on the role of particular T cell subsets (1). Interestingly, B cell depletion using anti-CD20 monoclonal Abs Oral dimethyl fumarate (DMF) has been recently approved for treat- effectively decreased MS disease activities without apparently ing patients with relapsing-remitting MS (RRMS) (2–4), yet the affecting the abnormal Ab levels in the CSF of MS patients (12– mode of action for DMF is still not fully understood. Although 15), suggesting that secreting pathogenic autoantibodies may emerging evidence has suggested that DMF can downregulate not be the primary mechanism by which B cells contribute T cell and myeloid cell proinflammatory responses (5–11), rela- to MS relapses. Indeed, we and others have shown that Ab- independent functions of B cells, such as Ag presentation and production of proinflammatory cytokines by functionally dis- *Neuroimmunology Unit, Montreal Neurological Institute, McGill University, Mon- tinct B cell subsets, are important contributors to MS disease † treal, Quebec H3A 2B4, Canada; Department of Neurology, Focus Program Trans- activity (16–23). lational Neuroscience and Immunotherapy, Rhine-Main Neuroscience Network, University Medical Center of the Johannes Gutenberg University Mainz, Mainz In this study we examined whether and how DMF may influence 55131, Germany; and ‡Department of Neurobiology, Harbin Medical University, both the phenotypes and functional response profiles of distinct NanGang District, Harbin 150086, Heilongjiang, China B cell subsets. We show that total B cell counts diminish substan- ORCIDs: 0000-0003-3594-4880 (M.G.); 0000-0002-1231-1928 (F.Z.); 0000-0002- tially following the initiation of DMF treatment, a decrease that 1346-3042 (P.S.G.). largely reflects the loss of circulating differentiated but not of Received for publication September 22, 2016. Accepted for publication November 14, 2016. immature transitional B cells in treated patients. In vitro treatment This work was supported by the research foundation of the Multiple Sclerosis Society with DMF mirrored the in vivo effects, directly inducing mature of Canada (A.B.-O.), Banque National Fellowship (R.L.), and National Natural Sci- B cell but not transitional B cell apoptosis. The functional analysis ence Foundation (Grant 81430035, R.L. and H.L.). further revealed that treatment with DMF (both in vivo and Address correspondence and reprint requests to Prof. Amit Bar-Or, Neuroimmunol- in vitro) decreased B cell expression of proinflammatory (GM-CSF, ogy Unit and Experimental Therapeutics Program, Montreal Neurological Institute a and Hospital, McGill University, 3801 University Street, Room 123, Montreal, QC IL-6, and TNF- ) but minimally impacted anti-inflammatory H3A 2B4, Canada. E-mail address: [email protected] (IL-10) B cell response profiles, associated not only with prefer- The online version of this article contains supplemental material. ential apoptosis, but also with reduced phosphorylation of STAT5/6 Abbreviations used in this article: DMF, dimethyl fumarate; HC, healthy control; and NF-kB in surviving B cells. Our study suggests that the ca- ICS, intracellular cytokine staining; MMF, monomethyl fumarate; MS, multiple scle- pacity of DMF to limit new MS inflammatory disease activity may, rosis; PI, propidium iodide; RRMS, relapsing-remitting MS. in part, relate to its ability to mediate an anti-inflammatory shift in Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 the balance of phenotypically and functionally distinct B cell subsets. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601649 692 DMF REGULATES THE BALANCE OF B CELL SUBSETS IN MS PATIENTS

interassay variability. Where indicated, magnetic bead sorting (Miltenyi Materials and Methods + Subjects and study design Biotec) was used to positively select CD19 B cells from fresh PBMC with purities routinely .98% as confirmed by flow cytometry. Isolated B cells A total of 13 patients (11 females, 2 males) with McDonald criteria– were plated in U-bottom 96-well plates at 2 3 105/well in a total volume of confirmed RRMS, mean age 41 (range 20–60), were prospectively fol- 200 ml of serum-free x-vivo medium (Lonza), and stimulated with soluble lowed at a single center in Montreal, Canada, prior to and following CD40L (1 mg/ml; Enzo Life Sciences), goat anti-human BCR cross- treatment initiation with DMF. Patients were assessed every 3 mo with linking Ab (Xab) (10 mg/ml; Jackson ImmunoResearch) with or without clinical review, physical examination, and expanded disability status score. IL-4 (20 ng/ml; R&D Systems) for 48 h, at which time supernatants were At study entry, patients had an average expanded disability status score of collected and frozen (270) for subsequent quantification of cytokine secretion 2.5 (range 1.0–4.0), preceding annualized relapse rate of 0.8 (0–2), and by ELISA and the cells were analyzed by flow cytometry (as described be- disease duration of 9.6 y (range 1–27 y). Of the 13 patients, 11 had pre- low). B cells were cultured in parallel wells in either medium alone, vehicle viously been treated with either IFN or glatiramer acetate, one had re- (DMSO), monomethyl fumarate (MMF), or DMF (Sigma Aldrich, Oakville, ceived a single dose of ofatumumab 18 mo prior to recruitment, and one ON, Canada). MMF and DMF were added to individual wells at concentra- was treatment-naive. All participants, including healthy control (HC) tions of 50 mM, with DMSO control added at the equivalent concentration. volunteers providing blood for in vitro studies, were recruited at the Montreal Neurological Institute and Hospital after providing informed Abs for flow cytometry analysis and intracellular cytokine consent as approved by the Montreal Neurological Institute and Hospital staining ethics review board. B cell immunophenotyping panels are listed in the Supplemental Table. Abs Sample processing and cell culture used to phenotype B cells were directed against: CD11c (B-ly6), CD20 (2H7), CD24 (ML5), CD27 (L128), CD38 (HB7), CD43 (1G10), CD80 (B High-quality PBMCs were isolated from all HC participants and from Montreal Neurological Institute B1), CD83 (HB15e), CD86 (2331(Fun-1)),

RRMS patients prior to and following treatment initiation with DMF. All HLA-DR (G46-6), IgD (1A6-2), IgG (G18-145), IgM (G20-127) and appro- Downloaded from steps of sample procurement, handling, PBMC isolation (by density cen- priate isotype controls, all purchased from BD Bioscience. IgA (IS11-8E10) trifugation using Ficoll; GE Healthcare), cryopreservation, and subsequent was purchased from Miltenyi Biotec. Abs for intracellular cytokine staining thawing followed the identical standard operating procedures developed (ICS) targeted: IL-6 (MQ2-6A3), IL-10 (JEF3-19F1), TNF-a (MAb11), and and validated by the experimental therapeutics program of the Montreal GM-CSF (BVD2-21C11), as well as appropriate isotype controls, all from BD Neurological Institute. Cryopreserved PBMC from individual patients, Bioscience. ICS involved 4 h stimulation with PMA (20 ng/ml; Sigma- collected serially, were thawed and cultured in batch, thereby eliminating Aldrich), Ionomycin (500 ng/ml; Sigma-Aldrich), and GolgiStop (Monensin; http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. B cell flow cytometry immunophenotyping gating strategy. PBMC were first gated based on their forward-scatter (FSC) and side-scatter (SSC). Doublets were excluded from the analysis using FSC-area (FSC-A) and FSC-height (FSC-H), as well as SSC-area (SSC-A) and SSC-height (SSC-H). Live cells were defined as negative for staining with Live/Dead marker. B cells were then gated as CD20+ CD32. Intracellular cytokine positive B cells was quantified as compared with the appropriate isotype controls. B1 cells are identified as CD43+ CD27+; mature B cells as CD24+/int CD382/int; and transitional B cells (Trans B) as CD24high CD38high. Within the mature B cell gating, naive B cells (nB) are identified as IgD+ CD272; class-switched memory B cells (CSM) as: IgA+ CD27+ or IgG+ CD27+; non–class-switched memory B cells (USM) as IgM+ CD27+; and double negative memory B cells (DNM) as IgD2 CD272. The Journal of Immunology 693

BD Bioscience) was followed by cell surface staining, then two washes and washing buffer (0.05% Tween 20, PBS) between each step. Hydrogen addition of a fixation/permeabilization buffer (Cytofix/Cytoperm; BD Biosci- peroxide and 3,39,5,59-tetramethylbenzidine (BD Bioscience) were then ence). Cells were then washed with the ICS washing buffer (BD Bioscience) added and the reaction was stopped by 0.01N H2SO4. The plates were then and ICS Abs (noted above) were added to the cell suspensions followed by two read by a Bio-Rad microplate reader (Model 550; Bio-Rad). additional washes with the ICS washing buffer. For apoptosis assays, cells were stained with Annexin V and propidium iodide (PI; BD Biosciences) following Statistical analysis the cell surface staining. All flow phenotyping was carried out by a single A Student paired t test was used for statistical comparisons between two operator who was blinded to the sample source and followed the same stan- groups and one-way ANOVAwas used for statistical comparisons between dardized immune phenotyping protocol (Fig. 1), using an LSR Fortessa flow more than two groups, as indicated in the figure legends. GraphPad Prism 6 cytometer (BD Biosciences) and FlowJo software analysis (Tree Star). was used to perform all the statistical analyses. A p value # 0.05 was ELISA considered statistically significant. Levels of secreted cytokines (GM-CSF, TNF-a, IL-6, and IL-10) within the frozen culture supernatants were quantified by OptEIA ELISA kit (BD Results Bioscience) based on the manufacturer’s protocols. Briefly, ELISA plates DMF treatment preferentially impacts mature and were coated with capture Ab at least 12 h in advance. After 1-h blocking differentiated B cells in vivo with blocking buffer (10% FCS, PBS), supernatant samples were added to the plate and incubated for 2 h at room temperature. Then, detection Ab To assess the impact of in vivo DMF treatment on B cell subsets, was added for 1 h at room temperature. Plates were washed with ELISA absolute counts of surface-defined B cell subsets (gated as in Fig. 1) Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. DMF preferentially targets mature/differentiated B cells in vivo. PBMC from RRMS patients (n = 12) were obtained pretreatment and then at least 3 mo after initiation of DMF treatment. Absolute counts of B cell subsets were defined using flow cytometry. Each line represents values for an individual patient studied pre- and post-DMF treatment, with the histograms reflecting average values. (A) Total B cells. (B) Naive B cells. (C) Memory B cells. (D) Naive/memory B cell ratio. (E) Un–class-switched memory B cells. (F) Class-switched memory B cells. (G) Double-negative memory B cells. (H) Transitional B cells. (I) Mature B cells. (J) Transitional/mature B cell ratio. Total B cell counts decreased following initiation of DMF treatment, which largely reflected losses of circulating differentiated (but not of immature transitional) B cells. *p , 0.05, **p , 0.01. ns, not significant. 694 DMF REGULATES THE BALANCE OF B CELL SUBSETS IN MS PATIENTS were quantified by flow cytometry within PBMC obtained from (Fig. 2H), which, together with reduced mature (CD24+/int CD382/int) patients with RRMS (n = 12) pretreatment and up to 12 mo after B cell numbers (Fig. 2I, p = 0.01), resulted in an increase of the initiation of DMF treatment. Total circulating B cell counts were transitional/mature B cell ratio (Fig. 2J, p = 0.02). These data indicate substantially reduced (by ∼45%) in DMF-treated MS patients that DMF preferentially impacts mature (especially memory) B cells (Fig. 2A, p = 0.01), reflecting decreased counts of both naive in vivo. All these changes were observed by the first (3 mo) post- (Fig. 2B, p = 0.04) and memory (Fig. 2C, p = 0.0011) B cell treatment assessment, and at 12 mo on treatment (Supplemental subsets. The memory B cells appeared more affected, resulting Figs. 1, 2), pointing to an early and persistent differential effect of in a small increase of the naive/memory B cell ratio following DMF treatment on distinct B cell subsets. treatment (Fig. 2D, p = 0.028). Among memory B cells, reduced counts were seen for all subsets, including class-switched memory DMF modulates the balance between proinflammatory and B cells, non–class-switched memory B cells, and double-negative anti-inflammatory B cell responses in treated patients memory B cells (Fig. 2E–G, p , 0.05). In contrast, immature Prior work has shown that B cells of untreated MS patients can transitional (CD24high CD38high) B cells were largely not impacted express abnormally high levels of IL-6, TNF-a, and GM-CSF as Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Differential impact of DMF on MS disease–implicated B cell subsets. (A–G) To detect ex vivo cytokine expression by B cells within PBMC, pre- and post-DMF treatment PBMC were briefly stimulated with PMA and ionomycin in presence of GolgiStop for 4 h. Flow cytometry and ICS was then used to detect cytokines (GM-CSF, IL-10, IL-6, and TNF-a) within B cells. (H–K) DMF impact on B cell expression of molecules implicated in mature B cell:T cell interactions (CD80, CD83, CD11c+, CD43+CD27+), were measured by flow cytometry. Each line represents values for an individual patient studied pre- and post-DMF treatment, with the histograms reflecting average values. *p , 0.05, **p , 0.01, ***p , 0.001. ns, not significant. The Journal of Immunology 695 well as deficient levels of IL-10 (20). We therefore considered survival and apoptosis. We found that DMF exposure resulted in whether and how DMF treatment might impact such MS disease– reduced survival of total B cells, which was due to apoptotic cell implicated cytokine-defined B cell subsets in treated patents. We death (Fig. 4A, 4B). These cell losses largely reflected apoptosis found that DMF treatment resulted in substantial decreases in the of mature B cell subsets, whereas, in contrast, survival of transi- frequencies of B cells expressing the proinflammatory cytokines tional B cells was minimally affected (Fig. 4C–F). MMF also IL-6, TNF-a, and GM-CSF (Fig. 3A–C). The counts of IL-10– induced B cell apoptosis but to a much lesser extent compared expressing B cells in these treated patients also tended to decrease, with DMF (Fig. 4A–F). Substantial DMF-induced apoptosis of although to a lesser extent (Fig. 3D), resulting in diminished B cells was also seen when DMF was added to B cells that were proinflammatory B cell cytokine profiles as indicated by the de- activated by combined stimulation through the BCR, CD40, and creased ratios of proinflammatory/anti-inflammatory B cell subsets IL-4 (a combination known to enhance B cell survival; Fig. 4G, (Fig. 3E–G). In keeping with this, DMF treatment also resulted in a 4H). Together, these results further support the concept that DMF substantially reduced expression of the B cell surface molecules treatment mainly impacts mature B cell survival, and that the CD11c, CD43, CD80, and CD83, known to play important roles in mechanism underlying their preferential losses reflects at least in mature B cell:T cell interactions (Fig. 3H–K, p , 0.001). part the enhanced susceptibility of these subsets to DMF-induced apoptotic cell death. DMF exposure in vitro induces selective B cell subset apoptosis that mirrors the preferential in vivo depletion of mature B cell DMF decreases proinflammatory B cell cytokine responses subsets partially through a pSTAT5/6 and NF-kB dependent mechanism To explore how DMF treatment may lead to preferential decreases Downloaded from of mature B cells in vivo, we exposed freshly isolated human HC Based on our earlier observation that in vivo DMF treatment re- peripheral B cells to either DMF or MMF and assessed B cell subset duces the proinflammatory cytokine response profiles of B cells in http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. DMF (but not MMF) induces mature B cell apoptosis in vitro. Purified human B cells were either left untreated or treated with vehicle (Veh), DMF, or MMF for 24 h. B cell apoptosis was detected by Annexin V and PI staining and quantified by flow cytometry. Early apoptotic B cells were defined as Annexin V+ PI2, whereas late apoptotic B cells were defined as Annexin V and PI double-positive cells. DMF preferentially induced mature B cell apoptosis (n = 9 independent experiments). (G and H) B cells were either left untreated or treated with vehicle (Veh), (A–F) DMF or MMF and stimulated with CD40L+aBCR+IL-4 for 48 h. B cell apoptosis was detected by Annexin V and PI staining and quantified by flow cytometry, as above (n = 5). *p , 0.05, **p , 0.01. ns, not significant. 696 DMF REGULATES THE BALANCE OF B CELL SUBSETS IN MS PATIENTS patients, we assessed the impact of in vitro exposure to DMF and cated that DMF may downregulate dendritic cell responses MMF on cytokine responses of activated B cells. We found that through NF-kB signaling (24, 25) and our own work recently DMF exposure substantially reduced B cell secretion of multiple showed that STAT5 and STAT6 signaling in B cells contributes cytokines as measured by ELISA and recapitulated the selective to induction of proinflammatory GM-CSF expression (20). We effects that in vivo treatment had on distinct cytokine-defined B cell therefore assessed whether DMF exposure may impact either of subsets, namely, causing marked reductions in the proinflammatory these signaling pathways. We observed that B cell exposure to B cell cytokine responses (IL-6, TNF-a, and GM-CSF), and a DMF strongly decreased both NF-kB (Fig. 6A) and STAT5/ lesser reduction in IL-10 responses (Fig. 5A–D). These differential STAT6 (Fig. 6B, 6C) phosphorylation in live activated B cells. effects again resulted in substantially decreased proinflammatory/ anti-inflammatory cytokine ratios expressed by the B cells ex- Discussion posed to DMF (Fig. 5E–G). As with the survival data, MMF ex- Oral DMF has been recently approved for treating patients with posure in vitro had little or no effect on B cell cytokine responses relapsing MS (3, 4, 26–29). The mode of action of DMF has not (Fig. 5A–G). been fully elucidated as it relates to MS disease mechanisms. We considered that the substantial reduction in proinflammatory Increasing evidence, particularly the success of B cell depleting cytokine secretion by B cells exposed to DMF was greater than therapy in limiting new MS disease activity (12–14), has pointed might be expected based on the degree of apoptotic cell death to B cells as relevant mediators of disease relapses. In this study previously noted. This raised the possibility that, in addition to we tested whether and how DMF may impact the B cell com- inducing apoptotic loss of B cells, DMF may modulate the cytokine partment in the context of MS. Using a series of iterative in vivo expression profile of viable cells. To address this, we examined the and in vitro studies, we first monitored the effect of DMF on B cell Downloaded from effects of DMF and MMF on B cell cytokine expression using ICS, subset phenotypes in treated patients and observed that mature and gated on live cells only. Indeed, exposure to DMF (and to a B cells (particularly memory B cells), but not transitional B cells, lesser extent to MMF) decreased proinflammatory B cell cytokine are preferentially lost in the circulation. This suggested that DMF expression in non-apoptotic cells (Fig. 5I). Prior work has indi- does not prevent the output of immature B cells from bone marrow, http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Both DMF and MMF decrease B cell proinflammatory cytokine secretion. Purified human B cells were either left untreated or treated with vehicle (Veh), DMF or MMF before stimulation with CD40L+aBCR+IL-4. Overall cytokine secretion [IL-6 (A), TNF-a (B), GM-CSF (C) and IL-10 (D)] by B cells was measured by ELISA (A–G) and cytokine expression by live B cells was analyzed by flow cytometry using ICS as noted previously. (H) Representative dot plot for assessing GM-CSF expression by activated viable B cells. Impact of DMF and MMF on viable B cell expression of GM-CSF (I) and TNF-a (J)inn = 5 independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. ns, not significant. The Journal of Immunology 697

FIGURE 6. DMF inhibits phosphor- ylation of STAT5/6 and NF-kB. Purified human peripheral B cells were either left untreated or treated with vehicle (Veh), DMF or MMF before stimulation with CD40L+aBCR+IL-4. Phosphory- lation of NF-kB(A and B), STAT6 (C and D) and STAT6 (E and F) within live cells were detected by flow cytometry (n = 3 independent experiments). *p , 0.05. ns, not significant. Downloaded from http://www.jimmunol.org/ but preferentially impacts more mature B cell subsets. A series of levels of costimulatory molecules and proinflammatory cytokines ex vivo and in vitro experiments further indicated that DMF may (16–18, 20, 32) — which can both more efficiently induce downregulate proinflammatory cytokine production from B cells, proinflammatory T cell responses — so the increase in naive/ both by preferential induction of their apoptotic cell death as well as memory B cell ratio after DMF treatment may be associated by limiting proinflammatory cytokine responses of viable B cells, with dampened ability of the B cell compartment to mediate T cell through inhibition of both pSTAT5/6 and NF-kB. activation. Finally, we show that DMF does not just affect the Several studies have recently shown that DMF treatment in proportions of phenotypically defined B cell subsets (e.g., transi- by guest on September 29, 2021 patients reduces circulating T cell (especially CD8+ T cell) counts tional versus mature; naive versus memory), but also mediates an (6–9). Decreases in total B cell counts have also been noted in anti-inflammatory shift in the remaining B cell cytokine re- DMF-treated patients (7, 9, 11). In vivo, the generation of mature sponses. Of particular interest in this regard is our observation B cells is partially dependent on T cells, such that decreased that, both in vitro and in vivo, DMF treatment results in an in- mature B cells with DMF treatment might indirectly reflect the creased ratio of IL-10+ B cells to GM-CSF+ B cells, a consequence known effect of DMF on T cells. However, our in vitro observa- of both preferential apoptotic cell death of proinflammatory B cells tions indicate that DMF can have important direct effects on B cell as well as downmodulation of proinflammatory cytokine expres- survival as well as modulation of the response profiles of surviving sion by surviving B cells (involving pSTAT5/6 and NF-kB sig- B cells. The prior T cell studies also noted that memory T cells are naling). Recently, a subpopulation of STAT5/6–dependent human more affected by DMF than naive T cells (6–9), which is similar B cell subset that expresses GM-CSF was found to be over- to our observation with B cells, suggesting that a common sig- represented in the circulation of patients with MS (20). An im- naling pathway (susceptible to DMF), may regulate the survival of balance in these patients between GM-CSF+ B cells and IL-10+ both memory B cells and memory T cells. B cells was found to be associated with a proinflammatory shift in Our implication of B cells as a relevant target for DMF’s ability their myeloid cell responses. Our current study suggests that the to limit CNS inflammation is supported by recent results indicating differential effects of DMF on functionally distinct B cell subsets, that DMF can decrease disease severity in a B cell–dependent and in particular an anti-inflammatory shift in the cytokine re- model of experimental autoimmune encephalomyelitis (30). Our sponses of remaining B cells, may contribute to its ability to limit data from treated patients and using human-derived B cells indi- new MS relapses. cates that DMF treatment could mediate an anti-inflammatory In conclusion, we have demonstrated that DMF particularly shift of B cell responses at several levels. First, we note that targets proinflammatory mature B cell subsets through preferential DMF has relatively little impact on transitional B cells although it induction of apoptotic cell death as well as downregulation of substantially impacts survival of mature B cells. Previous studies pSTAT5/6 and NF-kB in surviving B cells. In the context of MS, have shown that transitional B cells can exhibit immune modu- these findings extend our understanding of DMF’s putative mode latory functions through the secretion of anti-inflammatory IL-10 of action in patients as potentially limiting new disease relapses (31). Our findings suggest that transitional B cells and mature through modulation of the B cell compartment. Of a broader B cells use different machinery to maintain their survival, an in- implication, our results point to different utilization of intracel- sight that may have useful implications for developing more se- lular signaling and survival pathways by functionally distinct lective B cell targeting therapy. We also note that DMF results in human B cell subsets, which may help to guide development of preferential losses of memory versus naive B cells. Compared therapies that more selectively target particular B cell subsets of with naive B cells, memory B cells are known to express higher interest. 698 DMF REGULATES THE BALANCE OF B CELL SUBSETS IN MS PATIENTS

Acknowledgments 16. Duddy, M. E., A. Alter, and A. Bar-Or. 2004. Distinct profiles of human B cell effector cytokines: a role in immune regulation? J. Immunol. 172: 3422–3427. We appreciate the important input of the Experimental Therapeutics Pro- 17. Duddy, M., M. Niino, F. Adatia, S. Hebert, M. Freedman, H. Atkins, H. J. Kim, gram at the Montreal Neurological Institute for judicious handling of patient and A. Bar-Or. 2007. Distinct effector cytokine profiles of memory and naive and control samples. human B cell subsets and implication in multiple sclerosis. J. Immunol. 178: 6092–6099. 18. Bar-Or, A., L. Fawaz, B. Fan, P. J. Darlington, A. Rieger, C. Ghorayeb, Disclosures P. A. Calabresi, E. Waubant, S. L. Hauser, J. Zhang, and C. H. Smith. 2010. Abnormal B-cell cytokine responses a trigger of T-cell-mediated disease in MS? The authors have no financial conflicts of interest. Ann. Neurol. 67: 452–461. 19. Barr, T. A., P. Shen, S. Brown, V. Lampropoulou, T. Roch, S. Lawrie, B. Fan, R. A. O’Connor, S. M. Anderton, A. Bar-Or, et al. 2012. B cell depletion therapy References ameliorates autoimmune disease through ablation of IL-6-producing B cells. 1. Dendrou, C. A., L. Fugger, and M. A. Friese. 2015. Immunopathology of mul- J. Exp. Med. 209: 1001–1010. tiple sclerosis. Nat. Rev. Immunol. 15: 545–558. 20. Li, R., A. Rezk, Y. Miyazaki, E. Hilgenberg, H. Touil, P. Shen, C. S. Moore, 2. Kappos, L., R. Gold, D. H. Miller, D. G. Macmanus, E. Havrdova, V. Limmroth, L. Michel, F. Althekair, S. Rajasekharan, et al; Canadian B cells in MS Team. C. H. Polman, K. Schmierer, T. A. Yousry, M. Yang, et al; BG-12 Phase IIb 2015. Proinflammatory GM-CSF-producing B cells in multiple sclerosis and Study Investigators. 2008. Efficacy and safety of oral fumarate in patients with B cell depletion therapy. Sci. Transl. Med. 7: 310ra166. relapsing-remitting multiple sclerosis: a multicentre, randomised, double-blind, 21. Molnarfi, N., U. Schulze-Topphoff, M. S. Weber, J. C. Patarroyo, placebo-controlled phase IIb study. Lancet 372: 1463–1472. T. Prod’homme, M. Varrin-Doyer, A. Shetty, C. Linington, A. J. Slavin, 3. Gold, R., L. Kappos, D. L. Arnold, A. Bar-Or, G. Giovannoni, K. Selmaj, J. Hidalgo, et al. 2013. MHC class II-dependent B cell APC function is required C. Tornatore, M. T. Sweetser, M. Yang, S. I. Sheikh, and K. T. Dawson, DEFINE for induction of CNS autoimmunity independent of myelin-specific antibodies. Study Investigators. 2012. Placebo-controlled phase 3 study of oral BG-12 for J. Exp. Med. 210: 2921–2937. relapsing multiple sclerosis. N. Engl. J. Med. 367: 1098–1107. 22. Shen, P., T. Roch, V. Lampropoulou, R. A. O’Connor, U. Stervbo, E. Hilgenberg, 4. Fox, R. J., D. H. Miller, J. T. Phillips, M. Hutchinson, E. Havrdova, M. Kita, S. Ries, V. D. Dang, Y. Jaimes, C. Daridon, et al. 2014. IL-35-producing B cells Downloaded from M. Yang, K. Raghupathi, M. Novas, M. T. Sweetser, et al; CONFIRM Study are critical regulators of immunity during autoimmune and infectious diseases. Investigators. 2012. Placebo-controlled phase 3 study of oral BG-12 or glatir- Nature 507: 366–370. amer in multiple sclerosis. N. Engl. J. Med. 367: 1087–1097. 23. Li, R., A. Rezk, L. M. Healy, G. Muirhead, A. Prat, J. L. Gommerman, and 5. Parodi, B., S. Rossi, S. Morando, C. Cordano, A. Bragoni, C. Motta, C. Usai, A. Bar-Or, MSSRF Canadian B cells in MS Team. 2016. Cytokine-defined B cell B. T. Wipke, R. H. Scannevin, G. L. Mancardi, et al. 2015. Fumarates modulate responses as therapeutic targets in multiple sclerosis. Front. Immunol. 6: 626. microglia activation through a novel HCAR2 signaling pathway and rescue 24. Peng, H., M. Guerau-de-Arellano, V. B. Mehta, Y. Yang, D. J. Huss, synaptic dysregulation in inflamed CNS. Acta Neuropathol. 130: 279–295. T. L. Papenfuss, A. E. Lovett-Racke, and M. K. Racke. 2012. Dimethyl fumarate 6. Longbrake, E. E., M. J. Ramsbottom, C. Cantoni, L. Ghezzi, A. H. Cross, and inhibits dendritic cell maturation via nuclear factor kB (NF-kB) and extracellular

L. Piccio. 2016. Dimethyl fumarate selectively reduces memory T cells in mul- signal-regulated kinase 1 and 2 (ERK1/2) and mitogen stress-activated kinase 1 http://www.jimmunol.org/ tiple sclerosis patients. Mult. Scler. 22: 1061–1070. (MSK1) signaling. J. Biol. Chem. 287: 28017–28026. 7. Spencer, C. M., E. C. Crabtree-Hartman, K. Lehmann-Horn, B. A. Cree, and 25. Gillard, G. O., B. Collette, J. Anderson, J. Chao, R. H. Scannevin, D. J. Huss, S. S. Zamvil. 2015. Reduction of CD8(+) T lymphocytes in multiple sclerosis and J. D. Fontenot. 2015. DMF, but not other fumarates, inhibits NF-kB activity patients treated with dimethyl fumarate. Neurol. Neuroimmunol. Neuroinflamm. in vitro in an Nrf2-independent manner. J. Neuroimmunol. 283: 74–85. 2: e76. 26. Moharregh-Khiabani, D., R. A. Linker, R. Gold, and M. Stangel. 2009. Fumaric 8. Tahvili, S., B. Zandieh, and Z. Amirghofran. 2015. The effect of dimethyl fu- Acid and its esters: an emerging treatment for multiple sclerosis. Curr. Neuro- marate on gene expression and the level of cytokines related to different T helper pharmacol. 7: 60–64. cell subsets in peripheral blood mononuclear cells of patients with psoriasis. Int. 27. Havrdova, E., M. Hutchinson, N. C. Kurukulasuriya, K. Raghupathi, J. Dermatol. 54: e254–e260. M. T. Sweetser, K. T. Dawson, and R. Gold. 2013. Oral BG-12 (dimethyl fumarate) 9. Gross, C. C., A. Schulte-Mecklenbeck, S. Klinsing, A. Posevitz-Fejfa´r, for relapsing-remitting multiple sclerosis: a review of DEFINE and CONFIRM. H. Wiendl, and L. Klotz. 2015. Dimethyl fumarate treatment alters circulating T Evaluation of: Gold R, Kappos L, Arnold D, et al. Placebo-controlled phase 3 helper cell subsets in multiple sclerosis. Neurol. Neuroimmunol. Neuroinflamm. study of oral BG-12 for relapsing multiple sclerosis. N Engl J Med 2012;367:1098- by guest on September 29, 2021 3: e183. 107; and Fox RJ, Miller DH, Phillips JT, et al. Placebo-controlled phase 3 study of 10. Michell-Robinson, M. A., C. S. Moore, L. M. Healy, L. A. Osso, N. Zorko, oral BG-12 or glatiramer in multiple sclerosis. N Engl J Med 2012;367:1087-97. V. Grouza, H. Touil, L. Poliquin-Lasnier, A. M. Trudelle, P. S. Giacomini, et al. Expert Opin. Pharmacother. 14: 2145–2156. 2015. Effects of fumarates on circulating and CNS myeloid cells in multiple 28. Bar-Or, A., R. Gold, L. Kappos, D. L. Arnold, G. Giovannoni, K. Selmaj, sclerosis. Ann. Clin. Transl. Neurol. 3: 27–41. J. O’Gorman, M. Stephan, and K. T. Dawson. 2013. Clinical efficacy of BG-12 11. Lundy, S. K., Q. Wu, Q. Wang, C. A. Dowling, S. H. Taitano, G. Mao, and (dimethyl fumarate) in patients with relapsing-remitting multiple sclerosis: Y. Mao-Draayer. 2016. Dimethyl fumarate treatment of relapsing-remitting subgroup analyses of the DEFINE study. J. Neurol. 260: 2297–2305. multiple sclerosis influences B-cell subsets. Neurol. Neuroimmunol. Neuro- 29. Hutchinson, M., R. J. Fox, D. H. Miller, J. T. Phillips, M. Kita, E. Havrdova, inflamm. 3: e211. J. O’Gorman, R. Zhang, M. Novas, V. Viglietta, and K. T. Dawson. 2013. 12. Hauser, S. L., E. Waubant, D. L. Arnold, T. Vollmer, J. Antel, R. J. Fox, A. Bar- Clinical efficacy of BG-12 (dimethyl fumarate) in patients with relapsing- Or, M. Panzara, N. Sarkar, S. Agarwal, et al; HERMES Trial Group. 2008. B-cell remitting multiple sclerosis: subgroup analyses of the CONFIRM study. depletion with rituximab in relapsing-remitting multiple sclerosis. N. Engl. J. J. Neurol. 260: 2286–2296. Med. 358: 676–688. 30. Schulze-Topphoff, U., M. Varrin-Doyer, K. Pekarek, C. M. Spencer, A. Shetty, 13. Kappos, L., D. Li, P. A. Calabresi, P. O’Connor, A. Bar-Or, F. Barkhof, M. Yin, S. A. Sagan, B. A. Cree, R. A. Sobel, B. T. Wipke, L. Steinman, et al. 2016. D. Leppert, R. Glanzman, J. Tinbergen, and S. L. Hauser. 2011. Ocrelizumab in Dimethyl fumarate treatment induces adaptive and innate immune modulation relapsing-remitting multiple sclerosis: a phase 2, randomised, placebo- independent of Nrf2. Proc. Natl. Acad. Sci. USA 113: 4777–4782. controlled, multicentre trial. Lancet 378: 1779–1787. 31. Blair, P. A., L. Y. Noren˜a, F. Flores-Borja, D. J. Rawlings, D. A. Isenberg, 14. Bar-Or, A., P. A. Calabresi, D. Arnold, C. Markowitz, S. Shafer, L. H. Kasper, M. R. Ehrenstein, and C. Mauri. 2010. CD19(+)CD24(hi)CD38(hi) B cells ex- E. Waubant, S. Gazda, R. J. Fox, M. Panzara, et al. 2008. Rituximab in relapsing- hibit regulatory capacity in healthy individuals but are functionally impaired in remitting multiple sclerosis: a 72-week, open-label, phase I trial. [Published systemic Lupus Erythematosus patients. Immunity 32: 129–140. erratum appears in 2008 Ann. Neurol. 63: 803.] Ann. Neurol. 63: 395–400. 32. Bar-Or, A., E. M. Oliveira, D. E. Anderson, J. I. Krieger, M. Duddy, 15. Cross, A. H., J. L. Stark, J. Lauber, M. J. Ramsbottom, and J. A. Lyons. 2006. K. C. O’Connor, and D. A. Hafler. 2001. Immunological memory: contribution Rituximab reduces B cells and T cells in cerebrospinal fluid of multiple sclerosis of memory B cells expressing costimulatory molecules in the resting state. patients. J. Neuroimmunol. 180: 63–70. J. Immunol. 167: 5669–5677. 2172 The Journal of Immunology

Corrections

Li, R., A. Rezk, M. Ghadiri, F. Luessi, F. Zipp, H. Li, P. S. Giacomini, J. Antel, and A. Bar-Or. 2017. Dimethyl fumarate treatment mediates an anti-inflammatory shift in B cell subsets of patients with multiple sclerosis. J. Immunol. 198: 691–698.

The third author’s first name was published incorrectly. The correct spelling of the author’s name is Mahtab Ghadiri. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900114

Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50