Isotype-Switched Autoantibodies Are Necessary to Facilitate Central

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Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda−/− and Ung−/− Mice This information is current as of September 26, 2021. Georgina Galicia, Dennis S. W. Lee, Valeria Ramaglia, Lesley A. Ward, Jennifer Y. Yam, Leslie Y. T. Leung, Rui Li, Marcus Handy, Junxian Zhang, Paulina C. Drohomyrecky, Eric Lancaster, Amit Bar-Or, Alberto Martin and Jennifer L. Gommerman Downloaded from J Immunol 2018; 201:1119-1130; Prepublished online 6 July 2018; doi: 10.4049/jimmunol.1700729 http://www.jimmunol.org/content/201/4/1119 http://www.jimmunol.org/

Supplementary http://www.jimmunol.org/content/suppl/2018/07/06/jimmunol.170072 Material 9.DCSupplemental References This article cites 55 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/201/4/1119.full#ref-list-1 by guest on September 26, 2021

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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 © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda2/2 and Ung2/2 Mice

Georgina Galicia,*,1 Dennis S. W. Lee,*,1 Valeria Ramaglia,* Lesley A. Ward,* Jennifer Y. Yam,* Leslie Y. T. Leung,* Rui Li,†,‡ Marcus Handy,†,‡ Junxian Zhang,‡ Paulina C. Drohomyrecky,*,x Eric Lancaster,‡ Amit Bar-Or,†,‡ Alberto Martin,* and Jennifer L. Gommerman*

B cell–depleting therapies have been shown to ameliorate symptoms in multiple sclerosis (MS) patients; however, the mechanism of action remains unclear. Following priming with Ag, B cells undergo secondary diversification of their BCR, including BCR Downloaded from class-switch recombination (CSR) and somatic hypermutation (SHM), with both processes requiring the enzyme activation- induced (cytidine) deaminase. We previously reported that activation-induced (cytidine) deaminase is required for full clinical manifestation of disease in an animal model of MS (experimental autoimmune encephalomyelitis; EAE) provoked by immunization with the extracellular domain of recombinant human myelin oligodendrocyte glycoprotein (hMOG). In this study, we investigated the role of CSR versus SHM in the pathogenesis of EAE. We found that passive transfer of class-switched anti-MOG IgG1 Abs into hMOG-primed Aicda2/2 mice is sufficient to fully rescue EAE disease. In addition, we found that the nature of the http://www.jimmunol.org/ Ag is an important determinant of EAE severity in Aicda2/2 mice because the lack of a diversified BCR does not affect the induction of EAE when immunized with the extracellular domain of rat MOG. To discriminate the effect of either CSR or SHM, we induced EAE in uracil DNA glycosylase–deficient mice (Ung2/2) that exhibit a defect primarily in CSR. We observed that 2/2 Ung mice exhibit milder clinical disease compared with control mice, concomitant with a reduced amount of anti-MOG IgG1 class-switched Abs that preserved normal affinity. Collectively, these results indicate that CSR plays an important role in governing the incidence and severity of EAE induced with hMOG but not rat MOG. The Journal of Immunology, 2018, 201: 1119–1130.

he presence of B cells within active lesions in the CNS as the function of B cells (as APCs), their products (Abs and cyto- by guest on September 26, 2021 well as in the cerebrospinal fluid of multiple sclerosis (MS) kines), and their derived cells (Ab-secreting cells) in MS patho- T patients suggests that B cells may be involved in the genesis are not yet completely understood. Moreover, little is known etiopathology of the disease (1, 2), and intrathecal oligoclonal IgG about how secondary diversification of the BCR impacts neuro- bands are a diagnostic marker of MS (3). More recently, the success inflammatory processes. Studies performed using animal models of of B cell depleting therapies in the treatment of relapsing-remitting neuroinflammation such as experimental autoimmune encephalo- (4) and primary progressive MS (5) has provided further evidence myelitis (EAE) provide opportunities to gain further insight into the that B cells play a critical role in the MS disease process. However, mechanism(s) of B cell participation in MS disease processes. The role of B cells in EAE depends on the nature of the Ag used to induce the disease. C57BL/6 mice (H2b) are an EAE-susceptible *Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, strain in response to immunization with myelin oligodendrocyte Canada; †Center for Neuroinflammation and Experimental Therapeutics, Perelman glycoprotein (MOG), and the most commonly used MOG com- School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; ‡Department of Neurology, Perelman School of Medicine, University of Pennsylva- ponent to induce EAE is the MOG35–55 peptide (6). In the x nia, Philadelphia, PA 19104; and Toronto General Hospital Research Institute, MOG35–55 peptide model, B cells are dispensable for the induction University Health Network, Toronto, Ontario M5G 2C4, Canada of the disease, and B cell–deficient mice develop robust clinical 1G.G. and D.S.W.L. contributed equally to this work. disease upon immunization with MOG35–55 (7). Furthermore, ORCID: 0000-0002-7210-8358 (R.L.). mice subjected to anti-CD20–mediated B cell depletion prior to Received for publication May 22, 2018. Accepted for publication June 2, 2018. disease induction with MOG35–55 peptide exhibit exacerbated This work was supported by Multiple Sclerosis Society of Canada Grant EG2683. disease, indicating that B cells may play a regulatory role during Address correspondence and reprint requests to Dr. Jennifer L. Gommerman, Uni- the priming phase of the disease (8). Alternatively, EAE can also versity of Toronto, 1 King’s College Circle 7310, Toronto, ON M5S 1A8, Canada. be induced by immunizing mice with the extracellular domain of E-mail address: [email protected] rodent MOG, including rat or mouse MOG (residues 1–125). The The online version of this article contains supplemental material. induction of EAE with rodent MOG proteins instigates a robust Abbreviations used in this article: AID, activation-induced (cytidine) deaminase; activation of MOG-specific B cells, germinal center formation, CSR, class switch recombination; DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; hMOG, human MOG; LFB, Luxol Fast Blue; MHCII, and active production of MOG-specific Abs (9, 10). Although Abs MHC class II; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; are not required for pathogenesis in response to rodent MOG RGB, red/green/blue; rMOG, rat MOG; ROI, region of interest; SHM, somatic hyper- proteins, in these models, activated B cells have been shown to mutation; UNG, uracil DNA glycosylase; WT, wild type. play a role in presentation of MOG Ag to pathogenic T cells in the Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 CNS (11) and in the periphery (12). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700729 1120 CLASS-SWITCHED Abs ARE REQUIRED FOR EAE

Although both mouse and rat MOG (rMOG) conserve the same acidified water (reverse-osmosis and ultraviolet-sterilized), and housed under a 12 h light cycle. During EAE, mice were placed on heat pads, and diet was encephalitogenic MOG35–55 domain, the extracellular domain of human MOG (hMOG; residues 1–120) differs from rodent MOG at supplemented with breeder mash (Teklad; Envigo [2919]) to help prevent severe weight loss. All animal experiments were conducted with ethical several residues, including a proline for serine substitution at posi- approval from the University of Toronto Faculty of Medicine Animal Care tion 42 (13). Induction of EAE with hMOG depends on the presence Committee. The majority of the experiments were performed first in sepa- of B cells, and in this model, autoantibodies produced by B cells rately caged mice and then were repeated with cocaged and/or littermate play a major pathogenic role (7, 14, 15). Indeed, adoptive transfer of mice to confirm results. Abs from hMOG, but not MOG35–55 immunized mice, was capable Ags and EAE induction of rescuing disease in B cell–deficient mice immunized with hMOG (14). Furthermore, both anti-CD19– and anti-CD20–mediated B cell Recombinant hMOG and rMOG were provided by Drs. C. Linington and N. Ruddle. Both proteins were synthesized and purified as previously reported depletion alleviate disease severity in this EAE model (16, 17). (27, 29). Mice 6–8 wk of age were immunized s.c. with 100 mg of either Upon antigenic stimulation, B cells undergo secondary Ab di- hMOG or rMOG emulsified 1:1 in IFA (Sigma-Aldrich) containing 4 mg/ml versification by two DNA modification processes: class switch re- Mycobacterium tuberculosis (H37RA; Difco). Mice also received two combination (CSR) and somatic hypermutation (SHM). CSR takes 400-ng i.p. doses of pertussis toxin (List Biological Laboratories) on the day of immunization and 48 h later. Mice were weighed daily, and clinical place in IgH switch regions located upstream of the C region genes, disease was assessed according to a modified scale previously published. whereas SHM occurs in the V region genes. These two processes Briefly, the Giuliani scale (30), which has been used in other studies critically require the enzyme activation-induced (cytidine) deami- (31, 32), measures mobility impairments in each limb and the tail. Each nase (AID) (18). In B cells, AID transcription is induced by T cell– limb is graded from 0 (asymptomatic) to 3 (complete paralysis), and the tail is graded from 0 (asymptomatic) to 2 (limp tail). We added assessment dependent CD40L/CD40 interactions and/or T cell–independent Downloaded from of the righting reflex, which is scored from 0 to 2. Zero was assigned for a stimuli such as the TLR4 agonist LPS (18). normal righting reflex, 1 for slow righting reflex, and 2 for a delay of more The effector functions of an Ab are determined by its isotype, and than 5 s in the righting reflex. Each criterion was measured in 0.5 incre- an Ab isotype is altered by CSR, during which AID mediates de- ments. Thus, the modified composite scores range from 0 (nonsymptoms) amination of deoxycytosines in the core of switch regions, leaving to 16 (fully quadriplegic mouse with limp tail and significantly delayed righting reflex). Side-by-side comparison of the two scales showed that we both DNA strands with a high density of deoxyuracils. Uracil DNA were able to detect similar differences in clinical outcome between WT 2/2 glycosylase (UNG) removes deoxyuracils, yielding abasic sites that and Aicda mice (data not shown). http://www.jimmunol.org/ lead to double-strand breaks (19, 20). The double-strand breaks are then ligated via the process of nonhomologous end-joining. 818c5 Ab production and purification The Ab affinity maturation process is driven by Ag-mediated Hybridoma 818c5 was kindly provided by Dr. C. Linington via Dr. N. Ruddle. selection of B cell clones that, by virtue of the SHM process, Hybridoma cells were cultured in 30 ml of RPMI 1640 medium supple- have the highest affinity for Ag. SHM is characterized by the mented with 10% FBS (Wisent, St. Bruno, Quebec, Canada), 1% penicillin/streptomycin, 1% L-glutamine, 1% sodium pyruvate, 1% nones- introduction of single nucleotide substitutions in DNA at the Ig sential amino acids, and 0.05 mM 2-ME in a 75-cm2 flask for 72 h. Initial variable regions. Similar to CSR, this process is facilitated by AID, culture was combined with 1 l of culture medium (supplemented RPMI which deaminates deoxycytosines transforming G:C into G:U 1640) in a cell culture spin flask. After 1 wk of hybridoma cell expansion, mispairs. Deoxyuracils are then excised by UNG yielding abasic supernatants were recovered and filtered (0.22 mm) for Ab purification. by guest on September 26, 2021 sites. During replication, polymerases are recruited that execute the 818c5 Abs were purified using Protein G column according to the manufacturer’s recommendations. Following purification, Abs were dialyzed insertion of dNTPs into the newly synthetized strand opposite the against PBS, and the final concentration was determined by NanoDrop. abasic site, resulting in transition or transversion mutations (21). Therefore, AID deficiency in mice and humans results in a com- Quantification of anti-MOG Abs plete absence of CSR and SHM and, consequently, a deficiency in Serum samples and CNS tissues were obtained at the chronic phase of the circulating Ig of all classes except unmutated IgM (22, 23). In disease. Blood was collected and centrifuged at 1500 3 g to obtain serum. contrast, deficiency of UNG differentially affects CSR and SHM; Brain and spinal cord supernatants were obtained after mechanical disag- gregation of CNS tissue in PBS. Anti-hMOG Abs were analyzed by ELISA mice lacking UNG retain normal Ig affinity maturation, but their methodology. Ninety six-well plates (Nunc) were coated overnight at 4˚C ability to produce switched Ig in response to immunization is with 1 mg/ml hMOG in PBS followed by four washes with PBS 0.05% impaired in response to viral infection (24). Tween 20. Plates were blocked with PBS 2% BSA for 2 h at room tem- AID is not required for clinical presentation of EAE induced by perature, and serial dilutions of each serum sample were added at 100 ml/ MOG immunization (25). In contrast, Aicda2/2 mice im- well and incubated for 2 h at room temperature. Subsequently, 1 mg/ml anti– 35–55 IgG1-HRP or anti–IgM-HRP (100 ml/well) (Becton Dickinson) was added munized with hMOG exhibit drastically reduced clinical disease and incubated 1.5 h at room temperature. After four washes, bound MOG- symptoms compared with wild type (WT) controls (26, 27). In this specific Abs were detected with 3,30,5,50-Tetramethylbenzidine. OD was study, we induced EAE in Aicda2/2 and Ung2/2 mice with MOG read at 450 nm. For quantitative evaluation of anti-hMOG, a standard curve proteins, a context that supports activation of B cells and their was prepared with 818c5 Abs and added in duplicate at the same time as serum samples. A representative WT sample with a known anti-MOG Ig functional participation in the disease process, to evaluate the concentration was added to each plate as an internal control. impact of BCR secondary diversification on the production of pathogenic Abs and on Ag presentation. Measuring anti-MOG affinity

The affinity of anti-MOG Abs was assessed by NH4SCN elution as pre- Materials and Methods viously described (33). Serum was premeasured for anti-MOG IgG1 con- Mice centration, and 0.2 mg/ml concentration was selected as it provided a dynamic range of values sufficient to compass from negligible elution with WT mice were obtained from Charles River Laboratories or derived from NH4SCN to completely stripped (compared with blank wells) at our se- +/2 +/2 +/2 +/2 2/2 intercrosses of Aicda 3 Aicda or Ung 3 Ung mice. Aicda lected concentrations of NH4SCN. Serum samples were diluted to a con- 2/2 mice were obtained from Tasuku Honjo, Kyoto University (22), and Ung centration of 0.2 mg/ml anti-MOG IgG1 and bound to plates coated with mice were obtained from H. Krokan (28), Norwegian University of Science hMOG for 2 h at room temperature, followed by four washes with PBS and Technology (28) (via Dr. J.D. Noia, Institut de Recherches Cliniques de 0.05% Tween 20. Following incubation with serum and washes, bound Abs Montreál), respectively. The genotypes of the above models were confirmed were then treated with increasing concentrations of NH4SCN (0, 0.7, 1.4, by PCR. All animals were housed and bred at the University of Toronto 2.1, 2.8, 3.5, 4.2 M) for 15 min at room temperature. Samples were sub- animal facilities under a specific pathogen-free, closed caging system, pro- sequently incubated with anti–IgG1-HRP (1:2000) (Becton Dickinson) for vided with a standard irradiated chow diet (Teklad; Envigo [2918]) and 1 h at room temperature, then washed three times. Remaining bound Abs The Journal of Immunology 1121 were detected with 3,30,5,50-Tetramethylbenzidine, and the OD was read day, cells were transiently transfected with DNA (MOG-GFP) with at 450 nm. Affinity was calculated as the concentration of NH4SCN re- Lipofectamine 3000 Reagent (Thermo Fisher Scientific) according to quired to remove 50% of the bound Abs. manufacturer protocols and incubated overnight. Serum samples from WT, Aicda2/2,orUng2/2 mice were diluted 1:160 with culture medium and Assessment of spinal cord immunopathology placed onto cover slips for 30–60 min. The cover slips were transferred to new 24-well plates and washed with PBS. Cells were fixed in 4% para- Immune-mediated inflammation was analyzed at the chronic phase of the 2 2 formaldehyde, permeabilized with 0.3% Triton X-100, and then blocked disease 26 d after rMOG immunization of Aicda / mice or the peak phase 2 2 with 5% goat serum. DAPI and Alexa Fluor 555 Rat Anti-Mouse IgG1 or Ung / of disease 17 d after hMOG immunization of mice. At the time of IgM (SouthernBiotech) were used to detect nuclei and Abs, respectively. sacrifice, mice were intracardially perfused with 30 ml of ice-cold PBS. Slides were mounted using Fluoromount-G (SouthernBiotech) and dried The spinal columns were excised and postfixed in 10% buffered formalin prior to observation under a fluorescent microscope. Slides were quantified for 1 wk prior to being processed into paraffin. Seven-micron paraffin + by manually counting the number of cellular events that were GFP and coronal sections of mouse spinal cord were mounted on Superfrost Plus + + Alexa Fluor 555 , expressed as a ratio to the total number of GFP events. glass slides (Knittel Glass) and dried overnight at 37˚C. Paraffin sections were deparaffinated in xylene and rehydrated through a series of ethanol Statistical analysis washes. Histology was performed using standard H&E and Luxol Fast Blue (LFB) stains to visualize inflammation and demyelination, respec- Statistical analyses were performed with GraphPad Prism (GraphPad tively. Consecutive spinal cord sections were used for LFB and H&E. For Software) by using the nonparametric two-tailed Mann–Whitney U test, or, mice subjected to rMOG EAE, these two stains were performed in two sets in the case of EAE clinical disease course, two-way ANOVAwas used. For of nonconsecutive spinal cord sections from the thoracic level, one of analysis of histological sections, the variability of distribution was assessed which was used for quantification. For mice immunized with hMOG, these by Shapiro–Wilk normality test. For non–normally distributed data, the two stains were performed in three sets of nonconsecutive spinal cord nonparametric Mann–Whitney U test was performed. A difference was sections, all of which were further used for quantification analysis. Red/ considered to be statistically significant when p # 0.05. Downloaded from green/blue (RGB) images of H&E and LFB stains from thoracic spinal cord sections were acquired at original magnification 34 and 320 using a light microscope (Axioscope; Zeiss). Quantitative analysis of stains was Results 3 Transfer of anti-MOG IgG Abs restores disease in performed at original magnification 20, using ImageJ 1.15s (National 2 2 Institutes of Health). The perivascular white matter area was the chosen Aicda / mice region of interest (ROI) for the quantitative analysis. The RGB images were separated into single color channels using the color deconvolution Immunization with hMOG leads to EAE with clinical symptoms plugin. The single color channels for hematoxylin and LFB were subjected beginning at approximately day 9–10 postimmunization (onset) and http://www.jimmunol.org/ to thresholding to create a mask that captures the specific hematoxylin or peaking at day 12–16 (acute phase), followed by a decline (post- LFB staining. Equivalent ROI on each spinal cord examined was outlined. acute phase) and then a period of residual disease (chronic phase). The area fraction measurement was applied to each ROI to quantify the B cells from Aicda2/2 mice do not express or produce class- percentage of thresholded staining. The scale represents the percentage of stained perivascular white matter area in each spinal cord examined. switched Abs, and the IgM present in these mice is of low affinity. Consistent with prior studies (26, 27), we confirmed that Aicda2/2 Flow cytometry mice immunized with hMOG develop milder EAE compared with 2/2 +/2 At different time points of the disease (peak or chronic phase, indicated for WT mice (Fig. 1A), and comparison of Aicda mice to Aicda each group of mice), mice were sacrificed and intracardially perfused with littermate controls revealed similar results (Fig. 1B). 30 ml of ice-cold PBS. Brain and spinal cord were disaggregated in PBS AID could have a function during EAE that is related to the by guest on September 26, 2021 buffer with a tissue grinder. Samples were filtered through a 70-mm cell production of pathogenic class-switched and/or hypermutated Abs. strainer (BD Falcon) to obtain a single-cell suspension. Homogenized tissue was resuspended in 30% Percoll solution and centrifuged 20 min at Alternatively, expression of a BCR that has undergone secondary 900 3 g. A pellet containing mononuclear cells was resuspended in FACS diversification may be required for T–B interactions in the germinal buffer (10% FBS, 0.02% NaN3 PBS). Cells were incubated with LIVE/ center. To test the first possibility, we asked if hypermutated class- DEAD Fixable Aqua Reagent (Life Technologies) to discriminate between switched Abs could rescue disease in Aicda2/2 mice. Accordingly, dead and live cell populations. Subsequently, cells were labeled with anti- CD4, anti B220, and anti-CD19 (eBioscience). For intracellular cytokine we performed a passive transfer of anti-MOG IgG1 Abs (clone detection, CNS-derived single-cell suspensions were stimulated with PMA 818c5) that specifically bind to conformational epitopes of the ex- (50 ng/ml; Sigma-Aldrich), ionomycin (0.5 mg/ml; Sigma-Aldrich), and tracellular portion of MOG (36). It has been previously shown that Brefeldin A for 5 h for detection of proinflammatory cytokines. After passive transfer of serum specific for hMOG by injections at the day stimulation, cells were stained extracellularly with anti-CD4, then fixed of MOG immunization and every 3–4 d for a 2 wk period is suf- and permeabilized with Perm/Wash solution (Becton Dickinson). Finally, cytokines were detected with anti–IL-17 and anti–IFN-g (eBioscience). ficient to promote EAE in hMOG EAE-resistant B cell–deficient Staining was analyzed using a BD FACS LSRFortessa flow cytometer and mice (14). However, to eliminate the possibility that any other FlowJo (Becton Dickinson). component in the serum of hMOG-immunized mice could be 2/2 Measuring T cell activation responsible for inducing EAE in Aicda mice, we elected to passively transfer the 818c5 mAb, a class-switched (IgG1) MOG- Mice were immunized as previously described and sacrificed at day 9 specific Ab. Accordingly, Aicda2/2 and WT mice were first postimmunization. At time of sacrifice, mice were perfused with 30 ml of ice-cold PBS. Cervical, inguinal, axillary, and brachial lymph nodes were immunized with hMOG. Subsequently, either purified 818c5 Abs or collected, and single-cell suspensions were made by physical agitation isotype control Abs were injected i.v. at day 4 and day 11 post- through a 70-mm cell strainer (BD Falcon). In a 96-well plate, 500,000 immunization. Compared with the mild EAE exhibited in untreated cells were seeded per well with 10% complete medium (supplemented Aicda2/2 mice (Fig. 1A, 1B), Aicda2/2 mice that received 818c5 RPMI 1640) along with 0, 1, 2, or 5 mg/ml hMOG and cultured at 37˚C Abs rapidly developed EAE (Fig. 1C). In agreement with the and 5% CO2 for 72 h. Following stimulation, 100 ml of supernatant was 2/2 taken to measure IL-17 and IFN-g proteins by ELISA (Invitrogen) augmented severity of EAE in Aicda mice that received 818c5 according to manufacturer protocols. Cells were kept to measure prolif- Abs, body weight loss at the peak of the disease was more prom- eration via pulsing with [3H]-thymidine (PerkinElmer), and incorporation inent for 818c5-treated Aicda2/2 mice compared with Aicda2/2 was evaluated 18 h after pulsing by harvesting the cell cultures onto filter mice treated with isotype control Abs. In contrast, WT mice that paper and measuring cpm using a b-counter. received 818c5 Abs did not develop more severe EAE compared Detecting Ab reactivity to conformational MOG with mice receiving an isotype control Ab (Fig. 1D). Together, these To determine Ab reactivity to conformational MOG epitopes, we used results indicate that circulating pathogenic anti-MOG IgG Abs are previously defined bioassays (34, 35). HEK293 cells were split into 24-well sufficient to rescue CNS autoimmune inflammation provoked by the 2/2 plates overnight with a coverslip at 37˚C and 5% CO2. On the following extracellular domain of hMOG in Aicda mice. 1122 CLASS-SWITCHED Abs ARE REQUIRED FOR EAE

FIGURE 1. Impaired hMOG- induced EAE in Aicda2/2 mice is restored by a passive transfer of anti-MOG IgG1 Abs. Clinical scores (A–D) and weight loss (C and D)of animals were measured daily following immunization until date of harvest. (A) Separately caged WT (n = 25) and Aicda2/2 (n = 26) mice or (B) littermates from Aicda+/2 3 Aicda+/2 parents (WT n =5; Aicda2/2 n = 5) were immunized with hMOG1–120.(C) Following immunization, Aicda2/2 mice (n =5) were treated with 200 mgofanti-

MOG IgG1 Ab (clone 818c5) or an isotype control (n = 5) at days 4 and 11 postimmunization. (D) Following Downloaded from immunization, WT mice (n =5) were treated with 200 mg of anti- MOG IgG1 Ab (clone 818c5) or an isotype control (n = 4) at days 4 and 11 postimmunization. Arrows indicate i.v. injections of 818c5 Ab or an http://www.jimmunol.org/ isotype control. (A) Represents data pooled over three separate experiments. (B–D) Data shown are from a representative experiment from at least two experiments with similar outcomes. Means and SEM are shown. Statistical signiﬁcance was determined by two-way ANOVA (*p . 0.05, ***p . 0.001). by guest on September 26, 2021

Induction of EAE with rMOG does not require secondary with rMOG developed EAE with roughly similar severity as WT diversification of the BCR mice (Fig. 2A). The incidence of disease was 100% in both groups Our data show that the pathogenic role of AID during EAE is due, at of mice, and the average day of disease onset was not significantly different. However, subtle differences in the early versus later least in part, to its effect on secreted Abs; however, passive transfer 2/2 of 818c5 Ab may have masked an additional cell-intrinsic function stages of disease were observed, with Aicda mice exhibiting for AID. Specifically, AID may play a role in coordinating T–B slightly lower cumulative disease scores from the peak of disease 2/2 interactions by presenting Ag to encephalitogenic T cells to op- into the chronic phase (Table I). As expected, Aicda mice timize T cell priming and/or effector function. Whereas hMOG produced similar relative titers of anti-MOG IgM Abs as WT mice induces the production of pathogenic Abs that can transfer disease but not class-switched anti-MOG IgG1 (Fig. 2B, Supplemental (9, 14, 15) because of an amino acid difference in the MOG Fig. 1A). 35–55 + part of the protein, in which a proline at position 42 supplants a During EAE, activated CD4 T cells as well as B cells migrate serine (13), rodent MOG immunization does not elicit pathogenic into the CNS (38). Twenty-six days following immunization, we Abs, yet the peptide must nevertheless be processed and pre- therefore characterized cellular infiltrates in the brain and spinal 2/2 sented on MHC class II (MHCII) to elicit an encephalitogenic cord of WT versus Aicda mice during the chronic phase of T cell response (10, 29). Thus, rodent MOG immunization EAE (see representative FACS, Supplemental Fig. 2). Enumera- provides an opportunity to address whether pathways of in- tion of immune cells infiltrating the CNS by FACS revealed that + terest are required for Ag presentation, independent of Ab B cell and CD4 T cell numbers in the CNS were comparable in 2/2 production. Indeed, it has been shown that B cells cooperate both the spinal cord and the brains of Aicda mice and WT with dendritic cells (DCs) to enhance EAE severity and that in mice (Supplemental Fig. 3). Furthermore, CD4+ T cells that a scenario in which there is a high frequency of MOG-specific secrete proinflammatory cytokines (IFN-g and IL-17) in the brain B cells, expression of MHCII only on B cells is sufficient to and spinal cord were not significantly altered between WT and 2 2 drive EAE (37). Aicda / mice (Supplemental Fig. 3). However, despite a lack of To test whether secondary diversification of the BCR is required clinical disparity (Fig. 2A), histological analysis of the spinal cord for Ab-independent roles of B cells such as Ag processing and revealed increased inflammation (Fig. 2C, 2D) and demye- 2 2 presentation, Aicda2/2 and WT mice were immunized with the lination (Fig. 2E, 2F) in WT mice compared with Aicda / mice. 2/2 extracellular domain of rMOG1–120. Aicda mice immunized In WT mice, cellular infiltration and demyelination could be The Journal of Immunology 1123 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. WT and Aicda2/2 mice exhibit clinically similar EAE in response to rMOG. (A) Separately caged WT (n = 9) and Aicda2/2 (n = 10) mice were immunized with rMOG1–120, and clinical scores were measured daily until day 26 postimmunization. Data show a representative experiment from two 2/2 2/2 separate experiments with similar outcomes. (B) Titers of anti-rMOG IgM (WT n =8;Aicda n = 10) and IgG1 (WT n =6;Aicda n = 10) Abs in serum were determined by ELISA. Corresponding dilution curves used to calculate titers can be found in Supplemental Fig. 1A. Coronal sections of formalin-fixed paraffin-embedded thoracic spinal cords from each WT (n = 4) and Aicda2/2 (n = 5) mouse were subjected to H&E staining with a representative example for each genotype shown in (C). Subsequent blinded quantification using one coronal thoracic section for each animal is shown in (D). Coronal sections of formalin-fixed paraffin-embedded thoracic spinal cords from each WT (n = 4) and Aicda2/2 (n = 5) mice were subjected to LFB staining with a representative example for each genotype shown in (E). Subsequent blinded quantification using one coronal thoracic section for each animal is shown in (F). Please refer to Materials and Methods for quantification details. Arrows indicate examples of cellular accumulation (C) or demyelination (E) throughout the white matter area of the spinal cord. Means and SEM (A) or means and SD (B, D, and F) are displayed. Statistical significance was determined by using a two-way ANOVA (A) or a Mann–Whitney U test (B, D, and F)(*p . 0.05, ***p . 0.001).

observed throughout the white matter, but in Aicda2/2 mice, autoantibodies are dispensable, AID does not influence the clinical cellular infiltration was largely restricted to some perivascular course of EAE or the accumulation of B cells or cytokine- areas, whereas little or no demyelination was observed. Collec- secreting T cells in the CNS, despite a minor (subclinical) role tively, these results indicate that, in a model in which pathogenic in inflammation and demyelination in the spinal cord. 1124 CLASS-SWITCHED Abs ARE REQUIRED FOR EAE

Table I. Clinical disease severity at different phases of EAE induced by rMOG

Cumulative Score Day 0–13 Cumulative Score Day 14–26 p Value Onset (d) Incidence (%) Number of Mice per Group WT 23.3 6 4.4 87.5 6 16.4 0.02 10.5 6 1 100 9 Aicda2/2 34.9 6 13.4 72.1 6 15 0.04 11 6 1 100 10

Unraveling the role of CSR versus SHM in EAE self-antigen, and to correlate EAE severity with SHM, we ex- 2/2 Following the deamination of cytosine via AID, UNG processes amined the affinity of anti-MOG IgG1 Abs in WT versus Ung deoxyuridines to create double-strand breaks or point mutations that mice. To do this, we used an elution assay whereby Abs specific are subsequently required to initiate CSR or SHM, respectively. for plate-bound MOG Ag were stripped off with increasing levels Whereas the mutation spectrum of the BCR in Ung2/2 mice is only of NH4SCN, with the rationale being that the highest affinity anti- modestly altered (20), Ab affinity in response to immunization is MOG Abs would be most resilient to NH4SCN stripping, as has been shown before (33). We therefore employed this assay on largely unaffected (24). In contrast, switch to other Ab isotypes in 2/2 Ung2/2 mice is strongly impaired, suggesting that UNG preferen- serum derived from WT versus Ung mice harvested 21 d tially affects CSR. To determine the contribution of either CSR or postimmunization with hMOG, a time-point when we could detect anti-MOG IgG in the serum of WT and Ung2/2 mice (albeit at SHM during EAE, we therefore examined hMOG-induced EAE in 2 2

/ Downloaded from Ung2/2 mice. We observed that Ung2/2 mice developed signifi- reduced levels in Ung mice). Adding equivalent amounts of cantly milder clinical disease compared with WT mice from onset anti-MOG IgG1 from serum of mice of either genotype, we found that the relative affinity of IgG1 Abs was similar in the serum of to the chronic phase of the disease (Fig. 3A). In a separate exper- 2/2 iment, we found that Ung+/2 heterozygous mice exhibited similar WT versus Ung mice (Fig. 5A, Supplemental Fig. 1B). disease to WT littermates (Fig. 3B) and no obvious differences in Using the 818c5 anti-MOG IgG1 as a standard, we next subsequent analyses (data not shown); therefore, readouts from WT quantified levels of anti-MOG IgG1 in the periphery (serum) and and Ung+/2 littermates were considered as WT. In agreement with CNS (brain and spinal cord). The latter was examined because Abs http://www.jimmunol.org/ the reduced clinical scores in Ung2/2 mice, we found significantly that access the CNS promote the liberation of self-antigens that less cellular infiltration (Fig. 3C, 3D) and demyelination (Fig. 3E, provoke T cell responses (39). Coincident with the reduction in 3F) in the spinal cord of Ung2/2 mice compared with WT mice. In clinical scores (Fig. 3A, 3B), decreased levels of class-switched anti-hMOG IgG1 in the serum (Fig. 5B), brain (Fig. 5C), and WT mice, cellular infiltration was found in perivascular areas, 2/2 dorsal columns, and ventral columns of the spinal cord, whereas spinal cord (Fig. 5D) were observed in Ung mice compared demyelination was present throughout the white matter. Alterna- with WT mice. Moreover, we observed increased titers of anti- 2/2 MOG IgM in the serum (Fig. 5B) and polyclonal IgM in the brain tively, cellular infiltration and demyelination in Ung spinal cords 2/2 were largely restricted to ventral columns. (Fig. 5C) of Ung mice compared with controls (Note anti- MOG IgM was undetectable in the CNS, data not shown). by guest on September 26, 2021 2/2 T cell responses are unaltered in Ung mice Taken together, our data suggests that EAE severity in response to To determine if the observed decrease in clinical symptoms and spinal hMOG immunization correlates with the production of class- cord pathology in Ung2/2 mice was due to the reduced T cell acti- switched autoantibodies but not alterations in SHM in response vation, we first characterized if T cells from Ung2/2 mice responded to hMOG immunization. similarly ex vivo when restimulated with hMOG in a T cell activation UNG is required for optimal levels of IgG Abs against assay (Fig. 4A, 4B). Despite the discrepancy in clinical disease, we conformationally intact MOG found that T cells isolated from the lymph nodes of Ung2/2 mice prior to disease onset did not exhibit defects in their proliferative Previous studies have shown that recognition of conformationally intact capacity or proinflammatory cytokine production capacity when MOG is tantamount to the pathogenicity of MOG-specific autoantibodies (40). To determine if Abs produced by WT, Ung2/2,and restimulated ex vivo against hMOG. Therefore, the decreased EAE 2/2 severity observed in Ung2/2 mice was not due to defects in T cell Aicda hMOG-immunized mice could recognize conformationally intact MOG, we used MOG-GFP–transfected HEK293 cells in an priming in the periphery. Next, we enumerated the immune infiltrates 2/2 2/2 in the brain and spinal cord of WT and Ung2/2 mice during EAE by in vitro assay. Application of serum from WT, Ung ,andAicda flow cytometry (see representative FACS, Supplemental Fig. 4). In hMOG-immunized mice to transfected cells expressing MOG-GFP the CNS, we found no difference in the absolute number of infil- revealed equivalent levels of anti-hMOG IgM (conformational) in + + the serum of all three genotypes (Fig. 6A, 6C). In contrast, although trating CD4 TcellorCD4 T cells producing proinflammatory 2/2 cytokines in the brain or spinal cord of WT mice compared with we found that IgG1 from the serum of hMOG immunized Ung Ung2/2 mice (Fig. 4C, 4D). Together, these results demonstrate that mice could also bind hMOG, the proportion of hMOG coated by IgG1 Abs from Ung2/2 serum was lower compared with WT neither T cell priming in the lymph node nor T cell effector function 2/2 in the inflamed tissue (CNS) is impacted by UNG. serum(Fig.6B,6D).Asexpected,serafromAicda exhibited minimal recognition of MOG-GFP, as these mice do –/– During EAE, Ung mice produce fewer class-switched not produce class-switched Abs. Therefore, although class- autoantibodies switched Abs from both WT and Ung2/2 mice have the As it was unlikely that defects in T cell activation/function were propensity to recognize and bind conformational MOG, responsible for impaired hMOG-induced EAE in Ung2/2 mice, we hMOG-immunized WT mice have a higher concentration of 2/2 next characterized the Ab responses against MOG in WT and anti-MOG binding IgG1 Abs compared with Ung mice. Ung2/2 mice. The observation that Ung2/2 mice exhibit normal affinity maturation of the B cell response has only been examined Discussion in the context of foreign Ag (24). To ascertain if Ung2/2 mice Recent clinical studies of MS demonstrate that B cells are im- exhibit impaired affinity maturation in response to a persistent portant in the etiopathology of relapsing-remitting MS and possibly The Journal of Immunology 1125 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 3. Ung2/2 mice develop mild clinical EAE symptoms in response to hMOG. (A)WT(n = 3) and Ung2/2 (n =9)or(B)WT(n = 6) and Ung+/2

(n = 11) mice were immunized with hMOG1–120, and clinical scores were recorded daily until day 16 postimmunization. Coronal sections of formalin-fixed paraffin-embedded thoracic spinal cords from WT (n = 9) and Ung2/2 (n = 9) mice were subjected to H&E staining with a representative example for each genotype shown in (C). Subsequent blinded quantification on three nonconsecutive sections for each mouse is shown in (D). Coronal sections of formalin- fixed paraffin-embedded thoracic spinal cords from WT (n = 9) and Ung2/2 (n = 9) mice were subjected to LFB staining with a representative example for each genotype shown in (E). Subsequent blinded quantification on three nonconsecutive sections for each mouse is shown in (F). Examples of cellular infiltration (C) and demyelination (E) are denoted by arrows. Please refer to Materials and Methods for quantification details. Data are representative of four experiments with similar outcomes (WT n =4;Ung+/2 n =4;Ung2/2 n = 3). Means and SEM (A and B) or means and SD (D and F) are displayed. Statistical significance was determined by using a two-way ANOVA (A and B) or a Mann–Whitney U test (D and F)(*p . 0.05, ***p . 0.001). also primary progressive MS, a form of MS disease that has few therapy (an inhibitor of BAFF/APRIL) was shown to be delete- therapeutic options (5). However, despite these clinical advances, rious for some MS patients (41), more precise therapies for we do not know which types of B cells are pathogenic in treatment of MS are needed. Our goal was to unravel the role of MS. Because targeting a broad range of B cell subsets by atacicept BCR secondary diversification processes in EAE by using genetic 1126 CLASS-SWITCHED Abs ARE REQUIRED FOR EAE Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021 FIGURE 4. T cell responses are unaltered in Ung2/2 mice during EAE. T cell proliferation (A) and cytokine production (B) in response to various 2/2 concentrations of hMOG1–120 were measured in total lymph node cells harvested from WT Ung mice at day 9 postimmunization with hMOG. Following culture, cells were pulsed with [3H]-Thymidine to determine proliferative capacity, whereas IFN-g and IL-17 were measured in the supernatants by ELISA. At day 21 postimmunization, the absolute number of CD4+ T cells and cytokine-expressing CD4+IFN-g+, CD4+IL-17+, and CD4+IFN-g+IL-17+ T cells were determined in the brain (C) and spinal cord (D). Representative flow cytometry plots can be found in Supplemental Fig. 4. Statistical significance was determined by Mann–Whitney U test. tools that target the molecular mechanisms of BCR secondary Aicda2/2 mice receiving a passive transfer of anti-MOG IgG. diversification (Aicda2/2 and Ung2/2 mice) as well as different Investigation of these possible explanations would be a future EAE models (hMOG versus rMOG immunization). Using these direction resulting from this work. Nevertheless, although we did tactics, we found that although CSR of the BCR is critical for the not rule out the microbiome in the anti-MOG IgG1 transfer generation of pathogenic Ab and disease severity induced by studies, we did show that similar to separately bred/maintained hMOG, secondary diversification of the BCR was not needed for Aicda2/2 mice, Aicda2/2 mice derived from Aicda+/2 3 Aicda+/2 Ag presentation of rMOG to encephalitogenic T cells. These re- breeders fail to develop EAE in response to hMOG immunization sults provide important new information on the role of secondary compared with Aicda+/2 littermate controls (Fig. 1B). Therefore, diversification of the BCR in EAE and, by extension, identify animal husbandry practices, which can significantly affect the IgG class-switched B cells as potential therapeutic targets for the composition of the microbiome (43), are not a factor in the re- treatment of MS. sistance of Aicda2/2 mice to hMOG-induced EAE. Interestingly, Aicda2/2 mice immunized with hMOG that re- We also found that in contrast to hMOG immunization, the ceived a passive transfer of class-switched anti-MOG IgG1 Abs induction of EAE with rMOG protein induces EAE in Aicda2/2 not only developed EAE but presented with a slightly exacerbated mice with similar incidence and severity of disease to that of WT clinical presentation of EAE in response to hMOG compared with controls. This implies that whereas secondary diversification of similarly treated WT mice. Although we do not yet understand the BCR is dispensable for processing and presentation of rMOG why this may be the case, there are several possible explanations. to encephalitogenic T cells, other APCs (such as DCs) are suffi- For example, Aicda2/2 mice provided with anti-MOG IgG1 still cient to present rMOG to T cells in the presence of Aicda2/2 fail to produce other Ab isotypes such as IgE and IgA, which B cells, and indeed, B cell–deficient mMT mice develop rMOG themselves may play a regulatory role during neuroinflammation. EAE that is comparable to WT mice (9). However, histological Alternatively, the microbiome of Aicda2/2 mice, which has been analyses of rMOG-immunized mice revealed less inflammation shown to be dysbiotic compared with the microbiome of Aicda+/+ and demyelination in Aicda2/2 mice compared with WT controls, mice (42), may have indirectly exacerbated EAE symptoms in and we also observed subtle differences in disease severity in the The Journal of Immunology 1127 Downloaded from http://www.jimmunol.org/

FIGURE 5. Ung2/2 mice exhibit an impaired class-switch autoantibody response in response to hMOG. (A) Serum from WT (n =5;Ung+/2 n = 8) and Ung2/2 (n = 10) mice was diluted to a starting concentration of 0.2 mg/ml anti-MOG IgG1 and added to plates coated with hMOG, followed by washes with increasing concentrations of a stripping buffer (NH4SCN) to generate an affinity index. (B) Serum was measured for relative titers of anti-MOG IgM by guest on September 26, 2021 (WT n =4;Ung+/2 n =2;Ung2/2 n = 12) and anti-hMOG IgG1 (WT n =3;Ung+/2 n = 11; Ung2/2 n = 17). (C) Supernatants from brain homogenates were measured for total IgM (WT n =5;Ung+/2 n =8;Ung2/2 n = 10) and anti-hMOG IgG1 (WT n =6;Ung+/2 n =8;Ung2/2 n = 10). (D) Supernatants from spinal cord homogenates (WT n =4;Ung+/2 n =4;Ung2/2 n = 5) were measured for total IgM and anti-hMOG IgG1. Statistical significance was determined by Mann–Whitney U test (*p . 0.05, **p . 0.01, ***p . 0.001). later chronic phase of rMOG-induced EAE. Thus, AID presum- autoreactive T cells in the meninges of the CNS. Autoantibodies ably still exerts some subtle effect on rMOG-induced disease provide a means for concentrated delivery of opsonized myelin trajectory. In transgenic systems in which DCs are engineered to Ags to both peripheral and local Fc receptor–bearing phagocytes, be the only APCs capable of priming T cells, EAE is impaired, which in turn can process myelin Ags and present them to T cells implying that under such circumstances, cells other than DC (such (39, 46). In agreement, autoantibodies derived from neuromyelitis as B cells) play a role in priming T cell responses to rMOG (44). optica and MS patients have the capacity to opsonize myelin Ags, Moreover, in the context of high precursor frequencies of anti- potentially contributing to disease pathogenesis (39, 46). Thus, in MOG B cells, MHCII expression only on B cells is sufficient to cases in which anti-myelin T cell responses are weak or CNS Ag induce EAE (37). Interestingly, EAE induced with the extracel- availability is limited, autoantibodies may play a crucial role in lular domain of mouse MOG (1–125) in mice bearing a transgenic CNS inflammation by focusing Ag to local APCs, thus providing a BCR specific for an irrelevant Ag exhibit normal clinical disease means for perivascular T cells to enter the CNS parenchyma at disease onset; however, the late chronic phase of the disease is (47, 48). Anti-MOG IgM Abs should be able to activate phago- reduced (10). This indicates that although a MOG-specific BCR is cytosis by binding complement receptor–3 (49) and provoke dispensable to induce EAE, MOG-specific B cells may be relevant phagocytosis by binding the Fca/mR (50). However, the produc- during the late chronic phase of EAE. We hypothesize that tion of low-affinity anti-MOG IgM Abs in Aicda2/2 mice, and Aicda2/2 mice display subtle differences in rMOG-induced EAE IgM of normal affinity in Ung2/2 mice, is clearly insufficient to clinical severity and spinal cord pathologic condition because of a propagate EAE in response to hMOG immunization. disease-modifying role of B cells that have undergone secondary In MS, it has been suggested that autoreactive Abs serve as diversification of their BCR later in disease. mediators of demyelination in CNS lesions in which Igs and Thus far, the study of autoantibody pathogenesis in EAE has complement are found (51), with a significant skew toward Vh4 been limited to the role of autoantibodies on demyelination via usage (52). Also, in vitro studies have revealed that circulating engagement of inflammatory mechanisms such as complement IgG Abs from a subset of MS patients can mediate demyelination activation (45). However, it has been recently described that (53). Furthermore, the presence of oligoclonal bands in the cere- myelin-specific autoantibodies can contribute to EAE patho- bral spinal fluid of MS patients has been associated with a larger genesis by making available scarce endogenous myelin Ags to volume of cerebral spinal fluid, larger white matter lesions, and 1128 CLASS-SWITCHED Abs ARE REQUIRED FOR EAE Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Class-switched autoantibodies detected in the serum of WT and Ung2/2 mice recognize conformational epitopes of MOG. HEK293 cells transfected with MOG-GFP were overlaid with serum from WT (n =8),Aicda2/2 (n =6),orUng2/2 (n = 11) mice, and IgM (A)orIgG1(B) Abs were detected by secondary Abs tagged with Alexa Fluor 555 (AF555). DAPI staining for nuclei and merged RGB color images are shown for reference. (C and D)GFP+AF555+DAPI+ cellular events were counted and represented as a ratio of total GFP+DAPI+ cells. Images were captured at original magnification 320. Statistical significance was determined by Mann–Whitney U test (**p . 0.01, ***p . 0.001). The Journal of Immunology 1129 smaller regional gray matter volume (54), whereas the absence of 16. Chen, D., S. J. Ireland, L. S. Davis, X. Kong, A. M. Stowe, Y. Wang, W. I. White, R. Herbst, and N. L. Monson. 2016. Autoreactive CD19+CD20- plasma cells oligoclonal bands correlates with a benign MS course with less contribute to disease severity of experimental autoimmune encephalomyelitis. disability (55). We add to this knowledge by showing that whereas J. Immunol. 196: 1541–1549. affinity maturation of anti-MOG Abs does not correlate with 17. Monson, N. L., P. Cravens, R. Hussain, C. T. Harp, M. Cummings, M. de Pilar Martin, L. H. Ben, J. Do, J. A. Lyons, A. Lovette-Racke, et al. 2011. Rituximab disease, the production of class-switched IgG anti-MOG Abs is therapy reduces organ-specific T cell responses and ameliorates experimental critically required for disease propagation. These observations autoimmune encephalomyelitis. PLoS One 6: e17103. suggest that reducing the concentration of circulating IgG class 18. Xu, Z., H. Zan, E. J. Pone, T. Mai, and P. Casali. 2012. Immunoglobulin class- switch DNA recombination: induction, targeting and beyond. Nat. Rev. Immunol. autoantibodies in specific subgroups of MS patients may relieve 12: 517–531. disease symptoms. 19. Shen, H. M., A. Tanaka, G. Bozek, D. Nicolae, and U. Storb. 2006. Somatic hypermutation and class switch recombination in Msh6(-/-)Ung(-/-) double- knockout mice. J. Immunol. 177: 5386–5392. Acknowledgments 20. Rada, C., G. T. Williams, H. Nilsen, D. E. Barnes, T. Lindahl, and M. S. Neuberger. We thank Dr. Javier Di Noia for sending us the Ung2/2 mice. We thank 2002. Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr. Biol. 12: 1748–1755. Dr. Nancy Ruddle for sending us the constructs of rMOG and hMOG and 21. Di Noia, J. M., and M. S. Neuberger. 2007. Molecular mechanisms of antibody Dr. Chris Linington for generating these constructs. We thank Dionne somatic hypermutation. Annu. Rev. Biochem. 76: 1–22. White in the Faculty of Medicine core flow cytometry facility for technical 22. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, and assistance, Dr. Bryant Boulianne for technical advice on the anti-MOG T. Honjo. 2000. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. affinity assays, and Dr. Kate Banks in the Division of Comparative Med- Cell 102: 553–563. icine for help and guidance with animal experiments. 23. Revy, P., T. Muto, Y. Levy, F. Geissmann, A. Plebani, O. Sanal, N. Catalan,

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1 .5 WT 4

-/-

1 .2 Aicda 3 0 .9 2

0 .6

OD [450nm] OD OD [450nm] OD 1 0 .3

0 .0 0 1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 Dilution Dilution

B 4 1 0 0

3 7 5

IgG1 hMOG - 2 5 0

OD [450nm] OD 1 2 5

0 0

0 1 2 3 4 5 anti %bound of 0 1 2 3 4 5 Molarity of NH SCN Molarity of NH4SCN 4 C Serum IgM

2 .5 WT -/- Ung 2 .0

1 .5

1 .0 OD [450nm] OD 0 .5

0 .0 1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0 Dilution

Supplemental Figure 1. Representative dilution curves of serum antibodies from WT, Aicda-/-, and Ung-/- following rMOG- or hMOG-induced EAE. (A) Sera were diluted until loss of signal was observed in an anti-rMOG IgM (WT n=8, Aicda-/- n=10) or IgG1 (WT n=6, Aicda-/- n=10) ELISA. Representative means and S.D. are plotted. Note: Aicda-/- IgG1 was not detected. (B) Sera (n=23) was subjected to anti-hMOG IgG1 ELISA and incubated with various concentrations of NH4SCN to determine dissociation of binding as reported by O.D. Binding curves for each mouse were estimated by 4-parameter logistic regression and translated to the percentage of hMOG-bound by anti-IgG1. (C) Sera was diluted until loss of signal was observed in an anti-MOG IgM ELISA (WT n=4, Ung+/- n=2; Ung-/- n=12). Representative means and S.D. are displayed.

A A

SSC SSC SSC

FSC-A SSC-W Aqua

H -

SSC

- IFN

CD4 IL-17

-/-

Aicda

B220

IFN

IL-17 CD19 Supplemental Figure 2. Representative FACS gating strategy for single cell suspensions from the CNS of WT and Aicda-/- mice following rMOG-induced EAE. Following EAE, single-cell suspensions of brain or spinal cords were stimulated for 5 h with PMA and ionomycin and treated with Brefeldin A. Cells were gated on lymphocytes, singlets, and then live cells prior to distinguishing by extracellular markers. CD4 was used to gate for T helper cells and B cells were identified as CD19+B220+. T helper cells were further examined for intracellular expression of IFN-γ and IL-17.

A B Brain B Cells + CD4 N.S. N.S. N.S.

1 2 0 4 1 5

)

3 3 3 1 2

9 0 3

6 0 2 Brain Brain 6

3 0 Spinal Cord 1

Absolute Count (x10 Count Absolute Absolute Count (x10 Count Absolute 0 0 (x10 Count Absolute 0 -/- -/- -/- W T A ic d a W T A ic d a W T A ic d a C Spinal Cord Spinal Cord Spinal Cord + + + + + CD4 CD4 IFN-γ CD4 IL-17 N.S. N.S. N.S.

6 0 2 0 0 .8

)

3 3

4 5 1 5 0 .6

3 0 1 0 0 .4

1 5 5 0 .2

Absolute Count (x10 Count Absolute (x10 Count Absolute Absolute Count (x10 Count Absolute 0 0 0 .0 -/- -/- -/- W T A ic d a W T A ic d a W T A ic d a

Supplemental Figure 3. T cell responses and B cell numbers are unaltered in the CNS of Ung-/- mice during EAE induced by rMOG. (A) CD19+B220+ B cells in the CNS. (B) Absolute numbers of CD4+ T cells were measured in the brain and spinal cord (C). Of CD4+ T cells found in the spinal cord, intracellular expression of IFN-γ and IL-17 was measured. Values (WT n=5, Aicda-/- n=5) are plotted as means with S.D. Statistical significance was determined by Mann-Whitney U test. (N.S. = Not significant.)

- A

SSC SSC SSC

FSC-A SSC-W Aqua

WT or H

- +/-

Ung

γ SSC

- IFN

IL-17 CD4

-/-

Ung B220

γ -

IFN

IL-17 CD19

Supplemental Figure 4. Representative FACS gating strategy for WT and Ung-/- mice following hMOG-induced EAE. Following EAE, single-cell suspensions of brain or spinal cords were stimulated for 5 h with PMA and ionomycin and treated with Brefeldin A. Cells were gated on lymphocytes, singlets, and then live cells prior to distinguishing by extracellular markers. CD4 was used to gate for T helper cells and B cells were identified as CD19+B220+. T helper cells were further examined for intracellular expression of IFN-γ and IL-17.